European Journal of Cancer - ECCO - European CanCer Organisation [PDF]

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European Journal of Cancer

26th EORTC – NCI – AACR Symposium on Molecular Targets and Cancer Therapeutics Barcelona, Spain, 18 – 21 November 2014 Proceedings Book

Amsterdam • Boston • London • New York • Oxford • Paris • Philadelphia • San Diego • St Louis

European Journal of Cancer Editor-in-Chief:

Editors: Basic and Preclinical Research: Drug Development: Early Breast Cancer: Advanced Breast Cancer: Gastrointestinal Cancers: Genitourinary Cancers: Lung Cancer: Gynaecological Cancers: Head and Neck Cancer: Sarcomas: Melanoma: Neuro-oncology: Epidemiology and Prevention: Paediatric Oncology: Founding Editor: Past Editors: Editorial Office:

Alexander M.M. Eggermont Institut Gustave Roussy Villejuif, France Richard Marais, Manchester, UK Ulrich Keilholz, Berlin, Germany Jordi Rodon, Barcelona, Spain Kathleen I. Pritchard, Toronto, Canada David Cameron, Edinburgh, UK Volker Heinemann, Munich, Germany Michel Ducreux, Villejuif, France Karim Fizazi, Villejuif, France Mary O’Brien, London, UK Ignace Vergote, Leuven, Belgium Kevin Harrington, London, UK Jean-Yves Blay, Lyon, France Dirk Schadendorf, Essen, Germany Roger Stupp, Zurich, Switzerland Jan Willem Coebergh, Rotterdam, The Netherlands Rob Pieters, Rotterdam, The Netherlands Henri Tagnon Michael Peckham, London, UK; Hans-Jo¨rg Senn, St Gallen, Switzerland; John Smyth, Edinburgh, UK Elsevier, The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK Tel: +44 (0) 1865 843590, Email: [email protected]

EDITORIAL BOARD CLINICAL ONCOLOGY J.-P. Armand (France) A. Ayhan (Japan) R. Blamey (UK) M. Bolla (France) J. Boyages (Australia) N. Bru¨nner (Denmark) F. Cardoso (Portugal) J. Cassidy (UK) M. Castiglione (Switzerland) L. Cataliotti (Italy) L. Cheng (USA) H. Cody (USA) R. Coleman (UK) A. Costa (Italy) J. De Bono (UK) M.J.A. De Jong (The Netherlands) E. de Vries (The Netherlands) A. Dicker (USA) R. Dummer (Switzerland) F. Eisinger (France) S. Erridge (UK)

G. Ferrandina (Italy) H. Gabra (UK) H. Gelderblom (The Netherlands) B. Hasan (Belgium) J.C. Horiot (Switzerland) C. Huber (Germany) R. Jakesz (Austria) J. Jassem (Poland) D. Jodrell (UK) V.C. Jordan (USA) A. Katz (Brazil) M. Kaufmann (Germany) I. Kunkler (UK) L. Lindner (Germany) P.E. Lønning (Norway) P. Lorigan (UK) K. McDonald (Australia) R. Mertelsmann (UK) F. Meunier (Belgium) T. Mok (Hong Kong) D. Nam (Korea)

P. O’Dwyer (USA) J. Overgaard (Denmark) N. Pavlidis (Greece) J. Perry (Canada) P. Price (UK) D. Raghavan (USA) J. Ringash (Canada) J. Robert (France) A. Rody (Germany) D. Sargent (USA) M. Schmidinger (Austria) S. Sleijfer (The Netherlands) P. Sonneveld (The Netherlands) A. Sparreboom (USA) M. van den Bent (The Netherlands) M. Van Glabbeke (Belgium) G. Velikova (UK) U. Veronesi (Italy) A. Vincent-Salomon (France) A. Voogd (The Netherlands) E. Winquist (Canada)

BASIC, PRECLINICAL AND TRANSLATIONAL RESEARCH A. Albini (Italy) P. Allavena (Italy) F. Balkwill (UK) M. Barbacid (Spain) M. Broggini (Italy) C. Catapano (Switzerland) J. Collard (The Netherlands) E. Garattini (Italy)

A. Gescher (UK) R. Giavazzi (Italy) I. Hart (UK) W. Keith (UK) J. Lunec (UK) D.R. Newell (UK) G.J. Peters (The Netherlands) A. Puisieux (France)

V. Rotter (Israel) M. Schmitt (Germany) C.G.J. Sweep (The Netherlands) G. Taraboletti (Italy) P. Vineis (UK) N. Zaffaroni (Italy)

D. Forman (France) A. Green (Australia) K. Hemminki (Germany) C. Johansen (Denmark) L.A. Kiemeney (The Netherlands) E. Lynge (Denmark) M. Maynadie´ (France) H. Møller (UK) P. Peeters (The Netherlands)

A.G. Renehan (UK) S. Sanjose (Spain) M.K. Schmidt (The Netherlands) I. Soerjomataram (France) H. Storm (Denmark) L.V. van de Poll-Franse (The Netherlands) H.M. Verkooijen (The Netherlands) E. de Vries (The Netherlands) R. Zanetti (Italy)

G. Chantada (Argentina) F. Doz (France) A. Ferrari (Italy) M.A. Grootenhuis (The Netherlands) K. Pritchard-Jones (UK)

L. Sung (Canada) M. van den Heuvel-Eibrink (The Netherlands) M. van Noesel (The Netherlands)

EPIDEMIOLOGY AND PREVENTION B. Armstrong (Australia) P. Autier (France) J.M. Borras (Spain) C. Bosetti (Italy) H. Brenner (Germany) L.E.M. Duijm (The Netherlands) J. Faivre (France) S. Franceschi (France)

PAEDIATRIC ONCOLOGY C. Bergeron (France) A. Biondi (Italy) E. Bouffet (Canada) M. Cairo (USA) H. Caron (The Netherlands)

European Journal of Cancer Aims and Scope The European Journal of Cancer (EJC) is an international multidisciplinary oncology journal, which publishes original research, reviews, and editorial comments on basic and preclinical cancer research, translational oncology, clinical oncology – including medical oncology, paediatric oncology, radiation oncology, and surgical oncology, and cancer epidemiology and prevention. The EJC is the official journal of the European Organisation for Research and Treatment of Cancer (EORTC), the European CanCer Organisation (ECCO), European Association for Cancer Research (EACR) and the European Society of Breast Cancer Specialists (EUSOMA). For a full and complete Guide for Authors, please go to http://www.ejcancer.com Advertising information. 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Subscriptions are accepted on a prepaid basis only and are entered on a calendar year basis. Issues are sent by standard mail (surface within Europe, air delivery outside Europe). Priority rates are available upon request. Claims for missing issues should be made within six months of the date of despatch. Orders, claims, and journal inquiries: please contact the Elsevier Customer Service Department nearest you: St. Louis: Elsevier Customer Service Department, 3251 Riverport Lane, Maryland Heights, MO 63043, USA; phone: (800) 6542452 [toll free within the USA]; (+1) (314) 4478871 [outside the USA]; fax: (+1) (314) 4478029; e-mail: [email protected]. Oxford: Elsevier Customer Service Department, The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK; phone: (+44) (1865) 843434; fax: (+44) (1865) 843970; e-mail: [email protected]. 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You can also check our Author FAQs at http://www.elsevier.com/authorFAQ and/or contact Customer Support via http://support.elsevier.com. Language (usage and editing services). Please write your text in good English (American or British usage is accepted, but not a mixture of these). Authors who feel their English language manuscript may require editing to eliminate possible grammatical or spelling errors and to conform to correct scientific English may wish to use the English Language Editing service available from Elsevier’s WebShop http://webshop.elsevier.com/languageediting/ or visit our customer support site http://support.elsevier.com for more information. Illustration services Elsevier’s WebShop (http://webshop.elsevier.com/illustrationservices) offers Illustration Services to authors preparing to submit a manuscript but concerned about the quality of the images accompanying their article. 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AACR-NCI-EORTC International Conference e on

MOLECULAR TARGETS AND CANCER THERAPEUTICS

The Premier International Meeting Featuring Novel Cancer Therapeutics +2!)!.ƫĆġĊČƫĂĀāĆƫđƫ5*!/ƫ+*2!*0%+*ƫ!*0!.ƫđƫ+/0+*Čƫ
We look forward to seeing you in Boston! ORGANIZING COMMITTEE

SCIENTIFIC COMMITTEE CO-CHAIRPERSONS

Carlos L. Arteaga, MD Vanderbilt-Ingram Cancer Center, Nashville, TN (AACR)

Levi A. Garraway, MD, PhD Dana-Farber Cancer Institute, Boston, MA, USA (AACR)

James H. Doroshow, MD National Cancer Institute-DCTD, Bethesda, MD, USA (NCI)

Lee J. Helman, MD National Cancer Institute, Bethesda, MD, USA (NCI)

Roger Stupp, MD University Hospital Zurich (USZ), Zürich, Switzerland (EORTC)

Jean-Charles Soria, MD, PhD Institut Gustave-Roussy, Villejuif, France (EORTC)

ABOUT THIS CONFERENCE Join us for the premier international meeting featuring novel cancer therapeutics. This meeting brings together nearly 3,000 academics, scientists, and pharmaceutical industry representatives from across the globe to discuss innovations in drug development, target selection, and the impact of new discoveries in molecular biology.

WHAT PAST ATTENDEES SAID... “Very exciting conference with excellent speakers. Great opportunity to broaden my knowledge of cancer biology and therapeutics.” “I felt stimulated to work in cancer and science again! Thank you!”

For more information and to bookmark this important event, go to www.AACR.org/Targets15

v

Table of Contents

Letter of Welcome

vii

Societies’ Profiles Conference Committees

ix xi

Acknowledgement General Information

xii xiii

Travel Grants

xvii

CME Accreditation Scientific Programme Programme Overview

xvii xviii

Programme Details Abstracts Late Breaking Abstracts

xx 1 193

Presenting Authors List Author Index

201 205

Subject Index

229

FUTURE MEETINGS

AACR – NCI – EORTC International Conference on Molecular Targets and Cancer Therapeutics Boston, MA, USA, 5 – 11 November 2015 28th EORTC – NCI – AACR Symposium on Molecular Targets and Cancer Therapeutics Munich, Germany, 18 – 21 October 2016

As a significant number of cancers in Europe can be attributed to smoking a strict no-smoking policy will be enforced within all areas used by the conference.

28TH EORTC-NCI-AACR SYMPOSIUM

EORTC NCI AACR

2016 ‘MOLECULAR TARGETS AND CANCER THERAPEUTICS’

18 – 21 OCTOBER 2016

28TH EORTC-NCI-AACR SYMPOSIUM ON ‘MOLECULAR TARGETS AND CANCER THERAPEUTICS’ The 28th EORTC-NCI-AACR Symposium on ‘Molecular Targets and Cancer Therapeutics’ will bring together academics, scientists and representatives from the pharmaceutical industry to discuss innovations in drug development, target selection and the impact of new discoveries in molecular biology. During the last few years, numerous innovative agents have been discovered as a result of tremendous developments in the understanding of the molecular basis of cancer. Further clinical progress in cancer treatment will be accomplished mainly through the conduct of translational research projects, efficient new drug development and the execution of large, prospective, randomised, multicentre cancer clinical trials. This requires a joint and global approach and early and optimal exchange of information. Therefore, the Symposium will ensure the maximum amount of interaction and discussion through an exciting range of plenary sessions and lively workshops.

MUNICH, GERMANY

The future of cancer therapy

For forthcoming announcements about the 28th EORTC-NCI-AACR Symposium on ‘Molecular Targets and Cancer Therapeutics’ please bookmark www.eortc.be, or contact the Secretariat directly: c/o ECCO – the European CanCer Organisation Avenue E. Mounier, 83 B-1200 Brussels Belgium Tel.: +32 (0) 2 775 02 01 Fax: + 32 (0) 2 775 02 00 E-mail: [email protected] www.ecco-org.eu/ENA

SAVE THE DATE

vii

Letter of Welcome

Dear Colleagues, It is our pleasure to invite you the 26th EORTC–NCI–AACR Symposium on Molecular Targets and Cancer Therapies. This annual meeting is taking place alternatingly in the USA and in Europe (since 1978) and is the premier international symposium on drug development, innovative approaches, targeted agents including biomarkers and translational research. Over 2000 delegates are exchanging ideas and results to foster drug and cancer therapeutic development, with a particular focus on early clinical application. For this edition, we have revamped the programme, by adding more slots for oral presentations populated by abstracts that contain brand new data that has never been presented before. We have particularly focused our efforts in bringing more clinical data by attracting additional original phase I/II trials. The latest in immunotherapy, oncolytic viruses, epigenetic targets as well as drug resistance will be discussed by eminent speakers in plenary and workshop sessions. This year, to compliment the poster presentations, we will have “posters in the spotlight” sessions, where selected poster presenters will be given a podium to present their posters and answer any questions from delegates. There will be ample opportunities to network during the welcome reception, evening poster viewing sessions and the DNA networking event which will be held in the Casa Llotja de Mar, located on the sea front in the historical centre of Barcelona. The 26th EORTC–NCI–AACR Symposium on Molecular Targets and Cancer Therapeutics is the place to meet new collaborators and old friends; gaining new insights that will aid the development of more effective treatments for the patients. We welcome you to the meeting!

Jean-Charles Soria Scientific Chair

Roger Stupp EORTC President

ix

Societies’ Profiles

The European Organisation for Research and Treatment of Cancer (EORTC)

The EORTC, based in Brussels, Belgium, unites European cancer clinical research experts from all disciplines for trans-national collaboration. It links a network of over 2,500 collaborators from all disciplines involved in cancer treatment and research in more than 300 hospitals in over 30 countries and offers an integrated approach to drug development, drug evaluation programmes and medical practices. EORTC Headquarters in Brussels handles some 30 protocols that are permanently open to patient entry, over 50,000 patients who are in follow-up, and a database of more than 180,000 patients. The ultimate goal of the EORTC is to improve the standard of cancer treatment by developing new agents and innovative approaches and to test more effective treatment strategies using commercially available drugs, or surgery and radiotherapy. EORTC studies have contributed to success stories in drugs development including the registration of several drugs by the United States Food and Drug Administration and the European Medicines Agency. The EORTC has a proven track record in establishing new standards, e.g. RECIST, QOL, etc., and in changing clinical practice. EORTC Headquarters staff provides full clinical, scientific, operational, quality assurance, and regulatory support for clinical and translational research projects, and augments this support with strong expertise in biostatistics, clinical study design and methodology, endpoint definition and analysis, etc. The EORTC fully supports the idea of Health Technology Assessment (HTA) becoming standard practice for the approval of new drugs or new indications and, moreover, has agreed that high quality clinical trials in the future should be HTA compliant. The role of pragmatic data and related methodology need to be developed within multi-stakeholder platforms, and with this goal in mind, the EORTC participates in projects such as those put forth by the Innovative Medicines Initiative. Such activities make the EORTC one of Europe’s leading players in transforming experimental discoveries into new treatments with a significant clinical impact. Alongside its own scientific and clinical programme, the EORTC collaborates with a number of research institutions including the US National Cancer Institute, the National Cancer Institute of Canada, and many other national and international research groups worldwide. Funders of the EORTC include the EORTC Charitable Trust (with the support of national cancer leagues), the “Fonds Cancer”, private donations and corporate sponsorship, annual grants allocated by BELSPO (the Belgian Federal Science Policy Office) and by the Belgian National Lottery, and, for specific research projects, the European Commission. Since the early 1970’s, EORTC headquarters has received continuous support from the US NCI. Support is also received from the pharmaceutical industry for specific studies. www.eortc.org

x

Societies’ profiles

The National Cancer Institute (NCI)

The National Cancer Institute (NCI) is part of the National Institutes of Health (NIH), which is one of 11 agencies that compose the United States (U.S.) Department of Health and Human Services (HHS). The NCI, established under the National Cancer Institute Act of 1937, is the Federal Government’s principal agency for cancer research and training. The National Cancer Act of 1971 broadened the scope and responsibilities of the NCI and created the National Cancer Program. Over the years, legislative amendments have maintained the NCI authorities and responsibilities and added new information dissemination mandates as well as a requirement to assess the incorporation of state-of-the-art cancer treatments into clinical practice. The National Cancer Institute coordinates the National Cancer Program, which conducts and supports research, training, health information dissemination, and other programs with respect to the cause, diagnosis, prevention, and treatment of cancer, rehabilitation from cancer, and the continuing care of cancer patients and the families of cancer patients. Specifically, the Institute: • Supports and coordinates research projects conducted by universities, hospitals, research foundations, and businesses throughout the U.S. and abroad through research grants and cooperative agreements. • Conducts research in its own laboratories and clinics. • Supports education and training in fundamental sciences and clinical disciplines for participation in basic and clinical research programs and treatment programs relating to cancer through career awards, training grants, and fellowships. • Supports research projects in cancer control. • Supports a national network of cancer centers. • Collaborates with voluntary organizations and other national and foreign institutions engaged in cancer research and training activities. • Encourages and coordinates cancer research by industrial concerns where such concerns evidence a particular capability for programmatic research. • Collects and disseminates information on cancer. • Supports construction of laboratories, clinics, and related facilities necessary for cancer research through the award of construction grants.

The American Association for Cancer Research (AACR)

Founded in 1907, the American Association for Cancer Research (AACR) is the world’s first and largest professional organization dedicated to advancing cancer research and its mission to prevent and cure cancer. AACR’s membership includes 34,000 laboratory, translational and clinical researchers; population scientists; other health care professionals; and cancer advocates residing in more than 90 countries. The AACR marshals the full spectrum of expertise of the cancer community to accelerate progress in the prevention, biology, diagnosis and treatment of cancer by annually convening more than 20 conferences and educational workshops, the largest of which is the AACR Annual Meeting with more than 17,000 attendees. In addition, the AACR publishes seven peer-reviewed scientific journals and a magazine for cancer survivors, patients and their caregivers. The AACR funds meritorious research directly as well as in cooperation with numerous cancer organizations. As the Scientific Partner of Stand Up To Cancer, the AACR provides expert peer review, grants administration and scientific oversight of individual and team science grants in cancer research that have the potential for near-term patient benefit. The AACR actively communicates with legislators and policymakers about the value of cancer research and related biomedical science in saving lives from cancer. For more information about the AACR, visit www.AACR.org

xi

Conference Committees

Organising Committee EORTC NCI AACR

Roger Stupp Jean-Charles Soria James Doroshow Lee Helman C.L. Arteaga Jeff Engelman

EORTC President Scientific Chair NCI President Scientific Co-Chair AACR President Scientific Co-Chair

Executive Scientific Committee Jean-Charles Soria (France), Scientific Chair Lee Helman (USA), Scientific Co-Chair Jeff Engelman (USA), Scientific Co-Chair Josep Tabernero (Spain) Anthony Tolcher (USA)

Kapil Dhingra (USA) Roger Stupp (Switzerland)

Scientific Committee Jeffrey Abrams Carlos L. Arteaga Udai Banerji Philippe Bedard Emiliano Calvo Lewis Cantley Anthony Chan Jerry M. Collins Barbara Conley Sara A. Courtneidge William Dahut Kapil Dhingra James Doroshow Susan Galbraith Levi A. Garraway James Gulley Toby Hecht Monika Hegi Lee J. Helman David Hong Axel Hoos Maria Koehler Scott Kopetz Shivaani Kumar Annette Larsen

USA USA UK Canada Spain USA Hong Kong USA USA USA USA USA USA United Kingdom USA USA USA Switzerland USA USA USA USA USA USA France

Emma M. Lees Elaine Mardis Paul Meltzer Gordon B. Mills Frits Peters Ruth Plummer Yves Pommier Victoria M. Richon Neal Rosen Jan Schellens Joel Schneider Jeffrey E. Settleman Alice T. Shaw Kevan Shokat David B. Solit Patricia S. Steeg Charles Swanton Josep Tabernero Beverly A. Teicher Anthony Tolcher Joseph E. Tomaszewski Matthew G. Vander Heiden Yi Long Wu James Zwiebel

USA USA USA USA Netherlands United Kingdom USA USA USA Netherlands USA USA USA USA USA USA United Kingdom Spain USA USA USA USA China USA

xii

Acknowledgement

EORTC, NCI and AACR would like to gratefully acknowledge the following companies for supporting the 26th EORTC–NCI–AACR Symposium on Molecular Targets and Cancer Therapeutics: Major Sponsor:

Silver Partners:

Grant Support:

EORTC, NCI and AACR would like to thank the following sponsors and exhibitors for their support to this meeting. This list reflects the companies/organisations confirmed at the time of going to print. Company/Organisation

Booth

Company/Organisation

Booth

Advance Cell Diagnostics

D1

Horizon Discovery

A10

Affymetrix

C9

Indivumed

B2

Almac

C6

Medical Prognosis Institute

C1

Biocrates Life Sciences

C4

Novella Clinical

A4

Cancer Research Technology

C2

Oncodesign SA

A5

Cellecta, Inc.

A15

Oncotest GmbH

B1

Champions Oncology

B9

OracleBio Limited

B3

Charles River

C10

PerkinElmer Inc.

C5

Crown BioScience

A2

Proqinase GmbH

B4

Cureline, Inc.

A6

Silicon Biosystems

A9

Dove Medical Press

A11

Studylog Systems

A1

Epistem Ltd

C3

Sysmex Inostics

C8

European Association for Cancer Research (EACR)

C11

vivoPharm

B13

Xentech

A3

GSK

A13

xiii

General Information

Symposium Secretariat

ECCO − the European CanCer Organisation Avenue E. Mounier, 83 B-1200 Brussels − Belgium E-mail: [email protected] During the Symposium, the Secretariat can be reached at +32 (0)2 880 15 21

Venue

Centre de Convencions Internacional Barcelona (CCIB) Rambla Prim 1−17 ES-08019 Barcelona − Spain Tel.: +34 (0)93 230 10 00

App

All attendees may download the free ECCO App on iPhone, iPad, or Android supported devices. Features include EORTC–NCI–AACR 2014 related information and news. The App contains the complete list of sessions, session types, speakers and exhibitors. Users can save their selected sessions, notes, favourites, as well as export sessions to their smartphone calendar. To download the App, search for ECCO cancer in iTunes or Google Play. Learn more at www.ecco-org.eu/app or use the QR code for direct download.

Badges

For security reasons, delegates are requested to wear their badge at all times during the Symposium. Delegates who have lost their badge can obtain a new one at the registration desk. A replacement fee of 75 EUR per participant will be charged.

Catering

Coffee breaks Coffee breaks courtesy of the organisers have been scheduled as follows: Tuesday 18 November: 14:45–15:15 Wednesday 19 November: 09:45–10:15 and 15:20–16:00 Thursday 20 November: 10:00–10:30 and 15.35–16:00 Friday 21 November: 10:30–11:00 Coffee breaks will take place in the catering areas of the exhibition except for Friday 21 November when the coffee break will take place in the poster area. Lunches Complimentary lunches will be served in the exhibition catering areas: Tuesday 18 November: 12:00–13:00 Wednesday 19 November: 12:00–13.15 Thursday 20 November: 12.30–13:30 During the poster viewing sessions, local tapas and drinks will be served. Delegates can also purchase food and beverages from the cash bar near the entrance of the Convention Centre. Free water fountains are located throughout the Convention Centre.

xiv

General information

Certificate of Attendance

To print your certificate of attendance after the Symposium, you will need your badge code number, so please keep your badge for this purpose. You can print your certificate of attendance via this link: http://www.ecco-org.eu/ENA1014 or in the Internet Zone located on the venue ground floor (as of Wednesday 19 November). Online certificates will be available until the end of December 2014. Please note that the Symposium Secretariat will not post certificates to participants after the event.

City Information

Barcelona Convention Bureau will operate a tourist and tour desk in the registration area as of Tuesday 18 November.

Cloakroom

A cloakroom is situated in the entrance hall near the registration area. Cloakroom Opening Hours Tuesday 18 November: 10:00–20:00 Wednesday 19 November: 07:30–19:30 Thursday 20 November: 07:30–19:30 Friday 21 November: 08:30–13:30 The price for this service is 2 EUR per item.

Exhibition

The EORTC–NCI–AACR 2014 Exhibition is held on the ground floor of the Convention Centre. Entrance is free for registered delegates. Exhibition Opening Hours: Tuesday 18 November: Wednesday 19 November: Thursday 20 November:

12:00–18:30 09:45–16:00 09:45–16:00

First Aid

A first aid room is located on level −1. In case of emergency, please inform the nurse on duty via +34 697 324 433 and/or dial internal the extension number 257 from any internal phone in the Convention Centre.

Insurance

The organisers of the EORTC–NCI–AACR 2014 Symposium do not accept liability for individual medical, travel or personal insurance. Participants are strongly advised to take out their own personal insurance policies. The organisers of the EORTC–NCI–AACR 2014 Symposium accept no responsibility for loss due to theft or negligence.

Internet WIFI access

General WIFI access is available throughout the Convention Centre. To access WIFI, activate the WIFI network on your laptop or device, select the network listed as ENA2014, and enter the password ENA2014.

Internet Zone

The official EORTC–NCI–AACR Internet Zone is available free of charge during the Symposium. The terminals provide you with the following services: Internet browsing, access to web-based mail, the Symposium searchable programme and exhibitor information.

General information

xv

Language and translation The official language of the Conference is English. No simultaneous translation is provided. Lost and Found

All enquiries should be directed to the registration helpdesk in the entrance hall. Participants are advised to mark their own Symposium bag and programme book with their name. The organisers accept no responsibility for loss due to theft or negligence.

Media

Press Office Opening Hours Tuesday 18 November: Wednesday 19 November: Thursday 20 November: Friday 21 November:

10:00–18:00 08:00–18:00 08:00–18:00 09:00–13:00

Entry to the Press Office is restricted to accredited media staff only. The Press Office is located in Room M221. Posters

Posters will be displayed in the exhibition area on the ground floor. Poster presentations: Poster presentations are scheduled on: Wednesday 19 November: 18:00–19:30 Thursday 20 November: 18:00–19:30 Friday 21 November: 09:00–10:30 Poster Viewing Poster presenters will present their work and answer questions from delegates. During this session, local tapas and drinks will be served.

Posters in the Spotlight Session

The Scientific Committee will make a special selection of posters which will be highlighted through short oral presentations (5 min + 5 min discussion each). Take this opportunity to discuss some of the hottest news at the Symposium! These spotlight sessions will take place in the exhibition area on Wednesday 19 and Thursday 20 November during the lunch break.

Registration

EORTC–NCI–AACR 2014 is open to all registered participants. Your official delegate name badge is required for admission to the Convention Centre and all Symposium events. For security reasons, participants are requested to wear their badge at all times. Registration Opening Hours: Monday 17 November: Tuesday 18 November: Wednesday 19 November: Thursday 20 November: Friday 21 November:

Registration Package

15:00–18:00 08:00–18:00 07:00–18:00 07:00–18:00 08:00–12:00

The full Symposium registration package includes: • Entry to all scientific sessions, and to the Welcome Reception on Tuesday 18 November; • Entry to the exhibition; • Proceedings book; • EORTC–NCI–AACR 2014 coffee breaks and complimentary lunches; • Internet access via the internet zone and WIFI access in the Convention Centre.

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General information

The day registration package includes: • Access to all scientific sessions on that day; • Entry to the exhibition; • Proceedings book (depending on availability); • EORTC–NCI–AACR 2014 coffee breaks and lunch on that day; • Internet access via the internet zone and WIFI access in the Convention Centre. Speaker Preview Room

The Speaker Preview Room is located on the ground floor. Speakers are requested to bring their PowerPoint presentations to the speaker preview room at least 4 hours before their session starts or one day in advance if the session starts early in the morning. To avoid breaks between speakers, NO laptop presentations are foreseen in session rooms. Speaker preview room opening Tuesday 18 November: Wednesday 19 November: Thursday 20 November: Friday 21 November:

Social Events

hours: 11:00–18:30 07:00–18:30 07:00–18:30 08:00–12:00

Welcome Reception All registered participants are invited to the Welcome Reception, organised on Tuesday 18 November, in the exhibition hall. The Welcome Reception starts at 17:30. Networking Event “It’s all about DNA” Thursday 20 November from 20:00. Casa Llotja de Mar, Passeig d’Isabel II, 1 08003 Barcelona Casa Llotja de Mar, located on the sea front in the historical centre of Barcelona, is one of the most significant and splendid monuments representing the economy and culture of the city. It was built in the second half of the 14th century, the most brilliant period of Catalan Gothic. Price per person: 65 EUR How to get there: From the Convention Centre, walk to the El Maresme/F`orum subway station and take the L4 towards Trinitat Nova. Get off at Barceloneta station. Head west on Pia del Palau toward Plarca de Pau Vila. Turn left onto Passeig d’Isabel II. The Casa Llotja de Mar will be on the right.

Social media

We recommend and support Twitter as the main social platform during the Symposium − tweet and follow the latest updates about the 26th EORTC– NCI–AACR Symposium on Twitter using #ENA2014 Find links, tutorials and tips: www.ecco-org.eu/social

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Travel Grants

Through the generosity of its sponsors, EORTC, NCI and AACR have awarded 15 travel grants to attend the 26th EORTC–NCI–AACR Symposium in Barcelona. Baxter, Merissa

USA

Mittal, Deepak

Australia

Bhattacharya, Bhaskar

Singapore

Pettitt, Stephen

United Kingdom

Campagne, C´ecile

USA

Rose, April

Canada

Choi, Min-ah

South Korea

Sahay, Debashish

France

Eun, Young-Gyu

South Korea

Sos, Martin

USA

Goldstein, Rebecca

USA

Willetts, Lian

Canada

Kim, Jisun

Korea

Zoni, Eugenio

Netherlands

Mantaj, Julia

United Kingdom

CME Accreditation The 26th EORTC–NCI–AACR Symposium on ‘Molecular Targets and Cancer Therapeutics’ is accredited by the European Accreditation Council for Continuing Medical Education (EACCME) to provide the following CME activity for medical specialists. The EACCME is an institution of the European Union of Medical Specialists (UEMS), www.uems.net The 26th EORTC–NCI–AACR Symposium is designated for a maximum of 18 hours of European external CME credits. Each medical specialist should claim only those hours of credit that he/she actually spent in the educational activity. The EACCME credit system is based on 1 ECMEC per hour with a maximum of 3 ECMECs for half a day and 6 ECMECs for a full-day event. European Accreditation is granted by the EACCME in order to allow participants who attend the above-mentioned activity to validate their credits in their own country. Through an agreement between the European Union of Medical Specialists and the American Medical Association, physicians may convert EACCME credits to an equivalent number of AMA PRA Category 1 Credits™. Information on the process to convert EACCME credit to AMA credit can be found at www.ama-assn.org/go/internationalcme Live educational activities, occurring outside of Canada, recognised by the UEMS-EACCME for ECMEC credits are deemed to be Accredited Group Learning Activities (Section 1) as defined by the Maintenance of Certification Program of The Royal College of Physicians and Surgeons of Canada.

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Scientific Programme – Overview

Tuesday18November

Wednesday19November

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Thursday 20 November

Friday 21 November

Auditorium 09.00 08.00

Plenary Session 4

10.00

Antibody-Based Therapies (ADC and others)

Poster Viewing (Poster Area)

10:30 Coffee Break

Coffee Break

10.30

Auditorium

Plenary session 5

12.30 Lunch 12.30-13.30 Including Posters in the Spotlight Session

13.30

Plenary Session 6

Proffered Paper Session

15.35 Coffee Break 16.00

Plenary Session 7 Novel Mechanisms for Drug Resistance

17.50 18.0019.30

Poster Viewing (Poster Area)

Networking Event

Posters Chemoprevention Clinical Methodology DNA Repair Modulation (including PARP, CHK, ATR, ATM) Drug Delivery Drug Design Molecular Targeted Agents I Paediatric Oncology Toxicology

Exhibition 09:45 - 16:00

11.00 Epigenetic Targets

Plenary session 8 Targeting RAS and Other Driver Oncogenes

13.00

Posters Drug Synthesis Molecular targeted agents II New Therapies with Pleiotropic Activity

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Scientific Programme – Details

Tuesday 18 November 2014 Opening Ceremony 13:00–13:15

13:15–14:00

Opening 13:00 13:05 13:10

Auditorium Remarks J.C. Soria (France) L.J. Helman (USA) J.A. Engelman (USA)

EORTC NCI AACR

Michel Clavel Lecture Chair: J.C. Soria (France)

Auditorium

13:15 Rational combination therapies for cancer R. Bernards (Netherlands) 14:00–14:45

Keynote Lecture Chair: A.M.M. Eggermont (France)

Auditorium

14:00 Immunocheckpoints − Gateway to Immunotherapy D. Chen (USA) Objectives: 1. Understand how cancer immunotherapy works, the cancer-immunity cycle and what makes it different from other forms of cancer therapy. 2. Understand what rationale approaches can be taken to monotherapy and combination therapy involving cancer immunotherapy. 3. Understand the role for biomarkers in understanding emerging immune biology, interpreting clinical results and identifying optimal treatment decisions. Key Messages: 1. Our understanding of how the immune system interacts with cancer is rapidly evolving. 2. Cancer immunotherapy approaches may be able to generate durable responses in patients with metastatic cancer. 3. Emerging biomarkers may be able to help us understand biology, define combination approaches and choose between therapeutic options. Plenary Session 1 15:15–17:30 Is the Genomic Landscape Changing the Outcome for Cancer Patients? Chairs: R. Stupp (Switzerland) and J. Tabernero (Spain) 15:15 Overview of academic precision medicine trials P. Bedard (Canada) 15:35 Lessons from SAFIR01 trial F. Andre (France) Main objectives: 1. Understand the pillars of personalised medicine. 2. Understand the main reasons of failures to deliver personalised medicine. 3. Understand what are the possible solutions to address these limitations.

Auditorium Abstract number

Scientific programme – details

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Key messages: 1. There is a need for randomised trials before implementing multigene tools for personalised medicine. 2. Drug availability and rules to interpret genome data are mandatory. 3. Genome analysis will not provide all the informations needed to deliver personalised medicine. 15:55 Translating gene expression signatures into clinical practice: prospects and challenges in the context of ‘next-generation medicine’ R. Dienstmann (USA) Key messages: 1. There are unique issues with high-dimensional data that represent obstacles to generating performant genomic signature classifiers and translating initial research findings into robust diagnostics. 2. Better understanding of the intrinsic gene expression-based subtypes of a histopathologically defined cancer, independent of their prognostic and predictive values, may also lead to new biological insights and eventually to development of novel therapies directed toward homogeneous molecular subsets. 3. A rational and focused approach to the evaluation of genomic markers is needed, whereby analytically validated assays are prospectively investigated in clinical trials with adaptive designs that take into consideration primary–metastatic site tumour heterogeneity and clonal evolution in the decisionmaking process. 16:15 Genomic characterisation of cancer: Case studies E.R. Mardis (USA) Key objectives: 1. Provide detailed description of the molecular assays being used to characterise the cancer mutations and their expression in RNA for individual patients. 2. Describe the analytical approaches that permit integrated analysis of mutations and RNA expression toward two therapeutic ends: (1) identification of small molecule therapies that may provide relief of tumour burden, (2) identification of immunoepitopes that may contribute toward a personalised vaccine strategy for individual patients. 3. Illustrate the two aforementioned objectives by detailed descriptions of specific case studies from our work in this area of applied cancer genomics. 16:35 Screening Patients for Efficient Clinical Trial Access (SPECTA) D. Lacombe (Belgium) Key messages: 1. Cancer drug development is undergoing profound changes and the path to registration will be substantially altered due to challenges of big data (omics). 2. The forms and the methods of cancer clinical research are evolving with the need to integrate new technologies to understand biology early on in development. New efficient platforms for patient access (molecularly defined subgroups) are needed. 3. Clinical oncology will need to take into account new type of guidelines for treatment decision with an increased role played by molecular advisory boards. 16:55 ORAL PRESENTATION: Feasibility of large-scale genomic testing to facilitate enrollment on genomically-matched clinical trials F. Meric-Bernstam, L. Brusco, S. Kopetz, M. Davies, M.J. Routbort, S.A. Piha-Paul, R. Alvarez, S. Khose, J. DeGroot, V. Ravi, F. Janku, D. Hong, Y. Li, R. Luthra, K.P. Patel, R. Broaddus, K. Shaw, J. Mendelsohn, G.B. Mills 17:10 General discussion J. Tabernero (Spain) 17:15 Faster execution of clinical trials − has bioinformatics the solution? G. McVie (Italy)

1

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Scientific programme – details

Wednesday 19 November 2014 Workshop 1 08:00–09:45

Auditorium Management of Toxicity of Molecular Targeted Agents Chairs: J.C. Soria (France) and P. Bedard (Canada) 08:00 Cardiac toxicities with MOA and TKI S. Ederhy (France) 08:20 Digestive and endocrine toxicities of checkpoint inhibitors N. Chaput (France) Main objectives: 1. To describe digestive toxicities (clinic/endoscopic/histological) in patients treated with anti-CTLA4 and/or anti-PD1. 2. To describe immune disorders in patients developing digestive toxicities (blood and colonic biopsies). Key messages: 1. Anti-CTLA-4 and/or anti-PD-1, monoclonal antibody treatment of cancer, are associated with several phenotypes of enterocolitis. 2. Intestinal inflammation observed in patients who were prescribed anti-CTLA-4, anti-PD1 or both, have distinct phenotypes: anti-CTLA-4 is associated with acute, often severe colitis, chronic duodenitis and less often with ileal and anal lesions. Anti-PD-1 seems to be associated with mild, chronic colitis. The combination of anti-CTLA-4 and anti-PD1 seems to lead to severe duodenal inflammation and severe colitis. 3. Anti-CTLA-4 colitis appears as a model of T-cell induced colitis in humans as suggested by the immune monitoring of patients. 08:40 Management of pulmonary toxicity due to targeted anticancer agents J. Porter (United Kingdom) 09:00 Ocular toxicity of MEK inhibitors and other targeted therapies J.P. Velazquez-Martin (Canada) Main objectives: 1. Identify the key agents associated with ocular toxicities. 2. Identify symptoms and clinical findings associated with ocular toxicity. 3. Learn the management, follow up and prognosis of these patients. Key messages: 1. Ocular toxicities from targeted therapies are frequent. 2. Symptoms range from none to moderate visual impairment. 3. Always consult with an ophthalmologist or retina specialist (preferable). 09:20 Discussion Workshop 2 08:00–09:45

Room 117 Cancer metabolism Chairs: C. Van Dang (USA) and G. Hardie (United Kingdom) 08:00 AMPK − opposing the metabolic changes in tumour cells G. Hardie (United Kingdom) Key messages: 1. The AMP-activated protein kinase (AMPK) is a highly conserved sensor of cellular energy status that is switched on by metabolic stresses causing depletion of cellular ATP, such as ischaemia (a frequent event within solid tumours). 2. Most tumour cells (and other rapidly proliferating cells) display elevated glucose uptake and glycolysis (the Warburg effect); this occurs in part because the TCA cycle is being used as an anabolic pathway providing precursors for biosynthesis, and can no longer satisfy the increased demand for ATP caused by activation of biosynthetic pathways.

Scientific programme – details

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3. By inactivating most biosynthetic pathways and promoting oxidative metabolism rather than glycolysis, AMPK opposes these metabolic changes; this explains why the pathway is often downregulated in tumour cells, but also why AMPK-activating drugs have potential in the treatment or prevention of cancer. 08:20 IDH1/IDH2 D.P. Schenkein (USA) Key messages: 1. Cancer metabolism, a newly validated area for oncology drug discovery. 2. Mutations in the metabolic enzyme IDH are present in a wide range of malignancies and increasingly recognised in pre-malignant conditions: the role of 2-HG as an oncometabolite. 3. Clinical trial results from the first IDH inhibitor, AG-221: significant single agent activity in AML, excellent safety profile, and a novel mechanism of action. 08:40 Targeting MYC-driven Glutaminolysis C. Van Dang (USA) 09:00 Identification of novel inhibitors of GLUT1 as potent cancer cell-killing agents A. Wise (United Kingdom) Key objectives: 1. Demonstrate chemical tractability of the glucose transporter GLUT1 as a drug target by identifying a lead series of potent and selective small molecule inhibitors. 2. Demonstrate in vitro validation of GLUT1 as a therapeutic target in cancer by utilising our novel GLUT1 inhibitors as tool compounds. 3. Demonstrate in vivo validation of GLUT1 as a therapeutic target in cancer by utilising our novel GLUT1 inhibitors as tool compounds. 09:20 Discussion Workshop 3 10:15–12:00

Auditorium “Liquid Biopsies” in Solid Tumours Chairs: K. Dhingra (USA) and A. Bardelli (Italy)

10:15 Application of CTC detection technologies in oncology clinical trials J.S. De Bono (United Kingdom) Key objectives: Showing that circulating blood biomarkers can have clinical utility as multi-purpose biomarkers as 1. Prognostic biomarkers. 2. Predictive biomarkers 3. Response and surrogate biomarkers. 10:35 Cancer mutation and transcriptome analysis in exosomes J. Skog (USA) Key objectives: 1. What is an exosome and what do they contain? 2. What is the benefit of analyzing exosome nucleic acids? 3. What do you need for successful analysis of exosomal RNA, and what types of analysis are possible? 10:55 cfDNA for detection of actionable cancer mutations C. Caldas (United Kingdom) 11:15 Precision medicine for colorectal cancers: liquid biopsies and preclinical models A. Bardelli (Italy) Key objectives: 1. Understanding the molecular bases of secondary resistance to EGFR blockade in CRC is necessary to design additional therapeutic options. 2. Molecular alterations in KRAS, NRAS, and MET are causally associated with the onset of acquired resistance to anti-EGFR antibodies in colorectal cancers.

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Scientific programme – details

3. Development of a diagnostic platform to detect ‘molecular resistance’ in the blood (liquid biopsy) months before clinical or radiographic evidences of disease progression. 11:35 Discussion Workshop 4 10:15–12:00

Room 117 Translational Research with Immunostimulatory Monoclonal Antibodies Chairs: J. Tabernero (Spain) and I. Melero (Spain)

10:15 The making of new immunotherapy agents A. Korman (USA) 10:35 Translational research of combined immunotherapies I. Melero (Spain) 11:05 Biomarkers for new immunotherapies M. Calahan (USA) 11:25 The interface of immunomodulation and vaccines A. Van Elsas (NL) Key messages: 1. Cancer vaccines enhance anti-tumour immune responses when used prophylactically, but lack robust efficacy in therapeutic setting. 2. Several immunomodulatory receptor-ligand pathways regulate T cell responses to cancer and vaccines. 3. Experimental combinations of cancer vaccines and immunomodulatory antibodies display efficacy in preclinical models of established cancer. 11:45 Discussion 12:15–12:45

Posters in the Spotlight Session Moderator: R. Plummer (United Kingdom)

Exhibition Hall

The following abstracts will be discussed: 364 (poster board P144), 161 (P155), 422 (P155). Professional Advancement Session 12:15–13:15 “Navigating Your Career with Confidence” − A Professional Advancement Session Organized by the AACR-Women in Cancer Research (WICR) Council Chair: P. LoRusso (USA)

Room 117

Feel and project more confidence when seeking new career opportunities. This session will include an inspirational Keynote Lecture from Susan M. Galbraith, MBBCh, PhD, Vice President of Oncology, AstraZeneca, as well as a panel of professionals discussing the ways that women scientists in particular can bolster their confidence and overcome insecurities in order to embrace new opportunities. The panel will address topics such as assessing which career opportunities are a good fit, improving a resume or portfolio in order to secure an interview, blending personal and career life once on the job, and more. Although all conference attendees are invited to attend this session, it is geared toward early-career female investigators. Welcome P. LoRusso (USA) Keynote S.M. Galbraith (United Kingdom) Panel Discussion Susan M. Galbraith (United Kingdom), M. Foti (USA), F. Meunier (Belgium) Audience Engagement (Q&A) Closing Remarks and Evaluation P. LoRusso (USA)

Scientific programme – details

Plenary Session 2 13:15–15:20 Proffered Paper Session Chairs: C. Arteaga (USA) and R. Plummer (United Kingdom)

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Auditorium Abstract number

13:15 Target validation as a crucial bottleneck in cancer drug discovery P. Workman (United Kingdom) Key objectives: 1. To illustrate the challenges of identifying and prioritising new cancer targets in the multiomics era and to show how target validation is a critical bottleneck in cancer drug discovery. 2. To exemplify the above with case histories from our recent research discoveries concerning potential molecular targets in the areas of oncogenic kinases and the HSP90 molecular chaperone pathway. 3. To set out criteria for robust target validation, including the use of genetic techniques and chemical probes, so as to give the best chance of clinical impact. 13:35 ORAL PRESENTATION: Safety and early evidence of activity of a first-in-human phase I study of the novel cancer stem cell (CSC) targeting antibody OMP-52M51 (anti-Notch1) administered intravenously to patients with certain advanced solid tumors A. Patnaik, P. LoRusso, P. Munster, A.W. Tolcher, S.L. Davis, J. Heymach, R. Ferraroto, L. Xu, A.M. Kapoun, L. Faoro, J.A. Lewicki, J. Dupont, S.G. Eckhardt

2

13:50 ORAL PRESENTATION: Afuresertib (GSK2110183), an oral AKT kinase inhibitor, in combination with carboplatin and paclitaxel in recurrent ovarian cancer S. Blagden, A. Hamilton, L. Mileshkin, M. Hall, T. Meniawy, S. Wong, S. Anandra, M. Buck, D. McAleer, B.A. Reedy, R.B. Noble, D.A. Smith, S.R. Morris, H. Gabra

3

14:05 ORAL PRESENTATION: Activity of galeterone in castrate-resistant prostate cancer (CRPC) with C-terminal AR loss: Results from ARMOR2 M.E. Taplin, K.N. Chi, F. Chu, J. Cochran, W.J. Edenfield, E.S. Antonarakis, U. Emmenegger, E.I. Heath, A. Hussain, V.C. Njar, A. Koletsky, D. Lipsitz, L. Nordquist, R. Pili, M. Rettig, O. Sartor, N.D. Shore, D. Marrinucci, K. Mamlouk, B. Montgomery

4

14:20 ORAL PRESENTATION: Mechanism based targeted therapy for hereditary leiomyomatosis and renal cell cancer (HLRCC) and sporadic papillary renal cell carcinoma: interim results from a phase 2 study of bevacizumab and erlotinib R. Srinivasan, D. Su, L. Stamatakis, M.M. Siddiqui, E. Singer, B. Shuch, J. Nix, J. Friend, G. Hawks, J. Shih, P. Choyke, W.M. Linehan

5

14:35 ORAL PRESENTATION: Imaging in cancer immunology: Phenotyping of multiple immune cell subsets in-situ in FFPE tissue sections J.R. Mansfield, C. Slater, C. Wang, K. Roman, C.C. Hoyt, R.J. Byers

6

14:50 LATE BREAKING ABSTRACT: Clinical safety and activity in a phase I trial of AG-120, a first in class, selective, potent inhibitor of the IDH1-mutant protein, in patients with IDH1 mutant positive advanced hematologic malignancies D.A. Pollyea, S. de Botton, A.T. Fathi, E.M. Stein, M.S. Tallman, S. Agresta, C. Bowden, B. Fan, M. Prah, H. Yang, K. Yen, R.M. Stone

1LBA

15:00 LATE BREAKING ABSTRACT: The identification of potent and selective inhibitors of oncogenes in 2LBA medullary thyroid carcinoma and lung adenocarcinoma disease models S. Fritzl, H. Small, B. Acton, S. Holt, G. Hopkins, S. Jones, A. Jordan, N. March, R. Newton, I. Waddell, B. Waszkowycz, M. Watson, D. Ogilvie 15:10 Discussion Late Breaking Abstracts C.L. Arteaga (USA) Plenary Session 3 16:00–17:50 Oncolytic Viruses Chairs: K. Harrington (United Kingdom) and L.J. Helman (USA) 16:00 T-VEC for the treatment of melanoma: Are we ready for prime time? H. Kaufman (USA)

Auditorium Abstract number

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Scientific programme – details

16:20 Poliovirus oncolytic immunotherapy of glioblastoma M. Gromeier (USA) Key objectives: 1. Discuss the role of mechanisms of viral tumour cell killing in oncolytic immunotherapy. 2. Define principles of efficacious oncolytic immunotherapy: explain the role of the innate antiviral response to oncolytic poliovirus. 3. Communicate the major clinical/radiographic findings from oncolytic immunotherapy of recurrent glioblastoma with recombinant poliovirus. 16:40 Measles and vesicular stomatitis virus strains as novel oncolytic platforms E. Galanis (USA) 17:00 Challenges in clinical development of oncolytic viruses K. Harrington (United Kingdom) 17:20 LATE BREAKING ABSTRACT: Initial report of a first-in-human study of the first-in-class fatty acid 3LBA synthase (FASN) inhibitor, TVB-2640 J. Infante, M. Patel, D. Von Hoff, A. Brenner, C. Rubino, W. McCulloch, V. Zhukova-Harrill, M. Parsey 17:30 LATE BREAKING ABSTRACT: A phase 1 study of first-in-class microRNA-34 mimic, MRX34, in 4LBA patients with hepatocellular carcinoma or advanced cancer with liver metastasis M. Beg, A. Brenner, J. Sachdev, M. Borad, J. Cortes, R. Tibes, Y. Kang, A. Bader, J. Stoudemire, S. Smith, S. Kim, D. Hong 17:40 Discussion Late Breaking Abstracts L.J. Helman (USA) 18:00–19:30 Animal Models Poster board

Poster Viewing

Poster area

Abstract number

P001 Mouse clinical trial − A new preclinical study concept using patient-derived xenografts V. Vuaroqueaux, C. Gredy, S. Gorynia, S. Baltes, H.H. Fiebig, T. Metz P002 Imaging growth and anti-cancer activity in orthotopic patient derived tumors M. Baugher, C. Bull, A. Cohen-Barnhouse, A. Flecha, M. Franklin, K. Guley, P. McConville, W.R. Leopold P003 Antineoplastic effects of auranofin in canine lymphoma D. Thamm, B.J. Rose, J.K. Shoeneman P004 mTOR inhibition with everolimus − a novel treatment option for head and neck cancer identified in a translational research study using patient-derived xenografts K. Klinghammer, J.D. Raguse, T. Plath, A.E. Albers, B. Brzezicha, A. Wulf-Goldenberg, U. Keilholz, J. Hoffmann, I. Fichtner P005 A panel of patient derived xenograft models of different haematological malignancies suitable for preclinical drug screening campaigns E. Oswald, C. Tschuch, K. Klingner, B. Hammerich, D. Lehnhard, C. Rentsch, M. L¨ubbert, H.H. Fiebig, J. Sch¨uler P006 Next generation sequencing (NGS) guided therapy prediction for the treatment of glioblastoma multiforme (GBM) J. Sarkaria, D.M. Ma, S.P. Peng, S.B. Byron, D.C. Craig, J.C. Carpten, M.B. Berens, B.O. O’Neill, N.T. Tran P007 Whole exome sequence analysis of canine transitional cell carcinoma of the bladder D.L. Duval, B. Hernandez, J. Brown, S.E. Lana, R. Page, K.L. Jones P008 Mixeno mouse models for in vivo evaluation of anti-human cancer immunotherapeutics J. Zhang, J. Qiu, M. Qiao, Q. Shi P009 Genetic and molecular validation of uterine sarcoma patient-derived xenograft models T. Cuppens, E. Hermans, J. Depreeuw, M. Moisse, T. Van Brussel, L. Coenegrachts, D. Lambrechts, F. Amant

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9 10

11

12

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Scientific programme – details

Poster board

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Abstract number

P010 Allografting improves the feasibility of genetically engineered mouse models (GEMM) for anti-cancer drug development K. Kukuk, K. Klingner, A.L. Peille, P. M¨uller, A. Zipelius, J. Sch¨uler

16

P011 Studies on glycoprotein expression differences between MCF-7 and MCF-7-Z J. Ner-Kluza, A. Drabik, M. Kubbutat, A. Lingnau, J. Silberring

17

P012 Establishment and characterization of a Merkel Cell carcinoma PDX panel: Screening for potentially useful therapies M.J. Wick, J. Meade, M. Nehls, T. Vaught, J. Carlile, A.W. Tolcher, D.W. Rasco, A. Patnaik, K.P. Papadopoulos

18

P013 Syngeneic models for developing cancer therapeutics targeting immune system L. Zhang, J. Zhang, Q. Shi

19

P014 miR-25 is a key regulator of prostate cancer invasiveness by modulation of the cross-talk between Notch and TGF-b signaling E. Zoni, A.F. van de Merbel, G. van der Horst, J. Rane, T. Visakorpi, E.B. Snaar, N. Maitland, G. van der Pluijm

20

Cytotoxics Poster board

Abstract number

P015 Pharmacogenomics of mithramycin in thoracic malignancies W. Figg, T.M. Sissung, C.J. Peer, D. Schrump

21

P016 Novel combination therapy, TAS-102 combined with the anti-EGFR antibody or the anti-VEGF antibody showed therapeutic benefit toward colorectal cancer xenografts K. Ishida, K. Sakamoto, N. Tanaka, K. Oguchi, K. Yamamura, A. Fujioka, F. Nakagawa, K. Matsuo, T. Utsugi

22

P017 Phase I study of lurbinectedin (PM01183) administered on days (D) 1 & 8 every 3 weeks (q3wk) in patients (pts) with solid tumors M.J. Ratain, L. Gore, S. Szyldergemajn, J. Diamond, D. Geary, C. Fernandez-Teruel, A. Soto-Matos, M. Sharma, A. Jimeno

23

P018 Androgen receptor (AR) expression in triple negative breast cancer (TNBC): results from a phase II neoadjuvant trial with carboplatin and eribulin mesylate in TNBC patients K. Siziopikou, V. Parini, V. Kaklamani

24

P019 An ING1b-derived peptide that inhibits cancer cell viability and promotes apoptosis A. Boyko, K. Riabowol

25

P020 NPD926, a small molecule inducer of reactive oxygen species, kills cancer cells via glutathione depletion T. Kawamura, Y. Kondoh, M. Muroi, M. Kawatani, H. Osada

26

P021 TAS-102 treatment results in high trifluridine incorporation into DNA with pyrimidine metabolic pathway markedly up-regulated in cancer K. Oguchi, K. Sakamoto, H. Kazuno, H. Ueno, K. Ishida, T. Yokogawa, K. Yamamura, R. Kitamura, K. Matsuo, T. Utsugi

27

P022 Characterization of the type of cell death induced by novel tambjamine analogs in lung cancer A. Rodilla Mart´ın, V. Soto-Cerrato, P. Manuel-Manresa, L. Korrodi-Greg´orio, R. Quesada, R. P´erezTom`as

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P023 TAS-114 is a novel dUTPase/DPD inhibitor, its DPD inhibition reduces capecitabine dosage but does not diminish therapeutic window in human tumor xenografts W. Yano, H. Kazuno, T. Yokogawa, K. Sakamoto, K. Yoshisue, T. Wakasa, M. Fukuoka, K. Matsuo, K. Noguchi, T. Utsugi

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P024 The fungal-derived cyclohexadepsipeptide Destruxin E exerts multifaceted anticancer and antiangiogenic activities R. Dornetshuber-Fleiss, P. Heffeter, T. Mohr, P. Hazemi, K. Kryeziu, C. Seger, W. Berger, R. Lemmens-Gruber P025 N-Myc amplification sensitizes tumor cells to inhibition by Danusertib, an Aurora kinase inhibitor P. Carpinelli, R. Ceruti, R. Alzani, C. Re, D. Ballinari, S. Cribioli, M. Russo, A. Degrassi, G. Texido, M. Ciomei, E. Pesenti, A. Montagnoli, A. Galvani P026 Replication stress is a determinant of synergy between gemcitabine and Chk1 inhibition S.B. Koh, A. Courtin, R. Boyce, B. Boyle, F.M. Richards, D.I. Jodrell P027 Combining the long-acting topoisomerase 1-inhibitor etirinotecan pegol with the PARP inhibitor rucaparib to provide anti-tumor synergy without increased toxicity U. Hoch, D. Charych P028 Phase I, dose-escalation study of the investigational drug D07001-F4, an oral formulation of gemcitabine HCl, in patients (pts) with advanced solid tumors or lymphoma C. Lin, W. Su, J. Lee, C. Hsu, A. Cheng, C. Lin, H. Ho, C. Huang, S. Hsueh, J. Yang P029 BRCA1 expression exploratory analysis in patients of the phase III trial of trabectedin vs. doxorubicin-based chemotherapy as first-line therapy in translocation-related sarcomas M. Aracil, P. Lardelli, A. Nieto, C.M. Galmarini P030 Suppression of metastasis and improvement of drug distribution by eribulin mesylate Y. Ozawa, K. Okamoto, Y. Adachi, M. Asano, K. Tabata, Y. Funahashi, J. Matsui P031 Effect of a microtubule-targeting drug on cell–cell contacts in bladder epithelial tumour cells L.M. Ant´on-Aparicio, R. Castosa, M. Haz, M. Blanco, M. Rodriguez, M. Valladares, A. Figueroa P032 The indolyl-chalcone CDD-026 induces cancer cell death through targeting of STMN1 and mitotic catastrophe B. Wegiel, Y. Wang, F. Jernigan, L. Sun P033 Phytochemical indole-3-carbinol synergizes strongly with fludarabine and induces p53-dependent and -independent cell death in chronic lymphocytic leukemia cells irrespective of their IGHV mutation state and treatment resistances G. Perez-Chacon, C. Martinez-Laperche, N. Rebolleda, B. Somovilla-Crespo, C. Mu˜noz-Calleja, I. Bu˜no, J.M. Zapata P034 Selectivity and mechanism of action studies for Polo Box-targeted, non-ATP based inhibitors of PLK1 M. Baxter, S. Craig, C. McInnes, M.D. Wyatt P035 Clinical activity of BIND-014 (docetaxel nanoparticles for injectable suspension) as second-line therapy in patients (pts) with Stage III/IV non-small cell lung cancer R. Natale, M. Socinski, L. Hart, O. Lipatov, D. Spigel, B. Gershenhorn, G. Weiss, S. Kazmi, N. Karaseva, O. Gladkov, V. Moiseyenko, J. Summa, G. Otterson P036 Interaction of SJG-136 with cognate sequences of oncogenic transcription factors J. Mantaj, P.J. Jackson, D.E. Thurston, K.M. Rahman P037 CDKN1A-mediated responsiveness of MLL-AF4-positive acute lymphoblastic leukemia to Aurora kinase-A inhibitors L. Hung, Y. Chen, H. Lin, M. Tsai, H. Hsieh, J. Chang, N. Chen, S. Yang, T. Chen P038 Metformin and its impact on gastric cancer patients survival after gastrectomy C.K. Lee, M.K. Jung, I.K. Jung, S.J. Heo, J.Y. An, H.I. Kim, J.H. Chung, W.J. Hyung, S.H. Noh, H.S. Kim, S.Y. Rha, H.C. Chung P039 Early preclinical study of BO-2094 for treatment of human colon cancer, in combination with 5-fluorouracil T.L. Su, T.H. Ou, M.H. Wu, Y.W. Lin, T.C. Lee P040 Broad-spectrum preclinical combination activity of eribulin combined with various anticancer agents in human breast cancer, lung cancer, ovarian cancer, and melanoma xenograft models M. Asano, J. Matsui, M.J. Towle, J. Wu, S. McGonigle, T. Uenaka, K. Nomoto, B.A. Littlefield

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Abstract number

P041 Lurbinectedin (PM01183) specifically targets RNA Pol II for degradation via the proteasome pathway in a transcription and TC-NER dependent fashion G. Santamaria, J.F. Mart´ınez-Leal, C. Cuevas, L.F. Garcia-Fernandez, C.M. Galmarini P042 Trabectedin and lurbinectedin are effective against leukemic cells derived from patients affected by chronic and juvenile myelomonocytic leukemia M. Romano, A. Gall`ı, N. Panini, L. Paracchini, L. Beltrame, E. Bello, S.A. Licandro, C. Cattrini, R. Tancredi, S. Marchini, V. Rosti, M. Zecca, M. Della Porta, A. Zambelli, C.M. Galmarini, E. Erba, M. D’Incalci P043 siRNA targeting of mitochondrial thymidine kinase 2 (TK2) sensitizes cancer cells to gemcitabine and increases mitochondrial toxicity C. Di Cresce, P. Ferguson, R. Figueredo, M. Rytelewski, S. Maleki Vareki, M.D. Vincent, J. Koropatnick P044 The effect of esomeprazole, a proton pump inhibitor, on the pharmacokinetics of the investigational Aurora A kinase inhibitor alisertib (MLN8237) in patients with advanced solid tumors or lymphomas X. Zhou, J. Nemunaitis, S. Pant, T. Bauer, A. Lockhart, M. Patel, B. Zhang, V. Kelly, C.D. Ullmann, M. Bargfrede, K. Venkatakrishnan P045 Development of rational combination therapy strategies to optimize treatment for GPNMB expressing Her2+ and triple negative breast cancer A. Rose, G. Maric, M.G. Annis, H. Smith, W.J. Muller, P.M. Siegel P046 Platinum(IV) derivatives of oxaliplatin: Cellular effects and in vivo potency S. G¨oschl, V. Pichler, E. Brynzak, P. Heffeter, U. Jungwirth, M.A. Jakupec, W. Berger, M. Galanski, B.K. Keppler P047 Adeno-associated virus (AAV) carrying diphtheria toxin a gene for pancreatic cancer therapy M. Chen, C.Y. Ho, M. Teng, H. Chen P048 MGMT methylation assessed by methyl-BEAMing technique is a prognostic and predictive biomarker in glioblastoma and metastatic colorectal cancer patients L. Barault, A. Amatu, F.E. Bleeker, C. Moutinho, A. Cassingena, F. Tosi, T. Venesio, M. Esteller, A. Bardelli, S. Siena, A. Sartore-Bianchi, F. Di Nicolantonio P049 Low, frequent doses of PM060184 induce remarkable in vivo antitumor activity P. Aviles, M.J. Guillen, P.P. Lopez-Casas, F. Sarno, O. Catalu˜na, P. Nu˜nez, C. Cuevas, M. Hidalgo P050 Radiosensitizing effect of sodium metaarsenite in a metastatic brain tumor model W.Y. Kang, Y.M. Park, S.J. Kim P051 Differential antitumor activity of trabectedin, lurbinectedin, Zalypsis and PM00128 against a panel of human cells deficient in transcription and NER factors V. Moneo, S. Avila, P. Mart´ınez, B. de Castro, S. Cascajares, C. Cuevas, L.F. Garcia-Fernandez, C.M. Galmarini P052 Pipecolidepsin A, Stellatolide A and Irvalec: New cyclodepsipeptides of marine origin with antitumor activity J.M. Molina-Guijarro, V. Moneo, J.F. Martinez-Leal, C. Cuevas, L.F. Garcia-Fernandez, C.M. Galmarini P053 Discovery of novel inhibitor of FOXO nuclear–cytoplasmic shuttling from natural products of marine origin F.J. Castillo Correa, N. De Pedro, L. Rodriguez Quesada, D. Oves Costales, J.R. Tormo, J. Martin, F. Reyes, O. Genilloud, F. Vicente, W. Link, B. Cautain P054 Sodium metaarsenite cytotoxic activity is associated with telomere length and many types of arsenic transporters in non-small cell lung cancer Y.M. Park, S.J. Kim P055 A panel of pediatric liver cancer patient-derived xenografts to improve stratification of children with hepatoblastoma M. Fabre, D. Nicolle, A. Gorse, O. D´eas, C. Mussini, L. Brugi`eres, M.R. Ghigna, E. Fadel, L. Galmiche-Rolland, C. Chardot, C. Armengol, J.G. Judde, S. Branchereau, S. Cairo

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P056 Antitumor and temozolomide-sensitizing effects of sodium metaarsenite in an orthotopic glioblastoma xenograft model W.Y. Kang, Y.M. Park, S.J. Kim

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P057 Radium-223 dichloride − Efficacy and mode-of-action in a mouse model of prostate cancer bone metastasis M.I. Suominen, K.M. Fagerlund, J.P. Rissanen, Y. Konkol, E. Alhoniemi, D. Mumberg, K. Ziegelbauer, S.M. K¨ak¨onen, J.M. Halleen, R.L. Vessella, A. Scholz

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P058 Nifuroxazide halogenic derivatives induce ROS-mediated apoptosis and display antitumor activity against metastatic melanoma C. Fernandez de Farias, M.H. Massaoka, N. Girola, C.R. Figueiredo, R.A. Azevedo, L.C. Tavares, L.R. Travassos

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P059 Phase II drug metabolism UGT1A enzyme affects cellular response of colon cancer cells to antitumor triazoloacridinone C-1305 treatment E. Augustin, E. Bartusik, A. Theus, B. Borowa-Mazgaj, Z. Mazerska

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P060 Clinical validity of new genetic biomarkers of irinotecan neutropenia: An independent replication study F. Innocenti, J. Ramirez, W. Qiao, A.J. de Graan, M.J. Ratain, R.H.N. van Schaik, R.H.J. Mathijssen, G.L. Rosner, D.J. Crona

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P061 Cytotoxic response as a result of the cross-talk between UGT mediated metabolism and modulation of UGT activity by C-1311 and C-1305 acridinone antitumor agents in selected solid tumor cell lines Z. Mazerska, M. Pawlowska, A. Bejrowska, M. Szydlowska, B. Fedejko, E. Augustin

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P062 Can the P53 status predict the outcome of Polo-like kinase 1 inhibition in non-small cell lung cancer cell lines? J. Van den Bossche, A. Wouters, C. Deben, V. Deschoolmeester, P. Specenier, P. Pauwels, M. Peeters, F. Lardon

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Drug Resistance and Modifiers Poster board

Abstract number

P063 AKT3 activation mediates resistance to HER2-targeted therapies in HER2-amplified breast cancer F. Carmona Sanz, F. Montemurro, V. Rossi, C. Verma, M. Berger, J. Baselga, M. Scaltriti

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P064 Antitumor activity of S116836, a novel tyrosine kinase inhibitor, against imatinib-resistant FIP1L1PDGFRa-expressing cells J. Pan, Y. Shen, K. Ding

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P065 Novel regulation of estrogen receptor transcription by the PI3K pathway E. Toska, M. Elkabets, A. Bosch, O. Litvin, M. Scaltriti, J. Baselga

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P066 Nrf2 as a molecular target in overwhelming chemoresistance in breast cancer therapy N. Samadi, F. Ramezani, M. Sabzichi

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P067 Differential pathway resiliency in response to MAPK inhibition in BRAF-mutant cancer M. Sos, R.S. Levin, J.D. Gordan, J.A. Oses-Prieto, J.T. Webber, M. Salt, B. Hann, A.L. Burlingame, F. McCormick, S. Bandyopadhyay, K.M. Shokat

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P068 Increased EDN1 expression mediates acquired resistance to the combination therapy of PI3K and MEK inhibitors for colorectal cancer B. Bhattacharya, S.H.H. Low, T. Benoukraf, M.L. Chong, K.X. Koh, R. Soong

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P069 Loss of PTEN leads to acquired resistance to the PI3Ka inhibitor BYL719: a case of convergent evolution under selective therapeutic pressure P. Castel, D. Juric, M. Griffith, O.L. Griffith, H.H. Won, B. Ainscough, H. Ellis, S. Ebbesen, I. Gopakumar, C. Quadt, M. Peters, D. Solit, S.W. Lowe, E.R. Mardis, M.F. Berger, M. Scaltriti, J. Baselga

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Scientific programme – details

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Abstract number

P070 Mixed lineage kinases activate MEK independently of RAF to mediate resistance to RAF inhibitors A.A. Marusiak, Z.C. Edwards, W. Hugo, E.W. Trotter, M.R. Girotti, N.L. Stephenson, X. Kong, M.G. Gartside, S. Fawdar, A. Hudson, W. Breitwieser, N.K. Hayward, R. Marais, R.S. Lo, J. Brognard P071 Tumor suppressive roles of miR-221 and miR-222 in lung cancer M. Sato, R. Yamashita, T. Kakumu, T. Hase, E. Maruyama, Y. Sekido, M. Kondo, Y. Hasegawa P072 A comprehensive in vitro screen to identify therapeutic candidates for inclusion with etoposide/platin combinations to improve treatment of SCLC D. Evans, R. Delosh, J. Laudeman, C. Ogle, R. Reinhart, M. Selby, T. Silvers, A. Monks, E. Polley, G. Kaur, J. Morris, B.A. Teicher P073 Quantitative proteomics as a tool to identify resistance mechanisms in erlotinib-resistant subclones of the non-small cell lung cancer cell line HCC827 K. Jacobsen, R.R. Lund, H.C. Beck, H.J. Ditzel P074 Determination of an oxidative stress gene signature in inflammatory breast cancer patient tumors and development of a novel redox modulatory strategy in overcoming chemotherapy resistance and mediating anti-tumor efficacy G.R. Devi, J.L. Allensworth, M. Evans, N. Ueno, D. McDonnell, F. Bertucci, S. Van Laere P075 Sensitization of triple-negative breast cancer to PI3K inhibition by cotargeting IGF1R K. de Lint, J.B. Poell, J. Vidal Rodriguez, H. Soueidan, W. Nijkamp, L.F. Wessels, R.L. Beijersbergen P076 Regorafenib resistance in colorectal carcinoma is associated with enhanced expression of type II interleukin 1 receptor and reversed by MEK/ERK inhibitor A.C. Mar, C.H. Chu, C.W. Shiau, T.C. Lee P077 Acquired resistance to BET bromodomain inhibitors is associated with modulation of the apoptotic signaling network P. Sandy, S. Nerle, A. Conery, C. Hatton, B. Bryant, R. Sims, E. Normant P078 Src family kinase activation is a compensatory survival mechanism for acquired resistance to EGFRTKIs in lung cancer cells M. Ono, K. Sonoda, K. Azuma, K. Watari, M. Molina, R. Rosell, M. Kuwano P079 Wnt secretion is required to maintain Wnt activity in colon cancer O. Voloshanenko, G. Erdmann, T.D. Dubash, I. Augustin, M. Metzig, C.R. Ball, H. Glimm, R. Spang, M. Boutros P080 Influence of EGFR exon 19 mutation subtypes on survival outcomes in advanced stage Asian nonsmall cell lung cancer patients receiving TKI therapy B. Chowbay, O. Singh, N. Sutiman, D.S.W. Tan, W.T. Lim, E.H. Tan P081 Critical difference in development of acquired resistance to MDM2 inhibitor SAR405838 in vitro and in vivo G. Hoffman-Luca, C.Y. Yang, J. Lu, D. Ziazadeh, D. McEachern, L. Debussche, S. Wang P082 Genome-wide drug sensitivity screens in haploid mouse embryonic stem cells S.J. Pettitt, D. Krastev, H. Pemberton, Y. Fontebasso, I. Bajrami, I. Kozarewa, J. Frankum, R. Rafiq, J. Campbell, R. Brough, A. Ashworth, C.J. Lord P083 Tie-2 regulates the stemness of prostate cancer cells K. Tang, M. Ling P084 Tumor infiltrating leukocyte subpopulations as a biomarker of response and resistance to targeted therapy in patients with BRAF mutation-positive metastatic melanoma M.C. Kelley, D.B. Doxie, A.R. Greenplate, H. Crandall, J.A. Sosman, J.M. Irish P085 4E-BP1 expression levels determine sensitivity of triple negative breast cancer cells to mTOR inhibitors K. Jastrzebski, B. Thijssen, J. Vidal Rodriguez, K. de Lint, C. Lieftink, L.F. Wessels, R.L. Beijersbergen P086 Role of ERK nuclear translocation in cisplatin-sensitive and -resistant ovarian cancer cells S. Dilruba, G.V. Kalayda, U. Jaehde

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P087 A stress induced early innate response causes multi-drug tolerance in melanoma D.R. Menon, S. Das, C. Krepler, A. Vultur, B. Rinner, S. Schauer, K. Kashofer, K. Wagner, G. Zhang, E. Bonyadi Rad, H.P. Soyer, B. Gabrielli, R. Somasundaram, G. Hoefler, M. Herlyn, H. Schaider

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P088 Elucidating mechanisms of resistance to FGFR inhibitors in endometrial cancer L. Packer, S. Byron, C. Mahon, D. Loch, A. Wortmann, K. Nones, S. Grimmond, J. Pearson, N. Waddell, P. Pollock

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Drug Screening Poster board

Abstract number

P089 Identification of synthetic lethality compounds from natural products for cancers K.W. Ng, K.S. Lee, V. Patel, E. Sundaramoorthy, N. Ayoub, X. Su, A. Venkitaraman, S.H. Teo

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P090 Evaluation of hormone therapies in a panel of breast PDX models: Relevance of ER status on sensitivity to letrozole and tamoxifen M.J. Wick, T. Vaught, L. Gamez, J. Meade, A. Diaz, K.P. Papadopoulos, D.W. Rasco, A. Patnaik, M. Beeram, A. Lang, A.W. Tolcher

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P091 Utilization of low passage adenoid cystic carcinoma PDX models to identify novel combination therapies M.J. Wick, J. Meade, T. Vaught, M. Nehls, J. Flores, J. Kaufman, A.W. Tolcher, D.W. Rasco, A. Patnaik, C.A. Moskaluk, K.P. Papadopoulos

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P092 Synergistic inhibition of HER2 positive breast cancer by triptolide and lapatinib P. Chalugun, J.S. Shim, P. Korangath, S. Sukumar, J.O. Liu

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P093 Combination screening of investigational oncology agents S. Holbeck, J.M. Collins, J.D. Doroshow

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P094 Small molecules selectively targeting breast cancer cells J. Sakoff, J. Gilbert, A. McCluskey

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P095 PIK3CA mutation-targeting compounds analyses using NCI60 cell line panel H. Bando, J. Lih, E.C. Polley, S.L. Holbeck, B. Das, D. Sims, T. Doi, A. Ohtsu, M. Williams, N. Takebe

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P096 Comparison of platinum/taxane and anthracycline-based therapies in ovarian PDX models: Correlating stage of biopsy collection and engrafting with in vivo drug sensitivity J. Meade, M.J. Wick, T. Vaught, L. Gamez, M. Farley, A. Moriarty, A.W. Tolcher, A. Patnaik, D.W. Rasco, A.J. White, K.P. Papadopoulos

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P097 The use of next generation sequencing (NGS) in the management of metastatic breast cancer (MBC): Defining a model for genomic-driven therapies L. Austin, M. Gooptu, T. Avery, R. Jaslow, J. Palazzo, M. Cristofanilli

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P098 Identification of inhibitors of tryptophan metabolizing enzymes for cancer immunotherapy by highthroughput screening G. Zaman, J.C.M. Uitdehaag, S. van Gerwen, N. Seegers, A.M. van Doornmalen, J. de Man, R.C. Buijsman

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P099 A platform to test multiple therapy options simultaneously in a patient’s own tumor N. Caffo, R. Klinghoffer

105

Immunotherapy (Immunecheckpoints, Vaccination, Oncolytic viruses, Cytokines) Poster board

Abstract number

P100 Beta-3 integrin inhibition reduces inflammatory cytokine release but not anti-cancer activity of oncolytic adenovirus in ovarian cancer A.K. Browne, L.A. Tookman, C.K. Ingemarsdotter, R. Bouwman, K. Pirlo, Y. Wang, K.M. HodivalaDilke, I.A. McNeish, M. Lockley

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Scientific programme – details

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Abstract number

P101 CIGB-247: Anti-VEGF therapeutic vaccine in patients with advanced solid tumors ´ F. Hern´andez-Bernal, J.V. Gavilondo, M. Ayala Avila, A.V. de la Torre, J. de la Torre, K.H. SelmanHousein, Y. Morera, M. Bequet-Romero, C.M. Valenzuela, Y. Martin P102 CD70 (TNFSF7), a receptor involved in acute immune modulation of viral infection, is frequently overexpressed in solid and hematological malignancies J. Jacobs, K. Zwaenepoel, P. Aftimos, C. Rolfo, S. Rottey, L. Ysebrant de Lendonck, K. Silence, A. Awada, A. Thibault, P. Pauwels P103 Functional activity, but not PD-1 expression level, differentiates primary CLL from healthy PD1+ T cells using SCNP S. Liang, L. Leung, S. Putta, D. Hotson, D. Rosen, R.E. Hawtin P104 IL-6/STAT3/Fra-1 signaling axis promotes colorectal cancer aggressiveness through epithelial– mesenchymal transition J. Shao, H. Liu P105 Identification of peptides which could block PD-1 checkpoint for NSCLC immunotherapy Y. Zhu, C. Li P106 A novel primary human tumor explant platform provides a preclinical translational link from tissue culture to the clinic G. Juan, K. Paweletz, E. Trueblood, J. Rossi, M. Damore, A. Anderson, R. Loberg P107 Preclinical activity and pharmacodynamic biomarkers of W014A, a PD-1 decoy peptide blocking both PD-1 immune checkpoint ligands, PD-L1 and PD-L2 C. Bailly, M. Broussas, M. Ramachandra, P.G. Sasikumar, K. Shrimali, S. Adurthi, M. Ramachandra, L.K. Satyam, A.A. Dhudashia, S. Dhodheri, K.B. Sunilkumar, N. Corva¨ıa, P. Ferre P108 Toll-like receptor 5 agonist entolimod as a potential anticancer immunotherapeutic agent L. Burdelya, C. Brackett, B. Kojouharov, J. Veith, A. Gudkov P109 Anti-metastatic activity via co-blockade of PD-1 and adenosine A2A receptor D. Mittal, A. Young, K. Stannard, M.W. Teng, B. Allard, J. Stagg, M.J. Smyth P110 Novel targets for antibody–drug conjugate therapy A.G. Grandi, S.C. Campagnoli, M.P. Parri, E.D.C. De Camilli, B.J. Jin, P.S. Sarmientos, G.G. Grandi, L.T. Terracciano, P.P. Pileri, G.V. Viale, R. Grifantini P111 “Arming” the chimeric oncolytic adenovirus enadenotucirev to deliver checkpoint inhibitors and other therapeutics directly to tumours B. Champion, P. Kodialbail, S. Illingworth, N. Rasiah, D. Cochrane, J. Beadle, K. Fisher, A.C.N. Brown P112 Major synergy between Coxsackievirus A21 (CAVATAK™) and radiotherapy or chemotherapy in bladder cancer G.R. Simpson, N. Annels, M. Ajaz, F. Launchbury, G. Bolton, A.A. Melcher, K.J. Harrington, G. Au, D. Shafren, H. Pandha P113 3-Bromopyruvate as an inducer of immunogenic cell death in colon cancer cells K.H. Jung, J.H. Lee, J.W. Park, C. Quach, K.H. Lee P114 Density of CD 8 +ve T cells & CD 56 +ve NK cells in follicular adenoma & papillary carcinoma of thyroid in Pakistani population J. Varda, N. Naseem, A.H. Nagi P115 A modified double-deleted vaccinia virus combining viral oncolysis and potential gene therapy as a novel therapeutic for atypical teratoid/rhabdoid tumors Y. Ruan, A. Narendran P116 Analysis of immune-response markers in resectable NSCLC M. Us´o, E. Jantus-Lewintre, R. Sirera, S. Calabuig-Fari˜nas, S. Gallach, E. Escorihuela, A. Blasco, R. Guijarro, C. Camps P117 Targeting tryptophan metabolism in human lung cancer J. Deshane, C. Schafer, Y. Wang, A. Sawant, T.H. Jin, D. Zhi, S. Ponnazhagan, S. Grant

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P118 A WT1-derived peptide protects against metastatic melanoma in a syngeneic model by in vivo immunomodulatory effects on dendritic cells M.H. Massaoka, C.R. Figueiredo, N. Girola, R.A. Azevedo, L.R. Travassos

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P119 Critical issues in the clinical development of oncolytic viruses − A regulatory perspective M. Schuessler-Lenz

125

P120 IMCgp100: A novel bi-specific biologic for the treatment of malignant melanoma W. Shingler, J. Harper, G. Bossi, D. Barker, J. Dukes, N. Liddy, S. Paston, T. Mahon, P. Molloy, M. Sami, E. Baston, B. Cameron, A. Johnson, A. Vuidepot, N. Hassan, Y. McGrath, B. Jakobsen

126

P121 Correlation of clinical activity of pembrolizumab (MK-3475) with immunohistochemical staining for programmed death-1 ligand (PD-L1) in 50% of tumor cells in a prospective non-small cell lung cancer (NSCLC) validation population E.G. Garon, N.A. Rizvi, N.B. Leighl, R. Hui, J.P. Eder, A. Patnaik, C. Aggarwal, L. Horn, A.S. Balmanoukian, M.A. Gubens, E. Felip-Font, E. Carceny Costa, J.C. Soria, M.J. Ahn, H.T. Arkenau, J.S. Lee, G. Robinet, G.M. Lubiniecki, J. Zhang, K. Emancipator, R. Rutledge, M. Dolled-Filhart, L. Gandhi

127

P122 T cell-mediated cancer immunotherapy through OX40 agonism M. Huseni, C. Du, J. Zhu, P. Pacheco-Sanchez, M. Moskalenko, H. Chiu, K. Dalpozzo, K. Totpal, L. Damico-Beyer, J. Kim

128

P123 Characterization of PD-L1 expression in circulating tumor cells (CTCs) of non-small cell lung cancer (NSCLC) patients R. Krupa, D. Lu, M. Harvey, J. Louw, A. Jendrisak, D. Marrinucci, R. Dittamore

129

P124 Preclinical results of ProCervix, a first in class, first in indication therapeutic vaccine targeting HPV16/18 infected women M. Esquerre, M. Bouillette-Marussig, A. Goubier, M. Momot, H. Keller, M. Bissery

130

P125 Zfra activates novel Hyal2+ CD3− CD19− memory spleen cells to block cancer growth, stemness, and metastasis in vivo N. Chang

131

P126 A novel anti-PDL1 antibody-based bifunctional protein with enhanced immunological activity Y. Wu, S. Martomo, Z. Zhong, D. Lu, Z. Polonskaya, X. Luna, Z. Zhang, H. Zhang, L. Witte, S. Waksal, Z. Zhu

132

P127 Analysis of immune microenvironment in resectable NSCLC: Prognostic value of regulatory and conventional T cell markers proportion M. Us´o, R. Sirera, S. Calabuig-Fari˜nas, A. Blasco, E. Pastor, R. Guijarro, E. Jantus-Lewintre, J. Forteza, C. Camps

133

P128 Epigenetic immunomodulation by SGI-110 combined with immune checkpoint blockade for new therapeutic strategies A. Covre, C. Fazio, H.J.M.G. Nicolay, P.G. Natali, P. Taverna, M. Azab, S. Coral, M. Maio

134

P129 AD-O64.3: IFN-g–TRAIL fusion protein. Use of two independent signaling pathways for a strong synergistic antitumor effect B. Zerek, J.S. Pieczykolan, S.D. Pawlak, P.K. Rozga, A. Pieczykolan, M. Szymanik, A. Jaworski, M. Galazka, K. Bukato, K. Poleszak, M. Teska-Kaminska, W. Strozek

135

P130 Tumor-infiltrating lymphocytes (TILs) following intratumoral administration of ONCOS-102 are associated with prolonged overall survival in last line solid tumor patients S. Pesonen, T. Joensuu, E. Jager, J. Karbach, C. Wahle, K. Kairemo, K. Partanen, R. Turkki, A. Hemminki, C. Backman, M. von Euler, T. Hakonen, T. Ranki, L. Vassilev, A. Vuolanto, M. Jaderberg

136

P131 Immune checkpoint blockade enhances measles virotherapy C.E. Engeland, R. Veinalde, C. Grossardt, S. Bossow, I. Shevchenko, V. Umansky, D.M. Nettelbeck, D. J¨ager, C. von Kalle, G. Ungerechts

137

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P132 Preclinical activity and safety of MGD006, a CD123xCD3 Bispecific DART® molecule for the treatment of hematological malignancies P. Moore, G.R. Chichili, L. Huang, H. Li, S. Burke, F. Chen, L. He, Q. Tang, L. Jin, S. Gorlatov, V. Ciccarone, S. Koenig, M. Shannon, R. Alderson, S. Johnson, E. Bonvini

138

P133 GBR1302: a BEAT® bispecific antibody for the treatment of HER2 positive cancers A. Croset, J. Macoin, R. Ollier, M. Pluess, C. Delon, D. Skegro, S. Blein, S. Hou, J. Back P134 Relationship between programmed death ligand 1 (PD-L1) expression and clinical outcome in patients (pts) with melanoma (MEL) treated with pembrolizumab (pembro; MK-3475) A. Daud, O. Hamid, C. Robert, F.S. Hodi, J.D. Wolchok, W.J. Hwu, J.S. Weber, R. Kefford, P. Hersey, A.M. Joshua, R. Joseph, T.C. Gangadhar, R. Dronca, A. Patnaik, H. Zarour, K. Gerigich, J. Lunceford, K. Emancipator, M. Dolled-Filhart, X. Li, P. Kang, S. Ebbinghaus, A. Ribas P135 Phase I study of ipilimumab with stereotactic radiosurgery for melanoma patients with brain metastases W. Shi, E. Wuthrick, K. Feeney, M. Werner-Wasik, D.W. Andrews, J.J. Evans, K. Judy, C. Farrell, A.P. Dicker P136 IL-17 as a novel molecular target for prostate cancer prevention A. De Angulo, R. Faris, C. Jolly, L. deGraffenried

139

Preclinical Models Poster board

140

141

142

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P137 Regional bias of intratumoral genetic heterogeneity of nucleotide repeats in colon cancers with microsatellite instability C. An, Y. Choi, M. Kim, J. Lee, N. Yoo, S. Lee P138 Association between interleukin 17/interleukin 17 receptor gene polymorphism and papillary thyroid cancer Y. Eun, H. Chung, Y.C. Lee P139 ARQ 087, a novel pan FGFR-inhibitor crosses the BBB (blood–brain barrier) and distributes to the brain of rats R. Savage, T. Hall, B. Schwartz P140 Establishment of patient-derived xenografts (PDX) models for triple negative breast cancer (TNBC) as a pre-clinical platform for drug development J. Thatte, M. Meza, J. Ricono, T. Broudy, C. Mirsaidi, P. Nair P141 An integrated approach for identifying E-cadherin synthetic lethality networks I. Bajrami, S.J. Pettitt, R. Brough, H. Pemberton, D. Kastrev, Y. Fontebasso, J. Frankum, J. Campbell, A. Ashworth, C.J. Lord P142 RAS synthetic lethal interactions from yeast to human cells S. van Wageningen, A. Prahallad, G. Heynen, R. Rothstein, R. Bernards P143 Quantitative mutational assessment of circulating tumor DNA using massively parallel deep sequencing in plasma and urine from advanced colorectal cancer patients J.C. Poole, C.R.T. Vibat, L. Benesova, B. Belsanova, S. Hancock, T.L. Lu, M.G. Erlander, M. Minarik P144 Pirin downregulates E-cadherin gene expression and contributes to EMT K. Komai, Y. Niwa, Y. Sasazawa, S. Simizu P145 Impact of EGFR amplification pattern on the expression of miRNA-200c in primary glioblastoma multiforme L. Mu˜noz Hidalgo, C. L´opez Gines, E. Serna, D. Monleon, R. Callaghan, R. Gil Benso, H. Martinetto, A. Gregori Romero, J. Gonzalez Darder, M. Cerda Nicolas P146 Preclinical characterization of MM-151, an oligoclonal antibody therapeutic that targets EGFR by three distinct mechanisms of action A. King, M. Sevecka, N. Gerami-Moayed, O. Burenkova, J. Kearns, G. Tan, C. Sloss, R. Bukhalid, U. Nielsen, B. Wolf

143

144

145

146

147

148 149

150 151

152

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P147 Moving beyond in vitro models and addressing the challenges of pooled RNAi screens in mouse xenografts D. Tedesco, K. Bonneau, M. Makhanov, D. Deng, P. Sun, A. Chenchik

153

P148 Drug response database with PDX tumor models in biomarker-driven multi-drug multi-arm clinical trial settings J. Jiang, T.F. Yu, Y. Yan, W. Du, T.T. Tan, L. Hua, J.L. Gu, X.Q. Yang, Z.H. Liu, X.K. Ye, Z. Gu

154

P149 SNIPER(TACC3) degrades TACC3 protein via the ubiquitin–proteasome pathway and induces apoptosis in cancer cells expressing a large amount of TACC3 N. Ohoka, K. Nagai, K. Okuhira, N. Shibata, T. Hattori, N. Cho, M. Naito

155

P150 Behaviour of platinum(IV) complexes with prodrug function in different models of hypoxia E. Brynzak, P. Heffeter, V. Pichler, M.A. Jakupec, B.K. Keppler

156

P151 Sensitive and specific detection of 1p/19q codeletion in gliomas by next generation sequencing E. Dubbink, P.N. Atmodimedjo, R.M. van Marion, J.M. Kros, M.J. van den Bent, W.N.M. Dinjens

157

P152 The development of a series of orthotopic solid tumour models of prostate, lung and ovarian cancer using optical and X ray imaging M. Batey, M. Brown, E. Bowden

158

P153 Neoadjuvant chemotherapy in breast cancer patients induces expression of miR-34a and miR-122 P. Freres, C. Josse, N. Bovy, M. Boukerroucha, I. Struman, V. Bours, G. Jerusalem

159

P154 Identification of fusion genes through kinome-centered RNA sequencing in different types of solid tumors L. Mittempergher, C. Sun, F.H. Groenendijk, A.J. Bosma, S.M. Willems, T. Sustic, I.J. Majewski, W. Grernrum, N.M. Davidson, I. de Rink, H.M. Horlings, W. Theelen-Engelsman, S.F. Chin, A. Oshlack, B.W. van Rhijn, M. van den Heuvel, M.S. van der Heijden, C. Caldas, R. Bernards

160

P155 Leveraging a novel DNA barcoding platform for integrated profiling and pharmacodynamic readouts C. Castro, V. Peterson, A. Ullal, S. Agasti, S. Tuang, N. Miller, M. Birrer, R. Weissleder

161

P156 Drug–drug interaction predictions for MLN2480, an investigational pan-RAF inhibitor, based on nonclinical data S.K. Balani, A. Bulychev, L. Cohen, M. Liao, C.Q. Xia, F. Wang, P. Li, B. LeClair, T. Bohnert, L. Gan, X. Zhou, V. Bozon, S. Prakash

162

P157 Interrogation of pharmacogenes in cancer patients using targeted DNA sequencing F. Innocenti, N. Gillis, J. Parker, N. Hayes, D. Eberhard, K. Richards, J.T. Auman, E. Seiser

163

P158 Augmentation of NAD+ by NQO1 activation attenuates cisplatin-mediated hearing impairment S. Yang, G.S. Oh, K.B. Kwon, R.K. Park, S.Y. Lee, S.R. Moon, H.S. So

164

P159 Effect of zoledronic acid on the post-translational modification of activated leukocyte cell adhesion molecule (ALCAM) in cancer cells R. Toth, G. Trombino, V. Castronovo, A. Bellahcene

165

P160 Pre-clinical and clinical activity of Anti-DLL4 (demcizumab) in combination with gemcitabine plus nab-paclitaxel in pancreatic cancer M. Hidalgo, A. Cubillo, R. Stagg, J. Dupont, Y. Wan-Ching, T. Hoey

166

P161 Genomic characterisation of 1003 cancer cell-lines G. Bignall, F. Iorio, P.A. Futreal, M.R. Stratton, P. Campbell, U. McDermott

167

P162 CDCP1 as a new marker of aggressiveness in triple-negative breast cancers M. Campiglio, F. Turdo, F. Bianchi, M. Sasso, L. De Cecco, P. Casalini, P. Gasparini, L. Forte, R. Agresti, I. Maugeri, G. Sozzi, E. Tagliabue

168

P163 BRAF mutation testing in cell-free DNA from plasma of patients with advanced cancers using a novel, rapid, automated molecular diagnostics prototype platform (Idylla™) F. Janku, H.J. Huang, B. Claes, G.S. Falchook, A. Naing, S. Piha-Paul, A.M. Tsimberidou, R.G. Zinner, D.D. Karp, S. Fu, V. Subbiah, D.S. Hong, J.J. Wheler, R.G. Luthra, S.P. Patel, E.S. Kopetz, E. Sablon, G. Maertens, R. Kurzrock, F. Meric-Bernstam

169

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P164 Novel, ultra-deep next-generation sequencing for BRAF mutation testing using small amount of cellfree DNA from plasma of patients with advanced cancers F. Janku, H.J. Huang, N.M. Ramzanali, X. Cai, R. Klausner, F. Meric-Bernstam, J.B. Fan

170

P165 Antagonistic interaction between gemcitabine and erlotinib is influenced by EGR1 (early growth response 1) transcription factor expression C.D. Hose, Y. Zhao, E.C. Polley, J. Fang, N.D. Fer, A. Rapisarda, B.A. Teicher, R.M. Simon, J.D. Doroshow, A. Monks

171

P166 Modulation of estrogen-dependent transcription by cohesin in MCF7 human breast adenocarcinoma cells T. Dasgupta, J. Antony, J. Rhodes, M. McEwan, M. Eccles, J. Horsfield

172

P167 Ras-mediated activation of mitogen-activated protein kinase pathway unleashes basement membrane damaging activity of serine protease hepsin T. Tervonen, S. Pant, D. Belitskin, J. Englund, K. N¨arhi, E. Verschuren, P. Kovanen, J. Klefstr¨om

173

P168 Combination of molecular and drug response data in patient-derived xenografts to assist patient stratification S. Cairo, O. D´eas, A. Beurdeley, V. Yvonnet, M.F. Poupon, J.G. Judde

174

P169 Functional analysis of [methyl-3 H]choline uptake in glioblastoma cells: Influence of anti-cancer and central nervous system drugs M. Inazu, C. Taguchi, T. Yamanaka, H. Uchino

175

P170 Resolvin D2 has mitogenic activity in estrogen receptor positive breast cancer cell lines via activation of estrogen receptor N. Al-Zaubai, C. Johnstone, M. Rizzacasa, A. Stewart

176

P171 Validation of 3D primary organoid cultures of colorectal carcinoma as discovery and validation platform for personalized cancer therapy P. Halonen, A. Kuijpers, B. Morris, B. Diosdado, S. Mainardi, R. Bernards, V. Verwaal, R. Beijersbergen

177

P172 Establishment of patient-derived xenografts (PDX) models for small cell lung (SCL) as a pre-clinical platform for drug development T. Broudy, J. Ricono, C. Mullins, C. Mirsaidi, P. Nair

178

P173 Effects of human breast cancer cells secreted factors on macrophage differentiation S. Coimbra de Sousa, R. Brion, J. M¨onkk¨onen, H. Joensuu, D. Heymann, J. M¨aa¨ tt¨a

179

P174 Identifying and monitoring somatic mutations in cell free DNA of patients with metastatic melanoma J. Wisell, C.M. Amato, W.A. Robinson

180

P175 Aberrant Wnt signaling activation in human cancers: In vitro and in vivo models to facilitate Wnt targeted drug development G. Liu, C. Dong, R. Zhang, L. Zhang, S. Qian, J. Cai, J. Zhang, J. Ning

181

P176 TRAP1 represents a key mediator of stemness and glycolytic metabolism in colorectal cancer cells G. Lettini, F. Maddalena, L. Sisinni, V. Condelli, L. Del Vecchio, M. Gemei, T. Notarangelo, M. Landriscina

182

P177 Spatio-temporal characterization of tumor growth and invasion A.M. Jimenez, O. Yogurtcu, M. Horn-Lee, P. Rao, S.X. Sun, D. Wirtz

183

P178 Molecular profiling of heterogeneous tumor cells A. Chenchik, D. Deng, K. Bonneau, M. Makhanov, M. Coram, G. Dolganov, S.S. Jeffrey

184

P179 Treatment of patient-derived NSCLC xenograft preclinical models using image-guided small animal irradiation N. Papadopoulou, A. McKenzie, J. King, M. Page, R. Kumari

185

P180 The use of Quantitative Textural Analysis imaging biomarkers to predict response to temsirolimus treatment in advanced HCC subjects R. Korn, R. Osarogiagbon, R. Newbold, D. Burkett, J. Sachdev

186

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P181 Targeted genomic profiling of penile squamous cell carcinoma using the Oncomine cancer research panel A.S. McDaniel, D. Hovelson, A. Cani, C.J. Liu, Y. Zhang, S. Sadis, S. Bandla, P. Williams, D. Rhodes, S.A. Tomlins

187

P182 RANBP2 knock-down is synthetic lethal with BRAF V600E in colon cancer L. Vecchione, V. Gambino, G. d’Ario, S. Tian, A. Schlicker, S. Mainardi, B. Diosdado, I. Simon, M. Delorenzi, C. Lieftink, R. Beijersbergen, S. Tejpar, R. Bernards

188

P183 Molecular and pharmacological characterization of primary mesothelioma tumor cell lines orthotopically xenografted in nude mice C. Pisano, A. Cole, A. Barbarino, E. Bianchino, M. Guglielmi, C. Melito, G. Mercadante, A. Porciello, A. Riccio, I. La Porta, S. Orecchia, R. Libener, L. Mazzucco, S. Licandro, M. Ceccarelli, F. D’Angelo, P. De Luca

189

P184 Differential chemosensitivity between CETCs and tumour spheroids in cancer patients with solid tumors D. Zimon, M. Pizon, E.L. Stein, U. Pachmann, K. Pachmann

190

P185 Homotypic and heterotypic cell signaling transduction using a dielectrophoresis microfluidic device M. Tellez Gabriel, D. Heymann

191

P186 Extensive characterization of patient derived colon cancer xenografts for preclinical biomarker identification V. Vuaroqueaux, F. Kiefer, P. Bronsert, A.L. Peille, B. Zeitouni, F. Foucault, T. Kees, J. Guo, J. Sch¨uler, H.H. Fiebig

192

P187 The identification of new therapies for ependymoma subgroups N. Boulos, J.D. Dapper, Y.T. Patel, M. DeCuypere, B. Bianski, K.M. Mohankumar, M.O. Jacus, B. Freeman III, K.D. Wright, A. Gajjar, A.A. Shelat, C.F. Stewart, R.K. Guy, R.J. Gilbertson

193

P188 Pancreatic ductal organoids as a new platform for drug discovery ¨ M. Ponz-Sarvise, V. Corbo, D. Ohlund, T. Oni, A. Handly-Santana, D. Engle, H. Tiriac, C. Chio, M. Feigin, L. Baker, C. Ardito-Abraham, Y. Park, C. Hwang, E. Elyada, K. Yu, H. Clevers, D.A. Tuveson

194

P189 A humanized mouse model for preclinical testing of molecules targeting immune checkpoints G. Baia, D. Vasquez, D. Ciznadija, D. Sidransky, A. Katz, K. Paz

195

P190 Neuropilin 2 (NRP2) modifies CXCL12/CXCR4 signaling and promotes lymph node metastases in colon cancer H. Schneider, P. H¨onscheid, S. Sch¨olch, C. Jakob, M. Muders

196

P191 Statistical aspects of kinetic analysis of gliomas with FDG-PET D. Hawe, F.R. Hernandez, S. Murphy, E. Wolsztynski, J. Huang, J. O’Sullivan, M. Muzi, J. Eary, K. Krohn, F. O’Sullivan

197

P192 Genetic and pharmacological inhibition of PIM-1 reduces tumor development in a K-Ras-driven mouse model of non-small cell lung cancer E. Aguirre, O. Renner, M.C. Rodriguez de Miguel, M.I. Albarran, A. Cebria, D. Cebrian, F. RamosLima, J. Pastor, C. Blanco-Aparicio

198

P193 Inhibitor of differentiation 1 (Id1) expression in lung cancer cells and liver microenvironment is required for liver metastasis (LM) development from non-small cell lung cancer (NSCLC) by regulating EMT-related and proliferation-related proteins E. Castanon, A. Soltermann, I. L´opez, M. Ecay, M. Collantes, J.M. Lopez Picazo, M. Ponz, C. Rolfo, A. Calvo, I. Gil-Bazo

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P194 The influence of different cMET and EGFR backgrounds on the cytotoxicity of cMET and EGFR small molecule inhibitors in vitro N. Van Der Steen, K. Zwaenepoel, C. Rolfo, E. Giovannetti, M. Castiglia, V. Deschoolmeester, A.P. Carreca, P. Germonpre, P. Pauwels

200

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P195 Transcriptome analysis of CD133-positive stem cells and prognostic value of survivin in colorectal cancer S. Kim, S.H. Park, J. Lee, Y.S. Park, H.C. Kim Radiation Interactive Agents Poster board

201

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P196 The novel microtubule-destabilizing drug BAL101553 (prodrug of BAL27862) sensitizes a treatment refractory tumor model to ionizing radiation A. Broggini-Tenzer, F. Bachmann, V. Vuong, A. Messikommer, K. Nytko-Karouzakis, T. O’Reilly, H.A. Lane, M.N. Pruschy

202

P197 Ionizing radiation induced phosphatidylserine externalization on endothelial cell surface − a potential target for vascular targeting Z. Zhao, M. Johnson, B. Chen, M. Grace, J. Ukath, V. Lee, M. Stoodley

203

P198 High-throughput functional screening identifies the flavoreductase POR as a principal determinant of sensitivity to the hypoxia-targeting prodrug SN30000 F.W. Hunter, Z. Shalev, J. Wang, J. Moffat, T. Katella, M. Koritzinsky, W.R. Wilson, B.G. Wouters

204

P199 Metformin to modulate AMP-kinase and enhance chemotherapy and radiotherapy in non-small cell lung cancer M.J. Troncone, S.M. Cargnelli, G. Pond, E. Tsiani, J. Wright, G. Steinberg, H. Skinner, L. Bo, J. Bradley

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P200 Identification of novel targets for radiosensitisation of non-small cell lung cancer by secretome analysis A. Sharma, S. Bender, O. Riesterer, A. Broggini-Tenzer, M. Pruschy

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P201 The enhancement of radiotherapy efficacy with docetaxel-titanate nanotubes as a new nanohybrid for localized high risk prostate cancer C. Mirjolet, J. Boudon, A. Loiseau, S. Chevrier, T. Gautier, R. Boidot, J. Paris, N. Millot, G. Crehange

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P202 Combined treatment of a DNA-PKcs inhibitor (NU7441) and ionizing radiation causes a differential mode of cell death in a panel of NSCLC cell lines and exhibits robust radiosensitisation D. Saha, Y. Lan, F. Hsu, V. Tumati, Z. Zhang, Y. Lin, B. Chen

208

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Thursday 20 November 2014 Plenary Session 4 08:00–10:00 Antibody-Based Therapies (ADC and others) Chairs: A. Tolcher (USA) and N. G¨okbuget (Germany)

Auditorium Abstract number

08:00 Antibody mediated payload delivery: Cautionary lessons from 20 years of clinical trials A. Tolcher (USA) Key objectives: 1. Understand the disconnect between activity with antibody drug conjugates (ADC) in preclinical models versus clinical results. 2. Understand the multiple challenges associated with ADCs as a drug from the 3 components: antibody, linker and payload. 3. Understand patient selection based on target and potential mitigation strategies when the target is also expressed on normal tissues. 08:20 Challenging the dogmas: Clinical efficacy of SN38 conjugated antibodies in solid tumours D.A. Goldenberg (USA) Key objectives: 1. Discuss current status and challenges of antibody-drug conjugates (ADC) in solid cancer therapy. 2. Supertoxic drugs used in ADC may compromise therapeutic index. 3. A moderate cytotoxic drug in an ADC may allow higher doses and a higher therapeutic index. 08:40 Targeted alpha particle therapy for haematologic malignancies J. Jurcic (USA) Key objectives: 1. List the advantages and disadvantages of targeted alpha-particle therapy. 2. Summarise the results of recent clinical trials using targeted alpha-particle therapy for the treatment for haematologic malignancies. 3. Describe the results of recent preclinical studies using alternative radioisotopes and pre-targeting strategies. 09:00 Bispecific T-cell engaging antibodies in acute leukaemia − Recent advances and future challenges Speaker: N. G¨okbuget (Germany) Key messages: 1. Relapsed and refractory ALL has a very poor outcome and there is an urgent medical need for new, alternative treatment options. 2. Blinatumomab is a bispecific antibody which represents a new treatment principle based redirected cell kill of CD19 positive target cells by T-cells. 3. Reponse rates are promising and the compound is currently studied in a randomised trial. 09:20 ORAL PRESENTATION: Pre-clinical and translational pharmacology, pharmacokinetics and pharmacodynamics for a humanized anti-OX40 antibody MOXR0916, a T-cell agonist in the treatment of solid tumors S. Sukumaran, J.M. Kim, M. Huseni, J. Ruppel, H. Taylor, K. Totpal, J. Zhu, C. Zhang, H. Chiu, E.G. Stefanich

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09:35 ORAL PRESENTATION: A phase 1 study of KTN3379, a human anti-ErbB3 monoclonal antibody in patients with advanced cancers P. LoRusso, T. LaVallee, L. Kimmel, C. Lubeski, R. Gedrich, C. Sidor

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09:50 General discussion A. Tolcher (USA)

Scientific programme – details

Plenary Session 5 10:30–12:30 Epigenetic Targets Chairs: K. Helin (Denmark) and T. Helleday (Sweden)

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Auditorium Abstract number

10:30 Histone demethylases and methyltransferases as novel targets for cancer therapeutics K. Helin (Denmark) 10:50 DOT1 and EZH2 targeted therapies R. Copeland (USA) Key messages: 1. EZH2 plays a critical role in normal B-cell maturation and is frequently dysregulated in germinal center-derived lymphomas. 2. EPZ-6438 is a potent and selective inhibitor of EZH2 that demonstrates robust and durable antilymphoma activity in preclinical animal models both as a single agent and in combination with standards of care. 3. EPZ-6438 is the first EZH2 inhibitor to enter human clinical studies and early observations from this clinical trial will be discussed. 11:10 Targeting non-oncogene addiction with MTH1 inhibitors T. Helleday (Sweden) 11:30 ORAL PRESENTATION: A novel synthetic lethal interaction between the histone mark H3K36me3 and checkpoint kinases S.X. Pfister, E. Markkanen, Y. Jiang, S. Sarkar, V. D’Angiolella, G. Dianov, A.J. Ryan, T.C. Humphrey

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11:45 ORAL PRESENTATION: Novel anti-tumor activity of targeted LSD1 inhibition by GSK2879552 H. Mohammad, K. Smitheman, G. Van Aller, M. Cusan, S. Kamat, Y. Liu, N. Johnson, C. Hann, S. Armstrong, R. Kruger

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12:00 LATE BREAKING ABSTRACT: Results of a first-in-man phase I trial assessing OTX015, an orally available BET-bromodomain (BRD) inhibitor, in advanced hematologic malignancies A. Stathis, B. Quesnel, S. Amorim, C. Thieblemont, E. Zucca, E. Raffoux, H. Dombret, Y. Peng, A. Palumbo, N. Vey, X. Thomas, M. Michallet, C. Gomez-Roca, C. Recher, L. Karlin, K. Yee, K. Rezai, C. Preudhomme, T. Facon, P. Herait

5LBA

12:10 LATE BREAKING ABSTRACT: Phase 1 first-in-human study of the enhancer of zeste-homolog 2 (EZH2) histone methyl transferase inhibitor E7438 as a single agent in patients with advanced solid tumors or B cell lymphoma V. Ribrag, J.C. Soria, L. Reyderman, R. Chen, P. Salazar, N. Kumar, G. Kuznetsov, H. Keilhack, L.H. Ottesen, A. Italiano

6LBA

12:20 Discussion Late Breaking Abstracts T. Helleday (Sweden) 12:45–13:15

Posters in the Spotlight Session Moderator: E. Calvo (Spain)

Exhibition Hall

The following abstracts will be discussed: 41 (poster board P035), 195 (P189) and one to be announced. Plenary Session 6 13:30–15:35 Proffered Paper Session Chairs: E. Calvo (Spain) and J. Doroshow (USA)

Auditorium Abstract number

13:30 ORAL PRESENTATION: A phase I dose-finding study of BI 853520, a potent and selective inhibitor of focal adhesion kinase (FAK), in Japanese and Taiwanese patients with advanced or metastatic solid tumors T. Doi, C.C. Lin, A. Ohtsu, J.C.H. Yang, K. Shitara, L.C. Pronk, A. Sarashina, A.L. Cheng

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13:45 ORAL PRESENTATION: Homologous recombination deficiency (HRD) score and niraparib efficacy in high grade ovarian cancer P. Haluska, K.M. Timms, M. AlHilli, Y. Wang, A.M. Hartman, J. Jones, A. Gutin, Z. Sangale, C. Neff, J. Lynchbury, L. Rudolph-Owen, M.A. Becker, S. Agarwal, K.M. Wilcoxen

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14:00 ORAL PRESENTATION: Updated clinical and preliminary correlative results of ARIEL2, a Phase 2 study to identify ovarian cancer patients likely to respond to rucaparib E. Swisher, J. Brenton, S. Kaufmann, A. Oza, R.L. Coleman, D. O’Malley, G. Konecny, L. Ma, M. Harrell, D. Visscher, A.W. Hendrickson, K. Lin, M. Raponi, E. Mann, H. Giordano, L. Maloney, L. Rolfe, I. McNeish

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14:15 ORAL PRESENTATION: Phase I study of panobinostat and fractionated stereotactic re-irradiation therapy (FSRT) for recurrent high grade gliomas W. Shi, Y.R. Lawrence, M. Werner-Wasik, D.W. Andrews, J.J. Evans, J. Glass, L. Kim, V. Bar Ad, Y. Moshel, K. Judy, C. Farrell, N.L. Simon, A.P. Dicker

216

14:30 ORAL PRESENTATION: Results of a phase I, open-label, multicentre study to assess the safety, tolerability, pharmacokinetics and preliminary anti-tumour activity of AZD9150 in patients with advanced/metastatic hepatocellular carcinoma Y.K. Kang, B.Y. Ryoo, T.Y. Kim, K.H. Lee, H.Y. Lim, S.J. Lee, M. Ikeda, T. Okusaka, S. Nadano, C.C. Lin, T.P. Poon, C.J. Yen, P. McCoon, F. Neumann, K. Vishwantahan, R. DuPont, P. Lyne

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14:45 ORAL PRESENTATION: Genomic analysis identifies novel drivers and targetable pathways in inflammatory breast cancer patient samples D.M. Moran, K. Rao, P. Bacon-Trusk, K. Pry, V. Weigman, V. Velculescu, M. Cristofanilli, S. Bacus

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15:00 ORAL PRESENTATION: Phase I trial evaluating the antiviral agent Cidofovir in combination with chemoradiation in cervical cancer patients: A novel approach to treat HPV related malignancies? E. Deutsch, A. Levy, R. Mazeron, A. Gazzah, E.A. Angevin, V. Ribrag, R. Balheda, A. Varga, C. Lhomme, C. Haie-Meder, J.C. Soria

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15:15 LATE BREAKING ABSTRACT: Phase 1 dose-expansion study of AMG 900, a pan-Aurora kinase inhibitor, in adult patients with advanced taxane-resistant solid tumors B. Markman, D. Mahadevan, S. Hurvitz, D. Kotasek, M. Shaheen, M. Carducci, O. Goodman, X. Jiang, V. Chow, G. Juan, G. Friberg, E. Gamelin, J. Desai

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15:25 Discussion Late Breaking Abstract J. Doroshow (USA) Plenary Session 7 16:00–17:50 Novel Mechanisms for Drug Resistance Chairs: U. Banerji (United Kingdom) and S.A. Courtneidge (USA)

Auditorium Abstract number

16:00 Genomic instability, diversity and resistance during cancer evolution N. McGranahan (United Kingdom) Key messages: 1. Driver mutations are often subclonal in non-small cell lung cancer (NSCLC), potentially comprising the efficacy of targeted therapy approaches. 2. A single NSCLC tumour may follow multiple distinct evolutionary trajectories simultaneously, with mutational processes varying over space and time. 3. NSCLCs have a long period of tumour latency prior to clinical detection. 16:20 Immune-scape to PD1/PDL1 blockade D. Chen (USA) Key objectives: 1. Understand what responses and durability of responses looks like for cancer immunotherapy and PD-L1/PD-1 inhibitors to date. 2. Understand potential reasons for primary resistance. 3. Understand potential reasons for secondary resistance.

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Messages: 1. Responses to PD-L1/PD-1 inhibitors appear highly durable, but not every patient responds and not every responding patient is “cured”. 2. Primary resistance may be driven by lack of adequate active anti-cancer T cell immune responses in tumours. 3. It is too early to tell what might account for secondary resistance, but it could involve factors that lead to fluctuations in immunity and a break in the cancer-immunity cycle. 16:40 EMT as mechanism of resistance to TKI J.P. Thiery (Singapore) Key objectives: 1. Malignant cells harbor numerous genetic alterations at the time of clinical detection. The mutational landscape is extraordinarily complex, exhibiting considerable heterogeneity within the primary tumour, in circulating tumour cells and in metastases. 2. These findings are in support of the original hypothesis of clonal evolution, and suggest that the current therapeutics strategies must be revisited to delay the onset of tumour refractoriness. 3. It is imperative to develop treatment strategies that do not strictly rely on specific activating mutations. In particular, one can leverage on the Epithelial–Mesenchymal Transition (EMT) status of a tumour, as it has an impact on the tumour’s potential to progress and resist treatment. This can be achieved by establishing an EMT score for each tumour along the EMT spectrum. Targeted therapeutics can then be used to move the tumour along the EMT spectrum rather than inhibiting its growth in order to improve its response to conventional therapeutics and to restore the body’s immune response. 17:00 The landscape of kinase fusions in cancer C. Lengauer (USA) Key messages: 1. We have performed a pan-cancer analysis of kinase fusions across all TCGA RNA-seq data (>7,000 samples). 2. Our analysis unveiled several new and recurrent kinase fusions. Overall, kinase fusions are a driver event in at least 3% of solid tumours. 3. These discoveries have profound and immediate implications for the diagnosis and treatment of cancer patients, as well as potential avenues for new drug discovery programmes. 17:20 ORAL PRESENTATION: Overcoming drug-resistance in multiple myeloma by XPO1 inhibitor combination therapy J. Turner, J. Dawson, S. Grant, K. Shain, C. Cubitt, Y. Dai, L. Zhoui, M. Kauffman, S. Shacham, D. Sullivan

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17:35 LATE BREAKING ABSTRACT: Activity of the PARP inhibitor olaparib in ATM-deficient gastric 8LBA cancer: from preclinical models to the clinic D. Hodgson, H. Mason, L. Oplustilova, C. Harbron, X. Yin, S.A. Im, H. Jones, L. Zhongwu, B. Dougherty, M. McLoughlin, A. Dickinson, A. Fielding, J. Robertson, W.H. Kim, C. Womack, Y. Gu, Y.J. Bang, A. Lau, J.C. Barrett, M.J. O’Connor 17:45 Discussion Late Breaking Abstract U. Banerji (United Kingdom) 18:00–19:30

Poster Viewing

Chemoprevention Poster board

Poster area

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P001 Docosahexaenoic acid along with modulation of actin binding proteins reduces cancer cell migration M. Ali, L.K. Rogers

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P002 Estimating predictive values of short-term morphologic assays of cancer chemoprevention for efficacy in animal tumor assays B. Dunn, V.E. Steele, R.M. Fagerstrom, C.F. Topp, D. Ransohoff, C. Cunningham, R. Lubet, L.G. Ford, B.S. Kramer

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P003 Preclinical assessment of nintedanib for chemoprevention in hepatocellular carcinoma V. Tovar, A. Moeini, S. Torrecilla, M. Higuera, J. Peix, I.M. Quetglas, L. Rodriguez-Carunchio, H. Cornella, M. Sole, J.M. Llovet

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P004 Synergic tumor growth suppression with carbohydrate-restriction diet and natural AMP-dependent protein kinase activators M. Choi, J. Lee

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P005 Molecular targets of interest to the NCI PREVENT cancer preclinical drug development program R.H. Shoemaker, B.K. Dunn, C. Suen, R.A. Lubet, D.L. Boring, B.D. Klein, M.S. Miller, V.E. Steele

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P006 Efficacy of cancer preventing drugs administered by intermittent dosing regimens V.E. Steele, C. Grubbs, C.V. Rao, R.A. Lubet

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Clinical Methodology Poster board

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P007 Multiplexed ICE COLD-PCR: A mutation detection methodology for achieving sensitivities of <0.01% using either Sanger or NGS G. Wu, B. Legendre, S. Cherubin, C. Cubrich, A. Dowers, S. Jensen, J. Gniffke, A. Kruempel, P. Krzycki, E. McCutchen, E. Montagne, S. Peterson, J. Pope, K. Scott, K. Richardson

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P008 Her2−3 heterodimer is a new and better than HER2 IHC score for clinical outcome prognosis G. Weitsman, P.R. Barber, K. Lawler, C. Gillett, N. Woodman, B. Kholodenko, L.K. Nguyen, T. Santra, B. Vojnovic, T. Ng

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P009 DNA methyltransferase 1 expression in human solid tumors and lymphomas by immunohistochemistry S.X. Yang, D. Nguyen, L. Rubinstein, S. Kummar, J.E. Tomaszewski, J.H. Doroshow

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P010 High correlation between clinical responses to first line AML patients treated with cytarabine and idarubicin and their pharmacological profiles in patient samples measured by ExviTech J. Ballesteros, P. Hernandez, D. Primo, A. Robles, A.B. Espinosa, E. Arroyo, V. Garcia-Navas, J. Sanchez-Fenoy, M. Jimenez, M. Gaspar, J.L. Rojas, J. Martinez-Lopez, J. Gorrochategui

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P011 Clinical pharmacodynamic assay development for the first in class investigational ubiquitin activating enzyme (UAE) inhibitor MLN7243 B. Baham´on, F. Gao, B. Stringer, Y. Yang, J. Shi, K. Burke, J. Huck, T. Traore, D. Bowman, H. Danaee, M. Millhollen, M. Hyer, N. Bence, Y. Ishii

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P012 Development of a targeted NGS assay system for patient enrollment to the NCI-MATCH study M. Williams, D. Sims, J. Lih, A. Datta, S. Hamilton, A.J. Iafrate, J. Sklar, S. Sadis, N. Takabe, J. Tricoli, J. Doroshow, B. Conley

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P013 Analytical validation and application of the MPACT assay, a next generation sequencing based targeted mutation detection assay for treatment selection C. Lih, D.J. Sims, R.D. Harrington, E.C. Polley, Y. Zhao, R.M. Simon, M.G. Mehaffey, T.D. Forbes, W.D. Walsh, V. Datta, B.A. Conley, A.P. Chen, S. Kummar, J.H. Doroshow, P.M. Williams

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P014 Kinetic analysis of dynamic 11 C-verapamil PET study: Compartmental v adaptive mixture models comparison F. Hernandez, D. Hawe, S. Murphy, J. O’Sullivan, E. Wolsztynski, J. Huang, M. Muzi, J. Eary, K. Krohn, F. O’Sullivan

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P015 Kinetic analysis of dynamic 11 C thymidine PET imaging studies: Compartmental and nonparametric approaches S. Murphy, D. Hawe, F. Hernandez, E. Wolsztynski, J. Huang, J. O’Sullivan, M. Muzi, J. Eary, K. Krohn, F. O’Sullivan

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P016 Whole genomic assay on endoscopic ultrasound-guided fine needle aspiration samples of unresectable pancreatic cancer J.M. Ha, K.H. Lee, J.K. Lee, K.T. Lee, W.Y. Park, J.S. Bae, J.K. Jung, D.H. Park, Y.K. Seong, E. Kim

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P017 Impact of phase 1 expansion cohorts on probability of success in phase 2 and time-to-drug-approval: analysis of 385 new drugs in oncology D. Bugano, K. Hess, L.L. Siu, F. Meric-Bernstam, A.R.A. Razak, D.S. Hong DNA Repair Modulation (including PARP, CHK, ATR, ATM) Poster board

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P018 Reversal of primary and acquired PARP-inhibitor resistance in BRCA-mutated triple-negative breast cancers by inhibition of transcriptional cyclin-dependent kinases (CDKs) S. Johnson, N. Johnson, D. Chi, B. Primack, C. Cruz, D. Stover, A.K. Greifenberg, S. Cao, K. O’Connor, J. Baselga, J. Balma˜na, V. Serra, M. Geyer, A. D’Andrea, E. Lim, G.I. Shapiro

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P019 Niraparib, a selective PARP 1/2 inhibitor, is efficacious in pre-clinical models of small-cell lung cancer Y. Wang, J. Ricono, K. Admunson, S. Agarwal, R.J. Fram, T. Broudy, K.M. Wilcoxen

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P020 The combination of Chk-1 and ATR inhibitor synergistically kills cancer cells K. Sanjiv, A. Hagenkort, P.M. Reaper, T. Koolmeister, O. Mortusewicz, N. Schultz, M. Scobie, U.W. Berglund, P.A. Charlton, J.R. Pollard, T. Helleday

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P021 Epigenetic loss-of-function BRCA1 mediates tumor cure by single dose radiotherapy C. Campagne, T.H. Thin, J.D. Fuller, K. Manova-Todorova, A. Haimovitz-Friedman, S.N. Powell, R.N. Kolesnick, Z. Fuks

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P022 BMN 673 as single agent and in combination with temozolomide or PI3K pathway inhibitors in small cell lung cancer and gastric cancer models Y. Feng, L.E. Post, R. Cardnell, L.A. Byers, B. Wang, Y. Shen

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P023 Modulation of PI3K/mTOR pathway following PARP inhibition in small cell lung cancer R.J. Cardnell, Y. Feng, L. Diao, Y. Fan, F. Masrorpour, S. Mukherjee, J. Shen, J. Wang, L.A. Byers

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P024 Development of xenoimplants from germline BRCA1/2 mutant breast cancer (BC) for the identification of predictive biomarkers, mechanisms of resistance against poly(ADP-ribose) polymerase (PARP) inhibitors and evaluation of novel therapies ´ P.M. Avil´es, C. Cruz, Y. Ibrahim, B. Morancho, P. Anton, J. Grueso, P. Cozar, M. GuzmAn, M.J. Guill´en, C. Galmarini, J. Arribas, J. Baselga, J. Balma˜na, V. Serra

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P025 Chk1 is a potential novel therapeutic target that regulates cell survival and potentiates chemotherapy in osteosarcoma (OS) models S.J. Strauss, P. Mistry, A. Mendoza, M. Robson, H. Holme, P. Nandabhiwat, B. Kwok, M. Qadir, R.B. Pedley, J.S. Whelan, P.H.B. Sorensen

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P026 PARP inhibitors trap PARP1 onto damaged DNA via catalytic inhibition and not by an allosteric mechanism T. Hopkins, L. Solomon, Y. Shi, L. Rodriguez, C. Donawho, E. DiGiammarino, S. Panchal, A. Olson, D. Stolarik, D. Osterling, W. Gao, E. Johnson, D. Maag

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P027 Selective inhibitors of nuclear export (SINE) block the expression of DNA damage repair proteins and sensitize cancer cells to DNA damage therapeutic agents T. Kashyap, M. Crochiere, S. Friedlander, B. Klebanov, W. Senapedis, E. Baloglu, D. del Alamo, S. Tamir, T. Rashal, D. McCauley, R. Carlson, M. Kauffman, S. Shacham, Y. Landesman

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P028 Use of ATR inhibitor in combination with topoisomerase I inhibitor kills cancer cells by disabling DNA replication initiation and fork elongation R. Joss´e, S.E. Martin, R. Guha, P. Ormanoglu, T. Pfister, J. Morris, J. Doroshow, Y. Pommier

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P029 Preclinical efficacy of the PARP inhibitor rucaparib (CO-338/AG014699/PF-01367338) in pancreatic cancer models with homologous recombination deficiencies (HRD) L. Robillard, K. Lin, P.P. Lopez-Casas, M. Hidalgo, T.C. Harding

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P030 Comprehensive genomic profiling of pancreatic acinar cell carcinomas identifies recurrent RAF fusions and frequent inactivation of DNA repair genes J. Chmielecki, K.E. Hutchinson, G.M. Frampton, Z.R. Chalmers, A. Johnson, C. Shi, J. Elvin, S.M. Ali, J.S. Ross, O. Basturk, S. Balsubramanian, D. Lipson, R. Yelensky, W. Pao, V.A. Miller, D.S. Klimstra, P.J. Stephens

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P031 A combined in vitro and mathematical modelling approach for understanding the impact of an inhibitor of ATR on DNA damage and repair after ionising radiation J. Yates, S. Checkley, L. MacCallum, R. Odedra, J. Barnes, A. Lau

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P032 The DNA damage response gene Schlafen 11 (SLFN11) is a transcriptional target of ETS transcription factors in Ewing’s sarcoma and other cancers Y. Pommier, S.W. Bilke, F. Sousa, M. Yamade, J. Murai, V. Rajapakse, L. Helman, P. Meltzer

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P033 Phase 1 correlative study of ARQ761, a b-lapachone analogue that promotes NQ01-mediated programmed cancer cell necrosis D. Gerber, Y. Arriaga, M.S. Beg, J.E. Dowell, J.H. Schiller, A.E. Frankel, R. Leff, C. Meek, J. Bolluyt, O. Fatunde, R.T. Martinez, P. Vo, F. Fattah, V. Sarode, Y. Zhou, Y. Xie, M. McLeod, B. Schwartz, D.A. Boothman

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P034 Molecular analysis in breast cancer: correlation with immunohistochemical classification and pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) S. Baulies, M. Gonzalez-Cao, N. Karachaliou, A. Rodriguez Capitan, M.A. Molina-Vila, M.T. Cusido, C. Teixido, S. Viteri, R. Fabregas, X. Gonzalez, R. Rosell

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Drug Delivery Poster board

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P035 Pre-clinical in vivo characterization of MLN7243, an investigational ubiquitin activating enzyme inhibitor, in solid tumor models T. Traore, J.H. Huck, J.S. Shi, D.S. Sappal, J.D. Duffey, Y.Y. Yang, E.K. Kadakia, A.C. Chakravarty, B.S. Stringer, Y.I. Ishii, R.G. Griffin, C.X. Xia, M.M. Milhollen, J.C. Ciavarri, P.F. Fleming, N.B. Bence, M.H. Hyer

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P036 Minicell packaged targeted delivery of shRNA to cancer cells M. Jivrajani, N. Shrivastava, M. Nivsarkar

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P037 Development of peptide-mediated drug delivery systems for colon cancer targeted imaging and therapy H.C. Wu, C.H. Wu, Y.H. Kuo

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P038 Pancreatic cancer cells expressing the OATP1B3 transporter show promising sensitivity to the highly cytotoxic microcystin-LR molecule V. Kounnis, G. Chondrogiannis, M.D. Mantzaris, D. Fokas, N.A. Papanikolaou, I. Sainis, E. Briasoulis

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P039 Factors that limit delivery of Cdk4/6 inhibitor palbociclib to GBM K. Parrish, J. Pokorny, R. Mittapalli, K. Bakken, J. Sarkaria, W. Elmquist

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P040 Hsp90 Inhibitor Drug Conjugates (HDC): Payloads and possibilities D. Chimmanamada, W. Ying, J. Zhang, D. Proia, T. Przewloka, J. Jiang, D. Vutukuri, G. Lu, S. Osman, S. Chen, J. Chu, P. Rao, D. Zhou, T. Inoue, L. Shin Ogawa, R. Singh, N. Tatsuta, A. Sonderfan, C. Cortis

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P041 Lesion characterization with ferumoxytol MRI in patients with advanced solid tumors and correlation with treatment response to MM-398, nanoliposomal irinotecan (nal-IRI) R.K. Ramanathan, R.L. Korn, J.C. Sachdev, G.J. Fetterly, G. Jameson, K. Marceau, V. Marsh, N. Raghunand, J. Prey, S.G. Klinz, J. Kim, E. Bayever, J.B. Fitzgerald

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P042 Improved cytotoxic activity of Nor-b-lapachone-loaded PLGA microcapsules in PC3M prostate cancer cell line C. Pessoa, A.C.S. Feitosa, M.P. Costa, F.C. Evangelista, F.A.M. Sales, I.S. Bomfim, E.N. Silva J´unior, G.G. Dias, V.N. Freire, W.S. Caetano

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P043 Controlled release of cisplatin using hyaluronic oligosaccharides-coated gold nanoparticles as an efficient delivery system applied to the treatment of pancreatic tumors H. Parkkola, L. Sobrevals Amieva, L. Vivero, R. Mi˜nana, J. Sendra P044 Vasculogenic mimicry in small cell lung cancer F. Trapani, R.L. Metcalf, R. Polanski, A. Fusi, C. Hodgkinson, D. Nonaka, M.J. Hendrix, C. Morrrow, F. Blackhall, K.L. Simpson, C. Dive P045 Pretargeted nanoparticles to deliver both chemotherapeutics and radiation for the treatment of lymphoma C. Fang, J.C. Jones, S.M. Frayo, M.H. Hylarides, M. Zhang, O.W. Press P046 Auxiliar treatment by targeting the extracellular matrix to improve drug delivery and therapeutic response E. Henke, F. Roehrig, H. Hoffmann, F. Escorcia, M. Wartenberg, S. Volova, S. Gaetzner, A. Rosenwald, S. Erguen, D.A. Scheinberg, Z.V.I. Granot P047 Novel adjuvant therapy with leptin peptide receptor antagonist-2 conjugated to nanoparticles (IONPLPrA2) to minimize chemoresistance in triple negative breast cancer T. Harmon, A. Harbuzariu, L. Yang, R.R. Gonzalez-Perez Drug Design Poster board

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P048 A potent and highly efficacious bivalent Smac Mimetic APG-1387 in Phase I clinical development J. Lu, S. Rong, H. Sun, L. Liu, D. McEachern, G. Wang, J. Wen, Y. Zhai, M. Guo, D. Yang, S. Wang P049 The NCI-60 as an effective tool for scaffold hopping: A phenotypic systems-based approach to the design of novel chemotherapeutics D.G. Wishka, V. Kumar, B. Teicher, G. Kaur, B. Fang, P. Risbood, M. Hollingshead, J. Zais, J. Morris P050 Discovery, development and optimization of low molecular weight EPH−ephrin protein–protein inhibitors M. Tognolini, C. Giorgio, I. Hassan-Mohamed, E. Barocelli, M. Mor, D. Pala, S. Russo, M. Incerti, A. Lodola P051 Data integration and graph analysis for cancer genomics and drug discovery B. Bernard, M. Miller, H. Rovira, I. Shmulevich P052 8-(1-Anilino)ethyl)-2-morpholino-4-oxo-4H-chromene-6-carboxamides as PI3Kbeta/delta inhibitors: structure–activity relationships and identification of AZD8186, a clinical candidate for the treatment of PTEN deficient tumours B. Barlaam, S. Cosulich, S. Degorce, M. Fitzek, S. Green, U. Hancox, C. Lambert-van der Brempt, J.J. Lohmann, M. Maudet, R. Morgentin, M.J. Pasquest, A. Peru, P. Ple, T. Saleh, M. Vautier, M. Walker, L. Ward, N. Warin P053 Significance of serine-167 and cysteine-129 residues in the active site of the immune-suppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) for the binding of novel inhibitors P. Tomek, B.D. Palmer, J.U. Flanagan, L. Ching P054 Novel cysteine derivatives for the next generation anticancer agents acting on KSP N. Ogo, J. Sawada, Y. Ishikawa, K. Matsuno, A. Hashimoto, A. Asai P055 Modulation of PIP2 levels through small molecule inhibition of PIP5K D. Andrews, S. Cosulich, N. Divecha, D. Fitzgerald, V. Flemington, C. Jones, D. Jones, O. Kern, E. MacDonald, S. Maman, J. McKelvie, K. Pike, M. Riddick, G. Robb, K. Roberts, J. Smith, M. Swarbrick, I. Treinies, M. Waring, R. Wood P056 An X-ray crystal structure-based understanding of the inhibition of the MDM2−p53 protein–protein interaction by isoindolinones B. Anil, E. Blackburn, T. Blackburn, S. Cully, J. Liu, C.J. Drummond, J.A. Endicott, B.T. Golding, R.J. Griffin, K. Haggerty, J. Lunec, D.R. Newell, C.H. Revill, C. Riedinger, A.F. Watson, Q. Xu, Y. Zhao, I.R. Hardcastle, M.E.M. Noble

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P057 The discovery and pre-clinical development of the first clinical stage EZH2-inhibitor, EPZ-6438 (E7438) K. Kuntz, H. Keilhack, R. Pollock, S. Knutson, N. Warholic, V. Richon, R. Chesworth, R. Copeland, M. Porter-Scott, C. Sneeringer, T. Wigle P058 Sentinel lymph nodes mapping of macrophage targeted mannosyl human serum albumin-indocyanine detected by combined color and near infrared fluorescence imaging system in esophagus Y. Quan, Y. Oh, J.I.H.O. Park, J. Park, J. Jeong, B. Kim, H. Kim P059 Discovery of multiple kinases inhibitors, DBPR114, as the novel anti-cancer agent H.P. Hsieh, C.C. Kuo, J.J. Chiu, T.A. Hsu, T.K. Yeh, C.T. Chen P060 A hybrid drug design approach to overcome imatinib resistance for treating leukemia Y.M. Wei, K.K.W. To, S.C.F. Au-Yeung P061 Novel hybrid drug design strategy to circumvent erlotinib resistance and to optimize its pharmacokinetic properties for treating lung cancer D.C.S. Poon, S.C.F. Au-Yeung, K.K.W. To P062 Membrane anchorage of Stat3 via artificial protein lipidation M. Avadisian, S. Fletcher, B. Liu, W. Zhao, J. Turkson, C. Gradinaru, P. Gunning P063 The development of the first selective inhibitors of the UBA5 enzyme to probe for E1 activity in diseased cells S.R. da Silva, S.L. Paiva, M. Bancerz, M. Geletu, A.M. Lewis, J. Chen, Y. Cai, H. Li, P.T. Gunning P064 Poly(ADP-ribose) glycohydrolase (PARG) inhibitors increase nuclear poly(ADP-ribose) after methylating DNA damage A. Jordan, B. Acton, E. Fairweather, N. Hamilton, S. Holt, J. Hitchin, C. Hutton, D. James, S. Jones, A. McGonagle, H. Small, K. Smith, A. Stowell, I. Waddell, B. Waszkowycz, D. Ogilvie P065 A nanomolar-potency small molecule inhibitor of the STAT5 protein A.A. Cumaraswamy, A. Lewis, M. Geletu, A. Todic, D.B. Diaz, X.R. Cheng, C.E. Brown, R. Laister, D. Muench, K. Kerman, H.L. Grimes, M.D. Minden, P.T. Gunning P066 Potent and selective non-sulfamate-containing small molecule inhibitors of the ubiquitin activating enzyme S. Paiva, S.R. da Silva, M. Bancerz, H. Quereshi, G.W. Xu, A.D. Schimmer, P.T. Gunning P067 The discovery and optimization of small molecule antagonists of the WDR5−MLL interaction R. Al-Awar, R.S. Al-Awar, M. Getlik, D. Smil, Y. Bolshan, G. Poda, G. Senisterra, H. Wu, A. AllaliHassani, G.A. Wasney, D. Barsyte-Lovejoy, L. Dombrovski, A. Dong, H. He, A. Seitova, I. Chau, F. Li, J.F. Couture, E. Kuznetsova, R. Marcellus P068 Synthetic isomalyngamide A analogs that inhibit breast cancer migration W. Li, T. Chang, C. Hung, C. Chen, S. Jao P069 Progress in drugging CYP1A1, 1B1 and CYP2W1 overexpressed in cancer K. Pors, V. Le Morvan, S. Travica, S.D. Shnyder, M. Sutherland, H.M. Sheldrake, M. Searcey, I. Johansson, S. Mkrtchian, P.M. Loadman, J. Robert, M. Ingelman-Sundberg, L.H. Patterson P070 Exosome analysis in cancer patients: From the preclinical towards the clinical application: Trial design I. Mertens, M. Castiglia, A.P. Carreca, G. Baggertman, M. Peeters, P. Pauwels, C. Rolfo Molecular Targeted Agents I Poster board

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P071 Aflibercept has anti-tumor activity in bevacizumab-escaping tumors of colorectal cancer: Molecular profiles and mechanisms C. Dib, R.G. Bagley, P. Mankoo, J. Pollard, J. Watters, M. Chiron P072 Salmonella typhimurium A1-R decoys quiescent cancer cells to cycle rendering them chemosensitive S. Yano, Y. Zhang, M. Zhao, Y. Hiroshima, S. Miwa, F. Uehara, H. Kishimoto, H. Tazawa, T. Fujiwara, R.M. Hoffman

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P073 Inhibition of the cell cycle regulated Cdc7 kinase pathway is an efficacious therapeutic approach for hematologic malignancies and solid tumors R. Santos, D. Shum, D. Carrillo, R. Zhang, M. Churchill, S. Mukherjee, R. Brentjens, C. Radu, T.J. Kelly, H. Djaballah, M.G. Frattini P074 Synthetic lethal screen identifies Aurora A as a selective target in HPV driven cervical cancer B. Gabrielli, F. Bokhari, M. Ranall, A. Stevenson, M. Murell, M. Kelly, S. McKee, G. Leggatt, T. Gonda, N. McMillan P075 Combining forces: Study of the cytotoxic effect of the MDM2 inhibitor Nutlin-3 in combination with CDDP in non-small cell lung cancer cell lines C. Deben, C. Rolfo, V. Deschoolmeester, A. Wouters, M. Peeters, I. Gil-Bazo, F. Lardon, P. Pauwels P076 A first-in-Asian phase I dose escalation study to evaluate the safety and pharmacokinetics of VS-6063 (defactinib), a focal adhesion kinase inhibitor in subjects with non-hematologic malignancies T. Shimizu, H. Aida, J. Horobin, M. Keegan, M. Padval, A. Poli, C. Hashii, K. Nakagawa P077 Genetic and pharmacologic inhibition of EPHA2 promotes apoptosis in NSCLC J. Chen, K. Amato, S. Wang, V. Youngblood, D. Brantley-Sieders, R. Cook, L. Tan, N. Gray P078 Met degradation by SAIT301, a Met monoclonal antibody, reduces the invasion and migration of nasopharyngeal cancer cells via inhibition of EGR-1 expression C. Kim, B.S. Lee, K.A. Kim, Y.J. Song, K.H. Cheong P079 Tyk2-src dependence of kidney cancer B. Krishnan, S.C. Hanna, H.L. Wilson, S.T. Bailey, J.S. Damrauer, T. Simamura, R.L. Levine, K.K. Wong, G.L. Johnson, W.Y. Kim

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P080 Preclinical pharmacologic characterization of GSK2849330, a monoclonal AccretaMab® antibody with optimized ADCC and CDC activity directed against HER3 N. Clarke, C. Hopson, A. Hahn, K. Sully, F. Germaschewski, J. Yates, C. Akinseye, B. Mangatt, Z. Jonak, C. Matheny P081 Using PK/PD/efficacy modeling to predict potential of AZD9291 to target brain metastases from advanced NSCLC with EGFR sensitizing mutations (EGFRm+) J. Yates, P. Ballard, S. Ashton, D. Cross, R. Dattani, M. Mellor, J. Wilson, P. Yang, L. Xie P082 FAK inhibitor VS-6063 (defactinib) targets mesothelioma cancer stem cells which are enriched by standard of care chemotherapy J. Pachter, V.N. Kolev, P. Baas, D.T. Weaver, Q. Xu P083 Genomic and epigenomic analysis identify potential therapeutic targets in luminal B breast cancer molecular subtype L. Addou Klouche, A. Belhadj, A. Bendaoud, M. Benyelles, I. Ziane, M. Chaffanet, D. Birnbaum

300

P084 PI3 -Kinase inhibition forestalls the development of drug resistance in BRAFV600E/PTENNull melanoma M. Deuker, V. Marsh Durban, W. Phillips, M. McMahon P085 Selinexor, a novel selective inhibitor of nuclear export, potentiates the antitumor activity of gemcitabine against pancreatic cancer by nuclear retention of p27 M. Amit, S. Kazim, H. Kazim, S. Shacham, M. Kauffman, M.P. Malafa P086 CC-115 inhibits DNA damage and repair pathways in vitro T. Tsuji, L. Sapinoso, S. Sankar, D.S. Mortensen, S. Xu P087 Stromal delivery of long Pentraxin-3 impairs FGF/FGFR-dependent tumor growth and metastasis A. Giacomini, E. Di Salle, D. Coltrini, S. Rezzola, M. Belleri, M. Presta, R. Ronca P088 Androgen deprivation therapy sensitizes prostate cancer cells to T-cell killing through androgen receptor dependent modulation of the apoptotic pathway S. Gameiro, A. Ardiani, A.R. Kwilas, R.N. Donahue, J.W. Hodge P089 RNA sequencing and in silico analysis identifies an unannotated antisense long non-coding RNA involved in cancer progression S. Inoue, K. Horie-Inoue, K. Ikeda

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306 307 308

309

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P090 Inhibition of Trk-driven tumors by the pan-Trk inhibitor RXDX-101 D. Anderson, M. Ciomei, P. Banfi, S. Cribioli, E. Ardini, A. Galvani, G. Li

310

P091 IGF-1R inhibition induced activation of Yes/SFK acts as a by-pass resistance pathway in rhabdomyosarcoma X. Wan, C. Yeung, C. Heske, A. Mendoza, L.J. Hlman

311

P092 The prognostic role of mesothelin expression and its association with KRAS mutation in advanced lung adenocarcinoma A. Thomas, Y. Chen, S. Steinberg, J. Luo, G. Giaccone, I. Pastan, M. Miettinen, R. Hassan

312

P093 Development of a one-step isolation platform for exosomal RNA and circulating cell-free DNA from cancer plasma samples D. Enderle, K. Brinkmann, T. Koestler, S. Bentink, C. Berking, J. Skog, M. Noerholm

313

P094 Optimal sequencing schedules for combining BRAF inhibition with BCL-2 inhibition A. Schalck, D.T. Frederick, M.R. Hammond, I. Ferreiro-Neira, Z.A. Cooper, J.C. Cusack, D.P. Lawrence, K.T. Flaherty, J.A. Wargo, R.J. Sullivan

314

P095 Biological characterization of TAS3681, a new type of androgen receptor antagonist with androgen receptor downregulating activity K. Minamiguchi, M. Seki, H. Aoyagi, T. Mori, D. Kajiwara, N. Masuko, R. Fujita, S. Okajima, Y. Hayashi, E. Sasaki, T. Utsugi

315

P096 Potent, partial agonists at ERalpha as selective estrogen mimics for treatment of tamoxifen-resistant breast cancer G. Thatcher, R. Xiong, H.K. Patel, J. Zhao, X. Liang, Y. Wang, M.E. Molloy, D. Tonetti

316

P097 IKKb inhibition suppresses sphere formation and self-renewal of lung cancer initiating cells T. Carneiro-Lobo, A.C.P. Salviatto, A.S. Baldwin, D.S. Basseres

317

P098 Novel therapeutic targets for ARID1A mutant ovarian clear cell carcinoma (OCCC) R.E. Miller, I. Bajrami, R. Brough, A. Konde, J. Campbell, R. Rafiq, A. Ashworth, C.J. Lord

318

P099 Genomic profiling of uterine leiomyosarcomas reveal frequent alterations in Akt/mammalian target of rapamycin (mTOR) pathway genes and other actionable genomic abnormalities linked to targeted therapies J.A. Elvin, Z.R. Chalmers, J. Chiemlicki, K.A.I. Wang, N. Palma, S.M. Ali, A. Huho, C.E. Sheehan, V.A. Miller, P.J. Stephens, J.S. Ross

319

P100 First-in-human study with ARQ 092, a novel pan AKT-inhibitor, in subjects with advanced solid tumors or recurrent malignant lymphoma M. Saleh, K. Papadpoulos, A. Arabnia, A. Patnaik, R.M. Stein, F. Chai, M. Lamar, R. Savage, G. Abbadessa, A. Tolcher

320

P101 Potent and selective inhibition of EZH2 by AU-2121 leads to significant tumor growth inhibition in mutant EZH2 dependent non-Hodgkin lymphoma S. Ahmed, K. Narayanan, A. Gadakh, S. Dodheri, S. Surendranath, S. Nathan, S. Mukherjee, S. Marappan, R. Sushmita, S. Dhadunia, K.M. Obilisetti, J. Anil, J. Reddy, K. Reddy, A. Basavaraju, C. Naik, C. Pandit, M. Ramachandra

321

P102 Novel alternative splice variants as potential biomarkers and therapeutic targets in aggressive prostate cancer in men of African descent S. Patierno, J. Freedman, B. Wang, N. Lee, D. George

322

P103 The development of short form of mimic microRNA for lung cancer therapy S. Ohno, K. Sudo, M. Kuroda

323

P104 Clinical pharmacokinetics (PK), translational PK/pharmacodynamics (PD), and exposure– adverse events (AEs) relationship of MLN2480, an oral investigational pan-RAF kinase inhibitor X. Zhou, A.J. Olszanski, M. Middleton, R. Gonzalez, M.J. Bargfrede, M. Kneissl, V. Boz´on, E. Gangolli, K. Venkatakrishnan, M. Patel, C. Zopf, D.W. Rasco

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P105 Identification and characterization of an irreversible inhibitor of CDK2 M. Martin, E. Anscombe, E. Meschini, D. Staunton, M. Geitmann, U.H. Danielson, L.Z. Wang, R. Mora Vidal, T. Reuillon, B.T. Golding, D.R. Newell, S. Wedge, M.E.M. Noble, J.A. Endicott, R.J. Griffin

325

P106 A novel non-ligand competing anti-EGFR antibody for cancer treatment X. Wang, S. Rust, F. Comer, V. Muniz-Medina, Q. Du, A. Yuan, K. Senthil, R. Fleming, R. Minter, S. Coats

326

P107 Combination of MDM2 antagonists with RAS pathway inhibitors in colorectal cancer B. Higgins, C. Tovar, K. Kolinsky, Y. Zhang, S.A. Middleton, G. Nichols, K. Packman, F. Su, L. Vassilev

327

P108 Jagged1 expression and its relevance in metastatic progression of breast cancers N. Bednarz-Knoll, A. Efstathiou, F. Gotzhein, H. Wilkman, V. Mueller, Y. Kang, K. Pantel

328

P109 A first-in-human study evaluating the safety and pharmacology of MM-151, a novel oligoclonal antiEGFR antibody combination in patients with refractory solid tumors M. Beeram, C. Lieu, W. Harb, L. Power, J. Kearns, R. Nering, V. Moyo, B. Wolf, A. Adjei

329

P110 Preclinical development of BIS-1602, first in class growth hormone releasing hormone antagonist Y.Y. Maxuitenko, N.L. Block, A.V. Schally, S.J. Reich, P. Goldstein

330

P111 Biomarker discovery and validation for assessing the response to cMet inhibition and functional inactivation S. Jung, S. Selzer, C. L¨oßner, K. Kuhn, U. Fiedler, A. Klotzb¨ucher, H.D. Zucht, S. Koncarevic, T. Prinz, D. Hertfelder, K.A. Boehme, H. Volkmer, M.H.G. Kubbutat, P. Budde, I. Pike

331

P112 Dual PI3K delta/gamma inhibition by RP6530 accentuates bortezomib activity in multiple myeloma cell lines S. Viswanadha, G. Babu, S. Veeraraghavan, S. Vakkalanka

332

P113 Short antisense oligonucleotides antagonize Lin28 and enable pre-let-7 processing and suppression of cell growth in human hepatocarcinoma cells M. Roos, G. Civenni, M. Lucic, D. Pavlicek, U. Pradere, H. Towbin, C.V. Catapano, J. Hall

333

P114 Aplidin: first in class compound targeting EEF1A in tumor cells A. Losada, J.F. Martinez, P. Moral, L. Carrasco, F. Gago, C. Cuevas, L.F. Garc´ıa-Fern´andez, C.M. Galmarini

334

P115 RICTOR amplification as a novel molecular target for the treatment of lung cancer H. Cheng, B. Piperdi, Y. Zou, A. Verma, X. Liu, E. Schwartz, C. Zhu, C. Montagna, B. Halmos, A. Chachoua, R. Perez-Soler

335

P116 The effect of food on the pharmacokinetics of the investigational Aurora A kinase (AAK) inhibitor, alisertib (MLN8237), in patients (pts) with advanced solid tumors or lymphomas X. Zhou, T.M. Bauer, S. Goel, J. Sarantopoulos, B. Zhang, V. Kelly, J. Mertz, K. Venkatakrishnan

336

P117 Sensitisation of HPV+ HNSCC to cytotoxic treatments by targeting the G2/M checkpoint with AZ1775 to improve survival A. Osman, N. Tanaka, A. Patel, J. Wang, A. Fitzgerald, T. Xie, M. Zhao, S. Jasser, M. Gadhikar, H. Skinner, M. Frederick, F. Johnson, J.N. Myers

337

P118 BM-1252 (APG-1252): a potent dual specific Bcl-2/Bcl-xL inhibitor that achieves complete tumor regression with minimal platelet toxicity L. Bai, J. Chen, L. Liu, D. McEachern, A. Aguilar, H. Zhou, C.Y. Yang, H. Wang, J. Wen, G. Wang, Y. Zhai, M. Guo, D. Yang, S. Wang

338

P119 NAMPT inhibition as strategy to impair tumor growth A. Cerezo, S. Jim´enez, E. Lospitao, N. Bravo, R. Campos-Olivas, C. Aguilera, M. Ca˜namero, R. Gilmour, S. Geeganage, G. Zhao, S. Velasco-Miguel

339

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P120 Preclinical activity of the VEGFR, FGFR and PDGFR inhibitor lucitanib in FGFR2 aberrant endometrial and gastric cancer models F. Guffanti, R. Chil`a, E. Bello, L. Ceriani, M. Zangarini, M. Zucchetti, C. Saba, A. Jacquet-Bescond, M. Pierrat, G. Damia

340

P121 Dinaciclib alters cell cycle dynamics and induces cell death in Soft Tissue Sarcomas N. Mulet Margalef, S. Rello-Varona, S. Garcia-Moncl´us, M. Fuentes-Guirado, O.M. Tirado, X. Garcia del Muro

341

P122 A phase 1/2 study evaluating the safety, pharmacokinetics and efficacy of ABT-414 in subjects with advanced solid tumors likely to over-express the epidermal growth factor receptor (EGFR) A. Tolcher, G. Goss, M. Gordon, L. Gandhi, K.P. Papadopoulos, D. Rasco, M. Pedersen, J. Fischer, W. Ames, H. Xiong, M. Dudley, W. Munasinghe, P. Ansell, K. Holen, E. Vokes

342

P123 Salmonella typhimurium A1-R effectively targets experimental breast cancer brain metastasis Y. Zhang, S. Miwa, N. Zhang, R.M. Hoffman, M. Zhao

343

P124 Search for new blood biomarkers for response to antiangiogenic therapy in non-small cell lung cancer patients A. Rodr´ıguez-Garzotto, M.T. Agull´o-Ortu˜no, S. Ponce, C.V. D´ıaz-Garc´ıa, A. Agudo-L´opez, C. P´erez, E. Prieto-Garc´ıa, H. Cort´es-Funes, L. Iglesias

344

P125 Interaction of plitidepsin with eEF1A in living tumor cells C. Garc´ıa, A. Losada, J.M. Molina-Guijarro, M.A. Sacrist´an, J. Martinez, C.M. Galmarini, M.P. Lillo

345

P126 Inhibition of Wnt pathway by novel thiazole-based Traf2- and Nck-interacting kinase (TNIK) inhibitor Y. Uno, H. Moriyama, S. Kashimoto, M. Masuda, M. Sawa, T. Yamada

346

P127 Can a link between the EGFR and cMET pathway in non-small cell lung cancer explain resistance against targeted therapies and open new therapeutic opportunities? N. Van Der Steen, K. Van Der Steen, K. Zwaenepoel, E. Giovannetti, M. Castiglia, A.P. Carreca, P. Pauwels, C. Rolfo

347

P128 Two NSCLC-PDXs with different EGFR exon 20-insertions respond differently to different TKIs H. Li, M. Yang, J. Cai

348

P129 Synergistic drug combinations that target beta-catenin-driven and MYC-driven cancers J.C.M. Uitdehaag, J.A.P. Spijkers-Hagelstein, J.A.D.M. de Roos, A.M. van Doornmalen, M.B.W. Prinsen, J. de Man, R.C. Buijsman, G.J.R. Zaman

349

P130 Oncogenic Ras mutants differentially utilize PLC-dependent calcium flux and PKC activation for MAPK signaling C. Pitt, F. McCormick

350

P131 hz515H7, a humanized antibody exerts its antitumor activity via antagonism of the CXCR4/SDF-1 axis, and through effector functions C. Klinguer-Hamour, M. Broussas, B. Akla, S. Berger, N. Boute, C. Beau-Larvor, A. Robert, J.F. Haeuw, L. Goetsch, C. Bailly, N. Corvaia

351

P132 UNC2025, a novel small molecule MerTK and Flt3 tyrosine kinase inhibitor, has therapeutic activity and promotes sensitivity to chemotherapy in animal models of acute leukemia D. Graham, D. DeRyckere, X. Wang, A.A. Hill, W. Zhang, S.V. Frye, H.S. Earp

352

P133 Protein expression for receptor activator of NFkB (RANK) and its ligand (RANKL) in non-small cell lung cancer (NSCLC) M. D’Arcangelo, S. Ekman, W. Dougall, D. Branstetter, M. Bergqvist, P. Liv, D. Chan, J. Botling, F. Hirsch

353

P134 PCR-based assay for BRAFV600 mutation analysis in ctDNA: clinical results from plasma and serum samples M. Gonzalez-Cao, C. Mayo de las Casas, M.A. Molina-Vila, L. De Mattos-Arruda, J.L. Manzano, E. Mu˜noz, J. Cortes, J.P. Berros, M. Sanmamed, A. Gonzalez, C. Alvarez, N. Karachaliou, N. JordanaAriza, S. Martin Algarra, R. Rosell

354

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P135 Altiratinib (DCC-2701): a balanced inhibitor of MET, TIE2, and VEGFR2 kinases that exhibits broad anti-tumor and anti-angiogenic activities D. Flynn, B.D. Smith, C.B. Leary, M.D. Kaufman, B.A. Turner, M.M. Hood, W.P. Lu, T.J. Rutkoski, T. Samarakoon, S. Vogeti, S.C. Wise, O. Rosen

355

P136 Discovery of novel pyrido[2,3-b]pyrazine as fibroblast growth factor receptor (FGFR-1, 2, 3 & 4) kinase inhibitors with nanomolar affinity P. Angibaud, O. Querolle, V. Berdini, G. Saxty, A. Cleasby, H. Colombel, I. Csoka, R. Gilissen, P. King, L. Meerpoel, C. Paulussen, I. Pilatte, V. Poncelet, D.C. Rees, B. Roux, V. Tronel, T. Verhulst, B. Wroblowski, C.W. Murray, J. Vialard

356

P137 Multi-color flow cytometry immunophenotyping for detection of CSC in NSCLC A. Martinez-Romero, J.M. Pardo, S. Tejedor, S. Calabuig Fari˜nas, R. Lucas, S. Figueroa, E. JantusLewintre, C. Camps, R. Farras

357

P138 Knockdown of beta-catenin with dicer-substrate siRNAs down-regulates Wnt/beta-catenin pathway signaling N. Pursell, W. Zhou, R. Diwanji, B. Holmes, N. Avitahl-Curtis, C. Dutta, H. Dudek, S. Ganesh, M. Abrams, W. Wang, B. Ying, D. Chen, S. Shui, U. Saxena, H. Yang, A. Shah, R. Arvan, M. Koser, C. Lai, B.D. Brown

358

P139 Correlation of tumour-associated macrophage, but not tumour-infiltrating lymphocyte, levels with progression-free survival in patients with metastatic renal cell carcinoma treated with axitinib J.A. Williams, J.F. Martini, B. Escudier, B.I. Rini, R.J. Motzer, J. Tarazi, S. Li, P.A. English

359

P140 Tumor suppressive microRNA-192 as a prognostic factor for recurrence of surgically resected nonsmall cell lung cancer J.H. Hong, K.S. Roh, E.K. Jeon, J.H. Kang, S.S. Suh, S.C. Lee

360

P141 Nanoformulations of the PARP inhibitors olaparib and BMN 673 for cancer nanotherapy S. Sridhar, S. Tangutoori, P. Baldwin

361

P142 Phenotypic plasticity in epithelial progenitors and mesenchymal carcinoma is regulated by Axl signaling A. Engelsen, K. Wnup-Lipinska, C. Tiron, F. Pelissier, T. Jokela, G. Haaland, G. Gausdal, T. Sandal, R. Frink, X. Liang, S. Hinz, L. Ahmed, M. Hellesøy, D. Mickelm, J. Minna, M. LaBarge, R. Brekken, J. Lorens

362

P143 Cytosolic p21 is a pharmacodynamic marker of CHEK1 and IKKe inhibition in ovarian cancer cells M. Kim, D.J. Min, G. Wright, C. Annunziata

363

P144 First-in-human phase 1 study of MLN2480, an investigational oral pan-RAF kinase inhibitor, in patients (pts) with relapsed or refractory solid tumors, including BRAF/NRAS-mutant melanoma M. Middleton, D.W. Rasco, A.J. Olszanski, P. Corrie, P. Lorigan, R. Plummer, J. Larkin, A. Pavlick, X. Zhou, Z. Yuan, E. Gangolli, M. Kneissl, V. Boz´on, R. Gonzalez

364

P145 Pim-1 kinase: Validated as a therapeutic cancer target for MYC-driven tumours O. Renner, Y. Cecilia, M.C. Rodriguez de Miguel, S. Peregrina, B. Garcia-Serelde, M.I. Albarran, A. Cebria, D. Cebrian, F. Ramos-Lima, A. Carnero, J. Pastor, C. Blanco-Aparicio

365

P146 Targeting colorectal and pancreatic cancer stem cells with the LGR5 monoclonal antibody BNC101 P. Chu, F. Shojaei, K. Smith, J. Norton, C. Walsh, J. Iglesias, C. Reyes

366

P147 Induction of apoptosis and inhibition of angiogenesis by novel fusion protein − AD-O54.9 as a new preclinical strategy in cancer treatment P. Rozga, B. Zerek, A. Pieczykolan, M. Galazka, K. Bukato, S. Pawlak, M. Szymanik, A. Jaworski, M. Teska-Kaminska, K. Poleszak, A. Grochot-Przeczek, W. Strozek, J. Pieczykolan

367

P148 NP137, the first humanized monoclonal antibody directed against netrin-1, exhibits antitumor activity by inducing dependence receptors-mediated cell death J.G. Delcros, B. Ducarouge, R. Abes, D. Goldschneider, B. Gibert, J.G. Blachier, D. Neves, P. Mehlen, A. Bernet, S. Depil

368

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P149 Diagnosis and molecular targeting for individualized treatment of patients with pre-neoplastic lesions and locally advanced cervical cancer P.M.A. Moreno-Acosta, A.R.R. Alfredo Romero-Rojas, A.H.S. Antonio Huertas Salgado, D.M. Diana Mayorga, N.M. Nicolas Morales, J.A. Jinneth Acosta, O.G. Oscar Gamboa, N.M. Nicolas Magne, M.M. Monica Molano

369

P150 Discovery of the genes that underpin the transition to malignant phenotype of breast tissues in a highly consanguineous region I. Gupta, S. Shanmuganathan, A. Ouhtit

370

P151 Selective targeting of head and neck cancer cells by perturbing reactive oxygen species homeostasis M. Kwon, J.W. Kim, E.H. Kim, J.Y. Park, S.Y. Kim, J.L. Roh

371

P152 Specific inhibition of hTERT expression by targeting common promoter mutations which cause quadruplex DNA instability D. Miller, A. Sokolova, S. Thomas, F. Rezzoug, J. Chaires, W. Dean, R. Buscaglia, J. Trent

372

P153 AKT2 gene amplification is a marker for sensitivity to allosteric but not ATP-competitive AKT inhibitors T. Abe, K. Ichikawa, R. Fujita, M. Okada, K. Tanaka, M. Ohkubo, K. Yonekura, T. Shimomura, T. Utsugi

373

P154 Neuregulin 1 (NRG1) expression is a predictive biomarker for response to AV-203, an ERBB3 inhibitory antibody, in human tumor models K. Meetze, S. Vincent, S. Tyler, E. Mazsa, A. Delpero, S. Bottega, D. McIntosh, J. Gyuris, Z. Weng

374

P155 MER as a novel therapeutic target in colorectal cancer K. Wong, A.C. Tan, T. Pitts, P. Klauck, S. Earp, S. Frye, X. Wang, D.K. Graham, S.G. Eckhardt

375

P156 Tumor-targeting Salmonella typhimurium A1-R enhances gemcitabine–bevacizumab efficacy on a patient-derived orthotopic xenograft (PDOX) pancreatic cancer nude mouse model Y. Hiroshima, M. Zhao, M.H.G. Katz, J.B. Fleming, S. Sato, T. Murakami, M. Yamamoto, F. Uehara, S. Miwa, S. Yano, M. Momiyama, Y. Zhang, A. Maawy, T. Chishima, K. Tanaka, M. Bouvet, I. Endo, R.M. Hoffman

376

P157 Plucked hair as a platform for monitoring pharmacodynamic and mechanistic consequences of clinical exposure to the Wnt/beta-catenin inhibitor PRI-724 G. Miele, B. Reed, E. Harrison, T. Mefo, J. Read, T. Senba, T. Odagami

377

P158 A first-in-human (FIH) safety and pharmacological study of SAR405838, a novel HDM2 antagonist, in patients with solid malignancies V. de Weger, M.P. Lolkema, M. Dickson, A. Le Cesne, A. Wagner, M. Merqui-Roelvink, A. Varga, W. Tap, G. Schwartz, G. Demetri, W. Zheng, G. Tuffal, S. Mac´e, H. Miao, J.H.M. Schellens, M. de Jonge

378

P159 Identification and rational in silico-based targeting of a novel mediator of metastatic breast cancer R. Clarkson, J. Soukupova, A. Wakefield, D. Turnham, W. Yang, C. Bordoni, A. Westwell, A. Brancale

379

P160 Induction of apoptosis with a novel dual cIAP1/XIAP antagonist in models of melanoma G. Ward, G. Chessari, C.N. Johnson, J. Lewis, S. Rich, N. Thompson

380

P161 Efficacy of specific FGFR inhibitors against gatekeeper resistance mutations and shared mechanism of cell death in FGFR2-dependent endometrial cancer cell lines L. Packer, S. Byron, S. Stehbens, D. Loch, F. Dehkhoda, S. Stephenson, P. Pollock

381

P162 Phase Ib study of oral dual-PI3K/mTOR inhibitor GDC-0980 in combination with capecitabine and mFOLFOX6 + bevacizumab in patients with advanced solid tumors and colorectal cancer L. Rosen, J. Goldman, J.M. Hubbard, M. Roos, J. Capdevila, J. Maynes, W. Lin, B. O’Keeffe, M. Lackner, J. Spoerke, J. Ware, B. Arnieri, E. Freas, S. Leong

382

P163 Identification of potent and selective tankyrase 1/2 inhibitors with activity in a subset of APC mutant colorectal cancer S.M. Guichard, Y. Zhang, D. Ferguson, A. Mazzola, H. Wang, L. Bao, S. Grosskurth, J. Johannes, M. Wagoner, M. Zinda, S. Fawell, E.J.E. Pease, A. Schuller

383

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P164 REDX04988, a novel dual B-RAF/C-RAF inhibitor and a potential therapeutic for BRAF-mutant colorectal cancer J. Rainard, R. Testar, R. Poonawala, H. Mason, P. Smith, H. Brooke, V. Huart, S. Frith, J. Ahmet, J. Hall, A. Morrison, M.A. Campbell, M. Bingham, R. Armer

384

P165 Noninvasive monitoring of acquired EGFR-T790M mutation and discovery of its heterogeneity in patients with advanced NSCLC treated with EGFR-TKI X. Ye, D. Zheng, M.Z. Zhang, Y. Sun, J.Y. Wang, J. Ni, H.P. Zhang, L. Zhang, J. Luo, J. Zhang, L. Tang, B. Su, G. Chen, G.S. Zhu, J.F. Xu, Y. Gu

385

P166 PI3K and MEK inhibitor combination toxicities and relative dose intensity: Vall d’Hebron experience A. Azaro, D. Marino, A. Garrido-Castro, C. Cruz, M. Alsina, J. Perez, R. Dienstmann, G. Argiles, C. Hierro, M. Berzosa, B. Adamo, J. Tabernero, J. Rodon

386

P167 Combination of the ERK inhibitor GDC-0994 with the MEK inhibitor cobimetinib significantly enhances anti-tumor activity in KRAS and BRAF mutant tumor models M. Merchant, J. Chan, C. Orr, J. Cheng, X. Wang, T. Hunsaker, M.C. Wagle, S.A. Huang, J. Tremayne, H. Ngu, M. Solon, J. Eastham-Anderson, H. Koeppen, L. Friedman, M. Belvin, J. Moffat, M. Junttila

387

P168 An integrated molecular analysis of lung adenocarcinomas identifies potential therapeutic targets among TTF1-negative tumors R.J. Cardnell, C. Behrens, L. Diao, Y. Fan, X. Tang, J.D. Minna, G.B. Mills, J.V. Heymach, I.I. Wistuba, J. Wang, L.A. Byers

388

P169 A phase 1, dose-escalation, first-in-human study of ARQ 087, an oral pan-FGFR inhibitor, in adult subjects with advanced solid tumors K. Papadopoulos, A. Tolcher, M. Kittaneh, A. Patniak, D. Rasco, G. Chambers, G. Newth, R. Savage, T. Hall, B. Schwartz, J. Kazakin, P. LoRusso

389

P170 Inhibitors of EZH2 act synergistically with type 1 interferon to induce a potent interferon-stimulated gene response, triggering apoptosis in diffuse large B-cell lymphoma B. Bradley, F. Zhao, C.C. Yuan, S. Balasubramanian, P. Iyer, C. Hatton, B. Bryant, E. Normant, P. Trojer

390

P171 LOXO-101, a pan TRK inhibitor, for the treatment of TRK-driven cancers S. Winski, B. Baer, D. Hartley, S. Rhodes, R. Wallace, S. Smith, N. Nanda, L. Kunkle, P. Lee, K. Bouhana

391

P172 Using modelling & simulation to integrate mouse PK−PD-efficacy with preliminary human PK data to inform the Phase II doses and schedule for the experimental c-Met inhibitor AZD6094 (Volitinib) R. Jones, A. Cheung, T. Coleman, P. Ballard, C. D’Cruz, A. Schuller, M. Frigault, Y. Gu, Y. Sai, S. Weiguo, Y. Ren, W. Qing, L. Lindbom, K. Petersson

392

P173 Small modification of ceritinib enhances the activity against ALK C.H. Park, C.H. Kang, H.J. Jung, H.R. Kim, C.O. Lee, H.K. Lee, S.U. Choi

393

P174 Sensitivity of acute myeloid leukemia cells to a urokinase-activated anthrax lethal toxin (PrAgU2/lf) is dependent on uPAR expression and phospho-MEK1/2 levels A. Bekdash, S.H. Liu, S.H. Leppla, A.E. Frankel, R. Abi-Habib

394

P175 cMet: Proof-of-concept clinical trial with volitinib in patients with advanced papillary renal cell cancer (PRCC) T.K. Choueiri, B. Escudier, S. Kumar Pal, E. Jonasch, D. Heng, T. Powles, H.T. Arkenau, E. Clark, C. D’Cruz, M. Frigault, A.F. Nash, S.R. Morgan

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P176 Gastrointestinal stromal tumor associated with neurofibromatosis type I T. Takahashi, T. Nishida, R. Nakatsuka, M. Kaneda, S. Hirota, Y. Miyazaki, Y. Kurokawa, M. Yamasaki, H. Miyata, K. Nakajima, S. Takiguchi, M. Mori, Y. Doki, S. Serada, T. Naka

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P177 Characterization of molecular targets of therapy in Non-Small Cell Lung Cancer (NSCLC) utilizing a liquid biopsy S. Greene, D. Lu, R. Krupa, M. Harvey, J. Louw, A. Jendrisak, N. Bales, D. Marrinucci, J. Gray, R. Dittamore P178 TAS-119 a selective inhibitor of Aurora A kinase, potentiates taxane therapy in breast and lung cancer models Y. Nakatsuru, A. Hashimoto, H. Sootome, K. Ito, M. Sakuragi, A. Miura, N. Oda, H. Hirai, T. Utsugi P179 Rho-GTPase, RAC1 and Cdc42 mediates Wnt−beta-catenin signals for metastasis associated phenotypes in TNBC: A proof of concept study N. Dey, P. De, B. Leyland-Jones P180 Optimization of novel pyrido[2,3-b]pyrazine based small molecule fibroblast growth factor receptor 1, 2, 3 & 4 (FGFR) inhibitors into a potential clinical candidate P. Angibaud, O. Querolle, V. Berdini, G. Saxty, A. Cleasby, H. Colombel, I. Csoka, N. Esser, R. Gilissen, L. Meerpoel, C. Paulussen, I. Pilatte, V. Poncelet, D.C. Rees, B. Roux, T. Verhulst, V. Tronel, B. Wroblowski, C.W. Murray, J. Vialard P181 TAS-121, a highly potent and mutant-specific EGFR inhibitor, overcomes T790M-acquired resistance with promising antitumor activity through specific inhibition of mutant EGFR signaling M. Kato, K. Miyadera, K. Ito, Y. Aoyagi, A. Hashimoto, K. Yonekura, Y. Iwasawa, T. Utsugi P182 Characterization of the oncogenic properties of mutant isocitrate dehydrogenase 1 (IDH1R132H) in human primary cells M. Barradas, L. Diezma, C.M. P´erez-Ferreiro, A. Cerezo, E. Lospitao, S. Peregrina, S. Jim´enez, N.A. Brooks, R. Torres, S. Geeganage, R. Gilmour, S. Velasco-Miguel P183 A novel dielectrophoretic microwell array system for detection and single cell analysis of circulating tumor cells from breast cancer patients T. Sawada, A. Morimoto, T. Mogami, K. Iijima, Y. Akiyama, K. Katayama, T. Futami, M. Yunokawa, K. Tamura, M. Watanabe, Y. Koh, F. Koizumi P184 Targeting PI3K somatic mutations reduces invasion and EMT in squamous cell carcinoma of the lung A. Cavazzoni, M. Bonelli, F. Saccani, S. La Monica, M. Galetti, C. Caffarra, D. Cretella, C. Fumarola, R. Alfieri, P.G. Petronini P185 Aurora kinases A and B are required for KRAS-induced lung cell oncogenicity E.O. Ozorio dos Santos, M.N. Aoki, E. Levantini, D.S. Bass`eres P186 Aflibercept (Zaltrap) directly attenuates the migration and invasion of colorectal cancer cells A. Bouygues, P. M´esange, M. Ayadi, V. Poindessous, M. Chiron, E. Dochy, T. Andr´e, A. de Gramont, A.K. Larsen P187 Structural basis for inhibition of ligand-dependent and -independent ErbB3 activation by KTN3379 D. Alvarado, S. Lee, E. Greenlee, G.F. Ligon, J.S. Lillquist, E.J. Natoli, J. Amick, Y. Hadari, J. Schlessinger P188 A potent and selective small molecule inhibitor of MCL-1 sensitizes DLBCL cell lines to the BCL-2 selective inhibitor ABT-199 D.C. Phillips, Y. Xiao, L. Lam, E. Litinovic, L. Roberts-Rapp, A.J. Souers, J.D. Leverson Paediatric Oncology Poster board

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P189 Transcription factor activating protein 2 beta (TFAP2B) mediates neuronal differentiation in neuroblastoma F. Ikram, S. Ackermann, F. Roels, R. Volland, B. Hero, F. Hertwig, H. Kocak, D. Dreidax, K.O. Henrich, F. Berthold, P. N¨urnberg, F. Westermann, M. Fischer P190 Pediatric Preclinical Testing Program (PPTP) Evaluation of the p53−MDM2 Antagonist MK-8242 M. Smith, M. Kang, P. Reynolds, R. Lock, H. Carol, R. Gorlick, A. Kolb, J. Maris, S. Keir, J. Wu, R. Kurmasheva, P. Houghton

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P191 Druggability of p16 deleted pediatric leukemia: The novel cell line POETIC3 identifies potential agents and drug combinations for mechanism based targeted therapeutics J. Reimer, A. Kovulchuk, Y. Ruan, R. Shah, A. Jayanthan, M. Perinpanayagam, T. Truong, I. AuerGrzesiak, J. Luider, O. Kovulchuk, T. Trippett, A. Narendran

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P192 The Pediatric Preclinical Testing Program (PPTP): Analysis of the first 10 years in vivo testing P. Houghton, R. Lock, H. Carol, R. Gorlick, A. Kolb, J. Maris, S. Keir, J. Wu, M. Kang, P. Reynolds, R. Kurmasheva, M. Smith

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P193 Next-generation sequencing identifies the mechanism of tumourigenesis caused by loss of SMARCB1 in malignant rhabdoid tumours M.A. Finetti, M. Selby, A. del Carpio Pons, J. Wood, B. Skalkoyannis, A. Smith, S. Crosier, S. Bailey, S. Clifford, D. Williamson

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P194 Genomic profiling using a clinical next generation sequencing (NGS) assay reveals genomic alterations to guide targeted therapy in advanced neuroblastoma patients S. Ali, E.M. Sanford, M.J. Hawryluk, J. Chmielecki, K. Wang, G.A. Palmer, N.A. Palma, D. Morosini, R. Erlich, R. Yelensky, D. Lipson, J.S. Ross, Y. Mosse, P.J. Stephens, J.M. Maris, V.A. Miller

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P195 Comprehensive next generation sequencing of solid tumors from 669 adolescents and young adults reveals a distinct spectrum of targetable genomic alterations D. Morosini, K. Wang, K. Wagner, B. Gershenhorn, R. Yelensky, D. Lipson, J. Chmielecki, S.M. Ali, J.S. Ross, P.J. Stephens, V.A. Miller

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P196 Results of phase I study of bolus 5-fluorouracil in children and young adults with recurrent ependymoma K.D. Wright, D.C. Turner, K.M. Haddock, M.O. Jacus, K.E. Harstead, S.L. Throm, V.M. Daryani, G.W. Robinson, G.T. Armstrong, A. Onar-Thomas, C.F. Stewart, A. Gajjar

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P197 Evaluating the activity of the p53−MDM2 inhibitor NDD0005 in Ewing sarcoma J. Pecqueur, B. Vormoor, Y. Zhao, H. Newell

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P198 Population pharmacokinetics of intravenous bolus 5-fluorouracil in a phase I trial for children and young adults with recurrent ependymoma D.C. Turner, K.M. Haddock, M.O. Jacus, K.E. Harstead, S.L. Throm, V.M. Daryani, C.F. Stewart, K.D. Wright

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P199 Targeted inhibition of casein kinase II (CK2) produces a strong therapeutic effect in pediatric leukemia S. Dovat, C. Song, C. Gowda, K.J. Payne

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P200 Analysis of genomic alterations in Ewing sarcoma (German cohort) reveals cooperating mutations and novel therapy targets G.H.S. Richter, K. Agelopoulos, E. Schmidt, K. von Heyking, B. Moser, H.U. Klein, U. Kontny, M. Dugas, K. Poos, E. Korsching, T. Buch, G. K¨ohler, C. R¨ossig, D. Baumhoer, H. J¨urgens, S. Burdach, W.E. Berdel, C. M¨uller-Tidow, U. Dirksen

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P201 Molecular profiling for factors predicting sensitivity or resistance to therapy in relapsed child cancer F. Saletta, C. Wadham, J. Byrne, D. Ziegler, G. McCowage, M. Haber, G. Marshall, M. Norris

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P202 CBL0137, a novel NFkB suppressor and p53 activator, is highly effective in pre-clinical models of neuroblastoma M. Haber, J. Murray, L. Gamble, A. Carnegie-Clark, H. Webber, M. Ruhle, D. Carter, A. Oberthur, M. Fischer, D. Ziegler, G.M. Marshall, K. Gurova, C. Burkhart, A. Purmal, A.V. Gudkov, M.D. Norris

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P203 RNA helicase A is essential for 1p36 gene KIF1Bb tumor suppression in neuroblastomas Z.X. Chen, K. Wallis, S.M. Fell, V.R. Sobrado, M.C. Hemmer, D. Ramsk¨old, Z. Choo, U. Hellman, R. Sandberg, R.S. Kenchappa, T. Martinsson, J.I. Johnsen, P. Kogner, S. Schlisio

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Toxicology Poster board

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P204 Nonclinical safety assessment of a humanized anti-OX40 agonist antibody, MOXR0916 R. Prell, W. Halpern, J. Beyer, J. Tarrant, S. Sukumaran, M. Huseni, R. Kaiser, D. Wilkins, S. Karanth, H. Chiu, J. Ruppel, C. Zhang, K. Lin, L. Damico-Beyer, J. Kim, H. Taylor

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P205 Evaluation of drug reactions to anti-neoplastic agents in Phase I clinical trials M. Bupathi, J. Hajjar, K. Hess, S. Bean, D. Karp, F. Meric-Bernstam, A. Naing

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P206 Serum levels of CCL22 and CCL25 might predict skin rash induction the commonest adverse event by bendamustine in the treatment of malignant lymphoma Y. Terui, R. Kuniyoshi, Y. Mishima, K. Hatake

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P207 Hematotoxicity potential of new drug candidates measured in hematopoietic progenitors in bone marrow samples J. Ballesteros, D. Primo, P. Hernandez, A. Robles, A.B. Espinosa, E. Arroyo, V. Garcia-Navas, J. Sanchez-Fenoy, M. Jimenez, M. Gaspar, J.L. Rojas, J. Martinez-Lopez, J. Gorrochategui

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Friday 21 November 2014 09:00–10:30

Poster Viewing

Plenary Session 8 11:00–13:00 Targeting RAS and Other Driver Oncogenes Chairs: J.A. Engelman (USA) and M. Barbacid (Spain)

Poster area Auditorium Abstract number

11:00 Targeting KRAS driven lung and pancreatic adenocarcinoma M. Barbacid (Spain) 11:20 Systematic interrogation of vulnerabilities in KRAS driven cancers W. Hahn (USA) Key objectives: 1. Understand the concept of synthetic lethality. 2. Understand systematic genetic screens to identify targets. 3. Understand the role of innate immune regulators in KRAS induced tumorigenesis. 11:40 Targeting synthetic lethal partners of KRAS J. Luo (USA) Main objectives: 1. To inform the participants about therapeutic strategies in targeting KRAS mutant cancer. 2. To inform the participants about the concept of synthetic lethality and its therapeutic utilities. 3. To provide the participants examples of synthetic lethal interactions with the KRAS oncogene. Take home messages: 1. Current difficulties in treating KRAS mutant tumours warrant exploration of new therapeutic strategies. 2. Synthetic lethality is a powerful approach for identifying functional vulnerabilities in cancer cells. 3. Synthetic lethal partners of the KRAS oncogene could be potential targets for drug discovery. 12:00 ORAL PRESENTATION: Clinical acquired resistance to combined RAF/EGFR or RAF/MEK inhibition in BRAF mutant colorectal cancer (CRC) patients through MAPK pathway alterations R. Corcoran, E.M. Coffee, E. Van Allen, L.G. Ahronian, N. Wagle, E.L. Kwak, J.E. Faris, A.J. Iafrate, L.A. Garraway, J.A. Engelman 12:15 LATE BREAKING ABSTRACT: Antitumor activity of ASP8273, an irreversible mutant selective EGFR-TKI, in NSCLC patients with tumors harboring EGFR activating mutations and T790M resistance mutation H. Murakami, H. Nokihara, T. Shimizu, T. Seto, A. Keating, A. Krivoshik, K. Uegaki, S. Morita, K. Nakagawa, M. Fukuoka

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12:25 LATE BREAKING ABSTRACT: Interim phase 2 results of study CO-1686-008: A phase 1/2 study 10LBA of the irreversible, mutant selective, EGFR inhibitor rociletinib (CO-1686) in patients with advanced non small cell lung cancer J. Soria, L.V. Sequist, J.W. Goldman, H.A. Wakelee, S.M. Gadgeel, A. Varga, H.A. Yu, B.J. Solomon, S.H. Ou, V. Papadimitrakopoulou, G.R. Oxnard, L. Horn, R. Dziadziuszko, B. Chao, A.I. Spira, S. Liu, T. Mekhail, S. Matheny, J. Litten, R.D. Camidge 12:35 LATE BREAKING ABSTRACT: Phase I study of the selective BRAFV600 inhibitor 11LBA encorafenib (LGX818) combined with cetuximab and with or without the a-specific PI3K inhibitor alpelisib (BYL719) in patients with advanced BRAF mutant colorectal cancer J. Tabernero, R. van Geel, J.C. Bendell, A. Spreafico, M. Schuler, T. Yoshino, J.P. Delord, Y. Yamada, M.P. Lolkema, J.E. Faris, F.A.L.M. Eskens, S. Sharma, R. Yaeger, H.J. Lenz, Z. Wainberg, E. Avsar, A. Chatterjee, S. Jaeger, T. Demuth, J.H.M. Schellens 12:45 Discussion Late Breaking Abstracts J.A. Engelman (USA)

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Drug Synthesis Poster board

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P001 Development of extracellular signal-regulated kinase 5 (ERK5) inhibitors for anti-cancer therapy S. Myers, N. Martin, R. Bawn, T. Blackburn, L. Barrett, T. Reuillon, B. Golding, R. Griffin, T. Hammonds, I. Hardcastle, H. Leung, D. Newell, L. Rigoreau, A. Wong, C. Cano

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P002 Design and structure–activity relationships of highly potent and bioavailable imidazolinone FASN KR domain inhibitors G. Bignan, R. Alexander, J. Bischoff, P. Connolly, M. Cummings, S. De Breucker, N. Esser, E. Fraiponts, R. Gilissen, B. Grasberger, B. Janssens, T. Lu, D. Ludovici, L. Meerpoel, C. Meyer, M. Parker, D. Peeters, C. Rocaboy, C. Schubert, K. Smans

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Molecular Targeted Agents II Poster board

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P003 Real-time pharmacokinetic (PK) results from an ongoing randomized, parallel-dose phase 1 study of onapristone in patients (pts) with progesterone receptor (PR)-expressing cancers F. Lokiec, J. Bonneterre, A. Italiano, A. Varga, M. Campone, T. LeSimple, A. Leary, V. Dieras, K. Rezai, S. Giacchetti, S. Proniuk, A. Bexon, E. Gilles, J. Bisaha, A. Zukiwski, P. Cottu

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P004 ODM-203, a novel, selective and balanced FGFR and VEGFR inhibitor with strong anti-tumor activity in FGFR- and VEGFR-dependent cancer models T. Holmstr¨om, A. Moilanen, T. Linnanen, G. Wohlfahrt, S. Karlsson, R. Oksala, T. Korjamo, M. Bj¨orkman, S. Samajadar, S. Rajagopalan, S. Chelur, K. Narayan, R. Ramachandra, T. Anthony, S. Ds, M. Ramachandra, P. Kallio

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P005 Genomic predictors of therapeutic sensitivity to TAS-119, a selective inhibitor of Aurora-A kinase H. Sootome, N. Fujita, A. Miura, T. Suzuki, H. Fukushima, S. Mizuarai, H. Hirai, T. Utsugi

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P006 Bipolar androgen therapy for men with castration sensitive and castration resistant prostate cancer: Reversing resistance and maintaining sensitivity to androgen ablative therapies S.R. Denmeade, E.S. Antonarakis, M.A. Eisenberger, M.A. Carducci, H. Wang, C.J. Paller, J.T. Isaacs, M.T. Schweizer

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P007 A pan-cancer tumor-derived epithelial-to–mesenchymal transition (EMT) signature determines patterns of drug sensitivity and enrichment in immune target expression following EMT M.P. Mak, P. Tong, L. Diao, P.K.S. Ng, Y. Fan, R.J. Cardnell, D.L. Gibbons, W.N. William, J.V. Heymach, K.R. Coombes, L.A. Byers, J. Wang

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P008 Monitoring activity of RXDX-101 in Phase 1/2 patients using a pharmacodynamic assay for TrkA activation D. Murphy, H. Ely, R. Patel, G. Wei, A. Diliberto, R. Shoemaker, J. Christiansen

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P009 PIM kinase inhibitor AZD1208 sensitises SCLC to BH3 mimetic AZD4320 R. Sloane, B. Bola, M. Lancashire, C. Hodgkinson, C. Morrow, K. Simpson, C. Dive

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P010 MM-131: A bispecific antibody that inhibits c-Met signaling through avid binding to the EpCAM tumor antigen B.D. Harms, A. Lugovskoy, A. Abu-Yousif, A. Fulgham, M. Geddie, S.V. Su, N. Kohli, B. Johnson, K. Masson, U.B. Nielsen, B. Schoeberl, G. MacBeath

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P011 PI3K/mTOR inhibitor VS-5584 targets cancer stem cells and prevents tumor regrowth after chemotherapy in preclinical models of small cell lung cancer V. Kolev, M. Padval, Q. Wright, J. Ricono, D. Weaver, J. Pachter, Q. Xu

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P012 Phenotypic alteration in a highly metastatic variant of the MDA-MB-231 cell line: role of Annexin A1 Y. Tu, E. Fietz, J. Cameron, A. Stewart

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P013 The role of methylation in metastasis of oral squamous cell carcinoma: understanding the OSCC methylome M. Clausen, L.J. Melchers, T. De Meyer, S. Denil, W. Criekinge, G.B. Wisman, J.L.N. Roodenburg, E. Schuuring P014 Targeting urokinase plasminogen activator for radioimmunotherapy using an antagonistic internalizing human antibody A. LeBeau, H.F. VanBrocklin P015 Notch3-targeted antibody drug conjugates have superior preclinical efficacy to Notch signaling inhibitors K.G. Geles, Y. Gao, L. Sridharan, A. Giannakou, T.T. Yamin, J. Golas, M. Charati, J. Lucas, K. Wang, S. Pirie-Shepherd, M. Roy, M. Follettie, A. Maderna, X. Li, L. Tchistiakova, H.P. Gerber, P. Sapra P016 Debio 1143 in combination with carboplatin and paclitaxel in patients with non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC) and platinum-refractory epithelial ovarian cancer (EOC). Preliminary results of a Phase I dose-escalation study C. Le Tourneau, I. Ray-Coquard, N. Isambert, C.A. Gomez-Roca, P. Cassier, M.P. Sablin, E. Ruits, B. Gavillet, C. Zanna, P. Fumuleau, J.P. Delord P017 MEK inhibition enhances gemcitabine efficacy by increasing MDM2-mediated ubiquitination and degradation of RRM1 F. Vena, E. Li Causi, T. Hagemann, J.A. Hartley, S. Goodstal, D. Hochhauser P018 The cancer stem cell inhibitors VS-6063 (defactinib) and VS-5584 exhibit synergistic anticancer activity in preclinical models of mesothelioma Q. Xu, W.F. Tam, C.M. Vidal, V.N. Kolev, Y. Kadariya, C.W. Menges, J.R. Testa, J.A. Pachter P019 Novel, quantitative in vivo shRNA screening approach identifies new molecular targets to block cancer metastasis L. Willetts, R. Paproski P020 Exposure to EGFR inhibitors influences release of extracellular vesicles by tumor cells R. van der Meel, S.M. van Dommelen, P. de Corte, M. Coimbra, W.W. van Solinge, P. Vader, R.M. Schiffelers P021 Cytokine induces MIR-424 expression and modulates SOCS2/STAT5 signaling pathway in oral cancer S.G. Shiah, H.Y. Peng, S.L.C. Jin, J.Y. Chang, C.C. Kuo P022 Precise gene editing of mutant NRAS using CRISPR to determine sensitivity to trametinib C. Hose, N.D. Fer, M. Burkett, J. Connelly, E. Harris, J. Lih, M. Williams, D. Evans, T. Silvers, A. Monks, R. Parchment, B.A. Teicher, J.H. Doroshow, A. Rapisarda P023 Correlative and updated clinical endpoint analysis of a multicenter phase II trial of selumetinib (AZD6244) plus erlotinib in chemotherapy-refractory advanced pancreatic adenocarcinoma (PDAC) A.H. Ko, A.H. Tempero, T.B. Bekaii-Saab, P. Kuhn, R. Courtin, S. Ziyeh, S. Tahiri, R.K. Kelley, E. Dito, A. Ong, R. Linetskaya, A. Talasaz, A.P. Venook, W. Korn P024 eIF2alpha phosphorylation determines the adaptation of tuberous sclerosis complex mutant cells to stress and their response to anti-tumor therapies A. Koromilas, C. Tenkerian, J. Krishnamoorthy, R. Kamindla, U. Kazimierczak, S. Wang P025 Plasma metabolomic signature of novel signal transduction inhibitors from preclinical identification to clinical validation J.E. Ang, R. Pandher, Y. Asad, D.J. Skene, P. Workman, S. Eccles, J. De Bono, S. Kaye, U. Banerji, S. Davies, F.I. Raynaud P026 Monitoring therapy response and resistance mutations in circulating RNA and DNA of plasma from patients with malignant melanoma D. Enderle, K. Brinkmann, T. Koestler, S. Bentink, K.T. Flaherty, J. Skog, M. Noerholm P027 Role of MDM2 as therapeutic target in gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) F. Briest, I. Grass, F. Christen, F. Lewens, H. Freitag, D. Kaemmerer, J. Saenger, M. Hummel, B. Siegmund, P. Grabowski

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Scientific programme – details

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P028 SF3B1 mutations induce disease relevant aberrant mRNA splicing in cancer and confer sensitivity to spliceosome inhibition S. Buonamici, K. Lim, J. Feala, R. Darman, K. Myint, E. Park, D. Aird, B. Chan, P. Fekkes, R. Furman, G. Keaney, P. Kumar, K. Kunii, X. Puyang, M. Thomas, Y. Mizui, M. Warmuth, P. Zhu, L. Yu, P. Smith P029 Preclinical pharmacology of AZD5312, a generation 2.5 antisense oligonucletotide targeting the androgen receptor with differentiated activity from enzalutamide B.R. Davies, A. Thomason, R. Ellston, H. Campbell, C. D’Cruz, A.M. Mazzola, T. Zhou, J. Schmidt, M. Jo, Y. Kim, A.R. McLeod, D. Blakey, N. Brooks P030 Combinational genome and proteome survey of therapeutic targets of hepatocellular carcinoma T. Yamada P031 Preclinical characterization of CC-115, a novel inhibitor of DNA-PK and mTOR kinase currently under clinical investigation D.S. Mortensen, K.E. Fultz, W. Xu, T. Tsuji, M. Hickman, M. Abbasian, G. Khambatta, B. Cathers, P. Worland, M.F. Moghaddam, J. Apuy, S. Richardson, J. Elsner, G. Shevlin, S. Perrin-Ninkovic, S. Canan, H.K. Raymon, R.K. Narla, S. Peng, S. Sankar P032 Molecular biomarkers of inflammatory signature in melanoma S. Ekmekcioglu, M. Shin-Sim, K. Tanese, V.G. Prieto, D.S. Hoon, E.A. Grimm P033 A phase I trial of dabrafenib (BRAF inhibitor) and pazopanib in BRAF mutated advanced malignancies S. Haraldsdottir, F. Janku, C. Timmers, S. Geyer, L.J. Schaaf, J. Sexton, J. Thurmond, V. VelezBravo, V.M. Stepanek, E. Bertino, K. Kendra, A. Mortazavi, V. Subbiah, M. Villalona-Calero, M. Poi, M. Phelps, M.H. Shah P034 Clinical sequencing of cancer in real-time by digital sequencing of cell-free DNA for tailoring targeted therapy in refractory cancer patients A. Talasaz, S. Mortimer, B.J. Schiller, G. Mei, S. Huang, D.S.B. Hoon, H. Eltoukhy P035 PTPN11 is a central node in intrinsic and acquired resistance to targeted cancer drugs A. Prahallad P036 Influence of warm and cold ischemia on molecular patterns in clinical biospecimen F.T. Unger, N. Lange, P.C. Uhlig, H. Juhl, K.A. David P037 Biomarker analysis in Phase 1b study of anti-cancer stem cell antibody Tarextumab (TAR) in combination with nab-paclitaxel and gemcitabine (Nab-P+Gem) demonstrates pharmacodynamic (PD) modulation of the Notch pathway in patients (pts) with untreated metastatic pancreatic cancer (mPC) A. Kapoun, E. O’Reilly, A. Cohn, J.C. Bendell, L. Smith, J.H. Strickler, W. Gluck, Y.W. Liu, B. Wallace, R. Tam, B. Cancilla, A. Brunner, D. Hill, L. Zhou, J. Dupont, C. Zhang, M. Wang P038 IGF2 drives IGF oncogenic signaling in HCC and emerges as a potential target for therapies I.M. Quetglas, R. Pinyol, D. Dauch, A. Portela, A. Villanueva, J. Peix, M. Higuera, A. Moeini, J. Zucman-Rossi, M. Esteller, V. Mazzaferro, L. Zender, J.M. Llovet P039 Two in one − Delivery of apoptotic signal into cancer cells by new class of TRAIL derived, fusion protein S.D. Pawlak, B.M. Zerek, P.K. Rozga, K. Bukato, M. Szymanik, M. Teska-Kaminska, A. Pieczykolan, A. Jaworski, M. Galazka, K. Poleszak, W. Strozek, J.S. Pieczykolan P040 Clinicopathologic implication of c-MYC gene copy number gain and overexpression in colorectal cancer K. Lee, Y. Kwak, G. Choe, W. Kim, D. Kim, S. Kang, H. Lee P041 OTX015, a novel BET-bromodomain (BET-BRD) inhibitor, is a promising anticancer agent for human glioblastoma L. Ouafik, C. Berenguer, M. Cayol, L. Astorgues-Xerri, M. Bekradda, E. Odore, K. Rezai, M.E. Riveiro, E. Cvitkovic P042 Transcriptional regulation of cancer stem cells marker CD133 by p53 E.K. Park, S.Y. Bang, S.A. Yi, J.W. Han

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P043 Screening of Champions predictive TumorGraft platform guides the clinical development of the selective dual BRAF-EGFR inhibitor CEP-32496 B. Ruggeri, M. Wabler, E. Bruckheimer, B. Wilkinson, B. Dorsey, S. Trusko, J. Friedman

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P044 Do we hit the target? Phospho-reactome measurements determine efficacy of targeted therapies L. van ’t Veer, M. Moro, Z. Chen, P.R.E. Lee, B. Pan, D. Brunen, A. Prahalled, R. Bernards, J.P. Coppe

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P045 FDG PET/CT as imaging biomarker in the era of molecular targeting therapies: sequential FDG PET/CT demonstrated biological response and acquisition of resistance to tyrosine kinase inhibitor therapy for renal cell carcinoma N. Nakaigawa, K. Namura, D. Ueno, U. Tateishi, T. Inoue, M. Yao

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P046 Potentiation of oxaliplatin in colon cancer by JNK inhibition I. Vasilevskaya, M. Selvakumaran, L. Cabal-Hierro, P. O’Dwyer

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P047 The synergistic anti-proliferative effect of combining the FGFR inhibitor, ARQ 087 with the AKT inhibitor, ARQ 092 in human cancer cell lines and PDX models E. Marchlik, Y. Yu, E. Chiesa, D. Dransfield

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P048 FGFR2 targeting with allosteric inhibitor RPT835 I. Tsimafeyeu, F. Daeyaert, W. Yin, J. Ludes-Meyers, M. Byakhov, S. Tjulandin

476

P049 Jab1/Csn5: a new player driving the resistance to HER2-targeted therapies for breast cancer F. Claret, T. Vu, T.J. Shackleford, J.L. Allensworth, Q. Zhang, F.J. Esteva, E. Drakos, R. Zhang, L. Tian, G.Z. Rassidakis

477

P050 Pharmacological disruption of the Astrocytic Elevated Gene-1 (AEG1) in anticancer intervention: PB0412_3 (PB03) as a first-in-class AEG1 interacting agent J. Jimeno, G. Acosta, C. Teixido, C. Olbiol, N. Karachaliou, M.A. Molina, O. Villaca˜nas, M. SanchezRonco, J. Bertran, A. Gimenez-Capitan, J.C. Monasterio, M. Taron, R. Rosell, F. Albericio

478

P051 The discovery and development of potent and specific anti-SialylTn antibodies for the treatment of solid tumors K. Meetze, D. Ghaderi, M. Zhang, I. Purnajo, J. Hermann, J. Fett, J. Behrens, A. Paula Galvao da Silva

479

P052 Identification of novel small molecules as selective PAK4 allosteric modulators (PAMs) by stable isotope labeling of amino acids in cells (SILAC) W. Senapedis, Y. Landesman, M. Schenone, B. Karger, S. Wu, S. Shacham, E. Baloglu

480

P053 In vitro and vivo evaluation of the pan FGFR inhibitor ARQ 087 and selective pan AKT inhibitor ARQ 092 in endometrial cancer: potential for combination therapy J. Meade, M.J. Wick, T. Vaught, R. Chavez, M. Rundle, K. Stanfield, B. Quattrochi, K.P. Papadopoulos, D.T. Dransfield, Y. Yu, E. Marchlik, E. Chiesa, A.W. Tolcher

481

P054 Repurposing the antihelminthic mebendazole as a hedgehog inhibitor A.R. Larsen, R.Y. Bai, J.H. Chung, A. Borodovsky, G.J. Riggins, F. Bunz

482

P055 Irreversible covalent pan-FGFR inhibitors are highly efficacious against FGFR-dependent cancers V.T. Phan, E. Verner, M. Gerritsen, J.M. Bradshaw, D.M. Goldstein, R.J. Hill, D. Karr, J. LaStant, P. Nunn, D. Tam, J. Shu, J.O. Funk, K. Brameld

483

P056 Treatment of advanced solid tumors with golvatinib (E7050) in combination with lenvatinib (E7080) E.L. Kwak, D. Juric, J.M. Cleary, G. Cote, J.F. Hilton, K.T. Flaherty, K. Wood, C. Rance, S. Barrett, G.I. Shapiro

484

P057 Glutathione S-transferases M1−5 reduce the aggressive behaviour in breast cancer by modulating the PI3K/AKT pathway A. Bergamaschi, S. Levy, A. Scott, J. Jeong, M. Kiefer, E. Beasley, J. Baker

485

P058 Development of small molecule activators of protein phosphatase 2A for the treatment of lung cancer J. Sangodkar, S. Mazhar, D. Kastrinsky, M. Ohlmeyer, G. Narla

486

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Abstract number

P059 EC-70124, a multi-kinase inhibitor, blocks NF-kB and STAT3 dependent signaling in prostate cancer stem cells G. Civenni, P. Costales, C. Garc´ıa-Incl´an, D. Albino, N. Longoni, L.E. Nu˜nez, F. Moris, G.M. Carbone, C.V. Catapano

487

P060 Development of a publicly accessible knowledgebase to facilitate decision support for clinical cancer genomics reporting K. Shaw, J. Zeng, A.M. Bailey, A. Johnson, V. Holla, E.V. Bernstam, J. Butts, J. Mendelsohn, G.B. Mills, F. Meric-Bernstam

488

P061 Development and clinical validation of a quantitative mass spectrometric assay for PD-L1 protein in FFPE NSCLC samples E. An, W. Liao, S. Thyparambil, J. Rodriguez, R. Salgia, I. Wistuba, J. Burrows, T. Hembrough

489

P062 Potent and selective inhibitors of the KRAS-signaling nanocluster protein, CNKSR1, block oncogenic KRAS signaling and mut-KRAS cell growth L. Kirkpatrick, G. Triana-Baltzer, M. Indarte, M. Scott, R. Lemos, G. Powis

490

P063 Development and characteristics of resistance to the HER family tyrosine kinase inhibitor AZD8931 V.G. Brunton, H. Creedon, M. Muir, T. Klinowska, K. McLeod, A. Byron

491

P064 Quantification of exportin-1 (XPO1) occupancy by selective inhibitors of nuclear export (SINE) M. Crochiere, B. Klebanov, E. Baloglu, O. Kalid, T. Kashyap, W. Senapedis, D. del Alamo, S. Tamir, D. McCauley, R. Carlson, M. Kauffman, S. Shacham, Y. Landesman

492

P065 Preclinical pharmacokinetic (PK)/pharmacodynamic (PD)/Efficacy modeling for MLN2480, an investigational pan-RAF kinase inhibitor, in A375 and SKMEL-2 human melanoma xenografts M. Patel, J. Chouitar, J. Mettetal, E. Gangolli, S. Balani, P. Shimoga, K. Galvin, W.C. Shyu, A. Chakravarty, C.J. Zopf

493

P066 LPA6 promotes growth and tumorigenicity of hepatocellular carcinoma via activation of PIM-3 protooncogene kinase C. Lopane, V. Goffredo, F. Dituri, F. De Santis, A. Filannino, R.C. Betz, Y.Y. Li, N. Mukaida, P. Winter, C. Tortorella, G. Giannelli, C. Sabb`a, A. Mazzocca

494

P067 Divergent androgen regulation of UPR pathways drives prostate cancer Y.J. Arnoldussen, M. Storm, X. Sheng, M. Tesikova, Y. Jin, H.Z. Nenseth, S. Zhao, I.G. Mills, L. Fazli, P. Rennie, B. Risberg, H. Wæhre, H.E. Danielsen, G.S. Hotamisligil, F. Saatcioglu

495

P068 Prevalence of MET amplification, MET expression, and MET-related genomic alterations in nonsmall cell lung cancer (NSCLC) A.L. Ang, H. Yang, A.A. Anderson, R. Tang, M.A. Damore, R.D. Loberg

496

P069 Preclinical evaluation of dimeric IAP proteins inhibitor APG-1387, in triple negative breast cancer both in vitro and in vivo G. Wang, P. Min, H. Wang, Z. Du, M. Wu, H. Dong, D. McEachern, L. Liu, Y. Lin, X. Lin, J. Wen, Y. Zhang, L. Gu, M. Guo, Y. Zhai, S. Wang, D. Yang

497

P070 CDK4/6 inhibitor (LY2835219) exhibits potent anti-tumor activity in human lung cancer cell lines with intact retinoblastoma J.W. Goldman, E.B. Garon, D. Conklin, D.J.L. Wong, B. Wolf, D.D. Silveira, N. Kamranpour, S. Pitts, R.S. Finn, D.J. Slamon

498

P071 Phase 2a study of copanlisib, a novel phosphatidylinositol-3-kinase (PI3K) inhibitor, in patients with relapsed/refractory, indolent or aggressive lymphoma M. Dreyling, P.L. Zinzani, K. Bouabdallah, D. Bron, D. Cunningham, K. Linton, C. Thieblemont, E. Van den Neste, U. Vitolo, J. Grunert, M. Giurescu, S. Mappa, B.H. Childs, F. Morschhauser

499

P072 Triple blockade with LEE011, everolimus, and exemestane in women with ER+/HER2− advanced/metastatic breast cancer: results from a Phase Ib clinical trial A. Bardia, C. Chavez-MacGregor, S. Modi, M. Campone, B. Ma, M. Kittaneh, L. Dirix, M. Motta, V. Zhang, S. Bhansali, M.L. Fjaellskog, M. Oliveira

500

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lxv

Abstract number

P073 Immunological STAT3 knockdown associated with anti-tumor activity in pre-clinical models translates to clinical samples, suggesting immune modulation contributes to the clinical activity of AZD9150, a therapeutic STAT3 ASO P. McCoon, R. Woessner, R. DuPont, K. Bell, M. Collins, L. Pablo, D. Lawson, P. Nadella, V. Jacobs, C. Womack, C. Reimer, D. Hong, J. Nemunaitis, Y.K. Kang, T.Y. Kim, H.Y. Lim, T. Okusaka, S. Nadano, C.C. Lin, P. Lyne P074 MI130004, an antibody–drug conjugate including a novel payload of marine origin: Evidences of in vivo activity P.M. Aviles, M.J. Guillen, J.M. Dominguez, M.J. Mu˜noz-Alonso, L.F. Garcia-Fernandez, M. Garranzo, V. Martinez, A. Francesch, S. Munt, C.M. Galmarini, C. Cuevas P075 UNC2025: A small molecule inhibitor of merTK with efficacy in mouse melanoma models S. Earp, D. Darr, A. Holtzhausen, A. Zimmermann, K. Clark, L. Hunter, N. Sharpless, X. Wang, S. Frye, D. Graham P076 Frequent loss-of-function mutations in MLK4 suppresses signaling in the JNK-cJUN-p21/p15 pathway to promote growth of colon cancer cells A. Marusiak, N. Stephenson, H. Baik, E. Trotter, Y. Li, E. Testoni, K. Blyth, S. Mason, L. Puto, C. Miller, T. Hunter, O. Sansom, J. Brognard P077 The anti-ErbB3 antibody, EV20, counteracts vemurafenib resistance in BRAF-mutated colorectal cancer stem cells G. Sala, P.R. Prasetyanti, D. Barcaroli, S. Volpe, E. Capone, C. Rossi, R. Carollo, M. Todaro, G. Stassi, J.P. Medema, S. Iacobelli, V. De Laurenzi P078 Phase (Ph) 1/2a study of TSR-011, a potent inhibitor of ALK and TRK, in advanced solid tumors including crizotinib-resistant ALK positive non-small cell lung cancer J. Sachdev, H.T. Arkenau, J.R. Infante, M.M. Mita, S.P. Anthony, R.B. Natale, S. Ejadi, K. Wilcoxen, V. Kansra, H. Laken, L. Hughes, R. Martell, G.J. Weiss P079 Amplification of D-type cyclin genes CCND2 and CCND3 confers an oncogene addiction phenotype in tumor cells and hypersensitivity to the CDK4/6 inhibitor LY2835219 S. Buchanan, X. Gong, M. Lallena, L. Chio, F. Merzoug, J. Dempsey, R. Beckman, C. Reinhard P080 A phase Ib trial of AMG386 and temsirolimus in patients with advanced sold tumors (PJC-008/NCI#9041) J.W. Chiu, S.J. Hotte, C.K. Kollmannsberger, D.J. Renouf, D.W. Cescon, D. Hedley, S. Chow, J. Moscow, M. Perry, I. Diaz-Padilla, D. Tan, H. Hirte, E. McWhirter, H. Chen, L.L. Siu, P.L. Bedard P081 Detecting gene amplification in circulating tumor cells of patients with gastric cancer for clinical trial Y. Mishima, S. Matsusaka, Y. Terui, M. Mikuniya, T. Takayama, N. Mizunuma, K. Hatake P082 Inhibition of STAT3 enhances the radiosensitising effect of temozolomide in malignant glioma cells in vitro and in vivo I. Kim, E. Choi, B. Cho, T. Han, S. Song, J. Kim, S. Paek

501

P083 ComboPredictor: identification of synergy biomarkers and enrichment within tumor sample populations M. Tomilo, A. Bankhead III, M.E. Urick, S. Eddy, R. Rickles, J. Ledell, E.T. Bowden

511

P084 Somatically mutated ABL1 represents an actionable and essential lung cancer survival gene E. Testoni, N.L. Stephenson, A.A. Marusiak, E.W. Trotter, A. Hudson, J. Brognard P085 Synergistic inhibition of ovarian and endometrial cancer cell lines using combined treatment of ARQ 092 and ARQ 087 in vitro and in vivo Y. Yu, E. Nakuci, E. Chiesa, C.R. Chen, E. Marchlik, D.T. Dransfield P086 Hypoxia inducible factor (HIF)-1a expression levels and p53 mutations are prognostic factors for survival in breast cancer patients treated with neoadjuvant chemotherapy M. Molina-Vila, S. Baulies, M. Gonzalez-Cao, N. Karachaliou, A. Rodriguez Capitan, S. Viteri, M. Cusido, R. Fabregas, C. Teixido, R. Rosell

512

502

503

504

505

506

507

508

509 510

513

514

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P087 TAS-115, a potent MET/VEGFR-targeted kinase inhibitor, is a new therapeutic approach for the treatment of bone metastasis of lung cancer A. Gomori, M. Sakuragi, A. Hashimoto, K. Ito, T. Haruma, T. Suzuki, H. Fujita, Y. Fujioka, K. Yonekura, T. Utsugi

515

P088 Different genetic profiles of resistant and sensitive patients with EGFR wild type NSCLC undergoing tyrosine kinase inhibitor (TKI) treatment P.U. Ulivi, E.C. Chiadini, A.D. Dubini, D.C. Calistri, M.P. Puccetti, M.A.B. Burgio, A.D. Delmonte, A.V. Verlicchi, A.G. Gamboni, M.P. Papi, W.Z. Zoli, L.C. Crin`o, C.D. Dazzi

516

P089 Identification and characterization of selective MELK kinase inhibitors P. Carpinelli, N. Amboldi, D. Ballinari, S. Re Depaolini, U. Cucchi, G. Canevari, M. Caruso, R. Galli, D. Donati, A. Isacchi, E.R. Felder, A. Montagnoli, A. Galvani

517

P090 The HSP90 inhibitor, AT13387, overcomes resistance to crizotinib and second generation ALK inhibitors A. Courtin, A. Smyth, K. Hearn, V. Martins, J. Lewis, N. Thompson, J. Lyons, N. Wallis

518

P091 AD-O53.2 − novel biological anticancer therapeutic agent with a dual mechanism of action K. Poleszak, J. Pieczykolan, S. Pawlak, B. Zerek, P. Rozga, M. Teska-Kaminska, M. Galazka, M. Szymanik, A. Jaworski, A. Pieczykolan, K. Bukato, W. Strozek

519

P092 Validation of Nanostring for FGFR1 gene expression analysis in squamous non-small cell lung cancer (sqNSCLC) tissue C. Rooney, C. Geh, V. Williams, C. Cresswell, K. Al-Kadhimi, M. Dymond, T. French, P.D. Smith, C. Barrett, E.A. Harrington, E. Kilgour

520

P093 Tisular expression of the EGFR and N-Glycolyl GM3 Ganglioside as recognized by ior egf/r3 and 14F7 Mabs in triple negative breast cancer A. Calvo P´erez, R.I. Alvarez Goyanes, E. Rengifo Calzado, S. Franco Odio, R. Camacho Rodr´ıguez

521

P094 Evaluation of the efficacy of melatonin in breast cancer metastasis mediated by ROCK-1 T. Borin, A.S. Arbab, L.C. Ferreira, G.B. Botaro, L.B. Maschio, G.M. Moschetta, N.N. Gon¸calves, G.R. Martins, D.A.P.C. Zuccari

522

P095 Antitumor activity of mTOR kinase and DNA-PK inhibitor CC-115 in a mouse model of glioblastoma H. Raymon, S. Peng, J. Katz, C. Zhao, C. Phan, M.F. Moghaddam, K.E. Fultz, S. Sankar, D.S. Mortensen, R.K. Narla

523

P096 Targeting MET for a differentiation therapy of rhabdomyosarcoma K. Skrzypek, A. Jagiello, B. Szewczyk, T. Adamus, K. Miekus, M. Majka

524

P097 Analysing Src and phosphoinositide 3-kinase pathway inhibition in a radiotherapy context − pathway interplay and therapeutic response E. Rowling, N. Burrows, P. Elvin, K. Williams

525

P098 A Phase I study of the dual, intravenous (IV) phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor PF-05212384 in combination with irinotecan in patients (pts) with colorectal cancer (CRC) and other advanced solid tumors Z. Wainberg, J. Tabernero, M.A. Maqueda, S. Leong, G. Del Conte, C. Britten, N. Brega, C. Davis, B. Houk, K. Pierce, J. Vermette, L. Siu, I. Brana

526

P099 Is CRAF required for the maintenance of KRAS mutant non-small cell lung cancer? P. Russell, J. Wickenden, K. Cadwallader, S. Maguire, J. Joel, M. Stockdale, A. Chicas, D. Banka, R. Darman, S. Perino, P. Fekkes, P. Smith, P. Zhu, S. Buonamici, J. Moore

527

P100 A procaspase activator shows preclinical promise for glioblastoma therapy G. Riggins, A. Joshi, P.J. Hergenrother

528

P101 The Cancer Research UK Stratified Medicine Programme: From national screening to national trial I. Mirabile, E. Shaw, C. Lindsay, I. Walker, P.W.M. Johnson

529

Scientific programme – details

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Abstract number

P102 Anti-angiogenic therapy induces T-lymphocyte infiltration associated with poor survival in metastatic renal cell carcinoma patients X.D. Liu, A. Hoang, L. Zhou, S. Kalra, A. Yetil, M. Sun, Z. Ding, S. Bai, P. German, X. Zhang, P. Tamboli, P. Rao, J. Karam, C. Wood, S. Matin, A. Zurita, N. Tannir, K. Sircar, E. Jonasch P103 In vitro and in vivo anti-tumor activity of ARQ 092, a potent and selective pan-AKT inhibitor Y. Yu, S. Cornell-Kennon, C.R. Chen, E. Marchlik, T. Isoyama, K. Tazaki, K. Fujiwara, D.T. Dransfield P104 Evaluation of a BRCAness signature as a predictive biomarker of response to veliparib/carboplatin plus standard neoadjuvant therapy in high-risk breast cancer: results from the I-SPY 2 trial A. Glas, J. Peeters, C. Yau, D.M. Wolf, A. Sanil, Y. Li, T. Severson, S. Linn, I-SPY 2 TRIAL Investigators, M. Buxton, A. DeMichele, N. Hylton, F. Symmans, D. Yee, M. Paoloni, L. Esserman, D. Berry, H. Rugo, O. Olopade, L.J. van ’t Veer P105 Detection of recurrent novel fusion transcripts from whole transcriptome sequencing of 120 primary breast cancer J. Kim, S.Y. Go, S.W. Kim, S. Lee, H.S. Lee, J. Park, M.J. Lee, H.G. Moon, D.Y. Noh, S. Kim, S. Kim, W. Han P106 Expression of c-MET in invasive meningioma S. Yun, J. Koh, K. Lee, A. Seo, K. Nam, G. Choe

530

531

532

533

534

P107 Tumor targeting and tissue distribution of solitomab (AMG 110; anti-EpCAM BiTE® ) in human EpCAM-positive tumor bearing mice F.J. Warnders, S. Waaijer, M.N. Lub-de Hooge, M. Friedrich, A.G.T. Terwisscha van Scheltinga, P. Deegen, S.K. Stienen, P.C. Pieslor, H.K. Cheung, J.G.W. Kosterink, E.G.E. de Vries P108 Shifts in microRNA expression pattern can facilitate the cancer cell stemness V. Halytskiy P109 Absolute quantitation of MET using mass spectrometry for clinical application: assay precision, stability, and correlation with MET gene amplification in FFPE tumor tissue F. Cecchi, W.L. Liao, S. Thyparambil, K. Bengali, J. Uzzell, M. Darflar, D. Krizman, J. Burrows, T. Hembrough, T. Veenstra, D.P. Bottaro, T. Karrison, L. Henderson, P. Xu, B. Rambo, S.Y. Xiao, L. Zhao, J. Hart, D. Catenacci P110 Development of a novel anti-tumor antibody targeting CXADR M. Kawada, H. Inoue, M. Kajikawa, M. Sugiura, S. Sakamoto, S. Urano, T. Masuda, A. Nomoto P111 Reversing the epithelial to mesenchymal transition with N-myc downstream regulated gene-1 and novel iron chelators in pancreatic cancer L. Fouani, Z. Kovacevic, D. Richardson

535

P112 Vemurafenib alters glucose utilization in BRAF-driven human melanoma cells M. Falck Miniotis, T. Delgado-Goni, S. Wantuch, P. Workman, R. Marais, M.O. Leach, M. Beloueche-Babari

540

P113 The abnormal bone remodeling associated with prostate cancer bone metastasis is attenuated by TAS115, the dual inhibitor for HGF/VEGF signaling H. Fujita, C. Matsumoto, K. Yonekura, K. Watanabe, M. Hirata, C. Miyaura, T. Utsugi, M. Inada P114 Tivantinib in combination with gemcitabine shows strong antitumor activity on mesothelioma cell lines and cytoskeletal effects via inhibition of actin M. Simonelli, P. Zucali, M. Suter, L. Rubino, A. Santoro, C. Carlo-Stella P115 MEK1/2 specific inhibitor, SMK-17 selectively induces apoptosis in b-catenin mutated tumors M. Kiga, A. Nakayama, Y. Sasazawa, Y. Shikata, H. Ikeda, E. Tashiro, M. Imoto P116 Preclinical studies of a dual Bcl-2/Bcl-xL inhibitor APG-1252 with strong anti-tumor efficacy and significantly reduced platelet toxicity H. Wang, G. Wang, Z. Du, M. Wu, D. McEachern, A. Aguilar, Y. Lin, X. Lin, J. Wen, L. Gu, M.I.N.G. Guo, Y. Zhai, S. Wang, D. Yang

541

536 537

538 539

542

543 544

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P117 MiRNAs related to KRAS mutational status in resectable non-small cell lung cancer S. Gallach, S. Calabuig-Fari˜nas, E. Jantus-Lewintre, D. Montaner, E. Escorihuela, J. Castellano, S. Figueroa, A. Blasco, C. Hernando, R. Guijarro, C. Camps

545

P118 The PI3Kbeta/delta inhibitor AZD8186 combines with the dual mTORC1/2 inhibitor AZD2014 to give comprehensive PI3K pathway inhibition and drive tumour regression in vivo B. Simon, U. Hancox, U. Polanska, L. Hanson, P. Dudley, R. Ellston, J. Maynard, M. Kraus, J. Curwen, T. Klinowska, L. Ward, F. Cruzalegui, S. Symeonides, K. Cronin

546

P119 Preclinical activity of Debio 1347, an oral selective FGFR1, 2, 3 inhibitor, in models harboring FGFR alterations A. Vaslin Chessex, C. Moulon, V. Nicolas-M´etral, A. M´en´etrey, H. Maby-El Hajjami, S. Rigotti, C. Zanna, G. Vuagniaux

547

P120 Development of a small molecule activator of protein phosphatase 2A for the treatment of prostate cancer K. McClinch, D. Callejas, M. Cooper, A. Stachnik, D. Kastrinsky, M. Ohlmeyer, M. Galsky, G. Narla

548

P121 A novel cancer marker and potential therapeutic target M.P. Parri, S.C. Campagnoli, A.G. Grandi, A.S. Santi, E.D.C. De Camilli, G.V. Viale, P.C. Chiarugi, L.T. Terracciano, P.P. Pileri, R. Grifantini

549

New Therapies with Pleiotropic Activity Poster board

Abstract number

P122 Nupharidine inhibits NF-kB activity, induces apoptosis and has synergistic cytotoxic activity with cisplatin and etoposide J. Gopas, J. Ozer, N. Eisner, D. Benharroch, A. Golan-Goldhirsh

550

P123 Phase 1B study of CC-486 (oral azacitidine) in tumors associated with a viral etiology D.D. Von Hoff, N. Isambert, J. Lopez-Martin, P.N. Munster, D.W. Rasco, J.C. Bendell, J.H.M. Schellens, J. Tomaro, R. Sarmiento, K. Liu, A. Nguyen, G.L. Bray, S.R. Hatty, J.F. DiMartino, C. Le Tourneau

551

P124 Discovery of an unexpected vulnerability of cancer stem-like cells via a functional dissection of EMT biology Y. Feng, E.S. Sokol, C.A. Del Vecchio, S. Sanduja, J.H. Claessen, T.A. Proia, D.X. Jin, F. Reinhardt, H.L. Ploegh, Q. Wang, P.B. Gupta

552

P125 PRPF6, a tri-snRNP spliceosome protein, regulates the alternative splicing of a distinct oncogenic ZAK variant and promotes colon tumor growth R. Firestein, A. Adler, M. McCleland, M. Yaylaoglu, Z. Zhang, J. Liu, Z. Jiang

553

P126 Dual EZH2 and EHMT2 histone methyltransferase inhibition increases biological efficacy in breast cancer cells R. Brown, M. Fuchter, E. Curry, I. Green, S. Kandil, F. Cherblanc, L. Payne, N. Chapman-Rothe, E. Shamsaei, N. Srimongkolpithak, J. Snyder, M. Vedadi

554

P127 CB-5083, a first in class inhibitor of the AAA-ATPase p97/VCP, induces irresolvable ER stress that results in antitumor activity in solid and hematological tumor models F. Yakes, R. Le Moigne, S. Wong, D.J. Anderson, S. Djakovic, E. Valle, M.K. Menon, J. Rice, B. Yao, F. Soriano, J. Wang, S. Kiss von Soly, M. Chesi, P.L. Bergsagel, H.J. Zhou, M. Rolfe, D. Wustrow

555

P128 Antagonizing microRNA mediated epigenetic reprogramming as therapeutic strategy for aggressive prostate cancer C. Dallavalle, D. Albino, G. Civenni, P. Ostano, M. Mello-Grand, R. Garcia-Escudero, G. Chiorino, C.V. Catapano, G.M. Carbone

556

P129 A rational approach for discovery of inhibitors of YAP−TEAD interaction L. Chene, A. Soude, C. Valaire, S. Delaporte, S. Jacquet, Y. Cambet, I. Braccini, M. Barth, C. Montalbetti, P. Broqua, C. Fromond

557

Scientific programme – details

Poster board

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P130 RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer H. Wendel, K. Singh, A. Wolfe, Y. Zhong, P. Drewe, J. Porco, J. Pelletier, G. R¨atsch P131 Development of a unique biologic for treating cysteine-dependent malignancies E. Stone, S. Cramer, A. Saha, S. Tiziani, J. Digiovanni, G. Georgiou P132 ZEN3365 is a novel BET bromodomain inhibitor for the treatment of hematologic malignancies and solid tumors K.G. McLure, R. Jahagirdar, O.A. Kharenko, E.M. Gesner, E. Campeau, D. Gilham, J. Wu, L. Tsujikawa, S. Attwell, C. Calosing, N. Sharma, J. Tobin, H.C. Hansen P133 Nonclinical characterization of the first in class investigational ubiquitin activating enzyme inhibitor MLN7243 in cellular and in vivo models of cancer in support of a phase I study M. Milhollen, M. Hyer, J. Ciavarri, T. Traore, D. Sappal, J. Huck, J. Shi, J. Duffy, J. Gavin, J. Brownell, Y. Yang, B. Stringer, Y. Ishii, E. Koenig, A. Lublinsky, R. Griffin, C. Xia, J. Powe, P. Fleming, N. Bence P134 Lysophosphatidic acid-induced breast cancer metastasis depends on LPA1/ZEB1/miR-21-activation pathway D. Sahay, R. Leblanc, J. Ribeiro, P. Clezardin, O. Peyruchaud P135 Hsp90 pharmacoproteomics: Harnessing pleiotropy for therapeutic synergy R. Goldstein, G. Chiosis, L.C. Cerchietti, A.M. Melnick P136 OTX015, a novel BET-BRD inhibitor is active in non-small-cell lung cancer cell (NSCLC) lines harboring different oncogenic mutations M. Riveiro, L. Astorgues-Xerri, N. Ijaz, M. Bekradda, R. Vazquez, R. Frapolli, A. Rinaldi, I. Kwee, E. Cvitkovic, E. Raymond P137 Preclinical evaluation of OTX015, a novel BET-BRD inhibitor, on small cell lung cancer (SCLC) cell lines N. Ijaz, L. Astorgues-Xerri, E. Odore, M. Bekradda, E. Cvitkovic, K. Noel, E. Raymond, M. Riveiro P138 GNS396 and analogues are potent new small molecules to target and kill chemotherapy-resistant subpopulation cells in acute myeloid leukemia F. Bassissi, R. Castellano, E. Josselin, C. Motersino, L. Pouyet, A. Goubard, A. Rostouin, G. Nicolas, S. Brun, J. Courcambeck, C. Dubray, T. Pr´ebet, N. Vey, A. B´eret, P. Halfon, Y. Collette P139 OTX015, a BET-bromodomain (BET-BRD) inhibitor, potentiates the in vitro effects of chemotherapy drugs and targeted agents in human leukemic cell lines L. Astorgues-Xerri, C. Canet-Jourdan, M. Bekradda, E. Cvitkovic, P. Herait, E. Raymond, M. Riveiro P140 The BET bromodomain inhibitor OTX015 shows synergy with several anticancer agents in preclinical models of mantle cell lymphoma (MCL) and multiple myeloma (MM) E. Bernasconi, C. Tarantelli, E. Gaudio, I. Kwee, A. Stathis, E. Riveiro, P. Herait, E. Cvitkovic, E. Zucca, F. Bertoni P141 Biological consequences of selective inhibition of the first BET bromodomain J.G. Seitzberg, M.H. Hansen, T.T.A. Kronborg, C.R. Underwood, V. Polyak, G. Friberg, B. Tonnesen, M. Nørregaard-Madsen, L. Teuber, T. Franch, S. Nielsen P142 Modulation of chromatin-related processes in DNA damage response as a potential strategy to treat acute myeloid leukemia K. Chomej, M. Lagergren Lindberg, D. Zong, T. Juntti, L. Kanter, L. Stenke, R. Lewensohn, K. Viktorsson, P. H˚aa˚ g P143 Novel potent inhibitors of the Histone Demethylase KDM1A: Synthesis, pharmacological evaluation and in vivo activity M. Varasi, O. Botrugno, A. Cappa, R. Dal Zuffo, P. Dessanti, A. Mai, A. Mattevi, C. Mercurio, G. Meroni, S. Minucci, F. Thaler, P. Trifiro’, S. Valente, P. Vianello, M. Villa P144 Oral panobinostat in patients with advanced tumors and impaired renal function: Relationship between pharmacokinetics and key safety parameters M. Porro, S. Sharma, P.O. Witteveen, M.P. Lolkema, D. Hess, H. Gelderblom, S.A. Hussain, E. Waldron, S. Valera, S. Mu

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P145 Suppression of c-Myc oncogene and hematological tumor growth by a novel benzazepine BET inhibitor K.A. Gelato, P. Lejeune, A.E. Fern´andez-Montalv´an, R. Neuhaus, N. Schmees, S. Siegel, H. Weinmann, V. Gekeler, B. Haendler P146 M-COPA, a Golgi disruptor, inhibits cell surface expression of MET protein and exhibits antitumor activity against MET-addicted cancers Y. Ohashi, M. Okamura, A. Hirosawa, A. Akatsuka, I. Shiina, K. Yoshimatsu, T. Yamori, S. Dan P147 SF3B1 mutations are associated with alternative splicing in ER-positive breast cancer S. Maguire, A. Leonidou, P. Wai, C. Marchio, C.K.Y. Ng, B. Weigelt, A. Sapino, A. Vincent-Salomon, J.S. Reis-Filho, R. Natrajan P148 Novel inhibitors of peritoneal seeding Y. Shen, X.L. Li, X. Lu, S. Kuwada P149 Characterization of the cellular mechanism of action of the first in class investigational inhibitor of the Ubiquitin Activating Enzyme, MLN7243 M. Milhollen, D. Sappal, J. Duffy, K. Hoar, J. Huck, P. Sha, E. Koenig, M. Hyer, J. Ciavarri, N. Bence P150 Invadopodia are required for cancer cell extravasation and are a therapeutic target for metastasis H.S. Leong, A.E. Robertson, K. Stoletov, S.J. Leith, C.A. Chin, A.E. Chien, M.N. Hague, A.L. Ablack, K. Carmine Simmen, V.A. McPherson, C.O. Postenka, E.A. Turley, S.A. Courtneidge, A.F. Chambers, J.D. Lewis P151 Eph/ephrin-B interactions modulate a BAFF-R/TACI dependent survival of chronic lymphocytic leukemia (CLL) cells mediated in vitro by bone marrow stromal cells L.M. Alonso-Colmenar, A.G. Zapata, P. Fortea, M.A. Flores, F. Ortu˜no, G. Soler, M.D. Garc´ıa, J. Garc´ıa-Cantalejo P152 Evaluation of the pan-BET-bromodomain inhibitor OTX015 as a single agent and in combination with everolimus (RAD001) in triple-negative breast cancer models R. V´azquez, L. Astorgues-Xerri, M.E. Riveiro, M. Di Marino, L. Beltrame, M. Bekradda, E. Cvitkovic, E. Erba, R. Frapolli, M. D’Incalci P153 Identification of genomic and chromatin features that predict transcriptional response to BET bromodomain inhibition J. Mertz, H.R. Huang, N. Follmer, A. Reddy, R. Centore, B. Bryant, C. Hatton, H. Franco, W.L. Krause, R. Sims III P154 Rational for targeting chromatin-modifying genes in clear-cell renal cell carcinomas G. Malouf, J. Zhang, D. Khayat, X. Su, J.P. Spano P155 The 8p11 amplicon in luminal breast cancers harbors multiple interacting epigenome modifying oncogenes: implications for epigenome-targeted therapy S. Ethier, J. Irish, R. Wilson, B. Turner P156 The correlation between EMT and cancer stemness in lung adenocarcinoma affects on its prognosis T. Sowa, T. Menju, T. Nakanishi, K. Shikuma, N. Imamura, T. Yamada, A. Aoyama, M. Sato, T. Sato, F. Chen, M. Sonobe, M. Omasa, H. Date, T. Sozu P157 The investigational HSP90 inhibitor ganetespib displays robust single agent activity in gastric cancer models both as monotherapy and in combination with standard of care therapeutics S. He, C. Zhang, J.P. Jimenez, J. Sang, M. Sequeira, D. Smith, J. Acquaviva, M. Nagai, R. Bates, D.A. Proia P158 Phase I study of pan-histone deacetylase inhibitor abexinostat in combination with cisplatin in patients with advanced solid tumors M. Campone, N. Isambert, R. Sabatier, H. Castanie, S. Zanetta, I. Sudey, F. Cantero, J. Pauly, E. Leroux, S. Malasse, A. Goncalves P159 Cellular pharmacokinetics and molecular pharmacodynamics studies of the BRD-BET inhibitor OTX015 in sensitive and resistant leukemic cell lines E. Odore, L. Astorgues-Xerri, M. Bekradda, E. Cvitkovic, P. Herait, F. Lokiec, K. Rezai, M. Riveiro

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P160 Neddylation as a therapeutic target in refractory pediatric malignancies: Evaluation of the activating enzyme inhibitor MLN4924 Y. Ruan, A. Jayanthan, T. Cooper, A. Narendran

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P161 N-myc downstream regulated gene 1 (NDRG1) as a novel anti-angiogenic and therapeutic target for VEGF/VEGF receptor signaling by vascular endothelial cells K. Watari, A. Shinoda, T. Shibata, A. Kawahara, T. Nakama, S. Yoshida, M. Kage, M. Kuwano, M. Ono

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P162 A dose dense schedule improves antitumor activity of trabectedin in myxoid liposarcoma with type III FUS-CHOP chimera S. Uboldi, R. Frapolli, E. Bello, S. Brich, F. Bozzi, R. Sanfilippo, P.G. Casali, A. Gronchi, C.M. Galmarini, J.M. Fernandez Sousa-Faro, S. Pilotti, M. D’Incalci

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P163 Mechanistic analysis of reversible FASN inhibition in preclinical tumor models identifies highly susceptible tumor types and enriches biomarker discovery for clinical applications T.S. Heuer, R. Ventura, J. Waszczuk, K. Mordec, J. Lai, M. Fridlib, R. Johnson, L. Hu, H. Cai, A. Wagman, M. O’Farrell, D. Buckley, G. Kemble

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P164 TAS-116, a highly selective inhibitor of heat shock protein 90a/b, inhibits tumor growth in biliary tract cancer mouse models S. Ohkubo, H. Muraoka, Y. Kodama, K. Ito, S. Ito, A. Hashimoto, C. Yoshimura, T. Utsugi

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P165 Strategies to overcome resistance to BET bromodomain inhibitor in KRAS/LKB1 mutant NSCLC M. Soucheray, E. Kikuchi, I. Pulido, E. Akbay, J.H. Becker, C.L. Christensen, N. Johnson, T.B. Patel, J. Carretero, K.K. Wong, T. Shimamura

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P166 Identification of novel EZH2 inhibitor scaffolds X. Espanel, L. Chene, A. Soude, S. Estevez, V. Adarbes, B. Loillier, B. Boubia, P. Masson, C. Montalbetti, P. Broqua, C. Fromond

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P167 Improving specificity of epigenetic therapy through combined targeting of DNA and histone methylation T. Sato, M. Cesaroni, J. Jelinek, J.P. Issa

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26 EORTC–NCI–AACR Symposium on Molecular Targets and Cancer Therapeutics

Tuesday 18 November 2014

Plenary Session 1

Tuesday 18 November 2014

Tuesday 18 November 2014

15:15–17:30

PLENARY SESSION 1

Is the Genomic Landscape Changing the Outcome for Cancer Patients? 1 ORAL PRESENTATION Feasibility of large-scale genomic testing to facilitate enrollment on genomically-matched clinical trials F. Meric-Bernstam1 , L. Brusco2 , S. Kopetz3 , M. Davies4 , M.J. Routbort5 , S.A. Piha-Paul2 , R. Alvarez6 , S. Khose2 , J. DeGroot7 , V. Ravi8 , F. Janku2 , D. Hong2 , Y. Li9 , R. Luthra5 , K.P. Patel5 , R. Broaddus10 , K. Shaw11 , J. Mendelsohn11 , G.B. Mills12 . 1 The University of Texas MD Anderson Cancer Center, Investigational Cancer Therapeutics, Houston Texas, USA; 2 The University of Texas MD Anderson Cancer Center, Investigational Cancer Therapeutics, Houston Texas, USA; 3 The University of Texas MD Anderson Cancer Center, GI Medical Oncology, Houston Texas, USA; 4 The University of Texas MD Anderson Cancer Center, Melanoma Medical Oncology, Houston Texas, USA; 5 The University of Texas MD Anderson Cancer Center, Hematopathology, Houston Texas, USA; 6 The University of Texas MD Anderson Cancer Center, Breast Medical Oncology, Houston Texas, USA; 7 The University of Texas MD Anderson Cancer Center, Neuro-Oncology, Houston Texas, USA; 8 The University of Texas MD Anderson Cancer Center, Sarcoma Medical Oncology, Houston Texas, USA; 9 The University of Texas MD Anderson Cancer Center, Biostatistics, Houston Texas, USA; 10 The University of Texas MD Anderson Cancer Center, Pathology, Houston Texas, USA; 11 The University of Texas MD Anderson Cancer Center, Institute for Personalized Cancer Therapy, Houston Texas, USA; 12 The University of Texas MD Anderson Cancer Center, Systems Biology, Houston Texas, USA Background: We sought to determine the feasibility of performing standardized somatic mutation analysis for patients with a variety of advanced cancer types at a major cancer center. We report the experience with the first 2000 patients who underwent testing on a genomic testing protocol, including the frequency of actionable alterations across tumor types, subsequent enrollment on clinical trials, and the challenges for trial enrollment. Materials and Methods: From February 2012 to July 2013, after informed consent, 2000 patients underwent genomic testing using a ‘hot-spot’ platform, an 11-gene Sequenom assay (251 patients) or 46gene Ion Torrent (Ampliseq) assay (1749 patients). Of the 46 genes tested, 35 were considered ‘potentially actionable’, as these genes might be directly or indirectly targeted by approved or investigational agents. TP53 was not considered actionable. Trials that require a genomic alteration for enrollment were considered ‘genotype-selected’, and biomarker-unselected trials using agents relevant to a genomic alteration were designated ‘genotype-relevant’; either type of trial was considered ‘genomically-matched’. Results: Of the 2000 patients, 38.4% had at least one potentially actionable alteration. The most commonly mutated potentially actionable genes were PIK3CA (12.8%), KRAS (11.3%), BRAF (7%), NRAS (4.2%), EGFR (1.8%), AKT1 (1.4%) and PTEN (hot-spot mutations, 1.2%). Patients with mutations in potentially actionable genes were more likely to be treated on clinical trials than those without mutations (30% vs 25%, p = 0.0046). Of the patients with mutations in potentially actionable genes, 7% were treated on genotype-selected trials, 5% were treated on genotype-relevant trials and 20% were treated on other therapeutic trials. Of 97 patients treated on genotype-matched trials, 67% had PIK3CA/AKT1/PTEN or BRAF mutations. Eighty-five patients with PI3K/AKT1/PTEN/BRAF mutations had a discussion about genotype-matched trials documented but were not enrolled; most common reasons were election of treatment closer-to-home (22%), non-investigational therapy (20%), poor performance status (13%), ineligibility for trials for other reasons (17%), stable disease (9%), lack of relevant trials (8%) or enrollment in other trials (6%). Discussion: Broad implementation of multiplex hot-spot testing is feasible and identifies potentially actionable alterations. Patients with actionable alterations are more likely to be enrolled on clinical trials; however, only a small portion of patients with actionable alterations were enrolled on genotype-matched trials. Novel solutions to increase awareness of test results and therapeutic implications, increased awareness of existing trials such as clinical trial alert systems, and increased access to targeted drugs through basket trials and novel just-in-time clinical trial systems are needed.

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26 EORTC–NCI–AACR Symposium on Molecular Targets and Cancer Therapeutics

Wednesday 19 November 2014

Plenary Session 2

Wednesday 19 November 2014

Wednesday 19 November 2014

13:15–15:20

PLENARY SESSION 2

Proffered Paper Session 2 ORAL PRESENTATION Safety and early evidence of activity of a first-in-human phase I study of the novel cancer stem cell (CSC) targeting antibody OMP-52M51 (anti-Notch1) administered intravenously to patients with certain advanced solid tumors A. Patnaik1 , P. LoRusso2 , P. Munster3 , A.W. Tolcher1 , S.L. Davis4 , J. Heymach5 , R. Ferraroto5 , L. Xu6 , A.M. Kapoun6 , L. Faoro6 , J.A. Lewicki6 , J. Dupont6 , S.G. Eckhardt4 . 1 South Texas Accelerated Research Therapeutics (START), San Antonio TX, USA; 2 Wayne State University Karmanos Cancer Institute Detroit MI, Center for Translational Therapeutics, Detroit MI, USA; 3 University of California San Francisco, Department of Medicine, San Francisco CA, USA; 4 University of Colorado-Denver, University of Colorado Cancer Center, Aurora CO, USA; 5 MD Anderson Cancer Center, Houston TX, USA; 5 Oncomed Pharmaceuticals Inc., Redwood City CA, USA Background: The Notch pathway plays a key role in embryonic development, the regulation of stem and progenitor cells, and is implicated in human cancer. Notch1 (N1) signaling is activated by various mechanisms including N1 activating mutations in certain solid tumors. OMP-52M51 is a humanized IgG2 antibody that inhibits the signaling function of N1. As such, OMP-52M51 is a novel anti-cancer agent that inhibits tumor growth through direct actions on tumor cells, including CSCs, and effects on tumor angiogenesis. Materials and Methods: A phase I dose escalation and expansion study was initiated in patients (pts) with certain advanced solid tumors (cholangiocarcinoma, breast (BC), colorectal (CRC), esophageal, gastric, pancreatic, and small cell lung cancers) that have rates of N1 activation between 12−29%. OMP-52M51 was administered intravenously to study safety, pharmacokinetics (PK), pharmacodynamics, preliminary efficacy, and to determine the maximum tolerated dose. The trial has a N1 IHC biomarker selected expansion cohort. Results: 20 pts have been enrolled in 5 cohorts at doses of 0.25, 0.5, 1, and 2.5 mg/kg every 4 weeks (Q4W) and 2.5 mg/kg every 3 weeks (Q3W). The most frequent adverse events (AE) were: mild to moderate diarrhea (75%), fatigue (65%), and nausea (40%). Grade 3 or higher AEs included diarrhea (20%), increased alkaline phosphatase (15%), and pain (15%). Diarrhea was manageable. Two pts experienced dose-limiting toxicity (DLTs) AEs with gr 3 fatigue (2.5 mg/kg Q4W) and gr 3 diarrhea (2.5 mg/kg Q3W). PK exhibited dose-dependent clearance with a T 12 of 4 days at 2.5 mg/kg. One pt with an N1 activating mutation in adenoid cystic carcinoma (ACC) had partial response (38% decrease in lesions) after 2 doses. 1 BC and 1 CRC pt had stable disease (110 days and 280 days, respectively). Sequencing the cell-free DNA from the CRC pt found a loss-of-function mutation in FBXW7, a negative regulator of N1. A prototype immunohistochemistry (IHC) test showed high levels of activated N1 in the ACC and CRC pts. Biomarker analyses revealed reductions in circulating tumor cells with OMP-52M51 treatment. The MTD has not been established. Conclusions: OMP-52M51 is generally well tolerated. Diarrhea is the primary toxicity of this antibody. Potential early efficacy consistent with the predictive biomarker hypothesis is noted. Enrollment continues. Updated efficacy, safety, and PK results will be presented. Clinical trial information: NCT01778439.

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3 ORAL PRESENTATION Afuresertib (GSK2110183), an oral AKT kinase inhibitor, in combination with carboplatin and paclitaxel in recurrent ovarian cancer S. Blagden1 , A. Hamilton2 , L. Mileshkin3 , M. Hall4 , T. Meniawy5 , S. Wong6 , S. Anandra2 , M. Buck5 , D. McAleer7 , B.A. Reedy7 , R.B. Noble7 , D.A. Smith8 , S.R. Morris9 , H. Gabra1 . 1 Imperial College Hammersmith Hospital Campus, Department of Medical Oncology, London, United Kingdom; 2 Royal Women’s Hospital, Department of Medical Oncology, Parkville, Australia; 3 Peter MacCallum Cancer Centre, Department of Cancer Medicine, East Melbourne, Australia; 4 Mount Vernon Cancer Centre, Department of Medical Oncology, Middlesex, United Kingdom; 5 Sir Charles Gairdner Hospital, Department of Medical Oncology, Perth, Australia; 6 Western Hospital, Department of Oncology, Footscray, Australia; 7 GlaxoSmithKline, GSK Alternative Discovery & Development, Upper Merion, USA; 8 GlaxoSmithKline, GSK Clinical Pharmacology, Modelling and Simulations; 9 GlaxoSmithKline, GSK Alternative Discovery & Development, Research Triangle Park, USA Background: Afuresertib is a highly selective oral pan-AKT kinase inhibitor in development for patients with hematologic and solid malignancies. Preclinically, AKT inhibition by afuresertib can reverse platinum resistance in ovarian cancer cell lines isolated from patients with platinum-resistant ovarian cancer. Materials and Methods: Study objectives were to evaluate the maximum tolerated dose (MTD), pharmacokinetics (PK), and clinical activity of afuresertib in combination with carboplatin & paclitaxel in patients (pts) with recurrent, platinum sensitive, resistant or refractory epithelial ovarian cancer (EOC). This two part study included a dose escalation (part1) and expansion phase (part2). Part 1 evaluated the PK and toxicity profile of increasing doses [50 mg-150 mg daily] of afuresertib with IV carboplatin (AUC 5) & paclitaxel (175 mg/m2 ) q3 weekly for up to 6 cycles followed by afuresertib monotherapy until progression. Efficacy was evaluated using GCIG CA125 and RECIST 1.1 criteria. Results: Part 1 has completed enrolment (n = 29; mean age 59), 23 of the 29 pts had platinum-resistant or refractory EOC with a median platinum-free interval of 4 months. Pts had a median of 3 prior therapies; 17% had 6 prior therapies. Dose-limiting-toxicities were grade 3 rash (n = 3, at 125 mg (1) and 150 mg (2)) and grade 3 syncope (1) at 125 mg afuresertib. MTD of afuresertib with carboplatin & paclitaxel was defined as 125 mg daily. The most frequent adverse events (AEs) regardless of relatedness were diarrhea (72%), nausea (72%) and fatigue (69%); all  Grade 2. The most common AEs  grade 3 were neutropenia (40%), hyperglycemia (10%), thrombocytopenia (7%), and ascites (7%). There was no clinically relevant PK interaction between afuresertib and paclitaxel. The ORR (overall response rate) regardless of dose was 60% (CA125) and 29% (RECIST). For those patients who received afuresertib at the MTD dose of 125 mg, the ORR was 50% by both GCIG CA125 and RECIST 1.1 criteria; the clinical benefit rate (RECIST partial responses plus stable disease of  6 months) was 63%. Conclusions: Afuresertib can be safely combined with carboplatin and paclitaxel at a MTD of 125 mg once daily. This triplet shows promising clinical activity in a heavily pretreated patient population. Part 2 is now open to pts with resistant or refractory EOC (in two separate cohorts) to further evaluate the safety and clinical efficacy of afuresertib within this treatment combination.

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Wednesday 19 November 2014

4 ORAL PRESENTATION Activity of galeterone in castrate-resistant prostate cancer (CRPC) with C-terminal AR loss: Results from ARMOR2 M.E. Taplin1 , K.N. Chi2 , F. Chu3 , J. Cochran4 , W.J. Edenfield5 , E.S. Antonarakis6 , U. Emmenegger7 , E.I. Heath8 , A. Hussain9 , V.C. Njar9 , A. Koletsky10 , D. Lipsitz11 , L. Nordquist12 , R. Pili13 , M. Rettig14 , O. Sartor15 , N.D. Shore16 , D. Marrinucci17 , K. Mamlouk18 , B. Montgomery19 . 1 Dana Farber Cancer Institute, Department of Medicine Harvard Medical School, Boston MA, USA; 2 BC Cancer Agency, Genitourinary Oncology, Vancouver BC, Canada; 3 San Bernadino Urological Associates, Urology, San Bernadino Ca, USA; 4 Urology Clinics of North Texas, Urology, Dallas Tx, USA; 5 Cancer Centers of the Carolinas, Oncology, Greenville SC, USA; 6 The Sidney Kimmel Comprehensive Cancer Center at John Hopkins, Genitourinary Oncology, Baltimore MD, USA; 7 Sunnybrook Health Sciences Centre, Oncology, Toronto ON, Canada; 8 Karmanos Cancer Institute Wayne State University, Genitourinary Oncology, Detroit MI, USA; 9 Univ of Maryland, Genitourinary Oncology, Baltimore MD, USA; 10 Center for Hematology-Oncology, Oncology, Boca Raton FL, USA; 11 Carolina Urology, Urology, Concord NC, USA; 12 Urology Cancer Center and GU Research Network LLC, Oncology, Omaha NE, USA; 13 Roswell Park Center Institute, Genitourinary Oncology, Buffalo NY, USA; 14 Institute of Urologic Oncology, Genitourinary Oncology, Los Angeles CA, USA; 15 Tulane University, Genitourinary Oncology, New Orleans LA, USA; 16 Carolina Urologic Research Center Atlantic Urology Clinics, Urology, Myrtle Beach SC, USA; 17 EPIC Sciences, Sciences, San Diego CA, USA; 18 Tokai Pharma, Medical Affairs, Cambridge MA, USA; 19 University of Washington, Genitourinary Oncology, Seattle WA, USA

Plenary Session 2 5 ORAL PRESENTATION Mechanism based targeted therapy for hereditary leiomyomatosis and renal cell cancer (HLRCC) and sporadic papillary renal cell carcinoma: interim results from a phase 2 study of bevacizumab and erlotinib R. Srinivasan1 , D. Su1 , L. Stamatakis1 , M.M. Siddiqui1 , E. Singer1 , B. Shuch1 , J. Nix1 , J. Friend1 , G. Hawks1 , J. Shih1 , P. Choyke1 , W.M. Linehan1 . 1 National Cancer Institute, Urologic Oncology Branch, Bethesda Maryland, USA This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

6 ORAL PRESENTATION Imaging in cancer immunology: Phenotyping of multiple immune cell subsets in-situ in FFPE tissue sections J.R. Mansfield1 , C. Slater2 , C. Wang1 , K. Roman1 , C.C. Hoyt1 , R.J. Byers2 . 1 PerkinElmer, LST, Hopkinton, USA; 2 University of Manchester, Pathology, Manchester, United Kingdom Background: There has been a rapid grown in the field of tumor immunobiology in recent years as a result of recent successes in cancer immunotherapies, and it is becoming clear that immune cells play many sometimes conflicting roles in the tumor microenvironment. However, obtaining phenotypic information about the various immune cells

Poster Session – Animal Models that play these roles in and around the tumor has been a challenge. Existing methods can either deliver phenotypic information on homogenous samples (e.g., flow cytometry or PCR) or morphologic information on single immunomarkers (standard IHC). We present here a methodology for delivering quantitative per-cell marker expression and phenotyping, analogous to that obtained from flow cytometry, but from cells imaged in situ in FFPE tissue sections. Materials: This methodology combines the sequential multi-marker labeling of up to 8 antigens using antibodies all of the same species in a single section; automated multispectral imaging (MSI) to remove problematic FFPE tissue autofluorescence and correct cross-talk between markers; and an automated analysis that can quantitate the per-cell marker expression, determine the cellular phenotype, count these cells separately in the tumor compartment and stroma, and provide high-resolution images of their distributions. A tissue microarray comprising 35 post-transplant lymphoproliferative disorder (PTLD) samples was immunostained for CD3, CD8 and FOXP3. Single FFPE slides from 9 HER2+ breast cancer patients receiving neoadjuvant chemotherapy were stained with fluorophores targeting cytokeratin, CD8, CD4, FoxP3, CD20 and PD-L1. MSI and analysis was used to enumerate specific phenotypes of cells, as well as to map their spatial locations. Results: MSI successfully captured and quantified multiple immune cell types in all tissues. Validation of singly stained versus multiply stained samples for PTLD showed excellent correlation (R > 0.9). Breast cancer patients not achieving a pathologic complete response (pCR), the density of both the CD8 (p = 0.03) and CD4 (p = 0.05) infiltrates in the stroma were significantly greater than in the tumor. For patients achieving a pCR, there was no significant difference in the densities of stromal and intratumoral CD8 (p = 0.11) or CD4 (p = 0.75) infiltrates suggesting that T cell infiltration into the tumor from the stroma is critical. Conclusions: Multispectral imaging allows different immune cell phenotypes to be visualized and quantified simultaneously in the same tissue section enabling further study of the relationships and distribution of these cells within the tumor and tumor microenvironment, and their spatial distribution and proximity to the tumor cells. This technology will enable improved understanding of the immune infiltrate in solid tumors thereby facilitating the rational design and use of immunotherapeutic agents in combination with standard systemic therapies.

Wednesday 19 November 2014

Poster Sessions Animal Models 7 POSTER (Board P001) Mouse clinical trial − A new preclinical study concept using patient-derived xenografts V. Vuaroqueaux1 , C. Gredy1 , S. Gorynia1 , S. Baltes1 , H.H. Fiebig1 , T. Metz1 . 1 Oncotest GmbH, Freiburg, Germany Patient tumor explants passaged in immunocompromised mice (patientderived xenografts, PDXs) represent the most commonly used system for preclinical efficacy testing of anti-cancer agents. The standard format for efficacy tests typically uses preselected PDXs and group sizes of 8 to 12 PDX-bearing mice to test the efficacy of a treatment relative to a vehicle control group. The strength of this approach is the high reliability of the efficacy data obtained. However, in the face of limitations of available resources this test format restricts the number of PDX models that can be tested. In many studies less than 10 PDX models are used which, in view of the genetic diversity of cancer, is often not satisfactory. The need for in vivo efficacy tests in broader PDX panels is addressed by the emerging mouse clinical trial (MCT) format. Ideally, this format relies on only one mouse per PDX model and treatment arm (referred to as xenopatient), thus enabling the investigation of efficacy in substantially larger panels of PDX models (typically 40 or 50 models which are not preselected) which collectively better mirror the inter-patient response heterogeneity observed in the clinic. However, given that all PDX models display some growth heterogeneity, results obtained for individual models are less reliable. To compare results obtained for individual PDXs in the standard and in the MCT format, in the present work five SoC drugs were tested in colorectal (cetuximab, oxaliplatin, irinotecan, 5-FU) and non-small-cell lung cancer (cetuximab, paclitaxel) PDXs. Dosing and schedules were adapted to clinical standards. Preliminary data for 17 PDX models suggest that

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in approximately 80% of cases the results obtained in the MCT format (single mouse trial) are in line with the results of the standard efficacy tests. Furthermore, in about 10% of cases the results obtained with both formats were similar by trend. The proportion of false positive or false negative results obtained with tests in the MCT format was below 5%. Our findings indicate that for the drugs tested here the risk of misjudging the sensitivity of a given PDX model based on MCT efficacy data is relatively low. This risk could probably be further lowered by increasing group sizes to three mice. For the identification of biomarkers which profits from accurate efficacy data such an intermediate format between the MCT and the standard format may be advantageous. 8 POSTER (Board P002) Imaging growth and anti-cancer activity in orthotopic patient derived tumors M. Baugher1 , C. Bull2 , A. Cohen-Barnhouse1 , A. Flecha2 , M. Franklin3 , K. Guley2 , P. McConville2 , W.R. Leopold4 . 1 Molecular Imaging Inc, Pharmacology, Ann Arbor MI, USA; 2 Molecular Imaging Inc, Imaging, Ann Arbor MI, USA; 3 Molecular Imaging Inc, Pharmaco-Imaging, Ann Arbor MI, USA; 4 Molecular Imaging Inc, Oncology, Ann Arbor MI, USA Background: Preclinical models that more closely mimic the clinical setting are being sought using patient-derived tumors (PDX). Utilization of PDX material in an orthotopic (OT) setting provides a preclinical model in a disease-relevant location. Here we describe the growth and treatment response data of OT pancreas, lung and breast PDX material with conventional and non-invasive imaging techniques. Materials and Methods: All patient-derived tumor material was obtained in collaboration with Oncotest GmbH. SCID beige mice were used with the appropriate material surgically implanted into the pancreas, directly injected into the left lung or directly injected into the mammary fat pad. All dosing is as shown. Results: OT implantation of pancreatic PDX material resulted in robust disease establishment with a near 100% take rate. We utilized MRI and Fluorescence Molecular Tomography (FMT) imaging to non-invasively track tumor progression. We found that different FMT probes had different capabilities for detecting tumor burden. Both MRI and FMT showed that this model was highly sensitive to treatment with docetaxel, where a significant number of mice had complete response and increased overall survival when compared to control mice. The NSCLC PDX material showed near 100% take rates as determined by CT evaluation. By CT we found a minor bevacizumab response, as is consistent with this drug not being an optimal VEGFR inhibitor in the mouse. PET imaging was performed and demonstrated FDG avidity. However, no drug response was observed. In the breast model, treatment with cyclophosphamide resulted in a robust response with approximately 34 of mice having no measurable tumors on day 77. In this setting, FDG PET and FMT imaging were utilized to determine metabolic activity and avidity. FMT reflected the decrease in tumor burden with treatment. At the time of imaging, no difference in FDG SUV values was observed with treatment. In all models, disease latency and tumor volume doubling times were consistent with expectations based on known subcutaneous data. Conclusions: We have shown the establishment of OT PDX pancreas, lung and breast models. The use of multi-modality imaging non-invasively tracked tumor burden over time and provided useful readouts of disease progression and drug treatment response in these more disease-relevant models. Future work continues to investigate the metastatic potential of OT PDX material and non-invasive imaging to track this. 9 POSTER (Board P003) Antineoplastic effects of auranofin in canine lymphoma D. Thamm1 , B.J. Rose1 , J.K. Shoeneman1 . 1 Colorado State University, Dept. of Clinical Sciences, Fort Collins, USA Background: Lymphoma (LSA) is a serious condition for which there remain unmet medical needs in humans and dogs. The gold complex auranofin has been utilized as a human therapeutic, primarily as an antirheumatic agent. Antiproliferative and pro-apoptotic activity has been observed in a variety of human tumor-derived cell lines, including carcinomas of the breast, head and neck, ovary, lung, and a variety of hematopoietic tumors, including LSA. Putative antitumor mechanisms include inhibition of NF-kB and STAT3 signaling, and induction of reactive oxygen species via thioredoxin reductase (TrxR1) inhibition. The dog is a well-established model for spontaneous LSA in humans, owing to striking similarity in biology and gene expression. Dogs with spontaneous tumors naturally develop therapy resistance and metastasis. In addition, tumor burdens in spontaneous canine tumors are more similar to humans than those in murine models, which may be important with regard to biologic factors such as hypoxia and clonal variation. The size of canine tumors

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also allows for serial imaging and tissue collection over time. The goal of the current study was to investigate the antineoplastic effects of auranofin in a panel of canine lymphoma-derived cell lines, and to identify potential biomarkers of drug activity for future translational studies in spontaneous canine LSA. Materials and Methods: 4 canine LSA-derived cell lines were incubated in varying concentrations of auranofin +/− bortezomib, elesclomol, or the conventional antineoplastic agents doxorubicin (DOX), CCNU and vincristine (VCR) for 24−72 hours. Relative viable cell number was assessed using MTS. Cell cycle distribution and apoptosis induction were evaluated cytometrically with propidium iodide staining and Annexin V/PI staining respectively. Induction of ROS was assessed using the redoxsensitive fluorescent dye CM-H2 DCFDA. Results: Auranofin induced dose-dependent antiproliferative effects in all canine LSA cell lines, with 50% inhibitory concentrations between 0.1 and 1 uM. These are similar to those observed in human hematopoietic tumor cells, and well within clinically achievable serum concentrations. Dose- and time-dependent apoptosis induction was observed, and additive to synergistic antiproliferative/propaptotic activity was observed with bortezomib and elesclomol, but not DOX, CCNU or VCR. Dose- and timedependent induction of ROS was observed following auranofin treatment. Conclusions: Auranofin-associated antitumor activity is observed in vitro in canine LSA cells, at pharmacologically achievable concentrations. This is similar to activity observed in human LSA and leukemia. Cooperative activity was observed with the targeted agents bortezomib and elesclomol. These data support translational evaluation of auranofin in canine LSA as a model for the human disease. 10 POSTER (Board P004) mTOR inhibition with everolimus − a novel treatment option for head and neck cancer identified in a translational research study using patient-derived xenografts K. Klinghammer1 , J.D. Raguse2 , T. Plath2 , A.E. Albers3 , B. Brzezicha4 , A. Wulf-Goldenberg4 , U. Keilholz5 , J. Hoffmann4 , I. Fichtner4 . 1 Charite´ University Medicine, Department of Hematology & Oncology, Berlin, Germany; 2 Charite´ University Medicine, Department of Maxillio-Facial Surgery, Berlin, Germany; 3 Charite´ University Medicine, Department of ENT, Berlin, Germany; 4 Experimental Pharmacology & Oncology GmbH, Berlin, Germany; 5 Charite´ University Medicine, Cancer Comprehensive Center, Berlin, Germany Background-Aims: We recently established a large panel of head and neck squamous cell carcinoma (HNSCC) patient-derived xenografts for exploration of novel treatment approaches and biomarker evaluation. Next to activation of the EGFR-RAS-RAF-ERK pathway the PI3K-AKT-mTOR axis is often amplified and activated in HNSCC. We evaluated the mTOR inhibitor everolimus in a panel of HNSCC PDX derived from 29 different patients. Treatment response of everolimus was compared to treatment response of cetuximab and further correlated to gene expression of mTOR pathway members such as mTOR, RPS6KB1, Akt1, FKBP1B and TSC1. Since PI3K mutation has been associated with pathway activation, we aimed to define the predictive role of PI3K mutations for everolimus response. Methods: Specimens from head and neck tumor surgery were transplanted subcutaneously to immunodeficient mice. Groups of 6 mice were treated with cetuximab or everolimus as single agent. A T/C (mean tumor volume of treatment versus control) of less than 50% was defined as biological meaningful activity of the treatment. Transcription of mTOR pathway members were analyzed in detail using RT-PCR. Sequencing analysis was accomplished on Illumina TruSeq Amplicon − Cancer Panel. Results: Response to treatment was very individual. Response rate of everolimus was 20/29 (68%) and for cetuximab 23/29 (79%). RPS6KB1 gene expression showed a trend to positive correlation with treatment response to everolimus (p = 0.0784). PI3K mutations were identified in 7 models at different gene regions. All but one model harbouring mutations within the PI3K gene showed a significant growth inhibition (6/7, 85%), when treated with everolimus. Activity is slightly lower in the cohort with PI3K wildtype (14/22, 63%). Conclusion: The study demonstrated a significant therapeutic activity of everolimus in the majority of our HNSCC models. Although not significant, analysis of mutations in PI3K and the expression of RPS6KB1 mRNA provide some first mechanistic evidence, that an activated PI3K/mTOR pathway may predict sensitivity. Analysis needs to be extended to a larger study group. In conclusion, we demonstrate a comprehensively characterized panel of head and neck cancer PDX models, which represent a valuable and renewable tissue resource for evaluation of novel compounds and associated biomarkers.

Poster Session – Animal Models 11 POSTER (Board P005) A panel of patient derived xenograft models of different haematological malignancies suitable for preclinical drug screening campaigns E. Oswald1 , C. Tschuch1 , K. Klingner1 , B. Hammerich1 , D. Lehnhard1 , 3 C. Rentsch2 , M. Lubbert ¨ , H.H. Fiebig4 , J. Schuler ¨ 4 . 1 Oncotest GmbH, In Vivo Tumorbiology, Freiburg, Germany; 2 University Hospital Basel, Division of Urology, Basel, Switzerland; 3 University Hospital Freiburg, Hematology and Oncology, Freiburg, Germany; 4 Oncotest GmbH, Freiburg, Germany In order to obtain a better understanding of hematological malignancies and to develop improved therapeutic strategies, the generation of functional and reproducible in vivo models is widely pursued. Here, we report the establishment of transplantable PDX models of acute myeloid and acute lymphoid leukemia (AML, ALL) and Non-Hodgkin Lymphoma (NHL) growing subcutaneously as well as in a disseminated fashion in immunocompromised mice. Bone marrow and peripheral blood cells from 18 AML/ALL patients were injected intratibially into NSG or NOG mice (n = 4−8/patient). Separately, tumor tissue from 27 prostate cancer patients and one lung cancer patient was transplanted either under the subrenal capsule or subcutaneously into NSG or NOG mice (n = 4−10/patient), leading to the outgrowth of nine NHLxenografts (eight from transplanted prostate cancer and one from transplanted lung cancer tissue). Tumor growth was monitored via determination of overall survival, flow cytometry analyses, caliper measurement where applicable and Immunohistochemistry (IHC). Transplantable models as well as primary in vivo cultures were further characterized by treatment with respective standard of care drugs (SoC). Six out of 18 AML/ALL patient-derived specimens engrafted in immunocompromised mice, following transplantation into murine bone marrow. Human leukemic cells were detected in murine peripheral blood, bone marrow and spleen by flow cytometry as well as by IHC. One T-ALL and one AML could also be propagated when injected subcutaneously. Two transplantable AMLs and one transplantable ALL were treated with SoC drugs decitabine, cytarabine and dexamethasone. Efficacy tests using primary AML cells were feasible although availability of tumor material limited experiment size to maximally 25−30 mice. The eight NHL xenografts derived from prostate cancer tissue were all Epstein-Barr-Virus-positive. Seven were diagnosed as diffuse large B-cell lymphomas (DLBCL), one is T-cell-derived. The NHL excised from the lung was diagnosed as a MALT lymphoma. All lymphomas were transplantable and showed stable growth when propagated subcutaneously. Subtyping of the DLBCL lymphomas by IHC revealed that four of them were of the ABC-subtype. Our results confirm that PDX models of haematological malignancies replicate the heterogeneity and other important clinical characteristics of the respective disease, and are valuable tools for preclinical drug testing and investigation of tumour biology. 12 POSTER (Board P006) Next generation sequencing (NGS) guided therapy prediction for the treatment of glioblastoma multiforme (GBM) J. Sarkaria1 , D.M. Ma1 , S.P. Peng2 , S.B. Byron2 , D.C. Craig3 , J.C. Carpten4 , M.B. Berens2 , B.O. O’Neill5 , N.T. Tran6 . 1 Mayo Clinic Cancer Center, Radiation Oncology, Rochester Minnesota, USA; 2 Translational Genomics Research Institute (TGen), Cancer and Cell Biology Division, Phoenix Arizona, USA; 3 Translational Genomics Research Institute (TGen), Neurogenomics Division, Phoenix Arizona, USA; 4 Translational Genomics Research Institute (TGen), Genomics Division, Phoenix Arizona, USA; 5 Mayo Clinic, Neuro-oncology, Rochester Minnesota, USA; 6 Translational Genomics Research Institute (TGen), Cancer and Cell Biology Division, Rochester Minnesota, USA Background: The genomic heterogeneity of glioblastoma likely underlies the low response rates (8−24%) for targeted agents among unselected populations. We tested whether NGS would be useful in identifying therapeutically-actionable genetic alterations; we sought to test if this translated into improved tumor control in a patient-derived GBM model. Methods: Tumor specimens from each patient (n = 11) that were used for analyte extraction contained between 70−80% viable tumor cellularity. Genome sequence coverage was more than 30× for both tumor and germline genomes; tumor RNA sequencing included over 100 million reads. NGS of paired tumor and germline DNA enabled detection of single nucleotide variations (SNVs), indels, translocations, intra-chromosomal rearrangements, and copy number alterations. A custom drug-matching workflow utilizing publically available databases and curated literature on reported drug effects, pharmacokinetics, and blood–brain barrier penetration was used to map individual gene alterations in tumors with

Poster Session – Animal Models associated drug-response relationships. One specimen was tested in vitro and in vivo using a matched, patient-derived xenograft model. Results: NGS for the 11 tumor panel found ~30% of cases with EGFR amplification, p16, and/or PTEN deletion. Novel potentially actionable targets included a TRIM54-FGFR3 fusion, STAG2 mutation, MDM2 amplification, KIF11 and KIF15 mutation, and BRAF amplification. GBM150 is derived from a recurrent tumor and harbors the TRIM54-FGFR3 fusion, and this line is the first to be analyzed for chemovulnerability. In a Cyquant proliferation assay, GBM150 were relatively resistant to TMZ (IC50 300 uM) but significantly more sensitive to pan-FGFR1−3 inhibitors as compared to GBM108: ponatinib IC50 0.3 uM vs. 1.2 uM; AZD4547 IC50 3 uM vs. >10 uM. Similarly, in a flank tumor regrowth study, established GBM150 tumors were randomized to therapy with placebo, temozolomide, ponatinib or AZD4547. While TMZ-treated tumors were marginally smaller than placebo treated tumors (mean tumor volume 1312±208 vs. 992±183 mm3 , 97 days after therapy initiation), both ponatinib or AZD4547 resulted in sustained tumor stasis with a mean tumor volume of 240±35 and 199±40 mm3 , respectively, at the same time-point. Conclusions: These results support the concept that NGS can be used to individualize treatment of GBM and highlight how corresponding patient derived xenograft models can be used to validate the accuracy of potential therapeutic predictions. 13 POSTER (Board P007) Whole exome sequence analysis of canine transitional cell carcinoma of the bladder D.L. Duval1 , B. Hernandez1 , J. Brown2 , S.E. Lana1 , R. Page1 , K.L. Jones2 . 1 Colorado State University, Clinical Sciences, Fort Collins, USA; 2 University of Colorado Cancer Center, Biochemistry and Molecular Genetics, Aurora, USA Transitional cell carcinoma (TCC) is the most common bladder cancer in both humans and their canine companions, accounting for approximately 2% of all diagnosed malignancies in both populations. Clinical presentation of human TCC is broken into 2 groups: superficial non-muscle-invasive TCC (~70% of cases) and muscle invasive TCC (~30% of cases) which is associated with a high risk of death from distant metastasis. The majority of canine TCCs are papillary infiltrative TCCs of intermediate to high grade at diagnosis. Similarities in risk factors, histopathology, sites of metastasis, and other common features indicate that canine TCC may serve as an excellent model for invasive human TCC. Previous studies of human TCCs have identified genetic defects that may aid in the diagnosis and therapy of human bladder cancer. To further assess the value of spontaneous canine TCCs as a model for human TCC, we have utilized whole exome sequencing to screen a panel of canine TCCs for cancer gene mutations that contribute to the pathogenesis and progression of canine bladder cancer. Genomic DNA was isolated from 11 archived canine TCCs, 3 matched normal tissue samples, and 2 canine TCC cell lines. Whole exome capture was conducted using the Agilent Sure-select insolution capture system designed for the canine genome, and the captured fragments were sequenced using an Illumina HiSeq2000 next generation sequencing platform. The sequences were mapped to the CanFam3.1 canine reference genome and single-nucleotide polymorphisms, insertions and deletions were identified using Freebayes. Somatic mutations were characterized and compared to the Cancer Gene Census (COSMIC). Similar to a variety of human cancers including bladder cancers, the mutation spectrum in these genes is dominated by C:G>T:A transitions. Nonsense, missense, and insertion/deletion mutations were identified in 75 genes shown to be drivers or repressors in human cancer. Pathway analysis (Pathway Studio) identified DNA damage, genomic instability, and chromatin remodeling as the top 3 cellular pathways affected. The 10 genes most frequently exhibiting potentially deleterious mutations were: MITF, KDM6A, MLLT6, AKAP9, C2orf44, ROS1, NSD1, BRAF, FANCD2, and PCM1. This mutation spectrum, including members of the RTK/Ras/Raf pathway, histone modifying and chromatin remodeling enzymes, indicates that similar activating pathways drive both human and canine transitional cell carcinoma of the bladder. 14 POSTER (Board P008) Mixeno mouse models for in vivo evaluation of anti-human cancer immunotherapeutics J. Zhang1 , J. Qiu2 , M. Qiao2 , Q. Shi1 . 1 Crown Biosciences, Cancer Pharmacolgy, Santa Clara CA, USA; 2 Crown Biosciences, Cancer Signaling, Santa Clara CA, USA The past few years have witnessed a renaissance in the field of cancer immunotherapy, relating largely to the clinical advances associated with the development of immunomodulatory agents, e.g. monoclonal antibodies targeting the immune inhibitory pathways (CTLA-4 and PD-1/PD-L1).

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Often, the preclinical efficacy assessments are based on the evaluation of surrogate anti-mouse target antibodies using mouse syngenic tumor models. However, this strategy is limited due to the fact it can only be used to test surrogate molecules, rather than directly evaluate the therapeutic molecules that target human targets. Here we set out to validate mouse models that harbor human immune cells by engrafting the immuno-deficient mice with human PBMC (the MixenoTM model), and use them for efficacy evaluation of the humanized anti-PD-1 antibody. PD-L1 high-expression human tumor cell lines are selected using Xenobase® and FACS analysis to develop the in vivo models. BMS-936558, a fully humanized anti-PD-1 IgG4 produced promising anti-tumor activity in the HCC827 lung cancer MixenoTM model. Based on the preliminary result, the MixenoTM models may be a useful tools in immunotherapeutic antibody development, and may greatly increase the clinical translatability of animal studies. 15 POSTER (Board P009) Genetic and molecular validation of uterine sarcoma patient-derived xenograft models T. Cuppens1 , E. Hermans1 , J. Depreeuw2 , M. Moisse2 , T. Van Brussel2 , L. Coenegrachts1 , D. Lambrechts2 , F. Amant1 . 1 Catholic University of Leuven, Gynaecologic Oncology, Leuven, Belgium; 2 Catholic University of Leuven-Vesalius Research Center VIB, Oncology Translational Genetics, Leuven, Belgium Background: Genetic and molecular heterogeneity within tumor types directly affects a patient’s response to a given compound, warranting more personalized therapeutic strategies based on predictive markers. However, for rare malignant tumors in particular, randomized trials are scarce, emphasizing the need for reliable pre-clinical tumor models. Traditional approaches for pre-clinical evaluation of cancer therapies rely on the use of xenograft models of commercially available cell lines. Whereas these models are human in origin, they are unlikely to fully recapitulate human cancers. In contrast, patient-derived tumor xenografts (PDTXs), established directly from patient’s tumors, may better preserve the initial tumor characteristics as they represent the heterogeneity and retain the most important genetic features of the original tumor. Here, we aim to establish and validate PDTX models for uterine leiomyosarcoma, a highly aggressive tumor arising from the myometrium. We explore the genetic and molecular stability of xenografted tumors, which will ultimately serve as pre-clinical models for individualized treatment. Materials and Methods: In collaboration with the PDTX platform, 8 uterine leiomyosarcomas (obtained from surgery after patient’s informed consent) were subcutaneously implanted into immune-deficient mice. Engrafted tumors were re-implanted for several generations (F1, F2, F3, etc.) and then compared to their original tumors by means of copy number analysis (SNP arrays-Illumina), tissue-specific marker expression (immunohistochemistry) and histology (H&E). Results: We have established 5 uterine leiomyosarcoma models (take rate >60%), of which one diploid and one tetraploid model have been analyzed at present. Both tumors retained their histologic features, as well as their expression of mesenchyme-specific (vimentin) and muscle-specific (desmin/caldesmon) markers. Further, copy numbers of the F4 xenograft derived from the diploid tumor were >99% identical to the original tumor. However, the F3 xenograft of the same model showed tetraploidy and some additional copy number changes. In the tetraploid PDTX model, 37% of all genes in F2 and F3 tumors had changed in copy number when compared to the original tumor. Comparing the F2 and F3 tumor revealed a copy number stability of only 56%. Hence, in both models, the later xenograft generation shows greater resemblance to the original tumor than to the prior xenograft from which it was derived. This finding strongly suggests the presence of subclones. Conclusions: We successfully established patient-derived tumor xenograft models for pre-clinical individualized therapy testing. Of note, the genetic stability and in particular the presence of subclones should be taken into account when selecting therapies based on genetic features. 16 POSTER (Board P010) Allografting improves the feasibility of genetically engineered mouse models (GEMM) for anti-cancer drug development ¨ 3 , A. Zipelius3 , J. Schuler ¨ 2. K. Kukuk1 , K. Klingner1 , A.L. Peille2 , P. Muller Oncotest GmbH, In Vivo Tumorbiology, Freiburg, Germany; 2 Oncotest GmbH, Freiburg, Germany; 3 University Hospital Basel, Basel, Switzerland

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Genetically engineered mouse models (GEMMs) represent an attractive system for preclinical research since GEMM tumors develop in the presence of a competent immune system. Unlike classical syngeneic mouse tumor models, in GEMMs the identity of some of the oncogenic

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alterations is known. Typical drawbacks of GEMM tumors include slow and heterogeneous tumor development, making the use of these models challenging for routine in vivo efficacy studies. These disadvantages can be avoided by grafting autochthonous GEMM tumors on mice of the parental strain. Here, we describe the establishment and characterization of such allografts from a non-small cell lung cancer (NSCLC) and two breast cancer models, among them an invasive lobular carcinoma (ILC) and a model carrying a Brca-1 alteration. Tumors were excised from their organ of origin and tumor pieces were implanted subcutaneously (s.c.) and orthotopically (o.t.) into C57BL/6N (NSCLC model) and FVB/N mice (breast cancer models). Arising tumors were passaged and compared to the original in-situ tumors molecularly, histologically, and immunohistochemically (e.g. Ki67 proliferation marker). In parallel, cell lines were established from the in-situ tumors. Subcutaneous allografts were passaged at least 8 times with stable and consistent tumor growth as documented by tumor-growth-kinetics and H&E staining of tumor sections. The allografts of the ILC model metastasized to multiple organs when transplanted o.t. For all three models, patho-histology of in-situ tumors and allografts was congruent and no signs of an immunological reaction against allografts were detected. IHC analysis revealed differences between Ki-67 and CD31 expression among different allografts representing the same GEMM. Efficacy tests with standard of care (SoC) drugs performed on allografted NSCLC tumors revealed significant anti-tumor activity of the dual PI3K and mTOR inhibitor. BEZ235 displayed good antitumoral activity both in monotherapy and in combination with the dual EGFR and HER2 inhibitor afatinib. The selective EGFR inhibitor erlotinib had no significant effect, which is in line with the low mEGFR expression detected. In conclusion, s.c. and o.t. transplantable allografts, which replicate key aspects of the human disease, were obtained from the investigated GEMMs and enable efficacy tests in immunocompetent mice in a suitable time frame. Further studies will elucidate the feasibility of these models for preclinical testing of immunomodulatory drugs. 17 POSTER (Board P011) Studies on glycoprotein expression differences between MCF-7 and MCF-7-Z J. Ner-Kluza1 , A. Drabik2 , M. Kubbutat3 , A. Lingnau3 , J. Silberring2 . 1 Uniwersity of Science and Technology, Department of Biochemistry and Neurobiology, Krakow, Poland; 2 AGH Uniwersity of Science and Technology, Department of Biochemistry and Neurobiology, Krakow, Poland; 3 ProQinase GmbH, Freiburg, Germany According to the American Cancer Society, in 2014, more than 230,000 women will be positively diagnosed for breast cancer in the US, demonstrating the urgent need for more promising anti-tumor drugs worldwide. The human breast cancer cell line MCF-7 has been commonly used in subcutaneous xenograft tumor models in order to screen for novel cancer drugs. By comparing two different variants of MCF-7 (MCF-7 and MCF7-Z), both authentic when analysed by STR profiling, we found completely different tumor growth characteristics in mice. We excluded the possibility that a loss in estradiol dependency was responsible for these differences, and started analysing the proteome of the MCF-7 tumors by an approach to discriminate between the glycosylation pattern. Among post-translational modifications glycosylation is one of the most important involved in tumor progression. The presented project is based on monitoring changes in glycoprotein profiles of two cell lines MCF-7 and MCF-7-Z. For the isolation of glycoproteins lectin affinity chromatography (LAC) was applied, followed by one-dimensional SDSPAGE electrophoresis. After digestion, peptides extracted from the gel were analyzed by nanoLC-MS/MS system. The identified glycoproteins were identified by bioinformatics tools, such as Osprey and Panther databases. Final part of the research is based on quantitative analysis using labelfree approach of the identified proteins using Bruker software called Profile Analysis. The research was supported by the The Polish National Science Center 2012/07/B/NZ4/01468, and EuroNanoMed ‘META’ 05/EuroNanoMed/2012. 18 POSTER (Board P012) Establishment and characterization of a Merkel Cell carcinoma PDX panel: Screening for potentially useful therapies M.J. Wick1 , J. Meade1 , M. Nehls1 , T. Vaught1 , J. Carlile1 , A.W. Tolcher1 , D.W. Rasco1 , A. Patnaik1 , K.P. Papadopoulos1 . 1 South Texas Accelerated Research Therapeutics, (START), San Antonio TX, USA Background: Merkel cell carcinoma (MCC) is an uncommon, aggressive neuroendocrine skincancer with a poor prognosis and few effective treatment options for advanced disease. Recent studies have identified

Poster Session – Animal Models a strong association between infection with the Merkel cell polyomavirus (MCPyV) and MCC transformation. MCPyV antigens have been shown to affect cell growth and proliferation through apoptosis and cell cycle pathways. Targeted therapies have shown promise in vitro and in vivo; however, their utility in the clinical setting is anecdotal. To better understand which targeted therapies are effective in MCC, we established and screened a panel of MCC patient-derived xenograft (PDX) models evaluating approved and investigational therapies. Methods: Methods: Merkel cell carcinoma START-PDX models were established in immune-deficient mice from primary or metastatic patient tissue and once established were confirmed by histologic comparative analysis and linked with patient treatment and outcome data. Drug sensitivity studies were performed evaluating each model to available targeted therapies including pazopanib, everolimus and sunitinib and investigational Akt, IAP and CDK 4/6 inhibitors. Study endpoints included tumor volume and time from treatment initiation with tumor growth inhibition, delay and regression reported at study completion. Results: Five MCC models evaluated in this project demonstrated differential responses to test agents; statistically significant (p < 0.05) tumor growth inhibition was reported with pazopanib. Drugs targeting the PI3K/mTOR pathway, including an Akt inhibitor and everolimus as single agents, were not effective in these studies. Conclusion: We have established a panel of MCC PDX models and evaluated a panel of approved and investigational therapies. We found differential responses to these therapies, with pazopanib the most active as a single agent. These models may be of utility in identifying effective single and combination regimens for MCC. 19 POSTER (Board P013) Syngeneic models for developing cancer therapeutics targeting immune system L. Zhang1 , J. Zhang1 , Q. Shi1 . 1 Crown Biosciences, Cancer Pharmacolgy, Santa Clara CA, USA Syngeneic tumor models have long been used in cancer research, from mechanistic study to developing cancer therapeutics, especially those that require intact immune system, such as antibody therapeutics that have ADCC effect. Recently, cancer immunotherapy reignited to become one of the most promising therapies, largely because of the success of the clinical studies of CTLA-4, PD-1/PD-L1 antibodies. Researchers are now believe there are many novel therapeutics, both small and large molecules, and many novel targets, to be discovered and developed in this field. To meet this demand, Crown has established a large collection of syngeneic models that covers most of the tumor types and mutational profiles. In addition, we’ve also profiled the models using anti-mouse PD1/PD-L1 antibodies. The syngeneic models display very different responses towards the immunotherapeutics, ranging from shrinking the tumor to stimulating the tumor growth. These results emphasize the need to carefully select models based on the development goals. A single agent development approach would require selecting the models with the best response, while a combination study design would require a model with suboptimal response. Our comprehensive list of syngeneic models and profiling data are essential in developing cancer immunotherapies that may one day benefit the patients. 20 POSTER (Board P014) miR-25 is a key regulator of prostate cancer invasiveness by modulation of the cross-talk between Notch and TGF-b signaling E. Zoni1 , A.F. van de Merbel1 , G. van der Horst1 , J. Rane2 , T. Visakorpi3 , E.B. Snaar4 , N. Maitland2 , G. van der Pluijm1 . 1 Leiden University Medical Center, Department of Urology, Leiden, Netherlands; 2 YCR Cancer research Unit, Department of Biology, York, United Kingdom; 3 Institute of Medical Technology, University of Tampere, Tampere, Finland; 4 University of Leiden, Department of Biology, Leiden, Netherlands Background: Prostate cancer is the most commonly diagnosed cancer in males and the second leading cause of death from cancer in men. Prostate cancer stem/progenitor cells (CSCs) have been shown to play a crucial role in carcinogenesis, metastasis and therapy resistance. Previously, we have shown that the ALDHhigh subpopulation of human prostate cancer cells is enriched for CSCs. ALDHhigh cells display strong clonogenicity and migration when compared to the more differentiated ALDHlow . Material and Methods: We performed microRNA (miR) expression profiling of ALDHhigh vs ALDHlow subpopulation of prostate cancer cells. We studied the functional effects of miR-25 on Notch signaling on mRNA and proteins and used Luciferase reporters to monitor the effect of miR-25 on TGF-b signaling and to validate the interaction between miR-25 and Notch1. Finally we used zebrafish model to demonstrate that

Poster Session – Cytotoxics miR-25 reduces dissemination of human prostate cancer cells in the intact organism. Results: In this study we have identified miRs that are differentially expressed between the metastatic subpopulation of PC-3M-Pro4 cells (ALDHhigh /a2high /CD44+ ) vs non-tumorigenic/non-metastatic ALDHlow . miR-25 was strongly downregulated in the ‘driver’ ALDHhigh CSC subpopulation. In both clinical prostate cancer specimens and prostate cancer cell lines we found that miR-25 is low/absent in CSC compartment and steadily increases during differentiation into luminal epithelial cells. We used Targetscan to discover novel miR-25 predicted targets and identified Notch1 as putative target of miR-25. Previously published studies have highlighted the role of Notch and Jag1 (Notch ligand) in skeletal metastasis. Interestingly, overexpression of miR-25 strongly decreases the expression of Notch1 and Jag1 together with other Notch downstream targets in two prostate cancer cell lines. Moreover, we found that miR-25 is able to decrease TGF-b signaling in prostate cancer cells and can block the induction of Jag1 driven by TGF-b. In line with these observations, we further demonstrate that miR-25 reduced metastasis by blocking the extravasation of human prostate cancer cells in vivo. Conclusion: Previously we have shown that miR-25 directly target av- and a6-integrins and strongly reduces migration of human prostate cancer cells. Here we show that miR-25 affects Notch pathway and can interfere with the TGF-b signaling, reducing the TGF-b induced expression of Jag1. Our data are supported by previous studies on the critical role of av-integrin in activation of TGF-b signaling and its role in skeletal metastasis. In conclusion, we suggest that miR-25 seems to be a master regulator of invasiveness in human prostate cancer through its interaction with Notch and TGF-b signaling pathways. The identification of miRs and functional validation of their target genes, is crucial for the elucidation of the mechanisms involved in tumor progression and bone metastasis formation in human prostate cancer.

Cytotoxics 21 POSTER (Board P015) Pharmacogenomics of mithramycin in thoracic malignancies W. Figg1 , T.M. Sissung1 , C.J. Peer1 , D. Schrump2 . 1 National Cancer Institute, Clinical Pharmacology Program, Bethesda, USA; 2 National Cancer Institute, Thoracic Oncology Section, Bethesda, USA Background: Mithramycin inhibits expression of the fusion oncogene EWS-FLI1, and is therefore under investigation for use in thoracic malignancies. One of the major obstacles to mithramycin therapy is significant liver toxicity and unknown plasma concentrations. Materials and Methods: To discover genetic variants that are associated with these obstacles, we used the Drug Metabolizing Enzymes and Transporters (DMET) genotyping array on germline DNA from 12 patients with various thoracic malignancies receiving mithramycin. We also developed a novel assay to detect mithramycin plasma concentrations in these patients. Results: Grade 3−4 ALT and AST rises were observed in 8 of 12 patients that typically occurred after three daily administrations. Mithramycin plasma concentrations remained at sub-therapeutic levels, and pharmacokinetic parameters were not related to the LFT rises (P > 0.061). DMET analysis revealed two polymorphisms that are related to LFT rises in genes responsible for hepatocellular bile transport, ABCB4 (encoding PC-flopase) and ABCB11 (encoding BSEP). Patients without LFT rises carried wild-type alleles at both of these sites, whereas those with LFT rises carried at least one variant allele [OR (95% CI) = 153.0 (2.6–9100); P = 0.0020]. When DMET results were applied to pharmacokinetic data, an increasing trend was observed in which patients carrying (TA)6 /(TA)6 at UGT1A1*2 had low AUC that became progressively higher in those carrying (TA)6 /(TA)7 and (TA)7 /(TA)7 respectively. Conclusions: Taken together, the data indicate that mithramycin likely causes liver toxicity by inhibiting bile acid flow. Mithramycin is known to inhibit hepatic transporter expression that we suspect causes progressive bile acid accumulation in hepatocytes and subsequent cellular damage. Mithramycin is also most-likely glucuronidated by the liver; thus, extensive UGT1A1 metabolizers are likely to have sub-therapeutic plasma concentrations.

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22 POSTER (Board P016) Novel combination therapy, TAS-102 combined with the anti-EGFR antibody or the anti-VEGF antibody showed therapeutic benefit toward colorectal cancer xenografts K. Ishida1 , K. Sakamoto1 , N. Tanaka1 , K. Oguchi1 , K. Yamamura1 , A. Fujioka1 , F. Nakagawa2 , K. Matsuo1 , T. Utsugi1 . 1 Taiho Pharmaceutical Co. Ltd, Tsukuba Research Center, Tsukuba, Japan; 2 Taiho Pharmaceutical Co. Ltd, Tokushima Research Center, Tokushima, Japan Background: TAS-102 (Lonsurf® ) consists of trifluridine (FTD) and tipiracil hydrochloride, thymidine phosphorylase inhibitor (TPI), at a molar ratio of 1:0.5. FTD is a thymidine analog and the antitumor component of TAS-102 and exerts its antitumor activity via FTD incorporation into DNA. FTD is activated to F3 dTMP by thymidine kinase and further phosphorylated to triphosphate form, F3 dTTP which is incorporated into DNA. TPI prevents degradation of FTD by thymidine phosphorylase in liver. TAS-102 was found to significantly improve overall survival of patients with metastatic colorectal cancer that is refractory/intolerable to the standard chemotherapies in a global Phase III study. We investigated TAS-102 based combination therapy with cetuximab (Cmab), panitumumab (Pmab), or bevacizumab (Bmab) that are key drugs for the treatment of metastatic colorectal cancer. Material and Method: Antitumor effects of TAS-102 combined with Cmab, Pmab or Bmab were evaluated in human colorectal cancer xenograft implanted SW48, HCT116 and HT-29. TAS-102 was administered orally twice daily for 14 days, and Cmab, Pmab or Bmab were administered twice a week intravenously or intraperitoneally. Tumor growth inhibition on day 15 and tumor growth delay was calculated from the tumor volume. Moreover, we measured accumulation of FTD and its phosphorylated forms in tumor with LC-MS/MS in the study of the combination with Bmab. Results: The antitumor effects of TAS-102 were enhanced by the combination with Cmab or Pmab toward KRAS wt tumor SW48, compared with monotherapy with TAS-102 or anti-EGFR antibodies as measured by both tumor growth inhibition and tumor growth delay. Similarly, tumor growth inhibition by Bmab plus TAS-102 was stronger than monotherapy toward both KRAS wt and mut tumors, SW48 and HCT-116 as well as in a BRAF mut tumor, HT-29. The tumor growth delays were also extended by Bmab combined administration in comparison with monotherapy. Interestingly, it was observed that the level of FTD and its phosphorylated forms in the tumor treated with TAS-102 plus Bmab were increased compared with TAS102 monotherapy. Conclusions: TAS-102 plus anti-EGFR therapy for KRAS wt tumors and TAS-102 plus Bmab therapy for tumors with KRAS and BRAF mutations could be effective combinations for colorectal cancer. TAS-102 based therapy may prove to be an effective alternative to standard 5-FU based chemotherapy in combination with antibody treatment for advanced colorectal cancer. 23 POSTER (Board P017) Phase I study of lurbinectedin (PM01183) administered on days (D) 1 & 8 every 3 weeks (q3wk) in patients (pts) with solid tumors M.J. Ratain1 , L. Gore2 , S. Szyldergemajn3 , J. Diamond2 , D. Geary1 , C. Fernandez-Teruel3 , A. Soto-Matos3 , M. Sharma1 , A. Jimeno2 . 1 The University of Chicago, Chicago, USA; 2 The University of Colorado, Denver CO, USA; 3 PharmaMar, Colmenar Viejo Madrid, Spain Background: PM01183 is a new anticancer agent with broad clinical activity against several solid tumors. The recommended dose (RD) when administered on D1 every three weeks (q3wk) is 4.0 mg/m2 or 7.0 mg as flat dose (FD). Reversible myelosuppression is its dose-limiting toxicity (DLT). Here we explored a D1&8 q3wk schedule. Material and Methods: Pts with solid tumors (other than colorectal cancer), adequate organ function and PS 0−1 were treated at 3 dose levels (DLs) using a 3+3 design, from 3.0 mg to 5.0 mg FD on D1&8 q3wk. Results: 21 pts were treated and 20 were evaluable: (M/F: 5/15); median age 63 years (39−75); BSA 1.8 m2 (1.6−2.5); albumin 3.8 g/dL (2.8−4.3); prior lines 5 (2−13). Most pts had ovarian (8, 38%), soft tissue sarcoma (STS) (5, 24%) or pancreatic/biliary tract (5, 24%) cancer. DL3 (5.0 mg FD/D1&8) was defined as the RD whereas 3 of 13 (23%) evaluable pts had DLT in Cycle 1: G4 neutropenia lasting >7 days (n = 2) and D8 omission and C2 delay due to toxicity (n = 1). Six pts (46%) received the full RD without delay, omissions or dose reductions; 5 (36%) pts required either a dose reduction or G-CSF prophylaxis from Cycle 2. No dose reductions or delays were needed in pts treated below the RD (n = 7). G4 neutropenia occurred in 43% of pts and one pt had G4 thrombocytopenia, at the RD. Other G3 events included nausea/vomiting and hyponatremia (1 each). Nonhematological G1/2 toxicity in 15% of pts: fatigue (64%), nausea (57%), anorexia (36%), vomiting (28%), pyrexia (21%) and ALT/AST increase (64% and 50%). At the RD, 8/13 pts had stable disease (4 months in 5 pts). Two previously progressing pts with STS and ovarian cancer were on study

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for 57+ weeks. At the RD (n = 14): mean (SD) clearance (CL) 18.1 (7.2) L/h; AUC 318 (123) h·mg/L; Cmax 90.5 (30.7) mg/L; t1/2 30.5 (13.6) h. No apparent association between CL and DLT or magnitude of neutropenia was found. Conclusions: The RD of PM01183 is 5 mg FD on D1&8 q3wk. At this RD, myelosuppression led to frequent dosing delays or omissions, but G-CSF support enabled the targeted dose intensity in some individuals. Two patients had prolonged disease stabilization lasting over a year. No signs of cumulative toxicity were observed. Interindividual variability in CL was high, but did not appear to be associated with toxicity. 24 POSTER (Board P018) Androgen receptor (AR) expression in triple negative breast cancer (TNBC): results from a phase II neoadjuvant trial with carboplatin and eribulin mesylate in TNBC patients K. Siziopikou1 , V. Parini2 , V. Kaklamani3 . 1 Northwestern University Feinberg School of Medicine, Pathology, Chicago IL, USA; 2 Robert H. Lurie Comprehensive Cancer Center, Pathology Core Facility, Chicago IL, USA; 3 Northwestern University Feinberg School of Medicine, Oncology, Chicago IL, USA Background: Triple negative (ER-negative/PR-negative/HER2-negative) breast cancer remains a therapeutic challenge for the oncologist since these carcinomas do not qualify for endocrine manipulation or HER2targeted therapy. Platinum agents were lately used in TNBC resulting in a 16−32% pathologic complete response (pCR) in neoadjuvant trials. Eribulin mesylate, a nontaxane microtubule dynamics inhibitor with a novel mechanism of action also showed clinical activity in breast cancer. In addition, different subgroups of TNBC were identified, with an AR expressing subgroup reported to be associated with a better prognosis. Our group recently reported that, in a phase II neoadjuvant trial, the combination of carboplatin and eribulin mesylate in patients with TNBC was well tolerated and showed significant efficacy. Our current study aimed to investigate the role of AR in this patient population of TNBC cases uniquely treated with a combination of carboplatin and eribulin mesylate. Materials and Methods: 30 patients with stage I-III TNBC were treated in a neoadjuvant setting with eribulin 1.4 mg/m2 over 2−5 min IV on days 1 and 8 plus carboplatin AUC6 over 30 min IV in Day 1 for four cycles. The expression of AR (Dako, AR 441) was assessed immunohistochemically in breast cancer specimens prior to treatment and at the time of definitive surgery. Staining was considered negative if <1%, low positive if 1−9% and positive if 10%. Results: 30 patients were diagnosed with TNBC. 26 (86.7%) had a grade III tumor and 4 (13.3%) a grade II tumor. No grade I cases were seen in this group. All patients underwent definite surgery and were evaluated for pathologic response. pCR was observed in 13 (43.3%); of the remaining patients 15 (51.7%) showed a residual cancer burden (RCB) II and 1 (3.5%) patient a RCBIII as per Symmans criteria. Of the 21 patients for whom pretreatment material was available 3 (14.2%) were positive for AR expression. Of the 17 patients without a complete response post-treatment, 2 were positive for AR expression. Of the 3 cases that were AR-positive pre-treatment, 2 remained positive post-treatment and 1 had a pCR. Conclusions: 1. Combination of carboplatin and eribulin mesylate resulted in a pCR in almost half (43.3%) of the TNBC patients. 2. Expression of AR does not appear to play a role in the promising response rate seen with this novel neoadjuvant combination. Additional studies are underway to further characterize the molecular mechanisms that drive response in this challenging subtype of breast cancer. 25 POSTER (Board P019) An ING1b-derived peptide that inhibits cancer cell viability and promotes apoptosis A. Boyko1 , K. Riabowol1 . 1 University of Calgary, Department of Biochemistry & Molecular Biology, Calgary, Canada The ING1b protein is a type II tumor suppressor and stoichiometric member of HDAC-containing protein complexes. ING1b contributes to regulation of gene expression, senescence and apoptosis. Mislocalization and decreased levels of ING1b are commonly observed in human tumors and cancer cell lines. Multiple independent studies show that ING1b overexpression promotes apoptosis in targeted cells. Since the inactivation of apoptosis pathways is frequent in cancer cells, modulating ING1b expression may serve as a viable approach for cancer therapy. We are defining ING1b regions necessary for apoptotic function in order to design minimal recombinant peptides with potent apoptosis-inducing properties. Based on the predicted protein structure and published data, we produced a number of expression constructs that encompass various portions of the

Poster Session – Cytotoxics ING1b protein. Following the initial screening stage, pro-apoptotic effects of the most successful expression constructs were further confirmed and quantified by western blotting using the levels of PARP cleavage, and by FACS analysis using the Annexin V assay. We have established that ING1b-derived peptides containing its third alpha helix (A3H) and nuclear localization sequence (NLS)/nucleolar translocation signal (NTS) domains are able to induce apoptosis at levels comparable to those of the full length ING1b as determined using Annexin V assays. The A3H-NLS/NTS peptide exhibited strong nucleolar localization, characteristic of full length ING1b. Cells overexpressing the full length ING1b and A3H-NLS/NTS peptide showed similar changes in cell morphology characteristic of apoptosis and exhibited increased levels of PARP cleavage. While the A3H region was necessary but not sufficient, the NLS/NTS domain was required, and partially sufficient, for induction of apoptosis. Adenoviral delivery of A3H-NLS/NTS peptide led to a drastic increase in the number of cells undergoing apoptosis and resulted in a strong dose-dependent inhibition of cancer cells viability that was independent of p53 status. The strong anti-tumorigenic effects were observed in a broad range of tested cancer cell lines including those of osteosarcoma, glioblastoma and breast cancer origin. The evaluation of the synergy between the A3H-NLS/NTS peptide and common chemotherapeutic agents is currently ongoing. Our longterm goal is to develop ING1b-based therapeutics that can be used as an adjuvant therapy in combination with the existing cancer treatments. 26 POSTER (Board P020) NPD926, a small molecule inducer of reactive oxygen species, kills cancer cells via glutathione depletion T. Kawamura1 , Y. Kondoh1 , M. Muroi1 , M. Kawatani1 , H. Osada1 . 1 Riken, Antibiotics Laboratory, Wako Saitama, Japan Background: Elucidation of the mechanisms of action of bioactive compounds may render a therapeutic regimen to be more successful. However, this process is generally time-consuming. By a combination of proteomic profiling and affinity purification, we recently elucidated the mechanism of action of a newly identified cytotoxic compound, NPD926. Here we report the mechanism of action of NPD926 and its effects on cancer cells. Material and Methods: Prediction of the mechanism of action of NPD926 was performed by ChemProteoBase profiling, which is based on the proteomic perturbation in HeLa cells induced by treatment with bioactive compounds. NPD926-binding proteins were purified using affinity matrices, separated by SDS-PAGE, and identified by MALDI-TOF-MS. Results: NPD926 showed cytotoxicity against 19 types of human cancer cell lines. ChemProteoBase proteomic profiling showed that NPD926 shared similarity with 1-chloro-2,4-dinitrobenzene, a substrate for glutathione S-transferase (GST). By affinity purification, an isozyme of GSTs was identified as a specific NPD926-binding protein. Based on these findings and the subsequent biochemical assays, we elucidated the mechanism of action underlying NPD926-induced cell death: (i) conjugation with glutathione as a substrate for GSTs, (ii) depletion of cellular glutathione, and subsequent (iii) generation of reactive oxygen species (ROS). NPD926 preferentially induced cytotoxicity against KRAStransformed fibroblast NIH3T3 cells, compared with their untransformed counterparts. Furthermore, NPD926 sensitized cells to inhibitors of system xc− , a cystine-glutamate antiporter considered as a potential therapeutic target in cancers including cancer stem cells. Conclusions: By the chemical biological methods, we elucidated the mechanism of action of NPD926. Induction of excessive levels of ROS by compounds has been considered as a potentially effective therapeutic strategy against cancer cells. Our present study shows the effectiveness of a newly identified ROS inducer, which targets glutathione metabolism, in cancer cells including those harboring oncogenic KRAS.

Poster Session – Cytotoxics 27 POSTER (Board P021) TAS-102 treatment results in high trifluridine incorporation into DNA with pyrimidine metabolic pathway markedly up-regulated in cancer K. Oguchi1 , K. Sakamoto1 , H. Kazuno1 , H. Ueno1 , K. Ishida1 , T. Yokogawa2 , K. Yamamura1 , R. Kitamura1 , K. Matsuo1 , T. Utsugi1 . 1 Taiho Pharmaceutical Co. Ltd., Tsukuba Research Center, Tsukuba, Japan; 2 Taiho Pharmaceutical Co. Ltd., Business Development Dept., Chiyoda-ku, Japan Background: TAS-102 (Lonsurf® ) consists of trifluridine (FTD) and tipiracil hydrochloride (TPI), at a molar ratio of 1:0.5. FTD is a thymidine analog and the antitumor component of TAS-102. TPI prevents degradation of FTD by thymidine phosphorylase in liver. TAS-102 was found to significantly improve overall survival of patients with metastatic colorectal cancer that is refractory/intolerable to the standard chemotherapies in a global Phase III study. The mechanism of FTD is considered to be based on its incorporation into DNA, however its intracellular metabolism is not completely defined. Here, we investigate the cellular uptake and phosphorylation of FTD, and substrate specificity of incorporation into DNA by DNA polymerase a. Material and Methods: Intra-cellular uptake of FTD was evaluated with nucleoside transporter specific inhibitors, NBMPR and dipyridamole. Substrate specificity for nucleoside kinase and nucleotide phosphorylase was evaluated with recombinant proteins. DNA elongation with FTD triphosphate (F3 dTTP) by DNA polymerase a was analyzed. The levels of FTD incorporated into DNA were analyzed with [3 H]FTD or LC-MS/MS. Results: FTD was transported via ENT1 and ENT2 into cytoplasm, and was phosphorylated by thymidine kinase 1 as effectively as dThd. F3 dTTP was not recognized by dUTPase, whereas FdUrd-triphosphate was degraded to monophosphate form. F3 dTTP was inserted at the opposite site of adenine by DNA polymerase a. DNA extension was effectively carried out at the site of inserted FTD, and was evident on the single strand DNA template inserted FTD.The level of FTD incorporated into DNA was much higher than that of FdUrd in vitro, and FTD remained in DNA after the FTD removal from culturing medium. In vivo study also showed high FTD accumulation in tumor implanted in nude mice. Nucleosomal abnormalities such as swollen nuclei and decreased hetero-chromatin were observed in the cells treated with FTD. Conclusions: These results indicate that FTD is recognized and activated through the thymidine salvage pathway highly up-regulated in tumor, and incorporated into DNA during DNA replicating phase like the natural substrate dThd. FTD might exert antitumor activity by nucleosomal abnormalities caused by high level FTD incorporation into DNA. These unique properties might underlie the clinical benefit of prolonged survival of colorectal cancer patients treated with TAS-102 after failure on prior 5-FU based chemotherapy. 28 POSTER (Board P022) Characterization of the type of cell death induced by novel tambjamine analogs in lung cancer A. Rodilla Mart´ın1 , V. Soto-Cerrato1 , P. Manuel-Manresa1 , L. Korrodi´ as ` 1 . 1 University of Barcelona, Gregorio ´ 1 , R. Quesada2 , R. Perez-Tom Department of Pathology and Experimental Therapeutics, Barcelona, Spain; 2 University of Burgos, Department of Chemistry, Barcelona, Spain Lung cancer is the leading cause of cancer death worldwide. Despite of new advances in diagnosis and clinical care, the success of standard treatments is still limited, especially in chemotherapy. Therefore, novel anticancer compounds with different mechanisms of action are eagerly needed. In this view, our research group proposes a new therapeutic strategy against cancer that involves modulating the intracellular pH (pHi). Cancer cells have a reversed pH gradient compared to normal cells, which allows cancer progression by promoting proliferation and evasion of apoptosis. Hence, anion transporter compounds, such as tambjamine analogs, have been selected in this study for their potential as anticancer agents through the modulation of the pHi. First, the effect of our compounds in cell viability was evaluated in several lung cancer cell lines by the MTT assay. A significant decrease was observed in most of them, and two compounds (3 and 9) were chosen for further studies. To test whether these compounds have a cytostatic effect, cytometry assays were performed and showed that tambjamines did not induce a significant cell cycle arrest. Then, to characterize the type of cell death, we analyzed different molecular markers related to apoptosis and autophagy by western blot and we observed some evidence of caspases activation and LC3II accumulation. Furthermore, it could be seen at the microscope, phase contrast as well as electron microscope, that the compounds also induced massive cytoplasmic vacuolization. We tested several markers to distinguish among different potential organelles, which could have undergone this phenomenon. We

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used LAMP1 and LC3II by immunofluorescence and we could exclude lysosomes and autophagosomes, respectively. The origin of the vacuoles could also be from the incorporation of extracellular fluid by endocytosis or macropinocytosis, or by the swelling of organelles such as mitochondria or endoplasmic reticulum, which we are trying to identify. Altogether, these results show that these anion transporters have potent cytotoxic effects in lung cancer cell lines, inducing some kind of cell death in which traits of several cell death processes are involved. Acknowledgements: This work was supported by a grant from the Spanish government and the EU (FIS PI13/00089) and a grant from La Marato´ de TV3 Foundation (65/C/2013). LKG holds a postdoctoral fellowship through the Portuguese Foundation for Science and Technology-FCT (SFRH/BPD/91766/2012). 29 POSTER (Board P023) TAS-114 is a novel dUTPase/DPD inhibitor, its DPD inhibition reduces capecitabine dosage but does not diminish therapeutic window in human tumor xenografts W. Yano1 , H. Kazuno1 , T. Yokogawa2 , K. Sakamoto1 , K. Yoshisue1 , T. Wakasa1 , M. Fukuoka3 , K. Matsuo1 , K. Noguchi1 , T. Utsugi1 . 1 Taiho Pharmaceutical Co. Ltd., Tsukuba Research Center, Tsukuba, Japan; 2 Taiho Pharmaceutical Co. Ltd., Business Development Dept., Chiyoda-ku, Japan; 3 Taiho Pharmaceutical Co. Ltd., CMC Center, Tokushima, Japan Background: Deoxyuridine triphosphatase (dUTPase) is a gatekeeper enzyme for uracil misincorporation and is considered to be an important factor for resistance to 5-fluorouracil (5-FU). Dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme for 5-FU degradation. TAS-114 is a firstin class oral dUTPase/DPD inhibitor and is under clinical development in combination with 5-FU prodrugs. Previously, it was reported that the combination of capecitabine and a selective DPD inhibitor could reduce the maximum tolerated dose (MTD) of capecitabine. However, the therapeutic window of capecitabine/DPD inhibitor was no better than capecitabine alone. In contrast, we found that the combination of capecitabine with TAS-114, a dual inhibitor of dUTPase/DPD, significantly enhanced the antitumor efficacy and improved the therapeutic window. Here, we report the mode of DPD inhibition by TAS-114, and roles of dUTPase inhibition and DPD inhibition in capecitabine/TAS-114. Material and Methods: Mode of DPD inhibition was determined by measuring 5-FU degradation in human liver S9 fraction. Plasma levels of 5-FU were measured by LC/MS for pharmacokinetics. The antitumor activity of capecitabine/TAS-114 was evaluated in xenografts of MX-1 and MC-2 (human breast cancers), and KB/TP (cancer cell stably overexpressed thymidine phosphorylase (TP)). MX-1 is a dUTPase inhibition sensitive tumor, and MC-2 and KB/TP are sensitive to capecitabine due to high TP expression. Results: TAS-114 possessed moderate DPD inhibitory activity and its inhibition mode was reversible. In combination with capecitabine, TAS114 increased 5-FU concentration in plasma due to DPD inhibition. This increase in 5-FU exposure led to reduction of capecitabine MTD in all tumor models. In both MC-2 and KB/TP xenograft models, which were highly sensitive to capecitabine, TAS-114 did not reduce maximal antitumor activity and therapeutic window of capecitabine, despite a reduction in the dosage. In the MX-1 xenograft model, which is inherently less sensitive to capecitabine, TAS-114 significantly enhanced antitumor activity of capecitabine, demonstrating the importance of dUTPase inhibition. Conclusions: TAS-114 is a moderate and reversible inhibitor of DPD. Its favorable profile of DPD inhibition reduces the MTD of capecitabine but does not diminish therapeutic window. Phase 1 study of capecitabine/TAS114 is ongoing and therapeutic efficacy as a dUTPase/DPD dual inhibitor can hopefully be demonstrated.

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30 POSTER (Board P024) The fungal-derived cyclohexadepsipeptide Destruxin E exerts multifaceted anticancer and antiangiogenic activities R. Dornetshuber-Fleiss1 , P. Heffeter2 , T. Mohr2 , P. Hazemi3 , K. Kryeziu4 , C. Seger5 , W. Berger2 , R. Lemmens-Gruber3 . 1 University of Vienna and Medical University of Vienna, Institute of Pharmacology and Toxicology and the Institute of Cancer Research Department of Medicine I and Comprehensive Cancer Center of the Medical University, Vienna, Austria; 2 Medical University of Vienna, Institute of Cancer Research Department of Medicine I and Comprehensive Cancer Center of the Medical University, Vienna, Austria; 3 University of Vienna, Department of Pharmacology and Toxicology, Vienna, Austria; 4 Medical University of Vienna, Institute of Cancer Research Department of Medicine I and Comprehensive Cancer Center of the Medical University, Vienna, Austria; 5 Leopold-Franzens University Innsbruck, Institute of Pharmacy Department of Pharmacognosy, Vienna, Austria Background: Destruxins − a group of secondary metabolites of the entomopathogenic fungus Metarhizium anisopliae − recently came into focus of interest as anticancer therapeutics for colorectal cancer. However, the knowledge on their anticancer effects is fragmentary. Consequently, the present study aimed to investigate the impact of the representative derivative Destruxin E (Dtx E) on human colon cancer cell growth and survival. Materials and Methods: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays and Western blot analyses were used to assess the cytostatic/cytotoxic activity of Dtx E on Caco-2 and HCT116 cells. Impact of cell cycle distribution was evaluated using PI-stainings and FACS analyses. Apoptotic effects were determined with DAPI stainings and Western blot analyses. Angiogenesis inhibition was evaluated using scratch and tube-formation assays. To investigate the impact on the intracellular redox potential flow cytometry with dihydroxyethidium (DHE) and combination studies with the radical scavenger N-acetyl cysteine (NAC) and the glutathione inhibitor BSO were performed. Results: The experimental data showed that Dtx E exerts potent antiproliferative activity in the nanomolar range in CaCo-2 and HCT116 cells. Moreover, Dtx E caused an imbalance of cell cycle distribution and the cytostatic/cytotoxic effects were shown to be widely p53-independent but reduced by p21- and bax-deletion, respectively. Cytotoxicity is based on intrinsic apoptosis induction and associated with phosphoinositide-3kinase (PI3K)/Akt pathway inhibition. Additionally, anticancer activity of Dtx E involves disturbance of the intracellular redox balance and finally, Dtx E inhibits the migration and tube formation of human endothelial cells indicating antiangiogenic potential. Conclusion: The data of the present study indicate that Dtx E represents a feasible, multifunctional anticancer drug candidate for preclinical development against colorectal cancer. 31 POSTER (Board P025) N-Myc amplification sensitizes tumor cells to inhibition by Danusertib, an Aurora kinase inhibitor P. Carpinelli1 , R. Ceruti2 , R. Alzani2 , C. Re1 , D. Ballinari1 , S. Cribioli2 , M. Russo2 , A. Degrassi2 , G. Texido2 , M. Ciomei2 , E. Pesenti2 , A. Montagnoli1 , A. Galvani1 . 1 Nerviano Medical Sciences Srl, Cell Biology, Nerviano (Milano), Italy; 2 Nerviano Medical Sciences Srl, Pharmacology, Nerviano (Milano), Italy Amplification of N-Myc is a driving mutational event in a subset of tumor types, particularly those of neural origin and neuroendocrine tumors such as neuroblastoma (NB), neuroendocrine prostate cancer (NEPC), small cell lung cancer (SCLC) and others. Thus, inhibition/reduction of N-Myc protein levels may be of therapeutic benefit for such tumor types but to date small molecules that specifically target this oncogene are not clinically available. Aurora A kinase has been shown to interact directly with N-Myc in neuroblastoma and to induce its stabilization by preventing binding of the Fbxw7 ubiquitin ligase, which mediates its ubiquitination and subsequent proteasomal degradation. Moreover, there is clear evidence that pharmacological intervention with small molecule inhibitors which target Aurora A represents a promising therapeutic approach for this tumor subtype. Danusertib (PHA-739358) is a small molecule ATP competitor that inhibits Aurora A, B and C kinases. Aurora kinases inhibition by Danusertib results in cell cycle block with concomitant inhibition of cell proliferation. The compound is in clinical investigation in both solid tumor and hematological malignancy settings. Here we report the in vitro and in vivo activity of Danusertib in N-Myc amplified tumor models. We demonstrate that in vitro, NB cell lines bearing amplified N-Myc display greater sensitivity to Danusertib than

Poster Session – Cytotoxics non-amplified cell lines. In such sensitive NB cell lines, inhibition of histone H3 phosphorylation, a marker of Aurora kinase inhibition, is associated with decreased levels of N-Myc and induction of apoptosis. In NB tumor xenograft models, Danusertib induced significant in vivo tumor growth inhibition, including tumor regression, accompanied by robust reductions of N-Myc protein levels, again concomitant with inhibition of phospho-histone H3 and induction of apoptosis. Additionally, Danusertib also displayed significant in vivo activity against the TRAMP, a transgenic mouse prostate carcinoma model, which displays several molecular and pathological features of NEPC. Here again, treatment with Danusertib induced decreased levels of N-Myc, associated with inhibition of histone H3 phosphorylation. Thus, Danusertib destabilizes N-Myc both in vitro and in vivo and therefore merits further investigation as a potential option for the therapy of N-Myc amplified neuroendocrine tumors, as well as other settings which are dependent on this oncogene. 32 POSTER (Board P026) Replication stress is a determinant of synergy between gemcitabine and Chk1 inhibition S.B. Koh1 , A. Courtin1 , R. Boyce2 , B. Boyle2 , F.M. Richards1 , D.I. Jodrell1 . 1 Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge, United Kingdom; 2 Sentinel Oncology Limited, Cambridge, United Kingdom Background: Chk1 plays multiple regulatory roles within the cell cycle and this study interrogates the mechanism of synergy between Chk1 inhibition and gemcitabine in cancer cells. Methodology: We evaluated the cytotoxicity of FS105, a Chk1-specific inhibitor (IC50 2.8nM), with gemcitabine in human and murine cancer cells. We then employed established mathematical models to quantitatively identify drug concentrations that yielded synergistic growth inhibition. Subsequent mechanistic studies, achieved by quantitative high-content imaging, immunofluorescence microscopy, flow cytometry and immunoblotting, were performed at the identified synergistic drug concentrations. Results: Synergy with FS105 was found at sub-GI50 concentrations of gemcitabine in all tested cell lines, e.g. Emax 91±3% growth inhibition at 3uM FS105+30nM gemcitabine in Panc-1 cells compared to Bliss predicted 33±1% growth inhibition. At these concentrations, we did not observe substantial premature mitotic entry, contrary to the prevailing model that inhibiting Chk1 abrogates the G2/M checkpoint. Downstream checkpoint effector CDK1 Y15 was not inhibited by FS105 at synergistic concentrations with gemcitabine. In agreement with this, significant S phase arrest with hallmarks of elevated replication stress and DNA damage such as ùH2AX was observed. For example, 63±6% MIA PaCa-2 cells were in S phase 24 hours after the combination treatment, compared to control (33±2%), FS105 alone (35±4%) and gemcitabine alone (43±4%). The induction of S phase arrest and ùH2AX by the combination was inhibited when the activity of replication initiator CDC7 was suppressed by PHA-767491. This observation is consistent with Chk1 role in regulating origin firing and our hypothesis that Chk1 inhibition synergises with gemcitabine via destabilisation of the replication machinery. Conclusions: This study elucidates how Chk1 inhibition with gemcitabine at synergistic concentrations perturbs predominantly the S-phase homeostasis in cancer cells. It also provides a molecular rationale to reassess the current scheduling paradigm of Chk1 inhibitors with DNA-damaging agents. 33 POSTER (Board P027) Combining the long-acting topoisomerase 1-inhibitor etirinotecan pegol with the PARP inhibitor rucaparib to provide anti-tumor synergy without increased toxicity U. Hoch1 , D. Charych2 . 1 Nektar Therapeutics, Development, San Francisco CA, USA; 2 Nektar Therapeutics, Research, San Francisco CA, USA Background: PARP inhibition sensitizes cells to DNA-damaging agents, particularly topoisomerase 1-inhibitors, where the combination shows synergy in nonclinical studies. However, in the clinic, the combination of either topotecan or irinotecan with PARP inhibitors showed severe hematologic toxicities, requiring dose reductions beyond optimal therapeutic effects. Etirinotecan pegol (NKTR-102) is the first long-acting Topoisomerase 1-inhibitor providing continuous exposure to active metabolite throughout the entire chemotherapy cycle with reduced peak concentrations, resulting in better efficacy and safety, including a low rate of Grade 3 neutropenia (11%). Here we present in vivo combination data of etirinotecan pegol and rucaparib in the MX-1 breast cancer model. Methods: Mice bearing BRCA1-deficient MX-1 breast cancer tumors (~100 mm3 ) received either vehicle, PO rucaparib, IV etirinotecan pegol, or a combination of etirinotecan pegol and rucaparib as follows: 30 or

Poster Session – Cytotoxics 150 mg/kg rucaparib qd×21; 10 or 50 mg/kg etirinotecan pegol q7d×4; or 30/10, 30/50, 150/10, or 150/50 mg/kg rucaparib/etirinotecan pegol (n = 10/group). Anti-tumor efficacy was evaluated by tumor growth delay (TGD) and regression responses. Results: Control tumors grew to the 2000 mm3 endpoint in a median of 25 days. Rucaparib at 30 and 150 mg/kg resulted in TGDs of 14 and 18 days, respectively. Tumor growth was slower following rucaparib but was continuous in all animals. Etirinotecan pegol at 10 mg/kg resulted in TGD of 32 days with one tumor-free animal. Any combination of etirinotecan pegol and rucaparib, and single-agent etirinotecan pegol at 50 mg/kg caused complete tumor regression in all animals that persisted for at least 10 weeks. All single-agent and combination doses were well tolerated without clinical signs or body weight loss, except rucaparib/etirinotecan pegol 150/50 where individual animals showed BW loss of 15% (group mean: 6%). Conclusions: Combined etirinotecan pegol and rucaparib showed clear synergy in the BRCA1-deficient MX-1 breast cancer model with 100% complete responses and no tumor regrowth, even 7 weeks after last dose. The combination was well tolerated. SN38 trough and rucaparib plasma exposure at the dose levels employed in mice are achieved clinically. Our results suggest that this combination may diminish previously observed toxicities of combined Topoisomerase 1- and PARP-inhibitors while improving efficacy and support pursuing further studies with etirinotecan pegol and PARP-inhibitors. 34 POSTER (Board P028) Phase I, dose-escalation study of the investigational drug D07001-F4, an oral formulation of gemcitabine HCl, in patients (pts) with advanced solid tumors or lymphoma C. Lin1 , W. Su2 , J. Lee1 , C. Hsu1 , A. Cheng1 , C. Lin3 , H. Ho3 , C. Huang3 , S. Hsueh3 , J. Yang1 . 1 National Taiwan University Hospital, Oncology, Taipei, Taiwan; 2 National Cheng Kung University Hospital, Oncology, Tainan, Taiwan; 3 InnoPharmax Inc., Taipei, Taiwan Background: This phase 1 dose-escalation trial, using a 3+3 design, was conducted to evaluate the safety, tolerability, pharmacokinetics (PK), and preliminary efficacy of D07001-F4, an oral formulation of gemcitabine HCl, in pts with advanced solid tumors or lymphoma. The primary objectives were to determine the MTD, characterize DLT(s) and define the recommended phase 2 dose of D07001-F4. Methods: Patients aged 20 yrs with PS 0−2 were eligible. Gemcitabine 5 mg IV in cycle 0 and D07001-F4 from 2 mg PO were given on days 1, 3, 5, 8, 10, and 12 of a 21-day cycle to determine the MTD based on DLTs in cycle 1. Plasma and peripheral blood samples were obtained for PK analysis in cycle 1. Results: As of May 2014, 15 pts (10 M, 5 F; median 59 years [range 42−67]) were enrolled into 5 dose escalation cohorts (2−30 mg). A median of 3 cycles were given (range 0−13+). So far, no DLTs occurred and the MTD has not yet been reached. Enrollment is ongoing. Two pts at cohort 4 (20 mg) experienced 3 drug-related G1 AEs (1 fever, 2 fatigue) and 2 pts at cohort 5 (30 mg) experienced 3 drug-related G1 AEs (1 vomiting, 2 nausea). PK results showed a dose proportional increase in exposure of dFdC after repeated dosing. Plasma dFdC was fast eliminated after dosing. The average Cmax 7474 pg/mL and AUC0−24 5603 hr·pg/ml at day 1 and Cmax 6588 pg/mL and AUC0−24 7926 hr·pg/ml at day 12 were observed at cohort 4 (20 mg). 10 pts had SD, 7 of whom were on study for 3 months. One ongoing pt (5 mg) with thyroid cancer had tumor shrinkage of 28%. Conclusions: From preliminary data, D07001-F4 appears to be well tolerated and has shown signs of anti-tumor activity in pts with advanced solid tumors. Accrual at the 30 mg dose level is ongoing and a recommended phase 2 dose is expected to be reached soon. Updated results will be presented. Clinical trial registration: NCT01800630 35 POSTER (Board P029) BRCA1 expression exploratory analysis in patients of the phase III trial of trabectedin vs. doxorubicin-based chemotherapy as first-line therapy in translocation-related sarcomas M. Aracil1 , P. Lardelli2 , A. Nieto2 , C.M. Galmarini1 . 1 PharmaMar S.A., R&D, Colmenar Viejo (Madrid), Spain; 2 PharmaMar S.A., Clinical Development, Colmenar Viejo (Madrid), Spain Background: BRCA1 mRNA and protein expression levels were analyzed in translocation-related soft tissue sarcoma (TRS) patients treated with trabectedin (arm A) or doxorubicin (arm B) as first-line therapy (phase III trial ET-C-002−07). Expression levels were analyzed in tumor tissue samples obtained at diagnosis with the aim to determine the potential role of BRCA1 as prognostic and/or predictive biomarker.

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Materials and Methods: A total of 83 samples with clinical and molecular data were subjected to biostatistical analysis in order to correlate the expression values with the clinical outcome (ORR, PFS and OS). PFS and objective tumor response were evaluated by an Independent External Review Committee according to RECIST v.1.0. The IHC staining intensity was evaluated by a pathologist and reported following a 4-level scale from 0 (no staining) to +3 (strong staining). Expression levels were categorized using as cutoff the median value. Results: In the trabectedin arm, lower BRCA1 protein expression (<3) was associated with higher tumor control (PR+SD) (91.7% for low BRCA1 vs. 57.1% for high BRCA1, p = 0.0095) and longer median PFS (not reached for low BRCA1 vs. 2.4 m for high BRCA1; p = 0.0013). In the doxorubicin arm, lower BRCA1 protein expression was associated with longer median PFS (18.9 m for low BRCA1 vs. 5.5 m for high BRCA1; p = 0.0156). In a multivariate analysis including the most common clinical variables, where treatment is not selected, BRCA1 protein expression correlated with a worse PFS (HR = 4.539, 95% CI: 1.941–10.613; p = 0.0005). Conclusions: The results point out the potential importance of BRCA1 in the clinical outcome of patients with TRS treated with trabectedin or doxorubicin. In particular, high protein expression of BRCA1 seems to be associated with a worse clinical outcome. Its potential prognostic and/or predictive value in TRS patients needs to be further evaluated. 36 POSTER (Board P030) Suppression of metastasis and improvement of drug distribution by eribulin mesylate Y. Ozawa1 , K. Okamoto1 , Y. Adachi1 , M. Asano1 , K. Tabata1 , Y. Funahashi2 , J. Matsui1 . 1 Eisai Co. Ltd., Oncology PCU, Tsukuba Ibaraki, Japan; 2 Eisai Co. Ltd., BPM CFU, Andover, USA Background: Eribulin mesylate (eribulin) has shown trends toward greater overall survival compared to progression-free survival in late stage metastatic breast cancer patients in the clinic. This finding suggests that eribulin may have additional, previously unrecognized antitumor mechanisms. To investigate this possibility, eribulin’s effects on microenvironment including tumor vessels were investigated and we found that eribulin demonstrated improvement in tumor perfusion through vascular remodeling in human breast cancer models. Material and Methods: For in vitro study, cells were cultured under 1% O2 and 0.1 mg/ml glucose. EMT markers were analyzed by western blot analysis. For in vivo model, after 15 days post-4T1 cell inoculation into the 3rd mammary fad pad of Balb/c mice, drug treatment was started, and after 28 days lung metastases were counted. In MDA-MB-231 model, treatments with capecitabine or eribulin were started on day 1. On day 12, eribulin treated mice were divided into no-treatment and capecitabine treatment groups. Results: The remodeling of tumor vessels evoked 2 possibilities, antimetastatic effect and enhancement of antitumor activity thorough the improvement of drug influx into tumors. Our data showed that hypoxic conditions induced EMT phenotype in breast cancer cells in vitro, and eribulin treatment reduced hypoxic markers in xenorafts. Furthermore, eribulin significantly reduced the lung metastasis in 4T1 OT model in which eribulin did not have any antitumor effect against primary tumor because of MDR over expression. We next investigated the effect of eribulin on improvement of drug influx into tumors. We compared the antitumor effects of capecitabine with or without pre-treatment of eribulin in MDA-MB-231 xenograft model. In eribulin pre-treated group, eribulin treatment induced clear tumor regression, and then, capecitabine treatment was started when tumor volumes returned to baseline prior to eribulin treatment. Interestingly, antitumor effect of capecitabine was enhanced in eribulin-pretreated group, compared to non-treated group. We think this enhancement of antitumor effect is the result of improvement of capacitabine influx into tumors. Conclusion: We have found the new effects of eribulin, anti-metastatic effect and improvement of drug influx into tumors via the remodeling of tumor vessels, using human and mouse breast cancer models. These preclinical finding may provide new insights into the clinical observations. 37 POSTER (Board P031) Effect of a microtubule-targeting drug on cell–cell contacts in bladder epithelial tumour cells 1 , R. Castosa2 , M. Haz2 , M. Blanco2 , M. Rodriguez2 , L.M. Anton-Aparicio ´ M. Valladares1 , A. Figueroa2 . 1 Complejo Hospitalario Universitario A ˜ (INBIC-CHUAC), Medical Oncology Unit, Coruna, Spain; 2 Instituto Coruna ´ Biomedica ´ ˜ Investigacion A Coruna-Complejo Hospitalario Universitario A ˜ (INBIC-CHUAC), Translational Cancer Research Group, Coruna, Coruna Spain

Background: Bladder cancer is a common malignancy affecting the genitourinary system that represents the fifth most common cancer in

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the world. Transitional cell carcinoma (TCC) represents 95% of these tumours. Vinflunine (VFL) is a microtubule-targeting drug that suppresses microtubule dynamics, showing anti-metastatic properties both in vitro and in living cancer cells. An increasing body of evidence underlines the influence of the microtubules dynamics on the cadherin-dependent cell– cell adhesions. In this report, we investigate the role of VFL on cell–cell contacts in bladder epithelial tumour cells. Methods: Human bladder epithelial tumour cell lines HT1376, 5637 and UMUC3 were used to analyse cadherin-dependent cell–cell adhesions under VFL treatment. VFL effect on growth inhibition was measured by using a MTT colorimetric cell viability assay. Western blot, immunofluorescence and transmission electron microscopy analyses were performed to assess the roles of VFL effect on cell–cell adhesions, epithelial-to-mesenchymal markers and apoptosis. The role of the proteasome in controlling cell–cell adhesion was studied using the proteasome inhibitor MG132. Results: We show that VFL induces cell death in bladder cancer cells and activates epithelial differentiation of the remaining living cells, leading to an increase of E-cadherin-dependent cell–cell adhesion and a reduction of mesenchymal markers, such as N-cadherin or vimentin. Moreover, while E-cadherin is increased, the levels of Hakai, an E3 ubiquitin-ligase for E-cadherin, were significantly reduced in presence of VFL. In 5637, this reduction on Hakai expression was blocked by MG132 proteasome inhibitor, indicating that the proteasome pathway could be one of the molecular mechanisms involved in its degradation. Conclusions: Our findings underscore a critical function for VFL in cell–cell adhesions of epithelial bladder tumour cells, suggesting a novel molecular mechanism by which VFL may impact upon EMT and metastasis. 38 POSTER (Board P032) The indolyl-chalcone CDD-026 induces cancer cell death through targeting of STMN1 and mitotic catastrophe B. Wegiel1 , Y. Wang1 , F. Jernigan1 , L. Sun1 . 1 Beth Israel Deaconess Medical Center, Surgery, Boston MA, USA Background: Patients with metastatic prostate cancer are still in needs of more effective treatments. Mitotic catastrophe is a tumor suppressive process that is driven by aberrant mitosis and in many cases associated with changes in microtubules stability. The activation of mitotic catastrophe is a highly desirable therapeutic endpoint and the focus of this study. The aim of this study was to investigate the anticancer activities of a series of novel indolyl-chalcones and validate their targets in inducing mitotic catastrophe in prostate cancer cells lines. Materials and Methods: We have performed screen of synthetic indolyl-chalcones in induction of apoptosis and inhibition of proliferation of cancer cells. We have applied multiple cell biology assays to test apoptosis (AnnexinV/PI staining, crystal violet staining), proliferation (BrDU incorporation assay, PI cell cycle analysis), targets analysis (PCR Real time profiler) and targets validation (real time PCR). We conducted molecular modeling analyses to develop a QSAR model. Results: We have identified and improved the potency of a series of novel indoly-chalcones. Our results indicated that the lead molecule CDD026 (Formula: C20H16FNO) at 1−10 mM induced early apoptosis without affecting incorporation of BrdU in S-phase, suggesting its critical role in interrupting late phases of cell cycle. In contrast, the non-indolyl analog CDD-023 inhibited cell cycle progression through G1/S phase in a dose dependent manner (1, 10, 25 mM) but did not induce significant cell death. We then focused our analysis on CDD-026, which at as low dose as 0.1 mM induced apoptosis in PC3 cells. In addition, CDD-026 demonstrated potent inhibitory activity in lung and other cancer cells. To evaluate the potential target for this drug, we have employed Cancer Pathway Finder PCR array (Qiagen) and identified Stathmin 1 (STMN1) and adrenomedullin genes as the most highly upregulated in response to treatment with CDD026. We have validated these targets by real time PCR in PC3 cells treated with CDD-026. Further, we showed that in contrast to taxanes that stabilized microtubules, CDD-026 destabilized microtubules. This effect may be dependent on STMN1 upregulation leading to mitotic catastrophe, which is currently being addressed in our studies. Conclusions: CDD-026 induces mitotic catastrophe in PC3 cells via upregulation of STMN1, which destabilizes microtubules and disrupts cell cycle.

Poster Session – Cytotoxics 39 POSTER (Board P033) Phytochemical indole-3-carbinol synergizes strongly with fludarabine and induces p53-dependent and -independent cell death in chronic lymphocytic leukemia cells irrespective of their IGHV mutation state and treatment resistances G. Perez-Chacon1 , C. Martinez-Laperche2 , N. Rebolleda1 , B. Somovilla3 Crespo3 , C. Munoz-Calleja ˜ , I. Buno ˜ 2 , J.M. Zapata1 . 1 Instituto de Investigaciones Biomedicas “Alberto Sols” CSIC/UAM, Madrid, Spain; 2 Hospital General Universitario and Instituto de Investigacion Sanitaria ˜ Gregorio Maranon, Servicio de Hematologia, Madrid, Spain; 3 Hospital Universitario de la Princesa, Servicio de Inmunologia, Madrid, Spain Background: Chronic lymphocytic leukemia (CLL) is the most common type of leukemia found in adults in Western countries, which still is lacking a cure. A variety of chemotherapy regimens are being used in CLL, but unfortunately patients will eventually become refractory to treatments and die. It is therefore a priority to develop new treatments and therapeutic approaches that improve patient prognosis and survival. Indole-3-carbinol (I3C) is a glucobrassicine derivative that is found in edible Cruciferae plants, such as the broccoli and the cabbage. Phase I and II clinical trials have demonstrated its antitumoral activity in precancerous cervix lesions and in vulvar epidermal neoplasia, while lacking significant toxicity. Materials and Methods: We have tested whether I3C was active against CLL cells. We used a cohort of CLL cells from 36 patients representing different Rai stages, IGHV mutation status, cytogenetic alterations, and responses to treatments. The effect of increasing concentrations of I3C, F-ara-A and combinations of both drugs (keeping a constant 50:1 ratio) on CLL cell survival was determined. CLL samples were grouped according to their characteristics. Results: Our results show that I3C potently reduced the viability of CLL cells with a LD50 of ~37 mM, while it was much less cytotoxic in PBMCs from normal donors (LD50 ~100 mM). Moreover, I3C showed strong synergy with F-ara-A in all cases studied (Table). Remarkably, I3C was able to synergize with F-ara-A even in p53-deficient CLL cells (70% TP53/ATMdeletions) and in CLL cells that showed in vitro resistance to F-ara-A. Indeed, the concentrations of F-ara-A required to kill 90% of CLL cells in the combined I3C/F-ara-A treatment resulted in an F-ara-A dose reduction index (DRI) of 31 for CLL cells harboring TP53/ATM deletions and of 82.5 for CLL cells showing in vitro resistance to F-ara-A. The combination of I3C + F-ara-A was equally active in CLL cells with mutated and unmutated IGHV and were also active in CLL cells from patients that have developed treatment resistances (patients that have received 2−4 treatments). Of note is that I3C could also synergize with vincristine and chlorambucil (Table). Conclusions: Our data indicate that I3C, alone or in combination with F-ara-A and other chemotherapeutic drugs, is a potent inductor of apoptosis in CLL in vitro. I3C strongly synergizes with F-ara-A in all types of CLL tested, including CLL from refractory patients and CLL cells with deficient p53 pathway. The striking DRI for F-ara-A in combination with I3C would likely reduce harmful secondary effects associated to fludarabine while having a similar or better effectiveness. Moreover, the low toxicity of I3C, already clinically tested, and its anti-CLL activity would also support its use as a novel neoadjuvant and adjuvant therapy in CLL, even in those patients with relapsed or refractory disease. 40 POSTER (Board P034) Selectivity and mechanism of action studies for Polo Box-targeted, non-ATP based inhibitors of PLK1 M. Baxter1 , S. Craig1 , C. McInnes1 , M.D. Wyatt1 . 1 University of South Carolina, Drug Discovery and Biomedical Sciences, Columbia, USA Polo-like Kinase 1 (PLK1) performs critical roles in the coordination of mitosis and is also an oncogene over-expressed in many cancer types. ATP-binding site inhibitors of PLK1 have progressed to clinical trials, and one was recently given FDA breakthrough status to treat acute myeloid leukemia. However, recent data suggests that there are major disadvantages to blocking the kinase activity of PLK1. First, PLK family specificity is an issue with ATP-competitive compounds because they commonly inhibit all PLK paralogs, including PLK3, a known tumor suppressor. Second, a single point mutant in the active site of PLK1 (Cys67Val) results in dramatic resistance to structurally distinct ATPcompetitive inhibitors (Burkard et al, ACS Chem. Biol. 2012, 7, 978– 981), suggesting that the emergence of resistance in the clinic against these agents is likely. We have validated the innovative REPLACE strategy by discovering non-ATP competitive and PLK1-selective inhibitors as an alternative therapeutic strategy for this important target. The Polobox domain (PBD) of each PLK is a phospho-peptide binding motif that determines substrate recognition and subcellular localization. Our approach targets the PBD of PLK1 to achieve desired selectivity and improve efficacy. Through modeling and design we produced series

Poster Session – Cytotoxics of fragment-ligated inhibitory peptides (FLIPs). We report three FLIPs with an octyl-benzamide group, having the following different amino acid C-terminus sequences: −PNGL, −AI, and −PL. In vitro binding to the PBDs of PLK1 and PLK3 was measured by a fluorescence polarization assay. The IC50 values for PLK1 calculated from the competition assay are 0.36 mM, 0.41 mM, and 1.2 mM, respectively, and all three are at least >1800fold more selective for PLK1. Initial testing in two cancer lines revealed respectable anti-proliferative activity for two of the FLIPs. In addition, we report the investigation of our PBD-inhibitors in cells expressing the mutant C67V PLK1 that is resistant to ATP-based inhibitors. We find that our compounds are equally active in cells expressing wild-type or C67V PLK1, whereas cells expressing C67V PLK1 are dramatically resistant to the ATPbased inhibitor, BI 2536. Current work is characterizing the mechanism of action for these FLIPs in cancer cells, including their ability to induce a G2/M cell cycle arrest, and induce aberrant mitotic phenotypes associated with PBD-inhibition. These exciting developments demonstrate the validity of our approach to produce drug-like lead PBD-inhibitors that are PLK1 selective and are active against tumors resistant to ATP-inhibitors. PBDinhibitors could ultimately be used in combination therapy regimens with ATP-based inhibitors for a dual attack on this important, clinically validated oncology target. 41 POSTER (Board P035) Clinical activity of BIND-014 (docetaxel nanoparticles for injectable suspension) as second-line therapy in patients (pts) with Stage III/IV non-small cell lung cancer R. Natale1 , M. Socinski2 , L. Hart3 , O. Lipatov4 , D. Spigel5 , B. Gershenhorn6 , G. Weiss7 , S. Kazmi8 , N. Karaseva9 , O. Gladkov10 , V. Moiseyenko11 , J. Summa12 , G. Otterson13 . 1 Cedars-Sinai Medical Center Samuel Oschin Comprehensive Cancer Center, Clinical Lung Cancer Program, Los Angeles, USA; 2 University of Pittsburgh Cancer Pavilion, Lung and Thoracic Malignancies Program, Pittsburgh, USA; 3 Florida Cancer Specialists, Clinical Research Director Drug Development Program, Fort Meyers, USA; 4 Republic Clinical Oncology Dispensary, Department of Medical Oncology, Ufa, Russian Federation; 5 Sarah Cannon Research Institute, Lung Cancer Research Program, Nashville, USA; 6 Cancer Treatment Centers of America Midwestern Regional Medical Center, Department of Clinical Research and Clinical Trials, Zion, USA; 7 Cancer Treatment Centers of America Western Regional Medical Center, Department of Clinical Research and Clinical Trials, Goodyear, USA; 8 Cancer Treatment Centers of America Eastern Regional Medical Center, Department of Clinical Research and Clinical Trials, Philadelphia, USA; 9 City Clinical Oncology Dispensary, Department of Medical Oncology, Saint Petersburg, Russian Federation; 10 Chelyabinsk Regional Clinical Oncology Dispensary, Department of Medical Oncology, Chelyabinsk, Russian Federation; 11 N.N. Petrov Research Institute of Oncologists, Department of Medical Oncology, Saint-Petersburg, Russian Federation; 12 BIND Therapeutics, Department of Clinical Development, Cambridge, USA; 13 Ohio State University Comprehensive Cancer Center, Thoracic Oncology Program, Columbus, USA Background: BIND-014 is a novel, polymeric nanoparticle containing docetaxel (D) targeted to prostate-specific membrane antigen (PSMA), a protein expressed on prostate cancer cells and on the vasculature of many non-prostate solid tumors. BIND-014 is anticipated to improve the clinical benefit of D by increasing its concentration and duration of exposure in tumors. In a phase 1 study, BIND-014 was generally well-tolerated and displayed anti-tumor activity at low doses and in tumors where solventbased D (sbD; Taxotere® ) has minimal activity. Material and Methods: APhase 2 study was conducted of BIND-014 administered by a 60-minute intravenous infusion at 60 mg/m2 on Day 1 of a 21-day cycle as second-line therapy after a Pt-containing regimen in pts with Stage III/IV NSCLC with characterized genomic status (EGFR mutation, ALK rearrangement, KRAS mutation). A 2-stage design for futility was incorporated with a maximum of 40 pts anticipated. The primary endpoint was ORR. Results: Forty pts were enrolled and received a median of 3 doses (range 1−12); the study is closed to enrollment with several pts continuing on study. To date, 33 pts are evaluable for response, 5 (15%) had PR and 12 (36%) had stable disease lasting  12 weeks (SD  12 wks). Among 8 pts testing positive for a KRAS mutation, 2 (25%) had PR and 3 (38%) had SD  12 wks. Among the 40 treated pts, 4 experienced an SAE classified as related to study drug (Gr 2 neutropenic fever [1 pt], Gr 2 DVT [1 pt], Gr 3 dyspnea [1 pt], Gr 3 hypoxia [1 pt]). There were 174 drug-related AEs, 102 (59%) of which were Gr 1. Drug-related AEs Gr 2 occurring in at least 3 pts included anemia (3 pts), dehydration (4 pts) and fatigue (5 pts). Two pts experienced Gr 3 lymphocytopenia and 2 pts experienced Gr 2 neutropenia. There was no evidence of hepatotoxicity and only 1 incident of peripheral neuropathy (Gr 3).

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Conclusions: BIND-014 administered at 60 mg/m2 on Day 1 of a 21-day cycle is clinically active and well-tolerated assecond-line therapy in Stage III/IV NSCLC pts with broad tumor mutation status. BIND-014 demonstrated a 63% disease control rate (25% PR + 38% SD  12 wks) in pts with KRAS mutations, a population generally unresponsive to sbD. Neutropenia, anemia and neuropathy, commonly observed with sbD, were significantly reduced with BIND-014. The potential safety and activity advantages of BIND-014 compared to historical sbD data warrant further evaluation in pts with NSCLC, including those with KRAS mutations. Final data will be presented. 42 POSTER (Board P036) Interaction of SJG-136 with cognate sequences of oncogenic transcription factors J. Mantaj1 , P.J. Jackson1 , D.E. Thurston1 , K.M. Rahman1 . 1 King’s College London, Institute of Pharmaceutical Sciences, London, United Kingdom The pyrrolo[2,1-c][1,4]benzodiazepines (PBDs) are sequence-selective DNA minor-groove binding agents. They possess a ‘soft’ electrophilic imine moiety at their N10-C11 position which can form an aminal linkage with the C2-NH2 group of a guanine base. The PBD dimer SJG-136 is presently in Phase II clinical trials in ovarian cancer and leukaemia. There is growing evidence that PBD monomers exert their pharmacological effects through transcription factor inhibition, for example GWL-78, a C8linked PBD-Py-Py conjugate, has been shown to block interaction of the transcription factor NF-Y and KMR-28−39, a GC sequence selective PBDMPB conjugate, inhibits the transcription factor NF-kB. The aim of this study is to investigate whether PBD dimers like SJG-136 also interact with transcription factor binding sequences and exert their antitumor activity through this mechanism in addition to previously reported DNA strand breakage, inhibition of endonucleases, RNA polymerases and arrest of replication fork. We have developed an ion pair reverse phase HPLC/MS analytical methodology and demonstrated for the first time the ability of the PBD dimer SJG-136 to bind to specific DNA consensus sequences of the transcription factors NF-kB, EGR-1, AP-1 and STAT3. As a dimer, SJG-136 can form intra-, inter-strand and mono-alkylated adducts with DNA. Interestingly, we have been able to observe that SJG-136 formed three distinct adducts with the STAT3 consensus sequence, while only single adducts were observed in the case of NF-kB-1, NF-kB-2, EGR-1 and AP-1. After completing the biophysical study, the effect of SJG-136 on the expression of STAT3 dependent genes was carried out by RTPCR, qPCR and Western Blot experiments using the STAT3 dependent breast cancer cell line MDA-MB-231. Our results demonstrate a significant dose dependent down regulation of STAT3 dependent genes cyclin D1, survivin, NNMT, Bcl-2, STAT3 and fascin. These findings have implications for understanding the mechanism of action of SJG-136 and can potentially explain the differences in activity of SJG-136 against various tumour cell lines. Future work will focus on establishing the correlation with transcription factors NF-kB, AP-1 and EGR-1 following a similar procedure. 43 POSTER (Board P037) CDKN1A-mediated responsiveness of MLL-AF4-positive acute lymphoblastic leukemia to Aurora kinase-A inhibitors L. Hung1 , Y. Chen2 , H. Lin3 , M. Tsai4 , H. Hsieh5 , J. Chang6 , N. Chen1 , S. Yang1 , T. Chen7 . 1 Institute of Bioinformatics and Biosignal Transduction, Department of Life Sciences National Cheng Kung University, Tainan, Taiwan; 2 Institute of Clinical Medicine and Division of Hematology/Oncology, Department of Internal Medicine National Cheng Kung University, Tainan, Taiwan; 3 Department of Pharmacology, National Cheng Kung University, Tainan, Taiwan; 4 Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha NE, USA; 5 Institute of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan Miaoli County, Taiwan; 6 Institute of Clinical Medicine and National Institute of Cancer Research, National Cheng Kung University and National Health Research Institutes, Tainan, Taiwan; 7 Institute of Clinical Medicine and Division of Hematology/Oncology Department of Internal Medicine, National Cheng Kung University Hospital, Tainan, Taiwan Background: The prognosis of patients with relapsed or resistant acute lymphoblastic leukemia (ALL) is quite poor, and resistance to chemotherapy in ALL cells remains a challenge to successful treatment. Overexpression of Aurora kinases is largely observed in many cancers, including hematologic malignancies. Aurora kinases have become attractive therapeutic targets to help overcome chemotherapy resistance. Methods: The expression of Aurora kinases and their activators was analyzed by Western blot analysis. Drug susceptibility was determined by MTT assay. Expression of CDKN1A was detected by Western blot

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and Q-PCR. The status of TP53 in ALL cells was determined by Sanger sequencing. Results: Nine ALL cell lines exhibited different susceptibilities to Aurora kinase inhibitors (AKIs). Cells sensitive to AKIs underwent apoptosis at an IC50 of approximately 10 to 30 nM and displayed a phenotype of Aurora-A inhibition, whereas cells resistant to Aurora kinase inhibitors (with an IC50 more than 10 mM) accumulated polyploidy, which may have resulted from Aurora-B inhibition. Drug susceptibility was not correlated with the expression level or activation status of Aurora kinases. RS4;11 and MV4;11 cells, which contain the MLL-AF4 gene, were both sensitive to Aurora-A inhibitors. CDKN1A might govern the drug responsiveness of ALL cell lines in a TP53-independent manner. Primary ALL cells with MLL-AF4 and CDKN1A expression were sensitive to AKIs. Conclusions: Our study suggests that Aurora-A kinase inhibitors may have clinical utility in MLL-AF4-positive ALL. CDKN1A can be used as a biomarker to determine drug responsiveness in MLL-AF4-positive ALL. 44 POSTER (Board P038) Metformin and its impact on gastric cancer patients survival after gastrectomy C.K. Lee1 , M.K. Jung1 , I.K. Jung2 , S.J. Heo1 , J.Y. An3 , H.I. Kim3 , J.H. Chung3 , W.J. Hyung3 , S.H. Noh3 , H.S. Kim1 , S.Y. Rha1 , H.C. Chung1 . 1 Yonsei Cancer Center, Division of Medical Oncology, Seoul, Korea; 2 Yonsei University College of Medicine, Department of Biostatistics, Seoul, Korea; 3 Yonsei Cancer Center, Department of Surgery, Seoul, Korea Background: One of the most widely used oral anti-diabetes agent, metformin has recently received attention as anti-cancer treatment with accumulating evidences. But study showing survival benefit of metformin for gastric cancer patients has never been reported. Patients and Methods: We conducted a retrospective study of gastric cancer patients who have undergone gastrectomy as curative aim from May 2003 to December 2010 at a single institution (Yonsei Cancer Center, Severance Hospital, Korea) based on electronic medical records. Survival analysis was done using Kaplan–Meier plot and Cox proportional hazards regression model. Results: Among 1974 gastric cancer patients who underwent gastrectomy as curative aim, 326 patients were diagnosed as diabetes and 132 patients were treated with metformin. During a median follow up duration of 5.5 years (IQR, 3.8−7.4 years), 381 (19.3%) patients died, and 302 (15.3%) patients died as a result of gastric cancer. Gastric cancer patients diagnosed with diabetes who were treated with metformin showed significant survival benefit compared to diabetic gastric cancer patients who were not treated with metformin (Overall survival: P = 0.021, HR = 0.584, 95% CI = 0.369–0.926; cancer-specific survival: P = 0.038, HR = 0.57, 95% CI = 0.334–0.975). Non-diabetes group (n = 1648) showed significant better recur-free survival, overall survival and cancer-specific survival compared to diabetes group (n = 326), and metformin usage prolonged diabetes patients’ survival comparable to that of non-diabetes patients. In multivariable analysis using Cox proportional hazard model with timedependent covariate, each cumulative 6 months of metformin use was significantly associated to decreased risk of recur, cancer-specific mortality and all-cause mortality (HR = 0.847, 95% CI = 0.769–0.934; HR = 0.850, 95% CI = 0.770–0.939; HR = 0.861, 95% CI = 0.794–0.933). Conclusions: Our results show that increased cumulative duration of metformin use decrease recur rate, all-cause and cancer-specific mortality among gastric cancer patients with diabetes who had undergone gastrectomy. Further prospective study of evaluating metformin for adjuvant therapy in gastric cancer will be needed. 45 POSTER (Board P039) Early preclinical study of BO-2094 for treatment of human colon cancer, in combination with 5-fluorouracil T.L. Su1 , T.H. Ou1 , M.H. Wu1 , Y.W. Lin1 , T.C. Lee1 . 1 Institute of Biomedical Sciences, Academia Sinica, Taipei City, Taiwan Among cancers, colon cancer is the second leading cause of cancer deaths in the United States. Every year, about 140,000 Americans are diagnosed with colon. Surgery, radiotherapy, and chemotherapy are the main strategies for treating CRC patients. However, the mortality risk associated with CRC is metastasis leading to the diminishing of systemic treatments. Therefore, there is an urgent need of finding a better agent to treat CRC. Recently, we have designed a series of novel water soluble and chemically stable phenyl N-mustard-benzenealkylamide conjugates. Of these derivatives, we found that compound BO-2094 exhibits a broad spectrum of antitumor activity against a panel of human leukemia and solid tumor cell lines in vitro and potent therapeutic efficacy in various tumor xenograft models. We found that BO-2094 exhibited potent antitumor

Poster Session – Cytotoxics activity in xenograft models. It also demonstrated that that the combination of BO-2094 and 5-FU at ratios of 1:3 persuaded synergistic cytotoxicity to HCT-116 cells in vitro. Notable, more than 98% of tumor suppression was achieved in HCT-116-bearing nude mice when co-treated compound BO-2094 (30 mg/kg, QD×6, iv. inj.) with 5-FU [75 mg/kg, every 7 days for 2 doses (Q7D×2), intraperitoneal injection (ip. inj.)]; all mice (n = 5). The early preclinical studies showed that BO-2094 has LD50 value of 115.4 mg/kg 14-day acute toxicity of in mice via a single administration by iv injection. This agent is likely to have no cardiac arrhythmic side effect based on hERG assay. BO-2094 showed high degree protein binding in rat plasma, indicating that can be as drug reserve and allows drug to be slowly released. Additionally, BO-2094 has an acceptable PK profile in rats. The present studies indicate that the combination of compound BO-2094 + 5-FU has great potential benefit for the treatment of advanced colon cancer. 46 POSTER (Board P040) Broad-spectrum preclinical combination activity of eribulin combined with various anticancer agents in human breast cancer, lung cancer, ovarian cancer, and melanoma xenograft models M. Asano1 , J. Matsui1 , M.J. Towle2 , J. Wu2 , S. McGonigle2 , T. Uenaka3 , K. Nomoto2 , B.A. Littlefield2 . 1 Eisai Co. Ltd., Oncology PCU, Tsukuba, Japan; 2 Eisai Inc., Oncology PCU, Andover MA, USA; 3 Morphotek Inc., Oncology, Exton PA, USA Eribulin is a pharmaceutically optimized, fully synthetic analog of the marine sponge natural product halichondrin B. As its mesilate salt (E7389, Halaven® ), eribulin is approved in 54 countries worldwide for treatment of certain patients with advanced breast cancer. Eribulin is a microtubule dynamics inhibitor that binds to high affinity sites on microtubule plus ends, a distinct binding profile compared to most other tubulin-targeted agents. Previous preclinical studies have shown that under monotherapy conditions, eribulin has broad spectrum anticancer activity against a wide variety of human cancer cell types in vitro and human tumor xenograft types in vivo. The present preclinical studies were undertaken to evaluate eribulin’s in vivo antitumor activity when combined with a variety of cancer drugs in various human tumor xenograft types. In xenograft models of triple negative (MDA-MB-231, MDA-MB-436, MX-1) and HER2+ (UISO-BCA-1) breast cancer, eribulin variously showed combination activity (defined as greater activity than either agent alone with acceptable tolerability) with bevacizumab, capecitabine, everolimus, and the experimental PARP inhibitor E7449, although it failed to show combination activity with doxorubicin in the triple negative MDA-MB-435 model. In xenograft models of non-small cell lung cancer (NCI-H1993, NCI-H322M, NCI-H522, PC-9), eribulin variously showed combination activity with erlotinib, gemcitabine, and two experimental drugs, the PI3K inhibitor BKM-120 and the multi-targeted tyrosine kinase inhibitor lenvatinib. Eribulin also showed combination activity with bevacizumab in the SKOV-3 ovarian cancer model, and with lenvatinib in the A375 melanoma model. Taken together, these preclinical results suggest that eribulin can exert combination activity in a variety of tumor types with acceptable safety profiles when administered together with a wide range of combination agents having different mechanisms of action. 47 POSTER (Board P041) Lurbinectedin (PM01183) specifically targets RNA Pol II for degradation via the proteasome pathway in a transcription and TC-NER dependent fashion G. Santamaria1 , J.F. Mart´ınez-Leal1 , C. Cuevas1 , L.F. Garcia-Fernandez1 , C.M. Galmarini1 . 1 PharmaMar S.A.U., Cell Biology Department, Colmenar Viejo (Madrid), Spain Lurbinectedin (PM01183) is a synthetic tetrahydroisoquinoline alkaloid currently evaluated as single agent and in combination in phase I and II clinical trials for solid tumors and hematological malignancies. In living cells, PM01183-DNA adducts stall replication and transcription giving rise to double strand breaks, inducing accumulation of cells in the S-phase of the cell cycle and triggering apoptosis. In this work, we examined the effects of PM01183 on the activity and stability of the RNA Pol II as well as on other factors of the transcriptional machinery, including TBP (TFIID), p62 (TFIIH) and XPD. Our results showed that PM01183 induced a rapid, time- and concentration-dependent degradation of RNA Pol II in a panel of different human tumor cell lines, including HCT-116 (colon), A549 (NSCLC), HeLa (cervix) and A763 (sarcoma). This degradative process was efficiently abrogated in the presence of transcriptional (DRB), ubiquitination (PYR41), and proteasome inhibitors (MG132), demonstrating that PM01183 specifically targets the transcriptionally active RNA Pol II for degradation via the proteasome pathway. In addition, it was also shown that the effect of PM01183 on the RNA Pol II was dependent on the presence of a functional TC NER repair machinery. PM01183 induced degradation of

Poster Session – Cytotoxics RNA Pol II in global NER (XPC) deficient cells, but failed to do it in TC NER (CSB, XPD and XPG) deficient cells. Importantly, these effects were confirmed to be specific for the Rpb1 subunit of RNA Pol II, since other subunits were not affected (Rpb2 and Rpb4) as well as other factors of the transcriptional machinery, such as TBP (TFIID), p62 (TFIIH), XPD or the RPA194 subunit of the RNA Pol I. Finally, it was also demonstrated that, contrary to what occurs after DNA damage with UV light, the transcription of p53 target genes important for DNA repair, including p21 or mdm2, was irreversibly inhibited after PM01183 treatment. Together, these results show the mechanism by which PM01183 inhibits trans-activated transcription process on tumor cells. 48 POSTER (Board P042) Trabectedin and lurbinectedin are effective against leukemic cells derived from patients affected by chronic and juvenile myelomonocytic leukemia M. Romano1 , A. Gall`ı2 , N. Panini1 , L. Paracchini1 , L. Beltrame1 , E. Bello1 , S.A. Licandro1 , C. Cattrini3 , R. Tancredi3 , S. Marchini1 , V. Rosti4 , M. Zecca5 , M. Della Porta2 , A. Zambelli3 , C.M. Galmarini6 , E. Erba1 , M. D’Incalci1 . 1 IRCCS − Istituto di Ricerche Farmacologiche Mario Negri, Oncology, Milano, Italy; 2 Fondazione IRCCS Policlinico San Matteo, Onco-Hematology, Pavia, Italy; 3 IRCCS Fondazione Salvatore Maugeri, Oncology, Pavia, Italy; 4 Fondazione IRCCS Policlinico San Matteo, Center for the Study of Myelofibrosis Research Laboratory of Biotechnology, Pavia, Italy; 5 Fondazione IRCCS Policlinico San Matteo, Pediatric Onco-Hematology, Pavia, Italy; 6 PharmaMar S.A., Research and Development, Colmenar Viejo, Italy Background: Chronic (CMML) and juvenile myelomonocytic leukemia (JMML) are mixed myelodysplastic/myeloproliferative (MDS/MPN) haematological diseases of elderly and childhood patients, respectively. Any kind of chemotherapy is unable to improve the outcome of these patients, so allogeneic stem cell transplantation remains the only curative option for MDS/MPN neoplasms. Trabectedin and its analogue lurbinectedin are DNA minor groove binders acting as modulators of transcription and interfering with DNA repair mechanisms. They cause selective depletion of tumor-associated macrophages and circulating monocytes, activating caspase-8-dependent apoptosis and TRAIL receptors (TRAIL-Rs) expression. These findings offer strong proof-of-concept evidence for monocyte targeting in humans and provide the rational for testing these two drugs in myelomonocytic leukemias. Material and Methods: Patient samples (20 CMML; 12 JMML) were collected from peripheral blood. The drugs cytotoxicity was evaluated by growth inhibition; apoptosis (Annexin-V/P.I), TRAIL-Rs and cleaved caspase-8 expression were investigated by flow cytometric assay. In vivo experiments were performed using athymic nude mice transplanted with MV-4−11 cells. Results: Both trabectedin and lurbinectedin showed a strong cytotoxic effect on malignant CD14+ monocytes in vitro and were able to induce marked apoptosis, at nanomolar concentrations, much more effective than azacitidine (5-AZA), used at micromolar concentrations as positive control. In addition, trabectedin and lurbinectedin were found to efficiently inhibit the in vitro growth of bone marrow hematopoietic precursors (CFU-GM) of CMML and JMML patients. In a CMML cellular model, the MV-4−11 cell line, we found an increased TRAIL-Rs and cleaved caspase-8 expression after trabectedin or lurbinectedin exposure suggesting that apoptosis induced by these drugs followed the extrinsic pathway. Preliminary in vivo experiments, in MV4−11 model, showed a marked antitumor activity of both trabectedin and lurbinectedin that was much higher than that of 5-AZA. Conclusions: Our data suggest that trabectedin and lurbinectedin exert a more specific cytotoxic effect on malignant CMML and JMML monocytes than other drugs commonly used in MDS/MPN diseases and this makes these two drugs good candidates for clinical studies in CMML and JMML. Studies are in progress to elucidate the molecular mechanisms behind these therapeutically antitumor effects of these drugs. 49 POSTER (Board P043) siRNA targeting of mitochondrial thymidine kinase 2 (TK2) sensitizes cancer cells to gemcitabine and increases mitochondrial toxicity C. Di Cresce1 , P. Ferguson2 , R. Figueredo2 , M. Rytelewski1 , S. Maleki Vareki1 , M.D. Vincent3 , J. Koropatnick1 . 1 Western University, Microbiology and Immunology, London, Canada; 2 London Regional Cancer Centre, Cancer Research Laboratories, London, Canada; 3 Western University, Oncology, London, Canada Background: Mitochondrial thymidine kinase 2 (TK2) preferentially phosphorylates thymidine to generate thymidine monophosphate (dTMP).

Wednesday 19 November 2014

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TK2 also phosphorylates deoxycytidine to generate dCMP, a precursor for dCTP. dCTP negatively regulates deoxycytidine kinase (dCK), which primarily phosphorylates deoxycytidine as well as the anticancer drug gemcitabine. Because phosphorylation by dCK is required to activate the drug, there is a therapeutic advantage to high dCK in tumour cells treated with gemcitabine. Antisense knockdown of TK2 could reduce TK2-produced dCMP, thus decreasing dCTP levels and its inhibition of dCK, and lead to increased dCK activity, gemcitabine activation, and anticancer effectiveness. Importantly, gemcitabine is a poor substrate for phosphorylation by TK2. Given the ability of TK2 to phosphorylate deoxycytidine and thus down-regulate dCK activity, we hypothesized that: (1) TK2 can mediate human tumour cell resistance to gemcitabine, and (2) antisense (siRNA) downregulation of TK2 can inhibit this mechanism of resistance. We also hypothesized that anti-TK2 siRNA-induced drug sensitization is mediated by mitochondrial damage. Materials and Methods: siRNA downregulation of TK2 followed by gemcitabine treatment of MCF7, HeLa and A549 human tumour cell lines (which express high, medium, and low levels of TK2, respectively). Cell lines were analyzed for proliferation (alamarBlue viability staining), levels of various mRNAs (qPCR) and proteins (immunoblotting), mitochondrial to nuclear DNA ratio (mtDNA:nDNA) (qPCR), and mitochondrial membrane potential (MitoTracker CMXROS and flow cytometry). Results: Downregulation of TK2 using siRNA sensitized MCF7 and HeLa cells (high and medium TK2 expressers, respectively) to gemcitabine, but did not sensitize A549 cells (low TK2 expresser). siRNA knockdown of TK1 and/or thymidylate synthase (TS) in combination with TK2-siRNA and gemcitabine did not cause further sensitization; this phenomenon is specific to targeting of TK2. The combined treatment with TK2 siRNA and gemcitabine increased dCK enzyme levels in TK2 high- and medium-expressing cell lines. The addition of TK2 siRNA treatment to gemcitabine treatment of TK2-expressing tumour cells specifically decreased mitochondrial DNA (mtDNA:nDNA) and mitochondrial activity. Conclusions: This is the first demonstration of a direct role for TK2 in resistance to gemcitabine or any other anticancer drug, and distinguishes TK2 from other dTMP-producing enzymes (cytosolic TK1 and TS). Decreased mitochondrial function is related to TK2 siRNA-induced sensitization to gemcitabine. 50 POSTER (Board P044) The effect of esomeprazole, a proton pump inhibitor, on the pharmacokinetics of the investigational Aurora A kinase inhibitor alisertib (MLN8237) in patients with advanced solid tumors or lymphomas X. Zhou1 , J. Nemunaitis2 , S. Pant3 , T. Bauer4 , A. Lockhart5 , M. Patel6 , B. Zhang7 , V. Kelly8 , C.D. Ullmann8 , M. Bargfrede9 , K. Venkatakrishnan9 . 1 Millennium: The Takeda Oncology Company, Clinical Pharmacology, Cambridge, USA; 2 Mary Crowley Medical Research Center, Oncology, Dallas, USA; 3 Oklahoma University Medical Center, Hematology/Oncology, Oklahoma City, USA; 4 Sarah Cannon Research Insitute, Tennessee Oncology, Nashville, USA; 5 Washington University, Dept of Med. Oncology Div. Med Onc Section, St. Louis, USA; 6 Florida Cancer Specialists, Sarasota Cattlemen, Sarasota, USA; 7 Takeda Pharmaceuticals International Co, Statistics, Cambridge, USA; 8 Takeda Pharmaceuticals International Co, Oncology Clinical Research, Cambridge, USA; 9 Takeda Pharmaceuticals International Co, Clinical Pharmacology, Cambridge, USA Background: Alisertib is an investigational, orally available, selective Aurora A kinase inhibitor currently in clinical development for multiple oncology indications. Alisertib enteric-coated tablets are designed to bypass the acidic gastric pH and delay dissolution until entry into the upper small intestine. Alisertib exhibits pH-dependent solubility. This study characterized the effects of esomeprazole, a proton pump inhibitor (PPI) on the pharmacokinetics of single-dose alisertib in advanced cancer patients (pts). Materials and Methods: Eligible pts were 18 years of age with ECOG PS 0−1. In this fixed-sequence, 2-cycle, open-label study, a single oral dose of alisertib 50 mg was administered on cycle 1, day 1 (C1D1) and cycle 2, day 8 (C2D8). Cycle 1 was 24 days, and cycle 2 was 31 days. Esomeprazole 40 mg (delayed release) was given once daily in cycle 2 from days 1−10. Plasma pharmacokinetic samples were collected predose and up to 72 hours post alisertib on C1D1 and C2D8. Ratios of geometric mean Cmax , AUC0−last , and AUC0−inf in the presence of esomeprazole (C2D8) versus in its absence (C1D1) were calculated and 90% confidence intervals (CI) were estimated. Secondary endpoints were safety and tolerability. Results: Of 25 pts enrolled (male, 44%; white, 88%; median age, 61 years; and mean weight, 82 kg), 18 pts were evaluable. Following a single, oral dose of alisertib, median Tmax was 3 and 4 hours in the presence and absence of esomeprazole, respectively. The geometric mean AUC0−inf of alisertib in the presence of esomeprazole was 129% of that in its absence (90% CI, 107–156; N = 17). The geometric mean Cmax in the presence of

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esomeprazole was 114% of that in its absence (90% CI, 96–136; N = 18). At data cutoff, treatment-related adverse events (AE) were reported in 24 pts (96%); the most common AEs were diarrhea (52%), alopecia (52%), neutropenia (36%), and fatigue (36%). Treatment-related grade 3/4 AEs were reported in 15 pts (60%); the most common (>10%) were neutropenia (36%) and leukopenia (12%). The observed AEs and their incidence were generally consistent with the alisertib safety profile observed to date. Conclusions: Esomeprazole produced an approximately 30% increase in alisertib systemic exposure. These results support the recommendation that gastric acid-reducing agents (e.g., PPIs, H2 receptor antagonists) be avoided in pts receiving alisertib. 51 POSTER (Board P045) Development of rational combination therapy strategies to optimize treatment for GPNMB expressing Her2+ and triple negative breast cancer A. Rose1 , G. Maric2 , M.G. Annis2 , H. Smith2 , W.J. Muller2 , P.M. Siegel2 . 1 McGill University Department of Medicine, Goodman Cancer Research ´ Centre, Montreal, Canada; 2 McGill University, Goodman Cancer ´ Canada Research Centre, Montreal, Background: Glycoprotein NMB (GPNMB) is a cell surface protein that promotes tumor growth and metastasis in murine breast cancer (BC) models. CDX011 is a GPNMB-targeted antibody drug conjugate that has shown robust clinical activity and is being investigated in late stage clinical trials for BC. Data from early clinical trials with CDX011 reveals that response rate is directly linked to tumoral GPNMB expression levels. The goal of this project is to define the prognostic and functional relevance of GPNMB in basal/triple-negative (TN)/Her2-positive BC, and to devise combination therapy strategies, which could improve CDX011 efficacy for the treatment of BC. Methods: GPNMB was ectopically expressed in breast cancer cells. Cell invasion was assessed by boyden chamber assays and tumor growth and spontaneous metastasis were assessed with in vivo mouse models. The Cancer Genome Atlas (TCGA) human breast cancer gene expression data (n = 1160) was interrogated for associations between GPNMB levels and intrinsic subtype and overall survival. Lapatinib-resistant cells were generated from several Her2+ cell lines. GPNMB mRNA levels were determined by qPCR and protein by immunoblot and FACS. Results: Ectopic GPNMB enhanced invasion by 2−4 fold, and in vivo tumor growth was enhanced ~2-fold in both Hs578T and NIC-Her2 models. NIC cells expressing GPNMB displayed a 4-fold increase in spontaneous lung metastases. In the TCGA data set GPNMB expression was significantly higher in basal and Her2 than it was in the luminal A and B subtypes. Tumors with high GPNMB expression were associated with significantly shorter overall survival (HR = 2.0295, 95% CI = 1.3476 to 3.0567. Treatment with kinase inhibitors (KI) EGFR/Her2-family inhibitors (gefitinib, lapatinib) led to significantly up-regulated GPNMB in basal-like BC cells: MDA-468, MDA-436, MDA-157, and Her2+ cells: SkBR3, HCC1954, BT474, MDA361. Moreover, Mek inhibition with trametinib led to increased GPNMB expression in both basal and Her2+ BC. This induction was transcriptionally mediated, and led to increased GPNMB protein at the cell surface. Moreover, 34 Lapatinib-resistant cells expressed higher levels of GPNMB than corresponding parental cells. Finally, CDX011 as a single agent caused a significant reduction in MDA-468 tumor growth. Conclusions: GPNMB is highly expressed in Her2+ and Basal breast cancer − it promotes tumor growth, invasion and metastasis and correlates with poor survival. As such it represents a promising therapeutic target. GPNMB is up-regulated by many KI that are currently used to treat BC. Our data suggest that combination of KI with CDX011 will lead to improved tumor regression; our efforts to test these combinations in vivo will be discussed. 52 POSTER (Board P046) Platinum(IV) derivatives of oxaliplatin: Cellular effects and in vivo potency 1 S. Goschl ¨ , V. Pichler1 , E. Brynzak1 , P. Heffeter2 , U. Jungwirth2 , M.A. Jakupec1 , W. Berger2 , M. Galanski1 , B.K. Keppler1 . 1 University of Vienna, Institute of Inorganic Chemistry, Vienna, Austria; 2 Medical University of Vienna, Institute of Cancer Research, Vienna, Austria

Platinum(IV) complexes are an attractive way to overcome some disadvantages of platinum(II)-based anticancer drugs such as severe side effects or resistances, because they are kinetically more inert to ligand substitution and their higher lipophilicity may improve the cellular uptake. Four platinum(IV) complexes derived from oxaliplatin reported herein have shown activity in the colorimetric MTT assay in vitro in HCT15, HCT116 and HCT116oxR cells (all colon carcinoma). The extent of induced apoptosis (AnnexinV-FITC/PI staining) and the influence of the complexes on the

Poster Session – Cytotoxics cell cycle were measured by flow cytometry. Two of the complexes (1 and 2) were tested in vivo in murine L1210 leukemia, and 2 was tested in a colon tumor (CT26) mouse model. The platinum levels in the organs were determined by ICP-MS measurements. These platinum(IV) compounds, mostly yielding IC50 values in the micromolar range, are up to 25 times more active in HCT116 cells than in the oxaliplatin resistant subline, whereas oxaliplatin is 66 times less potent in HCT116oxR cells. After 24 h incubation with 2 a strong increase of the G2 /M phase fraction and a decrease of the S phase fraction was found in HCT15 cells, whereas the effects were somewhat less pronounced in HCT116 cells. After 24 h incubation with 1 an up to 5-fold increase of apoptosis was found in HCT116 but not in HCT15 and HCT116oxR cells. 48 h incubation of 1 or 2 resulted in a total increase of up to 50% and 15% apoptosis in HCT15 and 50% and 25% in HCT116 cells, respectively. In L1210 murine leukemia an increase in life span up to 60% was observed. 2 was tested in immuno-competent and immunodeficient mice to determine whether it is active in a solid tumor model and whether the immune system has an influence on the efficiency as found for oxaliplatin. The immune system seems to be relevant for activity because 2 was found to be active in Balb/c but not in SCID mice, although the tumor platinum levels in both mouse models are the same. Overall, these results demonstrate the high potency of modified platinum(IV) compounds. For a deeper insight into their suitability as prodrugs activated by reduction, their reduction properties and their activity under hypoxic conditions in 2D and 3D cell culture will be studied.

53 POSTER (Board P047) Adeno-associated virus (AAV) carrying diphtheria toxin a gene for pancreatic cancer therapy M. Chen1 , C.Y. Ho1 , M. Teng1 , H. Chen1 . 1 Virovek Incorporation, R&D, Hayward, USA Pancreatic cancer is an aggressive malignancy with morbidity rates almost equal to mortality rates because of the current lack of effective treatment options. Here, we describe a DNA-based therapy for pancreatic cancer using the AAV vector that expresses the diphtheria toxin A chain (DT-A) under the control of the survivin gene regulatory sequences. We produced the AAV vectors carrying DT-A coding sequence that was disrupted by the human growth hormone intron so that its expression was abolished during AAV vector production in insect cells and used these vectors for the study. In vitro experiments showed that the AAV vector was effective in inhibiting protein synthesis in pancreatic carcinoma cell lines but not in normal cell line. In vivo experiment results demonstrated tumor growth arrest in a xenograft mouse model for pancreatic cancer in a dose-dependent manner. Differences in tumor size between the control and high dose group were significant (P = 0.015). These AAV vectors provide a new option for cancer gene therapy. The protocol and any amendment(s) or procedures involving the care and use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of CrownBio prior to conduct. During the study, the care and use of animals were conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). Table 1. Antitumor activity of test article AAV6-DTA in the treatment of subcutaneous PANC-1 human pancreatic cancer xenograft model T/C (%)

T-C (days) at 786 mm3

P valueb

Group 1. AAV6-SURV-GFP 1.5×1012 vg/mouse 1720±258 Group 2. AAV6-SURV-DTA 1.5×1012 vg/mouse 786±234 Group 3. AAV6-SURV-DTA 1.5×1011 vg/mouse 808±218

−

−

−

45.72

12.5

0.015

46.98

10.5

0.017

Group 4. AAV6-SURV-DTA 1.5×1010 vg/mouse

60.84

6

0.072

Treatment

a Mean±SEM; b vs. vehicle control.

Tumor size (mm3 )a on day 48

1046±301

Poster Session – Cytotoxics 54 POSTER (Board P048) MGMT methylation assessed by methyl-BEAMing technique is a prognostic and predictive biomarker in glioblastoma and metastatic colorectal cancer patients L. Barault1 , A. Amatu2 , F.E. Bleeker3 , C. Moutinho4 , A. Cassingena2 , F. Tosi2 , T. Venesio5 , M. Esteller4 , A. Bardelli6 , S. Siena2 , A. Sartore-Bianchi2 , F. Di Nicolantonio6 . 1 Candiolo Cancer Institute − FPO IRCCS, Experimental Clinical Molecular Oncology, Candiolo, Italy; 2 Ospedale Niguarda Ca’ Granda, Department of Hematology and Oncology, Milan, Italy; 3 Academic Medical Center University of Amsterdam, Department of Clinical Genetics, Amsterdam, Netherlands; 4 Bellvitge Biomedical Research Institute (IDIBELL), Cancer Epigenetics and Biology Program (PEBC), Barcelona, Spain; 5 Candiolo Cancer Institute − FPO IRCCS, Investigational Clinical Oncology, Candiolo, Italy; 6 University of Torino, Department of Oncology, Candiolo, Italy Background: O6-methylguanine DNA methyltransferase (MGMT ) silencing by promoter methylation is a common alteration found in different cancer types. This has been shown to be both a prognostic and a predictive marker of sensitivity to alkylating agent-based therapy like dacarbazine and temozolomide in glioblastoma. However in other malignancies its value remains controversial. This might be due to sampling issues, tumor heterogeneity or to the use of inadequate detection methods. In this study, we present a new assay to reliably measure MGMT methylation both in tumor and plasma samples. Material and Methods: Methylation of MGMT has been assessed by an ultra-sensitive digital PCR technique, in which a two-step PCR is followed by detection via fluorocytometer (Methyl-BEAMing). Results were compared to two other commonly used techniques (Methylation Specific PCR, MSP and pyrosequencing). Two samples datasets have been evaluated: tumors from a cohort of 98 newly diagnosed glioblastoma patients from the pre-temozolomide era, and specimens from a cohort of 68 metastatic colorectal cancer patients treated with dacarbazine in a phase II clinical trial (DETECT-01 trial, EUDRACT number 2011–002080−21). The prognostic and/or predictive value of MGMT methylation has also been evaluated. As a proof of concept, the three methods were assessed in a subset of colorectal cancer patients’ plasma derived DNA to evaluate their performance as a liquid biopsy test. Results: Methyl-BEAMing showed high reproducibility across independent experiments, as well as high sensitivity (up to 0.09% methylation detected) and specificity. In the glioblastoma cohort, Methyl-BEAMing methylated status (>50%) was associated with a decreased hazard ratio for death (HR = 0.35; p < 0.0001) compared to MSP (HR = 0.54; p = 0.006) or pyrosequencing (HR = 0.61; p = 0.059). In mCRC, tissue where tumor heterogeneity is possibly higher, both Methyl-Beaming and pyrosequecing assays provided better prediction of objective response to dacarbazine than MSP. Progression free survival was also improved in metastatic colorectal cancer with methylated status when samples were assessed with Methyl-BEAMing (p = 0.0012) or pyrosequencing (p = 0.0005). Quantitative evaluation of MGMT methylation in circulating tumor DNA was effective with Methyl-BEAMing. Conclusions: MGMT methylation testing based on BEAMing technology outperforms commonly used methods and might allow the non-invasive follow-up of patients, upon alkylating agent treatments using blood circulating DNA. 55 POSTER (Board P049) Low, frequent doses of PM060184 induce remarkable in vivo antitumor activity P. Aviles1 , M.J. Guillen1 , P.P. Lopez-Casas2 , F. Sarno3 , O. Cataluna ˜ 4, P. Nunez ˜ 4 , C. Cuevas4 , M. Hidalgo3 . 1 PharmaMar S.A., Preclinical, Colmenar Viejo (Madrid), Spain; 2 Centro Nacional de Investigaciones ´ Oncologicas and Hospital de Madrid, Colmenar Viejo (Madrid), Spain; 3 ´ Centro Nacional de Investigaciones Oncologicas and Hospital de Madrid, Madrid, Spain; 4 Pharmamar S.A., Precl´ınica, Colmenar Viejo (Madrid), Spain Background: PM060184 is a synthetic marine-derived compound originally isolated from the marine sponge Lithoplocamia lithistoides. PM060184 induces disorganization and disruption of the microtubule network as well as aberrant mitotic spindle multipolarization and chromosome missegregation. These effects give rise to prometaphase arrest and formation of multinucleated cells. Then, cells enter to caspase-driven apoptosis or are arrested in a pseudo-senescent state. PM060184 is currently under evaluation in Phase I clinical studies in patients with advanced cancer diseases. The objective of the present work was to explore the in vivo anticancer efficacy of PM060184 administered at low daily doses (0.5 to 2 mg/kg).

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Material and Methods: Athymic female nu/nu mice were subcutaneously implanted with different tumors: MDA-MB-231 (breast), H460 (NSCLC) and several pancreas patient-derived (AVATAR) tumors, namely JH010, JH-015, Panc-291, and Panc-039. Tumor (ca. 300 mm3 ) bearing animals (N = 6−10/group) were randomly allocated to receive PM060184 or placebo. Treatments (0.5 to 2 mg/kg, iv) were administered daily for 20 consecutive days. Antitumor effect was calculated using DT/DC (%), defined as a percentage of the change in tumor size for treated (T) and placebo (C) groups during the placebo-treated survival time (D). Complete tumor regression (CR) was defined when tumor volume <63 mm3 for 2 or more consecutive measurements. Results: The treatment with PM060184 produced lowest DT/DC values as summarized in the table. Tumor

Breast

MDA-MB-231

NSCLC

H460

Pancreas

JH-024 JH-010 JH-015 Panc-291 Panc-039

Daily dose (mg/kg)

Minimal DT/DC (%)

On Day

2.0 1.0 0.5 2.0 1.0 0.5 2.0 2.0 2.0 2.0 2.0

7.3 14.1 24.0 5.5 7.8 17.7 19.7 14.8 38.9 12.0 6.1

21 21 21 11 11 11 21 21 28 24 28

Conclusion: The treatment with PM060184 at low, frequently given doses demonstrated significant in vivo antitumor activity in breast, NSCLC and pancreas xenografted tumors. 56 POSTER (Board P050) Radiosensitizing effect of sodium metaarsenite in a metastatic brain tumor model W.Y. Kang1 , Y.M. Park2 , S.J. Kim2 . 1 Institute for refractory cancer research Samsung medical center, Samsung Biomedical Research Institute, Seoul, South Korea; 2 Komipharm International Inc. Co., Pharmaceutical Division, Seoul, South Korea Background: Brain metastases are found in about 10% of lung cancer patients at the time of diagnosis, and about 40% of all lung cancer patients develop brain metastases during their disease progression. The chemotherapy is limited because of little or no effectiveness due to the blood–brain barrier. The radiation therapy is the most frequently used, and sensitizing agents, which synergize with radiation, can improve the efficacy of the therapy. Material and Methods: Sodium metaarsenite (KML001® ) is an orally bioavailable arsenic compound that has entered phase I/II clinical trials in solid tumors and hematopoietic malignancies. In this study, we elucidated the radio-sensitizing effect of sodium metaarsenite (KML001® ) in an animal model of metastasis of lung cancer to the brain. Results: The clonogenic assay showed that treatment with sodium metaarsenite (KML001® ) inhibited clone formation in radio-sensitive (H23) and radio-resistant lung cancer cells (A549 and PC14PE6) in a concentration-dependent manner. The combined irradiation and sodium metaarsenite (KML001® ) treatment significantly reduced colony formation in H23 (p < 0.01), A549 (p < 0.05) and PC14PE6 lung cancer cells (p < 0.05), compared with the radiation alone group. In the metastatic brain cancer model with H23 cells, sodium metaarsenite (KML001® ) treatment (5 mg/kg/day) and radiation therapy (5 Gy) showed 54.5% and 67.6% reduction in tumor volume, respectively, compared with control group (p < 0.001 vs. control). The combined irradiation and sodium metaarsenite (KML001® ) treatment induced 88.1% decrease in tumor volume (p < 0.001 vs. control). In the metastatic brain cancer model with PC14PE6 cells, the single irradiation (15 Gy) and the combined irradiation (15 Gy) and sodium metaarsenite (KML001® ) treatment (7 mg/kg/day) significantly increased median survival day of the mice to 22 and 26 days, respectively, compared to control group (median survival day = 19) (p < 0.001). The combination improved survival significantly with regard to the radiation only group (p < 0.001). Conclusions: This study demonstrated that sodium metaarsenite (KML001® ) may have potential as an alternative therapeutic agent,

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especially in combination with radiation therapy in lung cancer patients with brain metastases and provide a rationale for testing the combination of sodium metaarsenite (KML001® ) and radiotherapy in the clinic.

59 POSTER (Board P053) Discovery of novel inhibitor of FOXO nuclear–cytoplasmic shuttling from natural products of marine origin

57 POSTER (Board P051) Differential antitumor activity of trabectedin, lurbinectedin, Zalypsis and PM00128 against a panel of human cells deficient in transcription and NER factors

F.J. Castillo Correa1 , N. De Pedro1 , L. Rodriguez Quesada1 , D. Oves Costales1 , J.R. Tormo1 , J. Martin1 , F. Reyes1 , O. Genilloud1 , F. Vicente1 , W. Link2 , B. Cautain1 . 1 Fundacion Medina, Screening and Target Validation, Granada, Spain; 2 Universidad do Algarave, Ciencias Biomedicas e Medicina, Faro, Portugal

V. Moneo1 , S. Avila1 , P. Mart´ınez1 , B. de Castro1 , S. Cascajares1 , C. Cuevas1 , L.F. Garcia-Fernandez1 , C.M. Galmarini1 . 1 PharmaMar S.A.U., Cell Biology Department, Colmenar Viejo (Madrid), Spain Trabectedin, lurbinectedin, Zalypsis and PM00128 are synthetic marinederived anticancer drugs that covalently bind to the minor groove of DNA through via a common pentacyclic skeleton. The compounds differ in an additional chemical moiety attached to the pentacyclic skeleton that protrudes out from the double helix. Trabectedin, lurbinectedin and Zalypsis present a tetrahydroisoquinole, a tetrahydro b-carboline and a trifluorocinnamic group, respectively, while PM00128 lacks such additional moiety. To gain insight into how the presence of those different chemical groups contribute to the selectivity of the four compounds, we tested their cytotoxicity profiles against a panel of 43 fibroblast cell lines derived from patients with genetic diseases caused by mutations in transcription/NER factors, including CSA, CSB, XPC, XPA, XPE, XPB, XPD, XPG. In general, trabectedin and lurbinectedin presented a similar, although not identical, response pattern against the cell panel. However, these two compounds have a different behavior when compared to Zalypsis and PM00128. Four out of seven fibroblast cells lines carrying truncations in the XPG endonuclease gene were more resistant to trabectedin than normal fibroblasts, with RI values ranging from 3.4 to 6.7. Also interesting, a cell line (GM15754) carrying a missense mutation (R112H) in the XPD helicase gene, showed a significant resistance to both trabectedin and lurbinectedin, showing a RI of 41.0. Thus, the proper functionality of XPG and XPD factors in transcription and/or DNA repair processes may be important for the antiproliferative action of these compounds Remarkably, the sensitivity profiles obtained with standard DNA binders, such as cisplatin and mitomycin C, were completely divergent to those of trabectedin and lurbinectedin. On the other hand, no significant differences in the sensitivity of these cell lines were observed against Zalypsis and PM00128, suggesting that the repair of the DNA damage generated by these compounds is not exclusively dependent on the NER pathway. In summary, the differences in the chemical moieties attached to the common DNA binding scaffold in these compounds, seemed to correlate with the differential biological activity found against different tumoral cells. 58 POSTER (Board P052) Pipecolidepsin A, Stellatolide A and Irvalec: New cyclodepsipeptides of marine origin with antitumor activity J.M. Molina-Guijarro1 , V. Moneo1 , J.F. Martinez-Leal1 , C. Cuevas1 , L.F. Garcia-Fernandez1 , C.M. Galmarini1 . 1 PharmaMar S.A.U., Cell Biology Department, Colmenar Viejo (Madrid), Spain Pipecolidepsin A and Stellatolide A, along with Irvalec, are three synthetic marine-derived cyclic depsipeptides that show antitumor activity in vitro against a wide variety of human tumor cell lines. In dose–response curves against a panel of 24 human cancer cell lines, representative of 11 different tissues, the compounds showed GI50 values in the low micromolar range. Using the mechanism of action of Irvalec as a model, the cellular effects of Pipecolidepsin A and Stellatolide A were evaluated. Preliminary studies with these compounds were conducted in A549 (NSCLC) and HCT-116 (colon adenocarcinoma) cell lines as tumor models. The results pointed out that all of them share a common set of cellular effects. Phase contrast and fluorescence microscopy as well as plate fluorimetry analyses, showed that the compounds caused very rapid cell membrane destabilization, causing rapid and dramatic morphological changes, including cell blebbing, severe swelling, and plasma membrane permeabilization (as detected by propidium iodide) that irrevocably lead to cell lysis and necrotic cell death. Interestingly, membrane damage was observed only after reaching a threshold concentration, variable depending on the compound, in the culture medium. Strikingly, all the compounds caused a notable inhibition of cell viability after 30 min exposure, indicating that their cellular effects were produced very rapidly upon treatment. The efficacy of Pipecolidepsin A and Stellatolide A is currently being evaluated in in vivo models. In summary, these results indicate that the compounds Pipecolidepsin A, Stellatolide A as well as Irvalec, exert their potent antitumor activity by inducing rapid and severe membrane damage in tumor cells.

The nucleo-cytoplasmic transport of certain tumor suppressor and oncoproteins is disrupted in cancer cells resulting in their aberrant subcellular localization and their respective inactivation or over-activation. This subcellular localization occurs actively via the nuclear pore complex that spans the nuclear envelope and is mediated by transport receptors. Regulation of the FoxO (forkhead box O) factors is receiving increasing attention as their activation in the nucleus has been linked to cell-cycle arrest and apoptosis, acting as tumor suppressors. Microbial natural metabolites have played a major role as one of the most important sources for the discovery of novel drugs. In this work, we applied a previously established high-throughput and high-content cellular-imaging assay that monitors the nuclear–cytoplasmic translocation of a GFP–FOXO3a fusion protein in U2OS cells to screen a library of extract from marine actinobacteria, one of the most efficient groups of secondary metabolite producers. A total of 3,300 extract, fractions and pure compounds were screened. The nuclear accumulation of fluorescence triggered by the pan-PI3K inhibitor, LY294002 (at 250 mM), was defined as 100% activity and used as a reference to define primary hits in U2foxRELOC assay. A final hit rate of 0.04% (13/3,300) was achieved; defining hits as non-toxic, specific FoxO Relocators. Two news FOXO relocators were isolated as pure compounds. MDN-XX is an alkaloid that has been previously described as a compound with antifungal activity. The other one is a new structure actually under characterization. As a general result, we validate the strategy used to identify activators of FoxO based on the screening of a collection of natural products, such as marine actinomycetes using the U2foxRELOC assay. Furthermore, the discovery of MDN-XX, with an IC50 of 10 nM, proves that marine actinomycetes can be used as an attractive and unexplored source of FoxO activators. 60 POSTER (Board P054) Sodium metaarsenite cytotoxic activity is associated with telomere length and many types of arsenic transporters in non-small cell lung cancer Y.M. Park1 , S.J. Kim1 . 1 Komipharm International Co. Ltd., Pharmaceutical Division, Shiheung, South Korea Background: Sodium metaarsenite (KML001® ) is an orally bioavailable arsenic compound that has entered phase I/II clinical trials in solid tumors and hematopoietic malignancies. The underlying mechanisms of anticancer effects of KML001® have been shown to be telomere poisoning. Because arsenic resistance could limit the efficacy of KML001® in patients with nonsmall cell lung cancers (NSCLC), expression of membrane pore proteins involved in arsenic influx into cells, energy-dependent transporters that detect and eject arsenic from cells, and arsenic-detoxifying machinery proteins need to be evaluated to predict the sensitivity and resistance of human non-small cell lung cancers to KML001® . Material and Methods: In this study, we characterized six NSCLC cells with regards to the cytotoxicity by KML001® and expression of arsenic influx/efflux machinery proteins. We profiled expression of human aquaglyceroporin (AQP) 1−9, glucose transporter(GLUT) 1−4, LRP and MRP1−3 in six NSCLC cell lines. In addition, the telomere restriction fragment (TRF) lengths were measured and compared among six NSCLC cell lines. Finally we examined in vivo antitumor activity of KML001® in two human NSCLC xenografts transplanted with H292 cells with a low IC50 and PC14PE6 cells with a high IC50 for KML001® . Results: MTT assay revealed that H292, H23, and H2009 were sensitive to KML001® (IC50 = 2.2, 2.4, and 9.8, respectively), whereas A549, PC14PE6, and H1792 were resistant to KML001® (IC50 = 29.6, 31, and 35, respectively). Arsenic influx activity for arsenic-sensitive cell lines was higher than that for arsenic-resistant cell lines. However, compared to arsenic-sensitive cell, arsenic-resistant cells showed a rapid reduction in arsenic accumulation over 24 h coupled with a increase in the rate of arsenic efflux. TRF length analysis demonstrated that the resistant A549, H1792, and PC14PE6 cells displayed longer telomeres compared to the sensitive H2009, H292 and H23 cells. H23 and H292 with higher expression levels of AQP9, AQP2, AQP3, GLUT3 and GLUT4 showed significantly enhanced arsenic uptake in compared to A549 and PC14PE6 cell lines with lower expression levels of these membrane pore proteins. Arsenic contents were significantly low in PC14PE6 and A549 cell lines with increased

Poster Session – Cytotoxics MRPs and LRP expression. In H292 and PC14PE6 NSCLC xenograft models, administration of 3.5 and 7 mg/kg KML001® significantly reduced tumor volume and tumor weight without additional toxicity to the mice. The combination with cisplatin and KML001® showed synergic inhibitory effects on the growth of H292 and PC14PE6 xenografts. Conclusions: These results suggest that telomere length, membrane pore proteins and drug transporters responsible for arsenic influx/efflux, such as AQPs, glucose transporters, LRP and MRPs may be used to predict the sensitivity and/or resistance of human NSCLC cells to KML001® and sodium metaarsenite (KML001® ) may have potential as an alternative therapeutic agent, especially in combination with cisplatin in NSCLC patients. 61 POSTER (Board P055) A panel of pediatric liver cancer patient-derived xenografts to improve stratification of children with hepatoblastoma 4 M. Fabre1 , D. Nicolle2 , A. Gorse2 , O. Deas ´ 2 , C. Mussini3 , L. Brugieres ` , M.R. Ghigna5 , E. Fadel6 , L. Galmiche-Rolland7 , C. Chardot8 , C. Armengol9 , J.G. Judde2 , S. Branchereau10 , S. Cairo2 . 1 Institut Gustave Roussy, Biology and Medical Pathology Department, Villejuif, France; 2 ˆ Hospital, Pathology Department, Xentech, R&D, Evry, France; 3 Bicetre ˆ Le Kremlin Bicetre, France; 4 Institut Gustave Roussy, Pediatric Oncology 5 Department, Villejuif, France; Marie Lannelongue Hospital, Pathology Department, Le Plessis Robinson, France; 6 Marie Lannelongue Hospital, Surgery Department, Le Plessis Robinson, France; 7 Necker Hospital, Pathology Department, Paris, France; 8 Necker Hospital, Pediatric Surgery Department, Paris, France; 9 Health Sciences Research Institute Germans Trias i Pujol (IGTP), Translational Liver Oncology, Badalona, Spain; 10 ˆ ˆ Bicetre Hospital, Pediatric Surgery Department, Le Kremlin Bicetre, France

Despite being the predominant type of pediatric liver malignancies, hepatoblastoma (HB), with a world-wide incidence of 1 case per million persons per year, is a rare tumor. The high rate (>60%) of b-catenin activating mutations places HB as one of the human tumors most tightly associated with activation of the Wnt/b-catenin pathway. Evidence for (epi)genetic origin of HB is provided by its association with congenital anomalies, Beckwith–Wiedemann syndrome, and familial adenomatous polyposis, a disorder caused by germline mutation of APC, involved in b-catenin degradation. Like other rare diseases, rare cancers are particular challenging due to their low incidence, particularly for the identification of novel therapies. The rarity and the heterogeneity of childhood liver cancers hamper the development of reliable research tools that recapitulate each disease. To tackle this issue, we have launched a program aimed at the constitution of liver cancer patient-derived xenografts (PDXs). At present, 13 HBs and 2 HB/hepatocellular carcinoma (HCC) transitional liver cell tumors (TLCTs) have been successfully grown in immunocompromised mice out of 48 tumors grafted. HB and TLCT PDXs maintain the histological features of primary human tumors, and the heterogeneity of AFP levels in mouse blood correlate with that observed in patients. Comparative analysis of the clinical parameters associated to tumors from which PDX could or could not be established has been performed. This analysis clearly shows that tumor take is associated with high AFP level post-chemotherapy, with low % of treatment-induced necrotic/fibrotic area in the resected tumor, and, most important, with poor prognosis. Among HB PDXs models that have been established several are, in accordance with the clinical history, resistant to cis platinum and doxorubicin-based standard of care. In vivo anti-cancer screening in histologically different HB/TLCT PDX subtypes show unique profiles of response to the drugs tested, and identified the combination irinotecan/ temozolomide as a promising second line combination for a subset of HB PDXs. The identification of molecular biomarkers predictive of HB response to irinotecan/temozolomide combination will be discussed. Development of a panel of childhood liver tumor PDXs will endow the scientific community with an innovative and versatile research tool that will decisively contribute to improve our understandings on pediatric liver malignancies. These models constitute an unperishable reservoir of biological samples that strongly recapitulate the human tumor biology, and they can be used in several research domains such as functional genomics, cancer stem cell biology and pharmacogenomics, notably for the identification of Wnt/b-catenin inhibitors. In the long run, improved knowledge in all these research fields will be translated in improved cures for kids.

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62 POSTER (Board P056) Antitumor and temozolomide-sensitizing effects of sodium metaarsenite in an orthotopic glioblastoma xenograft model W.Y. Kang1 , Y.M. Park2 , S.J. Kim2 . 1 Institute For Refractory Cancer Research Samsung Medical Center, Samsung Biomedical Research Institute, Seoul, South Korea; 2 Komipharm International Inc. Co., Pharmaceutical Division, Shiheung, South Korea Background: Glioblastoma is one of the most malignant types of primary brain tumors and the treatment of glioblastomas remains difficult in that no contemporary treatments are curative. Temozolomide (TMZ) was approved and has been used for the treatment of glioblastoma as adjuvant or concomitant therapy with radiotherapy. Material and Methods: Sodium metaarsenite (NaAsO2 , KML001® ) is an orally available arsenic compound that has entered phase I/II clinical trials in solid tumors and hematologic malignancies. In this study, we investigated the antitumor activity and TMZ-sensitizing effects of KML001® in glioblastoma cell lines and a glioblastoma orthotopic xenograft mice model. Results: KML001® inhibited colony formation in TMZ-sensitive (U87MG, U373MG) and TMZ-resistant (U138MG and U251MG) glioblastoma cells in a concentration-dependent manner. The combined KML001® and TMZ treatment significantly decreased clonogenecity of glioblastoma cells, compared with TMZ treatment only (p < 0.05 or less). The combinational treatment of KML001® and TMZ increased the amount of cleaved PARP and caspase-3 that occur at the onset of apoptosis, in U87MG- and U251MG-glioblastoma cells, when compared with TMZ treatment only. Immunocytochemistry (ICC) assay showed that the combinational treatment increased g-H2AX levels which is a DNA damage response factor in U87MG and U251MG-glioblastoma cells, compared with KML001® and TMZ treatment only. The administration of 5 mg/kg of KML001® inhibited tumor growth (46%) and induced apoptosis (p < 0.05 vs. Control) in the U87MG-glioblastoma orthotopic xenograft mice model. And the combination therapy of KML001® and TXM significantly reduced tumor mass volume in the U87MG-glioblastoma orthotopic xenograft model, compared with KML001® monotherapy (p < 0.05). In the U251MGglioblastoma orthotopic xenograft mice model, the combination significantly decreased tumor volume, compared with KML001® treatment only group (p < 0.05) and TMZ treatment only group (p < 0.05). Conclusions: In conclusion, KML001® showed similar or more potent antitumor effect than TMZ. The combinational treatment of KML001® and TMZ was synergistic or additive in a glioblastoma orthotopic xenograft mice model. These results provide a rationale for clinical testing of the combination therapy of KML001® and TMZ. 63 POSTER (Board P057) Radium-223 dichloride − Efficacy and mode-of-action in a mouse model of prostate cancer bone metastasis M.I. Suominen1 , K.M. Fagerlund1 , J.P. Rissanen1 , Y. Konkol1 , 3 E. Alhoniemi1 , D. Mumberg2 , K. Ziegelbauer2 , S.M. Kak ¨ onen ¨ , J.M. Halleen1 , R.L. Vessella4 , A. Scholz2 . 1 Pharmatest Services Ltd, Turku, Finland; 2 Bayer Healthcare, Global Drug Discovery TRG-Onc/GT, Berlin, Germany; 3 University of Turku, Department of Cell Biology and Anatomy, Turku, Finland; 4 University of Washington, Departments of Urology and Microbiology, Seattle, USA Background: Radium-223 dichloride (Xofigo® ) is an alpha-emitting calcium mimetic that improves overall survival in prostate cancer patients with bone metastases (ALSYMPCA trial, NCT00699751, Parker et al. NEJM 18:213−33, 2014). Here, we clarified radium-223 dichloride modeof-action in an osteoblastic prostate cancer mouse model mimicking the devastating bone metastasis seen in prostate cancer patients. Material and Methods: The therapeutic effects of radium-223 dichloride were investigated in a clinically relevant patient-derived prostate cancer xenograft model exhibiting PSA expression, osteoblastic growth and systemic metastasis when inoculated into the bone marrow cavity. Human LuCaP 58 prostate cancer cells were inoculated into a tibia of 6−8-week-old male SCID mice (n = 17−18/group) and radium-223 dichloride (300 kBq/kg, iv injection) or vehicle control were administered once serum PSA levels reached 1 ng/ml and again 4 weeks later. X-rays and serum samples were obtained biweekly and at sacrifice 6 weeks after the first dose. Micro-CT measurements were performed on the tibiae (n = 8−11 mice/group) prior to preparation of decalcified paraffin sections for histomorphometry and immunohistochemistry. Soft tissues were collected for histology to observe visceral tumors, followed by macroscopical evaluation. Results: The inhibitory effect of radium-223 dichloride on tumor-induced osteoblastic bone growth was clearly visible in x-rays and in tibial weight

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measurement. Radium-223-treated mice exhibited reduced bone volume and surface in addition to decreased tumor volume compared to untreated animals. Metastatic spread to visceral organs was evident in 53% and 33% of vehicle- or radium-223 dichloride-treated mice, respectively. Importantly, both serum PSA and a bone formation marker PINP were reduced in radium-223 dichloride-treated mice. Moreover, the significantly lower PSA values were apparent as early as two weeks following the first dose, indicating that in addition to reducing osteoblastic bone growth, radium223 dichloride constrains tumor growth in metastatic prostate cancer. Conclusions: In conclusion, radium-223 dichloride (Xofigo® ) therapy exhibits a dual mode-of-action that impacts on tumor cells and on tumorinduced bone reaction, both important players in the destructive vicious cycle of osteoblastic bone metastasis in prostate cancer. 64 POSTER (Board P058) Nifuroxazide halogenic derivatives induce ROS-mediated apoptosis and display antitumor activity against metastatic melanoma C. Fernandez de Farias1 , M.H. Massaoka1 , N. Girola1 , C.R. Figueiredo1 , R.A. Azevedo2 , L.C. Tavares3 , L.R. Travassos1 . 1 Federal University ˜ Paulo, Department of Microbiology Immunology and Parasitology, of Sao ˜ Paulo, Brazil; 2 Butantan Institute, Department of Biochemistry and Sao ˜ Paulo, Brazil; 3 University of Sao ˜ Paulo, Department Biophysics, Sao ˜ Paulo, Brazil of Biochemical and Pharmaceutical Technology, Sao Background: Metastatic melanoma is a highly aggressive and therapyresistant malignancy. At the metastatic stage the disease is highly resistant to conventional therapy, and the 5-year survival is rather low. Therefore, novel therapeutic interventions are stimulated. Nifuroxazide is a widely used antibiotic, and its mechanism of action depends on ROS production. In this work we report on the antimelanoma activity of two halogenic derivatives of nifuroxazide.

Material and Methods: Cell viability was examined by MTT assay on B16F10-Nex2 murine melanoma cells after treatment with N-Br and N-I. Superoxide anion production was evaluated by dihydroethidium (DHE) assay in B16F10-Nex2 cells co-incubated or not with N-acetylcysteine (Nac). Chromatin condensation was analyzed by Hoescht 33342 staining. DNA degradation was evaluated in B16F10-Nex2 cells lysed in TELT buffer and subjected to electrophoresis in 1% Agarose gel. Apoptosis, in B16F10-Nex2 cells, was evaluated by annexin V-positive cells, measured by FACS. Mitochondrial membrane permeability of melanoma cells, coincubated with Nac and treated with N-Br and N-I, was assessed using the fluorescent probe tetramethylrhodamine ethyl ester (TMRE), and then analyzed by FACS. Therapeutic efficacy was examined in a syngeneic model of metastatic melanoma using C57BL/6 mice endovenously injected with B16F10-Nex2 cells and treated i.p. with N-Br and N-I. After 14 days, animals were sacrificed and the lungs were examined for metastatic colonization. Results: Both derivatives induced cell death in vitro in a dose-dependent manner. The IC50 values were 16mM and 12mM for N-Br and N-I, respectively, against murine melanoma cells. In the presence of Nac cytotoxicity of both compounds decreased. Furthermore, treatment with N-Br and N-I increased superoxide anion production by 87.5% and 55.65%, respectively. Co-incubation with Nac completely inhibited ROS production upon treatment with both compounds. Apoptosis cell death was suggested by a ladder pattern of DNA fragmentation and a drug concentration-dependent phosphatidyl serine surface translocation. Moreover, TMRE staining showed that N-B and N-I were able to induce the loss of mitochondrial membrane potential in a time-dependent manner, which was inhibited by Nac within 6 h of treatment. Hoescht staining confirmed that cells treated with both compounds exhibited chromatin condensation as in apoptotic nuclei. Remarkably, i.p. doses of N-Br and N-I were protective against metastatic melanoma, raised by endovenous injection of B16F10-Nex2 tumor cells. Conclusion: Nifuroxazide halogenic derivatives induce apoptosis in melanoma cells in a ROS-dependent manner at micromolar doses. Both N-Br and N-I were protective against metastatic B16F10-Nex2 melanoma in a syngeneic model. Supported by Fapesp no. 2014/05107-8.

Poster Session – Cytotoxics 65 POSTER (Board P059) Phase II drug metabolism UGT1A enzyme affects cellular response of colon cancer cells to antitumor triazoloacridinone C-1305 treatment E. Augustin1 , E. Bartusik1 , A. Theus1 , B. Borowa-Mazgaj1 , Z. Mazerska1 . 1 Gdansk University of Technology, Pharmaceutical Technology and Biochemistry, Gdansk, Poland Background: The essential limitation of a drug’s effectiveness in a living organism is the individual’s level of enzymes, which catalyze drug metabolism. Furthermore, the expression level of metabolic enzymes in tumor tissues (in vivo) or tumor cells (in vitro) may affect the final effect of the drug treatment. C-1305 is a promising antitumor agent with high activity against many experimental cellular and tumor models. Studies on C-1305 metabolism indicated that the compound is conjugated by several uridine diphosphate-glucuronyltransferase (UGT) isoforms, the most active being extrahepatic UGT1A10. Here, we investigated whether and how UGT1A isoenzyme affects the cellular response of colon cancer cells following C-1305 treatment. Materials and Methods: C-1305 activity was evaluated in two human colon carcinoma cell lines, HT29 (UGT1A positive) and HCT116 (UGT1A negative). To determine whether UGT1A expression affects the final cellular response upon C-1305 treatment, HT29 and HCT116 cells were exposed to EC50 concentration for 24 h to 120 h. DAPI staining was used to analyze cellular morphology. Flow cytometry for annexin V/PI assay and subG1 DNA fraction was applied to identify apoptosis. Analysis of cellular morphology and expression of SA-b-galactosidase were performed to identify cells with senescence-like phenotype. Acridine orange staining was performed to detect acidic vesicular organelles (AVOs) as a marker of autophagy. Results: Treatment of colon cancer cells with C-1305 for 72 h suppressed cell proliferation with EC50 concentration equal to 0.5 mM for HT29 and 0.8 mM for HCT116 cells, respectively. C-1305 induced apoptosis, necrosis and mitotic catastrophe in both cell lines as evidence by chromatin condensation, the presence of multinucleated cells, phosphatydilserine externalization and presence of sub-G1 fraction. In all tests, the number of apoptotic cells was low and increased in a time-dependent manner and reached ~23% after 120 h of drug exposure in both cell lines. Starting from 72 h, HCT116 cells which did not die by apoptosis or necrosis, underwent cellular senescence, preceded by autophagy. Interestingly, pharmacological inhibition of autophagy by chloroquine (10 mM) sensitized HCT116 cells to C-1305 and profoundly intensified the senescence phenotype. HT29 cells did not undergo cellular senescence. Moreover, rapid autophagy observed in those cells after 24 h decreased following prolonged drug treatment. Conclusions: Our results indicate that in colon cancer cells treated with C-1305, the lack of UGT1A expression promotes autophagy followed by cellular senescence. UGT1A status had no effect on apoptosis, mitotic catastrophe or necrosis induced by C-1305. 66 POSTER (Board P060) Clinical validity of new genetic biomarkers of irinotecan neutropenia: An independent replication study F. Innocenti1 , J. Ramirez2 , W. Qiao3 , A.J. de Graan4 , M.J. Ratain2 , R.H.N. van Schaik4 , R.H.J. Mathijssen4 , G.L. Rosner5 , D.J. Crona6 . 1 University of North Carolina at Chapel Hill, Linberger Comprehensive Cancer Center & Center for Pharmacogenomics and Individualized Therapy Eshelman School of Pharmacy, Chapel Hill NC, USA; 2 University of Chicago, Department of Medicine, Chicago IL, USA; 3 MD Anderson Cancer Center University of Texas, Houston TX, USA; 4 Erasmus University Medical Center, Erasmus MC Cancer Institute, Rotterdam, Netherlands; 5 Johns Hopkins University, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore MD, USA; 6 University of North Carolina at Chapel Hill, Center for Pharmacogenomics and Individualized Therapy Eshelman School of Pharmacy, Chapel Hill NC, USA Purpose: This study aimed to provide evidence for the clinical validity of germline genetic variants previously associated with irinotecan neutropenia and pharmacokinetics. Methods: Ten germline variants from six genes, whichassociated with absolute neutrophil count (ANC) nadir and/or irinotecan pharmacokinetics in a discovery cohort of 78 cancer patients, were genotyped in an independent replication cohort of 108 cancer patients. Patients from both cohorts received single-agent irinotecan every three weeks. Pharmacokinetic data and neutrophil counts were collected during cycle 1 of treatment. Associations between the germline variants and ANC nadir or irinotecan pharmacokinetics were analyzed using linear regression. Univariate and multivariate models adjusted for sex, age, irinotecan dose, and ANC nadir models also adjusted for baseline ANC.

Poster Session – Drug Resistance and Modifiers Results: For ANC nadir, we replicated UGT1A1*93, UGT1A1*28, and SLCO1B1*1b. In both univariate and multivariate models, SLCO1B1*1b had a protective effect against neutropenia, independent of the effect of either UGT1A1*28 or UGT1A1*93. For irinotecan AUC0−24 , we replicated ABCC2 −24C>T and HNF1a 79A>C; however ABCC2 −24C>T predicted only a small fraction of the interindividual variability in irinotecan exposure, and HNF1a 79A>C had a 75% lower estimate of effect compared to the discovery cohort. For SN-38 AUC0−24 and the glucuronidation ratio, we replicated UGT1A1*28, UGT1A1*93, and ABCC1 1684T>C; however ABCC1 1684T>C had an 85% lower estimate of effect compared to the discovery cohort. Conclusion: In addition to UGT1A1*28, this study independently validated UGT1A1*93 and SLCO1B1*1b as new predictors of irinotecan neutropenia. Further demonstration of the clinical utility of SLCO1B1*1b and UGT1A1*93 will help further optimize irinotecan therapy for cancer patients. 67 POSTER (Board P061) Cytotoxic response as a result of the cross-talk between UGT mediated metabolism and modulation of UGT activity by C-1311 and C-1305 acridinone antitumor agents in selected solid tumor cell lines Z. Mazerska1 , M. Pawlowska1 , A. Bejrowska1 , M. Szydlowska1 , B. Fedejko1 , E. Augustin1 . 1 Gdansk University of Technology, Department of Pharmaceutical Technology and Biochemistry, Gdansk, Poland Background: UDP-glucuronyltransferases (UGTs, EC 2.4.1.17) transform nearly 35% drugs to metabolites, which are usually of lower toxicity or limited therapeutic effects. Anticancer agents are of special interest in this respect, because the glucuronidation may represent a mechanism of intrinsic drug resistance and there were reported the differences in UGT expression between normal and tumour tissues. Moreover, the UGT level and activity in normal and tumour cells can be modulated by other chemotherapeutics, what is crucial in antitumor multidrug therapy. Considering above we aimed here to know whether antitumor acridinone derivatives C-1305 and C-1311 are glucuronidated in selected solid tumour cell lines and what is the impact of UGT1A10 overexpression on cytotoxicity of these compounds. We also answered whether C-1305 and C-1311 are able to influence UGT enzymatic activity. Materials and Methods: The studied compounds, 5-diethylamino-8hydroxyimidazoacridinone, C-1311, and 5-dimethylamino-8-hydroxytriazoloacridinone, C-1305, were developed in our laboratory, and C-1311 was selected for II phase of clinical studies. Cell lines of human hepatoma, HepG2, colon tumours, HCT116 and HT29 and breast cancer, MCF-7, were studied and they (except HT29) were tranfected with UGT1A10 vector by electroporation. UGT1A10 was selected as the most effective UGT isoenzyme towards studied compounds in vitro. Metabolic glucuronidation in tumour cells was followed by HPLC analysis. The concentration of standard UGT substrates 7-hydroxy-4-(trifluoromethyl)coumarin (7-TFK) and 7-ethyl10-hydroxycamptothecin (SN-38) were the markers of UGT activity. Results indicated that both compounds were metabolized in HT-29 wild type cells (natural UGT expression) and in UGT1A10 overexpressed tumour cells to O-glucuronides on 8-hydroxyl group. Cytotoxicity of C-1311 in MCF-7 cells overexpressed with UGT was lower than in MCF-7 wild type, whereas it was not changed in HepG2 and HCT116. At the same time the activity of C-1305 was slightly, but significantly, higher in the presence of higher level of UGT in HepG2 and MCF-7 cells. Compounds C-1305 and C-1311 were similar in the profile of UGT modulation. They activated UGT only at low concentration and after short time of incubation, whereas higher concentration and long time resulted in the inhibition of UGT mediated metabolism. Conclusions: The cross-talk between the glucuronidation and UGT induction/inhibition demonstrated here seems to be responsible for the fact that the final influence of UGT on cytotoxicresponse depended on the type of tumor cells and the type of compound. Nevertheless, the results showed that glucuronidation of C-1305 antitumor agent in MCF-7 cells did not result in lower cytotoxicity, as is usually observed for majority of drugs, but we even observed the activation effect of C-1305 glucuronide toward tumour cells. 68 POSTER (Board P062) Can the P53 status predict the outcome of Polo-like kinase 1 inhibition in non-small cell lung cancer cell lines? J. Van den Bossche1 , A. Wouters1 , C. Deben2 , V. Deschoolmeester2 , P. Specenier3 , P. Pauwels4 , M. Peeters3 , F. Lardon1 . 1 Antwerp University, Oncology, Antwerp, Belgium; 2 Antwerp University, Pathology, Antwerp, Belgium; 3 Antwerp University Hospital, Oncology, Antwerp, Belgium; 4 Antwerp University Hospital, Pathology, Antwerp, Belgium Introduction: Polo-like kinase 1 (Plk1), a key regulator of multiple steps during mitosis, is considered as one of the most promising therapeutic

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targets for mitotic intervention due to its overexpression in a number of human malignancies, including non-small cell lung cancer (NSCLC). TP53 is the most frequently mutated gene in human cancer and plays a role in many cellular processes including cell growth and response to DNA damage. Previous studies suggest a negative interplay between Plk1 and P53, leading to the hypothesis that P53 status might be a predictive biomarker for response to Plk1 inhibition. As such, we investigated the cytotoxic effect of the small molecule inhibitor BI6727 (volasertib) in a series of NSCLC cell lines differing in P53 status. Material and Methods: Three isogenic NSCLC cell lines (A549 (P53 wt), A549-NTC (empty vector control, p53 wt) and A549–920 (P53 deficient)) were generated using a P53 GIPZ shRNA lentiviral vector. The CRL5908 NSCLC cell line, harboring a P53 mutation (R273H), was included too. All cell lines were treated with BI6727 (0−85nM) for 24 or 72 hours. Treated cells were incubated under both normal and reduced oxygen conditions (hypoxia, <0.1% O2 ). Cell survival was assessed using the sulphorhodamine B (SRB) assay and IC50 values were calculated using WinNonlin software. The effect of Plk1 inhibition (0−10nM) on cell cycle distribution of A549 cells was determined flow cytometrically using the Vindelov method. Results: Treatment with BI6726 established a dose-dependent growth inhibition under both normoxic and hypoxic conditions. As shown in Table 1, a significantly reduced sensitivity to volasertib was observed in P53 deficient cells compared to p53 wt cell lines (p < 0.001). For example, in A549–920 cells, 24 hours of Plk1 inhibition (normoxia) resulted in an IC50 value of 27.59±5.77 nM in contrast to 17.87±0.40 nM for A549-NTC cells. Table 1 demonstrates also a decreased effect of BI6727 under hypoxic conditions (p < 0.001). However, this could not be observed in the P53 mutant cell line. Considering the cell cycle distribution under normoxic conditions after treatment with 10nM volasertib, a significant G2 /M phase block was induced in the A549 cell line (p < 0.022). Furthermore, a sub-G1 peak could be observed when treated with high concentrations of BI6727, suggesting induction of apoptosis. Similar experiments will be performed in the cell lines lacking functional P53 in order to detect possible differences in cell fate after Plk1 inhibition. Conclusion: Our results show that A549 NSCLC cells with functional P53 seem to be more sensitive to Plk1 inhibition compared to isogenic P53 deficient cells. Further experiments are warranted to further clarify this observation. In addition, combination studies of volasertib with conventional chemotherapeutics and radiotherapy will be initiated. Table 1. IC50 values ± standard deviations after 24 and 72 hours of treatment with volasertib under both normoxic and hypoxic conditions Cell line

A549 NTC 920 5908

Normoxia 24 h

72 h

Hypoxia 24 h

72h

15.44±2.49 17.87±0.40 27.59±5.77 61.90±3.94

18.44±1.83 17.33±1.32 28.00±6.32 72.38±9.87

79.91±15.41 66.39±14.31 115.46±27.60 76.43±10.83

54.46±5.58 63.89±22.30 112.52±34.19 68.66±9.83

Drug Resistance and Modifiers 69 POSTER (Board P063) AKT3 activation mediates resistance to HER2-targeted therapies in HER2-amplified breast cancer F. Carmona Sanz1 , F. Montemurro2 , V. Rossi2 , C. Verma3 , M. Berger1 , J. Baselga1 , M. Scaltriti1 . 1 Memorial Sloan-Kettering Cancer Center, New York City, USA; 2 Institute for Cancer Research and Treatment, Candiolo, Italy; 3 A STAR’s Bioinformatics Institute (BII), Singapore, Singapore Background: HER2 targeted therapies have improved the clinical benefit of HER2-amplified breast cancer when diagnosed before metastatic spread. Nevertheless, a substantial percentage of patients with advanced HER2 positive disease are intrinsically refractory to these agents, and many initially responding patients acquire resistance over time. Activation of PI3K/AKT pathway via mutation of PIK3CA or loss of PTEN has been associated with resistance to anti-HER2 therapy, and combination regimes with PI3K inhibitors are now under investigation in the clinical setting. We hypothesize that also aberrations in AKT3 concur in limiting the sensitivity to anti-HER2 agents. Material and Methods: We used targeted exome-sequencing to analyze clinical specimens obtained from HER2-amplified breast cancer patients

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treated with trastuzumab and/or lapatinib monotherapy. Further protein modeling studies and functional in vitro and in vivo assays were used to validate our findings. Genetically engineered AKT 1/2 null cells were also used to assess the contribution of AKT3 activation in the context of drug resistance. Results: We have identified gain-of-function genomic alterations in AKT3 associated with therapy refractoriness in HER2-amplified breast cancer patients treated with anti-HER2 agents in a chemotherapy-free setting. Specifically, copy-gain and genomic mutation were associated with intrinsic resistance to trastuzumab or lapatinib. Protein modeling predicts an activating role for the mutation identified, providing a mechanism for pathway activation beyond those previously described. Treatment combination with specific AKT inhibitors might improve treatment efficacy and overcome resistance to HER2-directed therapy. Conclusions: Genomic analyses of selected patient biopsies provide relevant biomarkers that can be used for clinical decision making. The involvement of AKT3 in the development of resistance to anti-HER2 agents provides an actionable target for efficient therapeutic intervention. Moreover, the confirmation of AKT3 as a reliable prognostic biomarker would improve patient stratification beyond the assessment of routinely assayed pathway alterations. 70 POSTER (Board P064) Antitumor activity of S116836, a novel tyrosine kinase inhibitor, against imatinib-resistant FIP1L1-PDGFRa-expressing cells J. Pan1 , Y. Shen2 , K. Ding3 . 1 Sun Yat-sen University Zhongshan Ophthalmic Center, State Key Laboratory of Ophthalmology, Guangzhou, China; 2 Sun Yat-sen University Zhongshan School of Medicine, Department of Pathophysiology, Guangzhou, China; 3 Guangzhou Institute of Biomedicine and Health Chinese Academy of Sciences, Key Laboratory of Regenerative Biology and Institute of Chemical Biology, Guangzhou, China The FIP1-like-1-platelet-derived growth factor receptor alpha (FIP1L1PDGFRa) fusion oncogene is the driver factor in a subset of patients with hypereosinophilic syndrome (HES)/chronic eosinophilic leukemia (CEL). Most FIP1L1-PDGFRa-positive patients respond well to the tyrosine kinase inhibitor (TKI) imatinib. Resistance to imatinib in HES/CEL has been described mainly due to the T674I mutation in FIP1L1-PDGFRa, which is homologous to the imatinib-resistant T315I mutation in BCR−ABL. Development of novel TKIs is imperative to overcome resistance to imatinib. We synthesized S116836, a novel TKI. In this study, we evaluated the antitumor activity of S116836 in FIP1L1-PDGFRa-expressing cells. The results showed that S116836 potently inhibited PDGFRa and its downstream signaling molecules such as STAT3, AKT, and ERK1/2. S116836 effectively inhibited the growth of the WT and T674I FIP1L1PDGFRa-expressing neoplastic cells in vitro and in nude mouse xenografts. Moreover, S116836 induced intrinsic pathway of apoptosis as well as the death receptor pathway, coincided with up-regulation of the proapoptotic BH3-only protein Bim-EL through the Erk1/2 pathway. In conclusion, S116836 is active against WT and T674I FIP1L1-PDGFRaexpressing cells, and may be a prospective agent for the treatment of HES/CEL. 71 POSTER (Board P065) Novel regulation of estrogen receptor transcription by the PI3K pathway 1

1

1

1

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E. Toska , M. Elkabets , A. Bosch , O. Litvin , M. Scaltriti , J. Baselga . 1 MSKCC, New York, USA Background: Alterations in the PI3K/Akt/mTOR pathway occur in up to 50% of estrogen receptor (ER) positive breast tumors. Clinical data showed that dual inhibition of the ER and mTOR pathways results in greater disease control. Although previous work has pointed to the existence of crosstalk between ER and certain nodes of the PI3K pathway, the mechanism by which both pathways regulate each other remains to be elucidated. Thus, we aim to investigate the mechanisms by which ER-dependent transcriptional program is regulated by the PI3K pathway. Material and Methods: ER-positive cell lines with different genomic alterations in the PI3K pathway were treated in vitro with the p110a-specific PI3K inhibitor BYL719. RNA expression was evaluated by gene expression microarray analysis and RT-PCR. Promoter occupancy of ER to its target genes was assessed by chromatin immunoprecipitation (ChIP) assay. A transposon activation mutagenesis screen was developed to identify modulators of resistance to PI3K inhibitors in ER-positive breast cancer cells. This approach employs the transfection of transposon containing CMV promoters that are randomly integrated in the genome and drive activation of nearby gene expression.

Results: We observed that inhibition of the PI3K pathway by BYL719 triggers ER gene expression, which in turn results in increased ERdependent transcriptional activity accompanied by an ER-driven re-wiring of the cell transcriptome. The transposon activation mutagenesis screen followed by high throughput sequencing successfully identified genes that regulate ER expression/activity and putatively cause resistance to PI3K inhibitors in ER+ breast cancer cell lines. Interestingly, among the genes identified we found a number of key transcription factors known to modulate ER expression. Importantly, these results were confirmed in breast cancer patients treated with BYL719. Conclusions: Our findings suggest that PI3K inhibition activates the ER pathway by modulating ER expression levels. Moreover, we have developed a transposon activation mutagenesis screen that identified a number of ER co-factors as possible responsible for resistance to PI3K inhibition. The molecular mechanisms by which these factors enter into play upon PI3K inhibition are currently being investigated. 72 POSTER (Board P066) Nrf2 as a molecular target in overwhelming chemoresistance in breast cancer therapy N. Samadi1 , F. Ramezani1 , M. Sabzichi1 . 1 RCPN, Clinical Biochimestry, Tabriz, Iran Background: Nuclear factor erythroid 2-related factor 2 (Nrf2) has been recognized as a transcription factor that control mechanisms of cellular defense response by regulation of three classes of genes, including, endogenous antioxidants, phase II detoxifying enzymes and transporters. Previous studies have revealed the role of Nrf2 in resistance to chemotherapeutic agents and high level expression of Nrf2 have found in many types of cancer. At physiological concentrations, luteolin as a flavonoid compound can inhibit Nrf2 and sensitized cancer cells to chemotherapeutic agents. We reported luteolin loaded in phytosome as an advanced nanoparticles carrier sensitized MDA-MB 231 cells to doxorubicin. Material and Methods: Luteolin-loaded phytosome was prepared by thin film hydration technique. Cell culture/cytotoxicity studies of luteolin, doxorubicin, and phytosome were performed by means of MDA-MB231 human cancer cells and MTT assay. Determination of the gene expression levels of Nrf2, HO1, NQO1 and MDR1, used by Real-Time polymerase chain reaction (RT-PCR). Results: In this study, we prepared nano phytosoms of luteolin to enhance the bioavailability of luteolin and improve passive targeting in breast cancer cells. Our results showed that co-treatment of the cells with nano particles containing luteolin and doxorubicin has the highest percentage of cell death in MDA-MB 231cells (p < 0.05). Furthermore, luteolin-loaded nanoparticles reduced Nrf2 gene expression at mRNA level in the cells higher than luteolin alone (p < 0.05). Similarly, expression of downstream genes for Nrf2 including Ho1 and MDR1 were reduced significantly (p < 0.05). Inhibition of Nrf-2 expression caused a marked increase in cancer cell death (p < 0.05). Conclusions: Taken together, these results suggest that phytosome technology can improve the efficacy of chemotherapy by overcoming resistance and enhances permeability cancer cells to chemical treatment and may thus be considered as a potential delivery system to increase the therapeutic protocols in cancer patients. 73 POSTER (Board P067) Differential pathway resiliency in response to MAPK inhibition in BRAF-mutant cancer M. Sos1 , R.S. Levin1 , J.D. Gordan1 , J.A. Oses-Prieto2 , J.T. Webber3 , M. Salt4 , B. Hann4 , A.L. Burlingame2 , F. McCormick4 , S. Bandyopadhyay3 , K.M. Shokat1 . 1 UCSF, Howard Hughes Medical Institute and Department of Cellular and Molecular Pharmacology University of California San Francisco, San Francisco CA, USA; 2 UCSF, Department of Pharmaceutical Chemistry University of California San Francisco California USA, San Francisco CA, USA; 3 UCSF, Department of Bioengineering and Therapeutic Sciences University of California San Francisco, San Francisco CA, USA; 4 UCSF, Helen Diller Family Comprehensive Cancer Center Department of Medicine University of California San Francisco San Francisco, San Francisco CA, USA Background: Despite the development of potent RAS-RAF pathway inhibitors, only a fraction of BRAF-mutant patients responds to the treatment with these drugs. Materials and Methods: Here, we applied a combined chemo-genomics and chemo-proteomics approach to gain insights into the dynamics of primary resistance signaling and to define novel cancer vulnerabilities in these tumors.

Poster Session – Drug Resistance and Modifiers Results: These analyses uncovered differential pathway resiliency in response to MAPK inhibition in BRAF-mutant cancer cells and identified how high-dose MEK inhibition as a viable strategy to overcome this source of resistance signaling. Mapping of global phosphorylation dynamics using orthogonal mass-spectrometry based methods revealed multi-layered MAPK pathway feedback-loop release and autocrine cytokine secretion as complementary drivers of adaptive signaling in these tumors. Conclusions: Overall, our data provide a kinome- and phosphoproteomewide view of the adaptive process of primary resistance to MAPK inhibitors in BRAF-mutant tumors that may be of importance for future clinical trials involving these targeted drugs. 74 POSTER (Board P068) Increased EDN1 expression mediates acquired resistance to the combination therapy of PI3K and MEK inhibitors for colorectal cancer B. Bhattacharya1 , S.H.H. Low1 , T. Benoukraf1 , M.L. Chong1 , K.X. Koh1 , R. Soong1 . 1 National University of Singapore, Cancer Science Institute, Singapore, Singapore Background: Previously we reported that acquired resistance (AQR) to the combination of BKM120 (PI3K inhibitor) and AZD6244 (MEK inhibitor) can be generated in PIK3CA and KRAS mutant HCT116 colorectal cancer (CRC) cells, in addition to AQR to the individual agents. Using gene expression analysis we demonstrated that the differentially expressed genes in the combination AQR cells are distinctly different to those expressed in single agent AQR and parental cells, with EDN1 (endothelin-1) gene being the most highly expressed (~33-fold). Currently, we generated AQR to the same combination in LoVo CRC cell line and explored determinants of resistance. Methods: LoVo CRC cells with KRAS mutation were exposed continuously with combined (1 IC50 dose of each agent) or single agent (2 doses of IC25 ) concentrations of MEK (AZD6244) and PI3K (BKM120) inhibitors, or DMSO (parental). Combination Indices (CI) was calculated by the method of Chou and Talalay. Differentially expressed genes were determined using Affymetrix Gene 1.0ST analysis. Results: LoVo cells with AQR to combination treatment were obtained after 12 months ([email protected] of ‘LoVoCR’ cells 2.5±0.3 vs parental 0.19±006, p = 0.03). LoVo cells with AQR to single agent treatment with AZD6244 (IC50 of ‘LoVoAR’ 67.1±0.02mM vs parental 5.1±0.01mM; p = 0.02) and BKM120 (IC50 of ‘LoVoBR’ 5.2±0.11mM vs parental 0.9±0.21mM; p = 0.002) were also generated. Consistent with our previous findings LoVoCR cells were resistant to apoptosis, wound healing response and cell cycle arrest compared with parental cells. Furthermore, these cells also displayed resistance to the combination of an alternative PI3K inhibitor (BYL719) and MEK inhibitor (GDC0973). No cross-resistance was observed with 5-FU or carboplatin in LoVoCR cells, arguing against a multi-drug resistance phenotype. Consistent with our gene expression data in HCT116 cells AQR to the combination (HCT116CR) cells a 7-fold overexpression of EDN1 was also observed in LoVoCR cells. Both HCT116CR and LoVoCR cells displayed significantly elevated phosphorylation levels of AKT and ERK compared to parental cells, suggesting some feedback mechanism. Silencing EDN1 by siRNA significantly reduced the proliferation rates of HCT116CR and LoVoCR cells compared to control siRNA, which was accompanied by a decrease in phosphorylation of AKT and ERK. Both the combination AQR cells were significantly sensitive to bosentan (endothelin receptor antagonist) induced growth inhibition (HCT116 IC50 = 67.4±0.9mM vs HCT116CR IC50 = 39.2±1.2mM; p = 0.01: LoVo IC50 = 83.2±2.5mM vs LoVoCR IC50 = 41.1±3.4mM; p = 0.03). Furthermore, in HCT116CR and LoVoCR cells the antagonism was converted to synergy in the presence of a non-growth inhibitory concentration (1/5th IC50 ) of bosentan. Finally, pre-stimulation with exogenous endothelin-1 conferred resistance to the combination in both parental HCT116 and LoVo cells, along with an increase in AKT and ERK phosphorylation. Current work aims to uncover the mechanism of transcriptional regulation of endothelin in AQR. Conclusions: EDN1 is a specific mediator of AQR to the use of the PI3K and MEK inhibitor combination in CRC, irrespective of the genetic lesions involved. Endothelin receptor antagonists can potentially circumvent such resistance.

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75 POSTER (Board P069) Loss of PTEN leads to acquired resistance to the PI3Ka inhibitor BYL719: a case of convergent evolution under selective therapeutic pressure P. Castel1 , D. Juric2 , M. Griffith3 , O.L. Griffith4 , H.H. Won1 , B. Ainscough5 , H. Ellis1 , S. Ebbesen6 , I. Gopakumar1 , C. Quadt7 , M. Peters7 , D. Solit1 , S.W. Lowe6 , E.R. Mardis5 , M.F. Berger1 , M. Scaltriti1 , J. Baselga1 . 1 Memorial Sloan Kettering Cancer Center, HOPP, New York, USA; 2 Massachusetts General Hospital, Cancer Center, Boston, USA; 3 Washington University School of Medicine, Department of Genetics, St Louis, USA; 4 Washington University School of Medicine, Department of Medicine, St Louis, USA; 5 Washington University School of Medicine, The Genome Institute, St Louis, USA; 6 Memorial Sloan Kettering Cancer Center, Cancer Biology and Genetics, New York, USA; 7 Novartis Pharma AG, Basel, Switzerland Background: Activating mutations of PIK3CA, the gene encoding the p110a subunit of PI3K, are frequent in breast cancer and selective inhibitors of this enzyme have shown promising clinical activity in breast tumors harboring these mutations. Material and Methods: We studied the case of a patient with metastatic breast cancer harboring a PIK3CA mutation that was treated in a clinical trial with BYL719, a highly selective PI3Ka inhibitor. The treatment resulted in partial tumor regression that lasted 9.5 months followed by progression and rapid death of the patient. A rapid autopsy was performed with collection of tissue samples from 16 different metastatic sites. We compared by whole genome and exome sequencing the original primary tumor, a rapidly progressing lung metastasis and a periaortic lesion that was still responding to BYL719 at time of death. Besides several common alterations, PTEN loss and a missense mutation were detected only in the lung metastasis. Using targeted exome sequencing we analyzed all the other available samples. Results: Strikingly, we observed a consistent loss in PTEN (via different mechanisms such as deletion, splice site mutation and frameshift mutations) in all the lesions refractory to BYL719 but not in the responding ones. We were able to build a dendrogram showing the phylogenetic evolution of the lesions and the evolutionary convergence of the PTEN alterations. To validate PTEN loss as a possible mechanism of acquired resistance to selective PI3Ka inhibition, we generated inducible PTEN shRNA clones starting from three different BYL719-sensitive cell lines. In all the studied models, induction of PTEN shRNA resulted in resistance to BYL719. Since PTEN deficient genetic models have been shown to rely on the b subunit of the PI3K holoenzyme, we tested whether the concomitant inhibition of both p110a and p110b was sufficient to revert the resistant phenotype. BKM120 (a pan-PI3K inhibitor) or the addition of AZD6482 (p110b inhibitor) to BYL719 re-sensitized the cells to BYL719. To expand our findings in vivo, we used a patient-derived xenograft (PDX) model generated from a lung PTEN-null non-responding lesion. Consistently, this PDX model was refractory to the antitumor activity of BYL719 but conserved sensitivity to BKM120 or the combination of AZD6482 and BYL719. In both cases, IHC analysis revealed a decrease in PI3K/AKT downstream effectors pAKT (473) and pS6 (240/4) staining with BKM120 or AZD6482+BYL719, but not with BYL719 alone. Preliminary analyses of other specimens collected from patients treated with BYL719 showed homozygous PTEN loss in another sample upon therapy progression. Conclusion: Taken together, the different mechanisms that inactivate PTEN in the tumor treated with BYL719 can be explained by convergent phenotypic evolution in a heterogeneous tumor and highlight the importance of PTEN and PI3Kb in acquired resistance to PI3Ka inhibitors. 76 POSTER (Board P070) Mixed lineage kinases activate MEK independently of RAF to mediate resistance to RAF inhibitors A.A. Marusiak1 , Z.C. Edwards1 , W. Hugo2 , E.W. Trotter1 , M.R. Girotti3 , N.L. Stephenson1 , X. Kong2 , M.G. Gartside4 , S. Fawdar1 , A. Hudson1 , W. Breitwieser5 , N.K. Hayward4 , R. Marais3 , R.S. Lo2 , J. Brognard1 . 1 Cancer Research UK Manchester Institute, Signalling Networks in Cancer, Manchester, United Kingdom; 2 Jonsson Comprehensive Cancer Center, Division of Dermatology, Los Angeles, USA; 3 Cancer Research UK Manchester Institute, Molecular Oncology, Manchester, United Kingdom; 4 QIMR Berghofer Medical Research Institute, Oncogenomics Research, Brisbane, Australia; 5 Cancer Research UK Manchester Institute, Cell Regulation, Manchester, United Kingdom Melanoma is a form of skin cancer that displays a particularly aggressive and malignant phenotype. Over 50% of melanoma cases involve mutation

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of the BRAF protein, and of these more than 90% carry the amino acid substitution V600E. This is an activating mutation that constitutively activates the ERK pathway, promoting survival and proliferation. BRAF inhibitors such as vemurafenib initially give good results, with a high rate of objective response and improved survival rates; however, resistance develops after around six months. We have identified a novel mechanism of resistance to BRAF inhibition, mediated by the mixed lineage kinase (MLK) family of proteins. MLKs are MAP3Ks, which are known to activate the JNK pathway. We observed that MLK1−4 can directly phosphorylate MEK in vitro and activate the ERK pathway in cells. Expression of MLK1−4 in melanoma cell lines harbouring BRAFV600E reactivates the ERK pathway and promotes cell survival despite treatment with BRAF inhibitors. Furthermore, MLKs are found to be upregulated in 9 of 21 melanoma patients with acquired drug resistance. Consistent with this observation, MLKs promote resistance to RAF inhibitors in mouse models, and contribute to acquired resistance in a cell line model. Lastly, we observe that a majority of MLK1 mutations identified in patients are gain-of-function mutations. In conclusion these results suggest a role for MLKs as direct activators of the MEK/ERK pathway with implications for melanomagenesis and resistance to RAF inhibitors. 77 POSTER (Board P071) Tumor suppressive roles of miR-221 and miR-222 in lung cancer M. Sato1 , R. Yamashita1 , T. Kakumu1 , T. Hase1 , E. Maruyama1 , Y. Sekido2 , M. Kondo1 , Y. Hasegawa1 . 1 Nagoya University Graduate School of Medicine, Department of Respiratory Medicine, Nagoya, Japan; 2 Aichi Cancer Center Research Institute, Division of Molecular Oncology, Nagoya, Japan Background: Bimodal roles of miR-221 and miR-222 microRNAs are reported in several types of human cancers. A previous study suggested their oncogenic role in invasiveness in lung cancer, albeit only one cell line (H460) was used. Material and Methods: To further evaluate involvement of miR-221 and miR-222 in lung cancer, we investigated the effects of miR-221 and miR222 overexpression on six lung cancer cell lines, including H460, as well as one immortalized normal human bronchial epithelial cell line, HBEC4. Results: miR-221 and miR-222 overexpression induced epithelial-tomesenchymal transition (EMT)-like changes in morphology as well as expression levels of EMT-associated genes in HBEC4 but did not confer EMT-associated oncogenic phenotypes, anchorage-independent growth and invasiveness. Consistent with the prior report, miR-221 and miR222 promoted growth in H460; however, miR-221 suppressed growth in four other cell lines with no effects in one, and miR-222 suppressed growth in three cell lines but promoted growth in two. Cell cycle and apoptosis analyses revealed that growth suppression by miR-221 and miR-222 occurred through intra-S-phase arrest and/or apoptosis resulting from DNA double strand breaks. Microarray analysis suggested that the JAK/STAT signaling pathway is involved in the apoptosis-induced by miR221 and miR-222. Finally, lung cancer cell lines transfected with miR-221 or miR-222 became more sensitive to the S-phase targeting drugs, possibly due to an increased S-phase population. Conclusions: Our data are the first to show tumor-suppressive effects of miR-221 and miR-222 on lung cancer, suggesting their potential as therapeutics for the disease. 78 POSTER (Board P072) A comprehensive in vitro screen to identify therapeutic candidates for inclusion with etoposide/platin combinations to improve treatment of SCLC D. Evans1 , R. Delosh1 , J. Laudeman1 , C. Ogle1 , R. Reinhart1 , M. Selby1 , T. Silvers1 , A. Monks1 , E. Polley2 , G. Kaur2 , J. Morris2 , B.A. Teicher2 . 1 Frederick National Laboratory for Cancer Research, MPB/DCTD, Frederick MD, USA; 2 NCI, DCTD, Rockville MD, USA Background: SCLC initially shows a good response to first line chemotherapeutic regimens but recurrent disease is highly resistant to treatment. Consequently, the SCLC 5-year survival rate is only 5−10%. In an attempt to improve therapeutic efficacy for SCLC, various combination treatments have been tested; however, the two-drug combination of etoposide and a platinum drug (carboplatin or cisplatin), with equivalent clinical activity, has remained the standard of care for 30 years. Improving the activity of the platin/etoposide backbone in SCLC, by adding a third, non-cross-resistant agent, has had little success. Empirical approaches in the clinic have not been able to evaluate the many investigational and approved agents that could serve as the third drug to make an efficacious triplet regimen. Materials and Methods: We established a panel of 67 human SCLC cell lines for use in single agent and combination screens. In phase 1, the

Poster Session – Drug Resistance and Modifiers SCLC lines were screened with ~100 approved anticancer drugs and 433 investigational agents. The single agent data were used to select agents for the combination screen seeking a third drug to add to the platin/etoposide backbone. The screen used fixed concentrations of carboplatin (3.7uM) and etoposide (0.3uM) and exposure time (96 h). The effect of simultaneously adding a third agent (from the approved and investigational small molecule collection), at varying concentrations, on the viability of each of the SCLC lines was examined. Results: Based on single agent activity in the primary screen of 67 SCLC lines, ~180 compounds from the Approved and Investigational Oncology Agent libraries were examined in the combination screen. The third agents were tested from a maximum concentration corresponding to the clinical CMax concentration with 3-fold serial dilutions over 8 dilutions. The in vitro screening data were analyzed using two methods: the Combination Index Method and the Bliss Independence model. Conclusions: The goal was to identify compounds producing greater-thanadditive cytotoxicity in the majority of SCLC lines. The results suggest that compounds targeting BCL2 and the JAK pathway improve the efficacy of the triplet across a number of cell lines. Combined with the gene expression and miRNA expression data for the lines, the data obtained will help identify novel therapeutic combinations, may help in matching patients with treatments and may identify novel molecular targets in SCLC. Funded by NCI Contract No. HHSN261200800001E. This research was supported in part by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. 79 POSTER (Board P073) Quantitative proteomics as a tool to identify resistance mechanisms in erlotinib-resistant subclones of the non-small cell lung cancer cell line HCC827 K. Jacobsen1 , R.R. Lund1 , H.C. Beck2 , H.J. Ditzel1 . 1 Molecular medicine, Cancer & Inflammation, Odense, Denmark; 2 Center for Clinical Proteomics, Clinical Biochemistry and Pharmacology, Odense, Denmark Background: Erlotinib (Tarceva® , Roche) has significantly changed the treatment of non-small cell lung cancer (NSCLC) as approximately 70% of patients show significant tumor regression when treated. However, all patients relapse due to development of acquired resistance, which in 43−50% of cases are caused by a secondary mutation (T790M) in EGFR. Importantly, a majority of resistance cases are still unexplained. Our aim is to identify novel resistance mechanisms − and potentially new drug targets − in erlotinib-resistant subclones of the NSCLC cell line HCC827. Materials and Methods: We established 3 erlotinib-resistant subclones (resistant to 10, 20, 30 mM erlotinib, respectively), and performed comparative quantitative proteomic analysis of these and the parental HCC827 cell line. The resistant subclones were examined both in absence and presence of erlotinib, and in biological triplicates on a Q-Exactive mass spectrometer. Only proteins identified with minimum 2 unique peptides and in minimum 2 of 3 replicates were used for further analysis. Results: Importantly, the resistant clones did not acquire the T790M mutation or other EGFR or KRAS mutations, potentiating the identification of novel resistance mechanisms in these subclones. We identified 2875 cytoplasmic proteins present in all 4 cell lines. Of these 87, 56 and 23 were upregulated >1.5 fold; and 117, 72 and 32 were downregulated >1.5 fold, respectively, in the 3 resistant clones compared to the parental cell line. By network analysis, we found cell survival, proliferation and migration to be induced, and apoptosis and adhesion to be repressed across the 3 resistant clones vs the parental cell line. Furthermore, networks involved in DNA repair and adherens junction signaling were inconsistently repressed in the 3 cell lines. More specifically, EGFR was consistently downregulated, indicating a bypass signaling mechanism to achieve resistance, and mitogen-activated protein kinase 1 (MAPK1) and synovial apoptosis inhibitor 1 (SYNV1) were upregulated. Conclusions: In conclusion, cancer-related networks such as proliferation and apoptosis were found to be regulated, supporting the validity of the model. EGFR was consistently downregulated and MAPK1 activated, indicating a bypass resistance mechanism, likely leading to activation of downstream proteins obviating EGFR. Generally, the overlap of regulated proteins between the 3 subclones was low, indicating the subclones have become resistant by different mechanisms.

Poster Session – Drug Resistance and Modifiers 80 POSTER (Board P074) Determination of an oxidative stress gene signature in inflammatory breast cancer patient tumors and development of a novel redox modulatory strategy in overcoming chemotherapy resistance and mediating anti-tumor efficacy G.R. Devi1 , J.L. Allensworth2 , M. Evans2 , N. Ueno3 , D. McDonnell4 , F. Bertucci5 , S. Van Laere6 . 1 Duke University, Surgery and Duke Cancer Institute, Durham NC, USA; 2 Duke University, Surgery, Durham NC, USA; 3 University of Texas MD Anderson, Oncology, Houston, USA; 4 Duke University, Pharmacology, Durham NC, USA; 5 Institut Paoli-Calmettes, Oncology, Marsellie, France; 6 General Hospital Sint-Augustinus, Oncology, Antwerp, Belgium Background: Cancer cells often have increased levels of reactive oxygen species (ROS); however, acquisition of redox adaptive mechanisms allows for evasion of ROS-mediated cell death. Chemotherapies including the anthracyclines, taxanes, alkylating agents, platinum compounds as well as radiation therapy all rely heavily on the induction of oxidative stress-induced apoptosis for their anti-tumor activities; thus, redox adaptation can confer resistance to many breast cancer therapies. Inflammatory breast cancer (IBC) is an advanced and distinct breast cancer subtype characterized by high rates of residual disease and regional recurrence despite advances in multimodality treatment. We hypothesize that the poor response of IBC tumors to common treatment strategies, many of which rely heavily on ROS induction, is the result of redox adaptation. Experimental Design: Metagene analysis was conducted to identify an oxidative stress response (OSR) gene signature in IBC cells treated with an acute dose of a strong ROS inducer and then allowed to recover. This OSR signature was applied to normal breast samples, pre-treatment IBC and nonIBC patient tumor samples to assess their oxidative stress response (OSR). A dithiocarbamate, disulfiram (DSF), currently approved as an antialcoholism drug was applied to therapeutically resistant IBC cells alone or in combination with copper (Cu); effects on viability, antioxidants, survival factors, and tumor progression were assessed. Results: Analysis of patient samples using the unique OSR metagene generated revealed significantly higher OSR scores in IBC tumor samples compared to normal or non-IBC tissue. In addition, we have identified a novel mechanism of DSF to act as a Cu ionophore in a Cu-transporter Ctr1-independent manner, target the Cu signaling redox axis in inducing oxidative stress-mediated apoptosis. This resulted in a potent inhibition of NF-úB signaling, aldehyde dehydrogenase activity, 3D mammosphere formation, in vivo orthotopic mammary tumor growth in murine IBC models and in overcoming chemotherapy resistance. Conclusions: These results indicate that IBC tumors are highly redox adapted, which may render them resistant to ROS-inducing therapies. DSF, through redox modulation, may be a useful approach to enhance chemoand/or radio-sensitivity for advanced BC subtypes where therapeutic resistance is a major challenge to durable responses to current standard of care. 81 POSTER (Board P075) Sensitization of triple-negative breast cancer to PI3K inhibition by cotargeting IGF1R K. de Lint1 , J.B. Poell1 , J. Vidal Rodriguez1 , H. Soueidan1 , W. Nijkamp1 , L.F. Wessels1 , R.L. Beijersbergen1 . 1 Netherlands Cancer Institute − Antoni van Leeuwenhoek, Molecular Carcinogenesis, Amsterdam, Netherlands Background: Targeted therapies have proven invaluable in the treatment of breast cancer, as exemplified by tamoxifen treatment for hormone receptorpositive tumors and trastuzumab treatment for HER2-positive tumors. In contrast, a subset of breast cancer negative for these markers (triplenegative breast cancer) has met very limited success with pathwaytargeted therapies. A large fraction of triple negative breast cancers depend on the PI3K pathway for proliferation and survival, however, inhibition of PI3K alone generally has limited clinical benefit. It is of interest to identify potential combination therapies that can enhance the response and thereby improve clinical outcome for triple-negative breast cancer. Materials and Methods: We have analyzed the response of a panel of triple-negative breast cancer cell lines to the PI3K inhibitor GDC-0941. We have generated a breast cancer cell line resistant to PI3K inhibition due to an adaptive response. We have applied large-scale pooled shRNA screening for the identification of genes required for cell proliferation under PI3K inhibition in the adapted triple-negative cell line. Results: We have identified IGF1R as crucial component of the adaptive response to PI3K inhibition. Knock-down of IGF1R potently restores the sensitivity of these cells to the PI3K inhibitor GDC-0941. Pharmacological inhibition of IGF1R prevents colony outgrowth of adapted cells. In treatment-naive cells IGF1R inhibition sensitizes cells to PI3K inhibition and induces apoptosis. In a subset of triple-negative breast cancer cell lines,

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IGF1R inhibition shifts the dose–response curve of GDC-041 up to 5-fold. In particular we find that breast cancer cell lines with high expression of IGF2-processing and signaling genes are responsive to the combination of PI3K and IGF1R inhibitors, whereas high expression of ERBB3 or mutated RAS are associated with a limited benefit of IGF1R inhibition. Conclusions: Our results suggest that combination treatment with PI3K and IGF1R inhibitors may prove beneficial over monotherapy in a subset of triple-negative breast cancers. 82 POSTER (Board P076) Regorafenib resistance in colorectal carcinoma is associated with enhanced expression of type II interleukin 1 receptor and reversed by MEK/ERK inhibitor A.C. Mar1 , C.H. Chu2 , C.W. Shiau3 , T.C. Lee1 . 1 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan; 2 Institute of Pharmacology, National Yang-Ming University, Taipei, Taiwan; 3 Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei, Taiwan Targeted therapy initially shows promising tumor regression but the development of drug resistance quickly results in failure of treatment. Among a batch of colorectal cancer (CRC) cell lines, we have preliminarily found that the expression levels of type II interleukin 1 receptor (IL1R2), an IL-1 decoy receptor, were closely associated with Regorafenib resistance. In addition, IL1R2 expression was associated with poor prognosis of patients with CRC cancer. Regorafenib was the newly approved multikinase inhibitor used for treatment of late-stage metastatic CRC by US FDA. In this study, we conducted experiments to elucidate the mechanism underlying which IL1R2 is involved in Regorafenib resistance. We first demonstrated that silencing of IL1R2 in HT29 cells overcame its resistance to Regorafenib, whereas ectopic expression of IL1R2 in HCT116 cells reduced its sensitivity to Regorafenib in both in vitro and in vivo systems. In addition, enhanced expression of IL1R2 was observed in Regorafenib-resistant DLD-1 colon cancer cells (DLD-1-R), which were established by growing DLD-1 cells in the presence of Regorafenib. In IL1R2 overexpressing cells, such as HT29, IL1R2-overexpressing HCT116, and DLD-1-R cells, Regorafenib treatment significantly resulted in activation of MEK/ERK signaling, which is crucial for survival. We also revealed that increased p-ERK levels in Regorafenib treated IL1R2overexpressing cells were likely due to decreased expression of MKP-3, one of the phosphatases of ERK. Furthermore, pretreatment of HT29, IL1R2-overexpressing HCT116, and DLD-1-R cells with MEK/ERK inhibitor U0126 significantly reversed their Regorafenib resistance in in vitro and in vivo systems. Taken together, our present study suggested that enhanced IL1R2 plays certain roles on Regorafenib resistance and the combination of Regorafenib and MEK/ERK inhibitor is a rationale regime to overcome Regorafenib resistance in CRC patients. 83 POSTER (Board P077) Acquired resistance to BET bromodomain inhibitors is associated with modulation of the apoptotic signaling network P. Sandy1 , S. Nerle1 , A. Conery2 , C. Hatton3 , B. Bryant3 , R. Sims2 , E. Normant1 . 1 Constellation Pharmaceuticals, Pharmacology, Cambridge, USA; 2 Constellation Pharmaceuticals, Biology, Cambridge, USA; 3 Constellation Pharmaceuticals, Bioinformatics, Cambridge, USA Background: Bromodomain and extraterminal (BET) family proteins function as chromatin readers that recognize and bind acetylated lysine residues on histones and play an essential role in the regulation of transcription of key oncogenes, including c-Myc and Bcl-2. Potent and selective small molecule inhibitors of BET bromodomains (BETi) induce growth arrest and apoptosis in a wide range of tumor types, in both in vitro and in vivo models, and are currently being evaluated in Phase I clinical trials. In this study we sought to model acquired resistance to long term BETi treatment and elucidate the underlying mechanisms in different tumor types. Materials and Methods: We used two orthogonal approaches to generate cell lines with reduced phenotypic sensitivity to BET bromodomain inhibition. In one, A375 melanoma cells were treated in culture with a BETi at a concentration where the majority of cells were eliminated by apoptosis. After several months, colonies able to proliferate in the presence of the inhibitor were obtained. In parallel experiments, mice inoculated with MV4−11 acute myeloid leukemia cells were dosed with a BETi at its maximum tolerated dose and the surviving tumors were serially transplanted into similarly treated recipient animals. Two BETi-resistant clones maintained decreased sensitivity to BETi in vitro when compared to parental cells in a 72-hour viability assay. Results: Detailed phenotypic analysis revealed an attenuated apoptotic response to high-dose BETi in the resistant clones as compared to parental cells. Transcriptional profiling and proteomic analysis revealed altered

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expression of Bcl-2 family members in both models, suggesting that two vastly different tumor types have converged upon the apoptotic signaling network as a mechanism of modulating the phenotypic response to BET bromodomain inhibition. Functional experiments demonstrated that these transcriptional changes are relevant in dictating the response to BETi. Conclusions: As BETi advance toward clinical use, it becomes increasingly important to understand potential mechanisms of acquired resistance and use this information to identify predictive biomarkers for maximal therapeutic benefit in patients. We will discuss strategies for stratifying patients based on the activity of the apoptotic signaling network, and present data on the use of pharmacological inhibition of the apoptotic response as a way to modify the phenotypic response to BETi. 84 POSTER (Board P078) Src family kinase activation is a compensatory survival mechanism for acquired resistance to EGFR-TKIs in lung cancer cells M. Ono1 , K. Sonoda1 , K. Azuma2 , K. Watari1 , M. Molina3 , R. Rosell4 , M. Kuwano5 . 1 Kyushu University, Dept. of Pharmaceutical Oncology Graduate Sch. of Pharmaceutical Sci., Fukuoka, Japan; 2 Kurume University School of Medicine, Dept. of Internal Medicine Div. of Respirology Neurology and Rheumatology, Kurume, Japan; 3 Pangaea Biotech, Barcelona, Spain; 4 Catalan Institute of Oncology, Barcelona, Spain; 5 Kyushu University, Lab. of Molecular Cancer Biology, Fukuoka, Japan Background: Most NSCLC patients harboring activating EGFR mutations benefit from treatment with EGFR-TKIs, but the clinical efficacy of EGFRTKIs is limited by the appearance of tumor drug resistance. Multiple kinase inhibitors of EGFR family proteins have been developed to overcome such drug resistance. To develop further personalized therapeutics and drug resistance modifiers, we should understand how drug resistance to EGFRTKIs including multiple kinase inhibitors is acquired at molecular basis. In our present study, we present a novel finding that Src family kinase activation is alternatively responsible for acquired drug resistance to afatinib in lung cancer cells harboring activating EGFR mutation. Our afatinibresistant clones does not harbor T790M, Ras mutation, PTEN loss, mutant EGFR loss, IGFIR activation, BIM and others. Materials and Methods: We have established afatinib-resistant subclones from human lung cancer cell lines, HCC827, harboring activating EGFR mutations after stepwise exposure to afatinib. We have characterized biochemical properties of drug-resistant subclones as compared with their drug-sensitive counterparts by Western blot, RT-PCR and microarray analysis. Results: We characterized two independent afatinib-resistant subclones isolated from HCC827. (1) Afatinib-resistant subclones, BR1−8 and BR2−3, showed markedly decreased expression of EGFR, HER2, HER3, c-Met and PDGFRb as compared with HCC827, and Akt phosphorylation was highly resistant to afatinib; (2) the activation of Src family kinase (SFK) was augmented in resistant subclones, and combination of afatinib with c-Src siRNA or with dasatinib suppressed cell growth and Akt phosphorylation; (3) among SFK, expression of Fyn was also upregulated in resistant subclones, and treatment with Fyn siRNA with afatinib partially suppressed Akt phosphorylation. In addition to above results, our recent relevant study showed that activation of Src along with enhanced expression of integrin b1, a2 and a5 was observed in erlotinib-resistant subclones derived from PC9. And also Src silencing or SFK inhibitor (dasatinib) restored erlotinib sensitivity through reduced activation of Akt. Conclusions: Together, our present study strongly support the novel finding that SFK activation including Src and/or Fyn could be one key mechanism responsible for acquired resistance to afatinib, and also that combination with SFK-targeted drug could be useful to overcome drug resistance to afatinib. 85 POSTER (Board P079) Wnt secretion is required to maintain Wnt activity in colon cancer O. Voloshanenko1 , G. Erdmann1 , T.D. Dubash2 , I. Augustin1 , M. Metzig1 , C.R. Ball2 , H. Glimm2 , R. Spang3 , M. Boutros1 . 1 Deutsches Krebsforschungszentrum, Division Signaling and Functional Genomics/B110, Heidelberg, Germany; 2 National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Department of Translational Oncology, Heidelberg, Germany; 3 University of Regensburg, Computational Diagnostics Group, Regensburg, Germany Aberrant activation of the canonical Wnt/b-catenin pathway occurs in almost all colorectal cancers (CRCs) and contributes to their growth, invasion and survival. It is widely believed that mutations in APC or b-catenin aberrantly activate Wnt signalling, independent of upstream ligands. Yet, several recent experimental findings have challenged this notion, for example

Poster Session – Drug Resistance and Modifiers secreted inhibitory proteins of the SFRP family are still able to attenuate Wnt signalling in colon cancer cells. Still the direct role of canonical Wnts in CRCs has never been studied. Surprisingly, we found that the Wnt cargo receptor Evi/Wls is highly expressed in CRCs as well as Wnt3 protein. We show that independent of the mutations in APC or b-catenin Wnt pathway activation is still responsive to stimulation by Wnt secretion and receptor-proximal signalling. Silencing of Evi leads to inhibition of clonal capacity of colon cancer cells and their proliferation. To identify pathways, which are synthetically lethal upon silenced Evi we performed the genome-targeted siRNA screen upon three different conditions − Wnt signalling high (shAPC), Wnt signalling normal for CRCs (shCtrl) and downregulated Wnt signalling (shEvi). We could not identify any pathways, which are synthetically lethal upon downregulation of APC but we found several pathways, which are synthetically lethal upon silencing of Evi. These results were confirmed by downregulation of Evi or b-catenin by siRNA and treatment with the drugs, which inhibits identified pathways. To conclude we showed that even in the presence of APC or b-catenin mutations CRCs are still dependent on upstream canonical Wnt signalling regulation. These findings potentially open new avenues for therapeutic interventions by targeting Wnt secretion via Evi/Wls. We identified several pathways, which are targeted by the drugs and are synthetically lethal upon inhibition of Wnt secretion in CRCs. 86 POSTER (Board P080) Influence of EGFR exon 19 mutation subtypes on survival outcomes in advanced stage Asian non-small cell lung cancer patients receiving TKI therapy B. Chowbay1 , O. Singh1 , N. Sutiman2 , D.S.W. Tan3 , W.T. Lim3 , E.H. Tan3 . 1 National Cancer Centre, Laboratory of Clinical Pharmacology Division of Medical Sciences, Singapore, Singapore; 2 The Academia SingHealth, Clinical Pharmacology Core, Singapore, Singapore; 3 National Cancer Centre, Department of Medical Oncology, Singapore, Singapore Background: Mutations in epidermal growth factor receptor (EGFR) gene have been shown to affect the treatment outcomes in patients. In this study we aimed to explore the influence of EGFR exon 19 mutation subtypes on survival outcomes in advanced stage Asian non-small cell lung cancer (NSCLC) patients receiving tyrosine-kinase inhibitor (TKI) treatment as 1st line therapy. Material and Methods: Of 937 Asian NSCLC patients, 148 patients with stage IIIB or IV, harboring EGFR exon 19 mutations and receiving TKI as 1st line treatment, were selected. The majority of the patients were of Chinese descent (83.1%), followed by Malays (10.8%), Indians (3.4%) and others (2.7%). The median age of patients was 60 years (range: 29−86 years). Kaplan–Meier analysis, log-rank test and Cox regression methods were implemented to evaluate the survival outcomes between groups Results: The EGFR exon 19 mutation subtype 15-nucleotide deletion (c.2236_2250del, ELREA) was observed most frequently (62.8%) followed by mixed insertion/substitution/deletion (23%), 18-nucleotide deletion (c.2240_2257del, 7.4%) and 15-nucleotide deletion (non-ELREA, 6.8%). Patients harboring exon 19 ELREA deletion and mixed insertion/substitution/deletion had significantly longer median overall survival (37.1 months), compared to those with c.2240_2257del [18 months; hazard ratio, 2.5; 95% CI (1.1−5.6); p = 0.027] and non-ELREA deletion [13 months; hazard ratio, 4.08; 95% CI (1.9−8.8); p < 0.001]. Similar trends were observed for progression free survival (PFS) outcomes. Patients harboring exon 19 ELREA deletion and mixed insertion/substitution/deletion had the longest median PFS (14.9 months) compared to those with c.2240_2257del [9.13 months; hazard ratio, 2.01; 95% CI (0.91−4.4); p = 0.08] and non-ELREA deletion [6.87 months; hazard ratio, 3.12; 95% CI (1.39–6.98); p = 0.006]. Conclusions: EGFR exon 19 mutation subtypes showed significantly different survival outcomes after 1st line TKI treatment in advanced stage NSCLC patients. Further studies are recommended in larger cohort of advanced stage NSCLC patients to improve the personalized therapy based on patients’ EGFR mutation status. 87 POSTER (Board P081) Critical difference in development of acquired resistance to MDM2 inhibitor SAR405838 in vitro and in vivo G. Hoffman-Luca1 , C.Y. Yang1 , J. Lu1 , D. Ziazadeh1 , D. McEachern1 , L. Debussche2 , S. Wang1 . 1 University of Michigan, Pharmacology, Ann Arbor MI, USA; 2 Sanofi, Oncology, Vitry/Seine, France SAR405838 is a potent and specific MDM2 inhibitor currently being evaluated in Phase I clinical trials for the treatment of human cancer as a single agent and in combination. In the present study, we investigated the acquired resistant mechanisms for SAR405838 using the SJSA-1

Poster Session – Drug Resistance and Modifiers osteosarcoma cell line in vitro and in vivo. The SJSA-1 cell line possesses an amplified MDM2 gene and wild-type p53 and is sensitive to SAR405838 and other MDM2 inhibitors in vitro and in vivo. In vitro treatment of the SJSA-1 cells with SAR405838 using two different treatment protocols resulted in acquired resistance to the drug. Analysis of these resistant cells showed that p53 is mutated in the DNA binding domain and cannot be activated by SAR405838. Treatment of mice bearing the parental SJSA-1 xenograft tumors with SAR405838 led to rapid tumor regression, but tumors eventually returned after treatment cessation. A number of cell lines were established by culturing the regrown tumors and, surprisingly, these sublines showed minimal loss of sensitivity to SAR405838 as compared to the parental SJSA-1 cell line. Consistently, analysis of thesesublines showed that p53 retains its wild-type status, with the exception of one subline, that harbored a single heterozygous mutation C176F. Computational modeling suggested that the p53 C176F mutant may still be capable of binding to target DNA to elicit gene transcription, which was confirmed by in vitro treatment with SAR405838. Xenografts of representative sublines lacking p53 C176F mutation obtained from regrown tumors treated with one-round SAR405838 were still responsive to SAR405838 treatment. Interestingly, after a second round of in vivo treatment with SAR405838, all sublines established from harvested tumors uniformly harbored a heterozygous C176F p53 mutation, which were still responsive to SAR405838, albeit with reduced sensitivity. Our study showed that the SJSA-1 tumor cells acquired profound resistance to MDM2 inhibitor SAR405838 in vitro and developed inactivating p53 mutations in its DNA binding domain. But in vivo, SJSA-1 tumor cells only acquired minimal or modest resistance when treated with either one- or two-rounds of SAR405838. Our study suggests that tumor cells respond differently to drug treatment under in vitro and in vivo environments and consequently develop different types of resistance. 88 POSTER (Board P082) Genome-wide drug sensitivity screens in haploid mouse embryonic stem cells S.J. Pettitt1 , D. Krastev1 , H. Pemberton1 , Y. Fontebasso1 , I. Bajrami1 , I. Kozarewa1 , J. Frankum1 , R. Rafiq1 , J. Campbell1 , R. Brough1 , A. Ashworth1 , C.J. Lord1 . 1 Institute of Cancer Research, Division of Breast Cancer, London, United Kingdom Background: We have screened a genome wide collection of mutants induced by transposon insertion in haploid cells with a variety of targeted agents and chemotherapies in order to find new genetic determinants of sensitivity. Materials and Methods: Haploid mouse embryonic stem cells were mutagenised with a piggyBac gene trap transposon. To enable accurate counting of mutants, a random 25-nt barcode was inserted into the transposon donor plasmid. This can be amplified in a minimal PCR step and the products analysed by next-generation sequencing, in a manner similar to pooled shRNA screens. Barcodes were associated with genomic insertion sites by inverse PCR. We generated a library of approximately 100,000 mutants and treated with DMSO (solvent control) or drug at SF50 dose. The mappings for barcodes showing significant change in abundance between treated and control arms were analysed and hits called based on the criteria of multiple insertions in a gene showing a phenotype. Results: In a screen for poly(ADP-ribose) polymerase (PARP) inhibitor sensitivity using the clinical agents olaparib and BMN 673, we detected known determinants of sensitivity including Brca2 and Cdk12. We identified loss of Ewsr1 as a novel determinant of PARP inhibitor sensitivity and showed that knockdown of EWSR1 in MCF7 breast cancer cells also sensitises to BMN 673. Since Ewing’s sarcoma cells with EWSR1-ETS fusions are reported to be PARP inhibitor sensitive, we are investigating a possible contribution of loss of wild type EWS function to this effect. We have also screened this library against 25 other drugs and will report our latest results. Conclusions: Pooled haploid mutant libraries can be used to identify genetic determinants of sensitivity at a genome-wide scale without prior assumptions of the mechanism. 89 POSTER (Board P083) Tie-2 regulates the stemness of prostate cancer cells K. Tang1 , M. Ling1 . 1 Institute of Health and Biomedical Innovation, Brisbane Qld, Australia Background: Prostate cancer (PCa) is the most commonly diagnosed male cancer in Western countries. Patients with advanced PCa are normally treated with hormone ablation therapy. This therapy is effective initially as PCa cells require androgen to grow and survive; however, the cancer cells eventually become androgen independent and develop metastatic, castration-resistant tumors. At this stage, chemotherapy and

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radiotherapy exhibit only small benefits. Ample evidence supports the idea that a rare population of cancer cells known as cancer stem cells (CSCs) is responsible for the development of treatment resistant and disease relapse. Unfortunately, little is known about the identity of these cells, making it difficult to target them. Here we reported that a rare population of PCa cells expresses the Tie-2 protein, a tyrosine kinase receptor that regulates the stemness and bone homing of hematopoietic stem cells. Material and Methods: Here, using fluorescence-activated cell sorting (FACS), we have isolated the Tie-2+ population from the PCa cell line PC-3. cDNA microarray analysis was then performed to characterize the gene expression profile of these cells. Meanwhile, cell adhesion assay was performed to examine the ability of the Tie-2+ cells to adhere to osteoblasts and endothelial cells. Furthermore, quiescent staining and drug sensitivity assay was carried out to examine whether Tie-2+ cells are more quiescent, and thus become resistant to chemotherapeutic drug. Finally, we have injected both Tie-2+ and Tie-2− PC-3 cells intracardiacly into the NOD-SCID mice to determine if Tie-2 expression promote prostate tumor metastasis under in vivo condition. Results: Characterization of Tie-2+ PCa cells revealed that these cells express higher level of prostate CSC markers when compared to the Tie-2− population. Meanwhile, Tie-2+ cells are highly adhesive to osteoblasts and endothelial cells, a characteristic necessary for the development of tumor metastasis. We also found that Tie-2+ cells are more quiescent and resistant to the chemotherapeutic drug cabazitaxel, further support that these cells possess CSC-like characteristics. More importantly, we found that Tie-2+ cells, but not the Tie-2− cells population, developed metastatic tumor in vivo. Conclusions: Our data suggested that Tie-2+ population represent the PCa stem cells population which play an importantly role in the development of drug resistance and prostate tumor metastasis. Thus, Tie-2 might be a novel therapeutic target for treatment of advanced PCa patients. 90 POSTER (Board P084) Tumor infiltrating leukocyte subpopulations as a biomarker of response and resistance to targeted therapy in patients with BRAF mutation-positive metastatic melanoma M.C. Kelley1 , D.B. Doxie2 , A.R. Greenplate2 , H. Crandall1 , J.A. Sosman3 , J.M. Irish2 . 1 Vanderbilt University, Surgical Oncology, Nashville TN, USA; 2 Vanderbilt University, Cancer Biology, Nashville TN, USA; 3 Vanderbilt University, Hematology-Oncology, Nashville TN, USA BRAF mutation-positive metastatic melanoma responds to BRAF and MEK targeted therapy, then progresses within 6−9 months. Many resistance mechanisms have been identified in tumor cells, but little is known about changes ocurring in the tumor microenvironment during resistance. Dysfunctional immune responses are linked to tumor progression, so we evaluated the tumor infiltrating leukocyte profile in melanoma metastases responding and progressing on targeted therapy. Samples were obtained during surgical excision of soft tissue metastases in a patient with BRAF V600E mutation-positive melanoma receiving therapy with a BRAF inhibitor (dabrafenib) and MEK inhibitor (trametinib). Responding lesions were defined by: stable reduction in tumor volume during therapy and the physical characteristics of the lesion. Novel high dimensional mass cytometry (CyToF) panels were used to characterize tumor and immune cell subsets. Machine learning tools (visNE) classified individual cells according to a multidimensional signature of 30+ proteins with roles in melanoma biology, immune regulation, and metastasis. Key biomarkers of melanoma and immune cell subsets included melanoma cell adhesion molecule (MCAM), NGFR, KIT, nestin, CD3, CD4, CD8, CD14, CD16, CD33, CD34, CD44, CD45, CD49F, MHC class II (HLA-DR), and chemokine receptors (CCRs & CXCRs) known to play a role in metastasis. Intracellular signaling (pERK, pAKT, pSTAT-1, 3, 5 and 6, pCREB), proliferation (Ki67) and apoptosis (cleaved caspease3) were also evaluated. Live cells were identified by length and DNA content and gated into melanoma cell (CD45−, MCAM+) and leukocyte (CD45+, MCAM−) populations. The abundance and phenotype of subpopulations of immune cells were compared in responding and progressing lesions. Responding lesions contained nearly 3 fold more T-cells than progressing lesions, and 63% of these cells were CD8+ effector T cells compared to 14% in progressing lesions. In contrast, progressing lesions contained almost 3 fold more MMHC-II expressing cells, including a nearly 2 fold increase in CD14−, CD33+, HLA-DR+ myeloid-derived suppressor cells (Table 1). In depth analysis of leukocyte subpopulations in additional melanoma metastases responding and progressing on therapy is underway. These preliminary findings confirm that maladaptive immune responses, including reduced CD8+ T-cell and increased myeloid subpopulations, are present in melanoma metastases progressing on BRAF targeted therapy. It is unclear if these changes occur in response to resistance developing within the tumor or are directly responsible for tumor progression. Further investigation of combinations of targeted therapy and immunotherapy

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Poster Session – Drug Resistance and Modifiers

are supported by these findings. Analysis of tumor infiltrating leukocyte subpopulations may be a useful biomarker for monitoring response to targeted therapy. Table 1. % CD45+ cells % MHC+ cells % CD3+ cells CD3+/MHC-II− CD3−/MHC-II+ CD14− CD14−/CD33+ CD8+ Responding 74* Progressing 27*

26* 73*

41 49

3.8 6.2

63* 14*

*p < 0.05.

91 POSTER (Board P085) 4E-BP1 expression levels determine sensitivity of triple negative breast cancer cells to mTOR inhibitors K. Jastrzebski1 , B. Thijssen1 , J. Vidal Rodriguez1 , K. de Lint1 , C. Lieftink1 , L.F. Wessels1 , R.L. Beijersbergen1 . 1 NKI-AvL, Molecular Carcinogenesis, Amsterdam, Netherlands Background: In contrast to other breast cancer subtypes, triple negative breast cancers are characterized by an absence of recurrent genetic alterations. However, alterations in the PI3K pathway are frequently observed, either through mutation or amplification of PIK3CA or the loss of the negative regulator PTEN. As a result, inhibitors of this pathway, including inhibitors of mTOR, the downstream target of the PI3K pathway, have been explored in the clinic. It has been found that mutational activation of PI3K represents an important determinant of sensitivity to mTOR inhibitors. Nevertheless, a number of PI3K wild-type cell lines also show sensitivity to these inhibitors, although the regulators of this response are still unknown. We set out to find such modulators of response to mTOR inhibitors in a panel of triple negative breast cancer cell lines initially using high throughput approaches. Material and Methods: We determined the sensitivity of a panel of triple negative breast cancer cell lines to AZD8055 (mTOR inhibitor) and BEZ235 (mTOR/PI3K dual inhibitor). This data was then used together with additional datasets generated for this panel, including RNA expression (by RNA-seq), (phospho-)protein levels (by Reverse Phase Protein Arrays) and mutational status (by post-capture DNA-seq), to carry out regression analysis to identify features correlated with response. Results: We found that the protein expression levels of 4E-BP1, a downstream target of mTOR involved in regulating cap-dependent protein synthesis, correlated with response to both inhibitors. In poorly responding cell lines expressing low levels of 4E-BP1, overexpression of the protein increased sensitivity. Conversely, in sensitive cell lines expressing high levels of 4E-BP1, knock-down of 4E-BP1 led to reduced sensitivity to mTOR inhibition. Conclusions: These results indicate that effective inhibition of protein synthesis downstream of PI3K/mTOR is an important determinant of mTOR inhibitor activity. Given that approximately 20% of breast cancer tumors annotated in The Cancer Genome Atlas show an amplification of the 4E-BP1 locus, such patients would likely benefit from mTOR inhibitor treatment. Furthermore, we hypothesize that mTOR inhibitor response can be improved even in patients expressing low 4E-BP1 by co-treating with agents that induce its expression. To this end, we are currently screening for genes and drugs that modulate 4E-BP1 levels. 92 POSTER (Board P086) Role of ERK nuclear translocation in cisplatin-sensitive and -resistant ovarian cancer cells S. Dilruba1 , G.V. Kalayda1 , U. Jaehde1 . 1 Institute of Pharmacy, Clinical Pharmacy, Bonn, Germany Background: The clinical use of the anti-cancer drug cisplatin is limited due to the rapid development of resistance against this drug. However, the mechanisms of resistance are not completely understood yet. Extracellular signal regulated kinase1/2 (ERK1/2) is activated in response to cisplatin treatment in ovarian cancer cells. Activated ERK1/2 induces the expression of survival genes upon translocation to nucleus, which may confer resistance to this drug. Objectives: This projects aims at investigating the importance of ERK1/2 nuclear translocation for cisplatin resistance in ovarian cancer cells. Methods: EFO27 and A2780 ovarian carcinoma cells and their corresponding cisplatin-resistant variants (EFO27/CDDP and A2780cis) were investigated. The cytotoxicity of cisplatin was estimated using the MTT assay. Phosphorylated ERK1/2 translocation to the nucleus was confirmed by immunofluorescence staining. Selected proteins were overexpressed by lipofectamine-mediated transfection. Phosphorylation

of ERK1 (T202/Y204) and ERK2 (T185/Y187) and transfected protein expression were detected by Western blot. Results: Cytotoxicity of cisplatin was assessed with and without inhibition of ERK1/2 by U0126. Whereas U0126 increased cisplatin sensitivity of EFO27 and EFO27/CDDP cells, the opposite effect was found in A2780 and A2780cis cells. Translocation of ERK1/2 to the nucleus in response to cisplatin was detected in both EFO27 cells, but not in A2780 cells. Further, PEA-15, a small protein which inhibits ERK1/2 translocation to the nucleus was used to investigate the relevance of ERK1/2 cytoplasmic sequestration for cisplatin resistance. PEA-15 was overexpressed in A2780cis cells and the cytotoxicity of cisplatin in transfected cells was measured. The pEC50 values obtained were 5.15±0.21, 5.04±0.15, 5.00±0.09 in A2780cis, A2780cis-HA (empty vector), and A2780cis-PEA-15wt (n = 4−6) respectively. Conclusion: ERK1/2 nuclear translocation differs between the ovarian cancer cell lines. Further studies will reveal the most suitable cell line pair to study the association between ERK1/2 nuclear translocation and cisplatin resistance. 93 POSTER (Board P087) A stress induced early innate response causes multi-drug tolerance in melanoma D.R. Menon1 , S. Das2 , C. Krepler3 , A. Vultur3 , B. Rinner4 , S. Schauer2 , K. Kashofer2 , K. Wagner4 , G. Zhang3 , E. Bonyadi Rad5 , H.P. Soyer1 , B. Gabrielli6 , R. Somasundaram3 , G. Hoefler2 , M. Herlyn3 , H. Schaider1 . 1 Translational Research Institute, Dermatology Research Centre, Woolloongabba, Australia; 2 Medical University of Graz, Institute of Pathology, Graz, Austria; 3 The Wistar Institute, Molecular Oncogenesis, Philadelphia, USA; 4 Medical University of Graz, Center for Medical Research, Graz, Austria; 5 Medical University of Graz, Dermatology, Graz, Austria; 6 Translational Research Institute, Diamantina Institute, Woolloongabba, Australia Background: Acquired drug resistance constitutes a major challenge for effective cancer therapies with melanoma being no exception. The dynamics of early drug resistance leading to permanent resistance are poorly understood. Material and Methods: Melanoma cell lines were exposed to molecular targeted inhibitors like BRAF or MEK inhibitors or chemotherapy at sublethal drug concentrations for over 90 days. Alternatively melanoma cells were exposed to hypoxic conditions or low glucose media. Cells surviving drug exposure, hypoxia or nutrient starvation were monitored for the expression of CD271, ALDH activity, differentiation markers, ABCB5, chromatin remodeling, histone demethylases and markers for angiogenesis to characterize cells exposed for a minimum of 12 days. Further gene expression analyses, RPPA analyses and in vivo tumorigenicity were performed in these cells. Results: Drug exposure, hypoxia or nutrient starvation leads to an early innate cell response in melanoma cells resulting in multi-drug resistance, termed induced drug tolerant cells (IDTC). Transition into the IDTC state seems to be an inherent stress reaction for survival towards unfavorable environmental conditions or drug exposure independent of any subpopulation or cancer stem cell. The response comprises chromatin remodeling, activation of signaling cascades, and markers proposed to be stem cell markers with higher angiogenic potential and tumorigenicity. These changes are characterized by a common increase in CD271 expression concomitantly with loss of differentiation markers such as melan-A and tyrosinase, enhanced ALDH activity and upregulation of histone demethylases. Accordingly, IDTCs show a loss of H3K4me3, H3K27me3 and gain of H3K9me3 suggesting activation and repression of differential genes. Drug holidays at the IDTC state allow for reversion into parental cells re-sensitizing them to the drug they were primarily exposed to. However, upon continuous drug exposure IDTCs eventually transform into permanent and irreversible drug resistant cells. Knockdown of CD271 or KDM5B decreases transition into the IDTC state substantially but does not prevent it. Conclusions: Our results suggest a phenotypic shift of parental cells to the induced drug tolerant cell (IDTC) state irrespective of a given subpopulation thus not representing cancer stem cells. Targeting IDTCs would be crucial for sustainable disease management and prevention of acquired drug resistance.

Poster Session – Drug Screening 94 POSTER (Board P088) Elucidating mechanisms of resistance to FGFR inhibitors in endometrial cancer L. Packer1 , S. Byron2 , C. Mahon1 , D. Loch3 , A. Wortmann1 , K. Nones4 , S. Grimmond4 , J. Pearson5 , N. Waddell4 , P. Pollock1 . 1 Translational Research Institute, Queensland University of Technology, Brisbane Qld, Australia; 2 Translational Genomics Research Institute, Phoenix, USA; 3 Queensland University of Technology, Brisbane Qld, Australia; 4 Institute of Molecular Biology, University of Queensland, Brisbane Qld, Australia; 5 QIMR Berghofer Medical Research Institute, Brisbane Qld, Australia Background: Fibroblast Growth Factor Receptor 2 (FGFR2) is mutated in 10−20% of endometrial cancer patients and is associated with a higher risk of recurrence. Preclinical studies by our lab and others have demonstrated FGFR2 inhibition is a viable therapeutic strategy in FGFR2mutant endometrial cancer. A significant clinical issue that we aim to address is that of acquired resistance to anti-FGFR therapies in the context of endometrial cancer. Materials and Methods: The endometrial cancer cell lines AN3CA and JHUEM-2 express mutant FGFR2 and are sensitive to FGFR inhibitors. We have generated subclones of AN3CA and JHUEM-2 cells resistant to the FGFR inhibitors PD173074 and NVP-BGJ398, respectively. Phosphoprotein arrays, gene expression profiling, exome sequencing and SNP analysis were performed to identify genes and signalling pathways that mediate resistance to FGFR inhibitors. Results: Loss of FGFR2 protein expression was identified in one resistant subclone, but the remaining subclones retain FGFR2 expression. Upregulation of downstream survival pathways such as pERK and pAKT was observed in some of the JHUEM-2resistant and AN3CAresistant subclones. To further characterise these subclones we are currently analysing data from exome sequencing, expression arrays and copy number analysis, to determine the molecular mechanisms of resistance in these cell lines. Like most endometrial cancer cells, AN3CA and JHUEM-2 cells have constitutive activation of the PI3K pathway, due to mutations in PIK3R1, PTEN or PIK3CA. Our results show that combined inhibition of the PI3K pathway and the MEK/ERK pathway by trametinib or an FGFR inhibitor causes cell death in these cells. Conclusions: This research will provide insight into possible combination therapies that may combat drug resistance in FGFR-mutant endometrial cancer.

Drug Screening 95 POSTER (Board P089) Identification of synthetic lethality compounds from natural products for cancers K.W. Ng1 , K.S. Lee1 , V. Patel2 , E. Sundaramoorthy3 , N. Ayoub3 , X. Su3 , A. Venkitaraman3 , S.H. Teo4 . 1 Cancer Research Initiatives Foundation, Drug Discovery Research Group, Selangor, Malaysia; 2 Cancer Research Initiatives Foundation, Nasopharyngeal Cancer Research Group, Selangor, Malaysia; 3 University of Cambridge, The Medical Research Council Cancer Cell Unit, Cambridge, United Kingdom; 4 Cancer Research Initiatives Foundation, Breast Cancer Research Group, Selangor, Malaysia Background: Many cancers, such as basal breast and serous ovarian cancers, are characterized by genomic instability, through the loss of function of TP53 and BRCA2. Until recently, there have been limited successes in developing efficient targeted therapies for these forms of solid cancers. In 2005, two research groups independently demonstrated that inhibition of Poly-(ADP-ribose)-polymerase (PARP) can selectively kill BRCA2-deficient cancer cells through synthetic lethality. However, no study has hitherto explored natural products that are synthetic lethal to the BRCA2-deficient cancers. Here, we describe the development of a cell-based assay to search for natural products with synthetic lethality properties. Methods: We have developed a cell-based assay using an isogenic pair of BRCA2-proficient and deficient cell lines. The sensitivity of the assay was evaluated using Olaparib, Mitomycin C and Irinotecan, either in isolation or in combination as ‘spike-in’ controls with natural product extracts, to investigate their inhibitory activities in the presence of potential interfering compounds. The assay was then used to screen for our in-house natural product library of plant, microbial and fungus extracts. Results: We report that this assay format is able to detect the selective cytotoxic effects of Mitomycin C, Olaparib and Irinotecan with 22, 17 and 16 fold difference of EC50 in BRCA proficient cells compared to that of BRCA deficient cells. When added as spike-in controls with natural product

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extracts, the detection limits were 1% (w/w) for Olaparib and Irinotecan and 0.1% (w/w) for Mitomycin C. Screening of our in-house library of 454 plant, 305 microbial and 62 fungus extracts, identified 2 plants, 1microbial, 6 fungus extracts with EC50 less than 5 mg/mL on the BRCA2 deficient cells and fold difference of >6. Conclusion: Our data represents the first systematic identification of potential synthetic lethal natural products using a cell based assay. We anticipate from our on-going work to unravel and identify the key chemical components of the 9 extracts conferring synthetic lethality properties. 96 POSTER (Board P090) Evaluation of hormone therapies in a panel of breast PDX models: Relevance of ER status on sensitivity to letrozole and tamoxifen M.J. Wick1 , T. Vaught1 , L. Gamez1 , J. Meade1 , A. Diaz1 , K.P. Papadopoulos1 , D.W. Rasco1 , A. Patnaik1 , M. Beeram2 , A. Lang2 , A.W. Tolcher1 . 1 South Texas Accelerated Research Therapeutics, (START), San Antonio TX, USA; 2 START Center for Cancer Care, San Antonio TX, USA Background: Breast cancer classification and treatment strategy is currently based on immunohistochemical staining and pathological scoring for the estrogen (ER), progesterone (PR) and ERBB2 (HER2) receptors. Based on threshold ER scores, breast cancers are designated hormonereceptor positive and treated with endocrine therapy including ER antagonists and aromatase inhibitors (AI). However, whether breast cancers with ER scores below threshold would benefit from endocrine therapy is unclear. To better understand the role of endocrine therapy in breast cancer, we stratified a panel of breast PDX models using ER percentage (ER%): ER<10% and ER10%. Drug sensitivity studies were performed evaluating models response to letrozole and tamoxifen and efficacy compared to the ER% for each model. Methods: Breast PDX models were developed in immune-deficient mice from primary or metastatic patient tissue and established models confirmed by histologic comparative analysis and linked with patient treatment and outcome data. Each model was assigned an ER% from the clinical biopsy score at the time of sample collection and engraftment. Drug sensitivity studies were performed evaluating models response to chronic dosing of letrozole or tamoxifen; study endpoints included tumor volume and time from treatment initiation as study endpoints. T/C values, growth delay and regressions were reported for each model. Results: Letrozole treatment resulted in statistically significant (p < 0.05) tumor growth inhibition in several ER<10% models including ST518 and ST1077 (ER = 0%) while several ER  10% models including ST565 (ER = 100%), developed from a chemo na¨ıve patient, was insensitive to therapy. Letrozole tested in HER2 (3+) models was inactive in ST1339 (ER = 0%) model but efficacious in ST340 (ER = 20%). Activity of tamoxifen was similar to letrozole in evaluated models. Conclusion: We evaluated a panel of breast patient-derived xenografts and compared model sensitivity to letrozole and tamoxifen with ER% in each model. Overall sensitivity of either agent did not correlate with higher ER% values, suggesting patients with ER% below threshold may benefit from these therapies in combination with novel targeted agents or approved standards of care. 97 POSTER (Board P091) Utilization of low passage adenoid cystic carcinoma PDX models to identify novel combination therapies M.J. Wick1 , J. Meade1 , T. Vaught1 , M. Nehls1 , J. Flores1 , J. Kaufman2 , A.W. Tolcher1 , D.W. Rasco1 , A. Patnaik1 , C.A. Moskaluk3 , K.P. Papadopoulos1 . 1 South Texas Accelerated Research Therapeutics, (START), San Antonio TX, USA; 2 Adenoid Cystic Carcinoma Research Foundation, (ACCRF), Needham MA, USA; 3 University of Virginia, Charlottesville VA, USA Background: Adenoid Cystic Carcinoma (ACC) is an uncommon cancer of the head and neck which typically originates in the salivary glands with limited treatment options and once metastatic no approved standard of care. To identify potentially useful therapies we utilize a panel of low passage ACC xenograft models for in vivo screening of FDA-approved and investigational therapies with potential benefit towards ACC. Based on these results, combination treatments are evaluated looking for additive or synergistic effects. Previously we reported activity of the multi-tyrosine kinase inhibitor dovitinib in models of ACC. However, whether combination with other targeted therapies would result in additive or synergistic activity was unclear. To test this we evaluated dovitinib in combination with two classes of agents with anecdotal clinical activity, an HDAC inhibitor and IGF-1R inhibitor; for these studies we utilized vorinostat and linsitinib. RNA and DNA-based sequence analysis was also performed on the ACC panel models to correlate sensitivity or resistance with molecular markers.

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Poster Session – Drug Screening

Methods: Low passage ACC models were established in immune-deficient mice from primary or metastatic patient tissue and once established were confirmed by histologic comparative analysis. Drug sensitivity studies were performed evaluating dovitinib, vorinostat and linsitinib. Study endpoints included tumor volume and time from treatment initiation with tumor growth inhibition, delay and regression reported at study completion. Results: To date over eighty FDA-approved and investigational therapies have been evaluated. In the current study additive effects were reported in dovitinib/vorinostat and dovitinib/linsitinib combination groups compared with single agents. Statistically significant (p < 0.05) tumor growth inhibition was reported in two models comparing the combination to single agents using a submaximal dovitinib concentration. Sequence based analysis is currently underway to correlate sensitivity and resistance to these and other therapies. Conclusion: Low passage ACC models have been used to identify agents potentially useful in the treatment of ACC. Currently we have identified preclinical combination benefit with dovitinib/vorinostat and dovitinib/ linsitinib combinations and RNA and DNA sequence based analysis is currently underway to correlate sensitivity and resistance to these and other therapies.

mechanisms of action produced supra-additive activity with the same compounds in the ‘Combo Set’. Examples of these interactions included: (1) ‘Test Agents’ inhibiting DNA damage repair checkpoints (p53, ATR, or GSK3-b) that all produced similar patterns of combination activity when combined with ‘Combo Set’ alkylating agents, and to a lesser extent with Topoisomerase poisons; (2) Wee 1 and Chk1 inhibitors that demonstrated optimal therapeutic benefit when combined with gemcitabine; this result was striking because gemcitabine, a member of the ‘Combo Set’, was an infrequent combination hit with other ‘Test Agents’; (3) EGFR inhibitors, when combined with dacarbazine, were all supra-additive in a single cell line: PC-3. We also found multiple antagonistic interactions, including: (1) Src inhibitors were antagonistic with ATRA and oxaliplatin in all three tested cell lines; (2) HDAC inhibitors were antagonistic with many antimetabolites in the ‘Combo Set‘. These results may aid in selection of combinations for further preclinical investigation, leading to clinical studies.

98 POSTER (Board P092) Synergistic inhibition of HER2 positive breast cancer by triptolide and lapatinib

J. Sakoff1 , J. Gilbert2 , A. McCluskey3 . 1 Calvary Mater Newcastle, Medical Oncology, Newcastle NSW, Australia; 2 Calvary Mater Newcastle Hospital, Medical Oncology, Newcastle NSW, Australia; 3 The University of Newcastle, Chemistry, Newcastle NSW, Australia

P. Chalugun1 , J.S. Shim1 , P. Korangath1 , S. Sukumar1 , J.O. Liu1 . 1 Johns Hopkins University, Pharmacology, Baltimore MD, USA HER2 positive breast cancers are poorly differentiated and have a higher potential for metastasis. Although trastuzumab and lapatinib have been used for the treatment of HER2 positive breast cancer, resistance of both drugs has dampened the long-term benefit of those drugs. As cancer has multiple genetic aberrations and can readily develop resistance to individual drugs with specific targets, we employed synergistic drug combination as a strategy to overcome drug resistance and to enhance to efficacy of existing drugs. From a screen of the Johns Hopkins Drug Library, we identified triptolide as a strong synergistic hit with lapatnib in HCC1954, a drug-resistant HER2-positive breast cancer cell line. The combination of lapatinib and non-toxic doses of triptolide synergistically inhibited the HER2 signaling pathway. We found that lapatinib increased both mRNA expression and the half-life of HER2 protein, whereas triptolide countered the effects of lapatinib by downregulating the expression of HER2, leading to the synergistic inhibition of HER2 signaling pathway. The synergy in cell proliferation was observed in all HER2-positive breast cancer cell lines regardless of drug resistance status, but was absent in HER2-negative ones. In mouse xenograft models, the combination of lapatinib and triptolide at their minimum effective doses almost completely inhibited the growth of the drug-resistant HER2-positive breast cancer cells. Together, these findings suggest that the combination of triptolide and lapatinib could be a promising new therapeutic regimen for drug-resistant HER2-positive breast cencer. 99 POSTER (Board P093) Combination screening of investigational oncology agents S. Holbeck1 , J.M. Collins2 , J.D. Doroshow3 . 1 National Cancer Institute, Rockville, USA; 2 National Cancer Institute, DCTD, Rockville, USA; 3 National Cancer Institute, DCTD, Bethesda, USA Treatment with combinations of drugs forms the backbone of most cancer treatment regimens. With the advent of newer therapies targeting specific drivers or molecular defects present in tumor cells come new opportunities and challenges in developing drug combinations. Many molecularly targeted drugs elicit profound responses in patients as single agents, but it is common for resistance to arise in a matter of months. There is thus an urgent need for strategies to develop combinations that incorporate these new agents. We have previously reported on our systematic approach to the screening of all pair-wise combinations of approved small molecule oncology drugs (~5000 drug pairs) in a panel of 60 human tumor cell lines, the NCI-60. Our combination screening has now been expanded to include ~9000 drug pairs, including many investigational agents, initially screened against a small panel of cell lines (3−5 lines). With some overlap between the 2 screens, a total of 12,660 unique drug pairs has been tested. In the current screen, we established a ‘Combo Set’ panel of ~70 approved and investigational drugs. Agents of interest (‘Test Agents’) were then evaluated in combination with each of the drugs in the ‘Combo Set’ in each of 3−5 cell lines. Additional testing in the NCI-60, and/or xenografts, was performed for 55 combinations with greater than additive activity in the smaller cell line panel. By examining multiple representatives of a mechanistic class as ‘Test Agents’, we identified interactions wherein ‘Test Agents’ with similar

100 POSTER (Board P094) Small molecules selectively targeting breast cancer cells

Breast cancer is one of the most common cancers among women. Despite advances in therapy, drug resistance is often induced, tumour selectivity is poor and drug off-targets culminate in limited efficacy. Indeed metastatic advanced breast cancer is deemed incurable. Better, more effective treatments for this disease are clearly needed. We have discovered a structurally distinct class of small molecules that target breast cancer cell lines while having little to no effect on normal breast cancer cells or on cell lines derived from other tumour types including colon, ovarian, lung, skin, prostate and pancreatic carcinomas, neuroblastoma and glioblastoma. Indeed these molecules show more than 500-fold selectivity towards breast cancer cells compared with other tumour types, while maintaining nM potency, as determined by the MTT growth inhibition (GI50 ) assay with GI50 values of 0.1−0.7uM (72 h exposure). Moreover, the sensitive breast cancer cell lines represent tumour types from the four main breast cancer classifications including ER+ luminal A (MCF-7, T47-D and ZR-75−1 cells), ER+ luminal B (BT-474), HER2+ (SKBR-3), and most importantly the Basal (triple negative MDA-MB-468 cells) classification which traditionally carries a very poor prognosis. Our novel class of molecules also retain activity in MCF-7/VP16 cells (GI50 0.2uM) which overexpresses the drug resistance ABCC1 gene. Only one breast cancer cell line has shown insensitivity, MDA-MB-231 (Basal triple negative), which unlike all of the other breast cell types has amplifying mutations in KRas and BRaf activity, a genotype found in less than 5% of breast cancer tumours. Furthermore, our novel compounds induce minimal effects on the growth of normal MCF10A breast cells. Cell cycle analysis in the most sensitive breast cancer cell line, MDAMB-468, shows an increase in the proportion of cells in the S-phase of the cell cycle within 24 h of exposure, concomitant with a decrease in the proportion of cells in the G1 -phase, and preceding an increase in the subG1 cell death population. The ability to specifically target breast tumours, while having little or no effect on normal breast cells, or other tumour types is a unique finding. Elucidating the mechanism controlling this phenomenon is now the focus of our research efforts. 101 POSTER (Board P095) PIK3CA mutation-targeting compounds analyses using NCI60 cell line panel H. Bando1 , J. Lih2 , E.C. Polley3 , S.L. Holbeck4 , B. Das2 , D. Sims2 , T. Doi5 , A. Ohtsu5 , M. Williams2 , N. Takebe1 . 1 National Cancer Institute National Institute of Health, Cancer Therapy Evaluation Program Division of Cancer Treatment and Diagnosis, Rockville, USA; 2 National Cancer Institute National Institute of Health, The Frederick National Laboratory for Cancer Research, Frederick, USA; 3 National Cancer Institute National Institute of Health, Biometric Research Branch Division of Cancer Treatment and Diagnosis, Rockville, USA; 4 National Cancer Institute National Institute of Health, Developmental Therapeutics Program Division of Cancer Treatment and Diagnosis, Rockville, USA; 5 National Cancer Center Hospital East, Department of Gastroenterology and Gastrointestinal Oncology, Kashiwa, Japan Background: The US National Cancer Institute 60 (NCI-60) human tumor cell line panel was developed as an in vitro drug-discovery tool, and it is a unique publicly accessible platform of various datasets. Through the CellMiner website, we could access genomic datasets of NCI-60. We could

Poster Session – Drug Screening also obtain the activities of various test compounds to the NCI-60 cell line panel from NCI Developmental Therapeutics Program (DTP) website. Using this capability, we screened for therapeutic agents against the cell lines with PIK3CA mutation, the second most frequently mutated actionable oncogene among various types of tumors. Material and Methods: Utilizing NCI-60 cell line GI50 (50% growthinhibitory levels) data of PI3K/AKT/mTOR pathway compounds, we calculated the differences in mean log GI50 between the PIK3CA mutated cell lines and non-mutated cell lines (delta log GI50) to investigate promising compounds. The volcano plot (the x-axis depicts delta log GI50 and the y-axis depicts statistical significance level) was used to search for the promising test compounds. Results: Seven cell lines had PIK3CA mutation (3 p.E545K, 3 p.H1047R, and 1 p.R38C mutation). We could collect GI50 data on 35 PI3K/AKT/mTOR targeting compounds (7 pan-class PI3K inhibitors, 7 isoform-selective PI3K inhibitors, 5 rapalogs, 3 mTOR kinase inhibitors, 6 panPI3K/mTOR inhibitors, and 7 AKT inhibitors). In our analysis, 1 panclass PI3K inhibitor, 2 isoform-selective PI3K inhibitors, 1 rapalog and 1 AKT inhibitor showed promising trends by both delta log GI50 and statistical significance. Conclusions: NCI-60 cell line panel may be a valuable tool to identify various actionable mutation–drug relationships and to discover promising drug candidates. 102 POSTER (Board P096) Comparison of platinum/taxane and anthracycline-based therapies in ovarian PDX models: Correlating stage of biopsy collection and engrafting with in vivo drug sensitivity J. Meade1 , M.J. Wick1 , T. Vaught1 , L. Gamez1 , M. Farley1 , A. Moriarty1 , A.W. Tolcher1 , A. Patnaik1 , D.W. Rasco1 , A.J. White2 , K.P. Papadopoulos1 . 1 South Texas Accelerated Research Therapeutics, (START), San Antonio TX, USA; 2 START Center for Cancer Care, San Antonio TX, USA Background: Standard first-line treatment for ovarian cancer is a platinumbased therapy often including a taxane, with various salvage therapies for recurrent disease. We and others have established and validated low passage ovary patient-derived xenograft (PDX) models from biopsies obtained at diagnosis and following clinical treatment progression; however, whether drug sensitivity to each model correlates to the clinical context was unclear. To test this we stratified thirty-nine ovary PDX models using clinical outcome data at the time of biopsy collection into four groups: chemo na¨ıve, first-line platinum responding, first-line platinum refractory and anthracycline pretreated. Drug sensitivity studies were performed, evaluating model response to platinum, platinum plus taxane and liposomal doxorubicin and results compared between each group. Methods: Ovary PDX models were developed inimmune-deficient mice from primary or metastatic patient tissue and established models confirmed by histologic comparative analysis and linked with patient treatment and outcome data. Drug sensitivity studies were performed evaluating models response to three regimens with tumor volume and time from treatment initiation as study endpoints. T/C values, growth delay and regressions were reported for each model. Results: For the thirty-nine models evaluated: 18/39 were from chemona¨ıve patients, 6/39 from first-line platinum responding, 10/39 from firstline platinum refractory and 5/39 from anthracycline pretreated patients. In these studies chemo-na¨ıve and first-line platinum responding models were sensitive to the platinum based regimens with few exceptions including ST036 which harbors known AKT and KRAS mutations. Most first-line platinum refractory models including ST419 and ST884 were resistant to platinum alone and platinum/taxane combination. Interestingly sensitivity to liposomal doxorubicin did not correlate with clinical pretreatment as several models from chemo-na¨ıve patients including ST024 and ST511 were insensitive while some models established from anthracycline pretreated patients including ST255B and ST467 were sensitive to the therapy. Conclusion: We have established a panel of ovary patient-derived xenografts and correlated model sensitivity to platinum and anthracyclinebased treatments to the clinical context at the time of biopsy collection and engraftment. Overall platinum sensitivity in models from chemo-na¨ıve and first-line platinum responding patients, with few exceptions, correlated with clinical response, irrespective of activating mutations. Preclinical response to anthracyclines was less predictive and not predicated on the clinical context.

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103 POSTER (Board P097) The use of next generation sequencing (NGS) in the management of metastatic breast cancer (MBC): Defining a model for genomic-driven therapies L. Austin1 , M. Gooptu1 , T. Avery1 , R. Jaslow1 , J. Palazzo2 , M. Cristofanilli1 . 1 Thomas Jefferson University Hospital, Medical Oncology, Philadelphia PA, USA; 2 Thomas Jefferson University Hospital, Pathology, Philadelphia PA, USA Background: Metastatic breast cancer is an incurable disease treated with palliative intent. The limited benefit from standard therapies could be related to the fact that the disease is biologically heterogeneous and not properly managed by current treatments. Increased understanding of the molecular complexity using next generation sequencing (NGS) allows for individual tumor mutation analysis which provides better selection of treatments based on genomic abnormalities driving the disease. Methods: We retrospectively analyzed 33 patients with advanced or metastatic breast cancer treated at Thomas Jefferson University, diagnosed between 2003–2013, on whom next generation sequencing (NGS) by Foundation One™ was sent for genomic analysis. Immediately actionable mutations for this analysis were considered those for which FDA-approved agents for breast cancer were identified. We also reported potentially actionable mutations which were defined as mutations with investigationaltherapeutic agents. Results: The majority of patients (88%) had inflammatory breast cancer (IBC). According to disease subtype, 36% were ER+/HER− (Luminal A), 18% ER+/HER+ (Luminal B), 15% ER−/HER+ (HER2+), and 30% were ER−/HER− (triple negative). NGS genomic analysis revealed a total of 78 unique mutations with a total of 178 mutational events. The five most commonly mutated genes in order of frequency were TP53 (55% of patients), MYC amplification (39%), PIK3CA mutations (36%), ERBB2 amplification (30%) and PTEN loss or mutation (15%). Eleven of 78 mutations (14%) were immediately actionable (based on our definition) and comprised of PIK3CA mutations/amplifications, ERBB2 amplifications, AKT mutations and PTEN loss/mutations. In total, 21 of 33 patients (64%) had at least one immediately actionable mutation and 97% (32) of patients had either an immediately actionable or potentially actionable mutation. The most common potentially actionable mutations were TP53, MYC amplification, FGFR1 amplification and MDM2 amplification. All but one of the HER2+ patients (IHC/FISH) had ERBB2 amplification on NGS. Interestingly, of the 10 patients that had ERBB2 amplification, 70% had a concomitant PIK3CA mutation and all had IBC. Two patients with ERBB2 amplification and PIK3CA mutations who had progressed on trastuzumab ((Herceptin® ) combinations were started on everolimus (Afinitor® ), Trastuzumab and vinorelbine (Navelbine). Conclusion: In conclusion, in this cohort of 33 patients, the Foundation One™ panel detected unique mutations of which 14% were immediately actionable and 97% of patients had at least one mutation that was immediately or potentially actionable. Actionable mutations were detected across all receptor groups and there was a high incidence on concomitant ERBB2 amplification and PIK3CA mutation. The Foundation One™ panel is a useful tool which can be utilized for tailoring targeted therapy for breast cancer patients. This warrants further investigation with a prospective trial to evaluate the clinical impact of this added information and its effect on decision making and clinical outcomes. 104 POSTER (Board P098) Identification of inhibitors of tryptophan metabolizing enzymes for cancer immunotherapy by high-throughput screening G. Zaman1 , J.C.M. Uitdehaag1 , S. van Gerwen1 , N. Seegers1 , A.M. van Doornmalen1 , J. de Man1 , R.C. Buijsman1 . 1 Netherlands Translational Research Center B.V., Oss, Netherlands Background: The amino acid tryptophan is an important regulator of the immune system by regulating the activation of T cells. Tumor cells express enzymes that oxidize tryptophan, thereby dampening the local T cell immune response against cancer cells. These enzymes are indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO). Despite the substantial evidence of the importance of IDO1 and TDO as small molecule drug targets, only a handful of different chemical scaffolds have been reported. The lack of chemical matter is explained by the lack of robust assays for high-throughput screening. We have developed new assays for IDO1 and TDO, which we have used to screen a compound library, and to characterize the selectivity of reference inhibitors. Materials and Methods: NFK Green™ is a chemical probe that specifically reacts with N-formyl kynurine, the reaction product of the enzymatic conversion of tryptophan by IDO1 and TDO. The reaction is quantified by measuring fluorescence on a 384-well plate multimode reader. Biochemical assays were developed for IDO1 and TDO using recombinantly expressed

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proteins, and a library of 87,000 diverse lead-like molecules was screened. NFK Green™ was also used to determine the tryptophan metabolizing activity in a collection of human cancer cell lines and was related to the expression levels of IDO1 and TDO by western blot analysis. Results: Biochemical and cell-based screening assays were developed for IDO1 and TDO using a new fluorescent read-out. High-throughput screening of libraries of small chemical compound libraries yielded novel selective inhibitors of either IDO1 or TDO. Side-by-side comparison of published reference compounds revealed significant, previously unnoted cross-reactivity of a widely used hydroxyamidine-based inhibitor of IDO1 (Compound 5l) with TDO. The selectivity of other reference IDO1 or TDO inhibitors was confirmed, leading to definition of a new tool compound set. Biochemical selectivity of compounds correlated with inhibition of cellular tryptophan metabolizing activity and expression of IDO1 or TDO. Conclusions: We have developed new biochemical and cell-based assays for IDO1 and TDO, to enable the identification of novel small molecule inhibitors and to support lead optimization. Side-by-side comparison of published inhibitors revealed novel, unanticipated cross-reactivity of IDO1 inhibitor scaffolds with TDO. 105 POSTER (Board P099) A platform to test multiple therapy options simultaneously in a patient’s own tumor N. Caffo1 , R. Klinghoffer1 . 1 Presage Biosciences Inc., Seattle, USA While investigational cancer drugs must ultimately be validated in clinical trials, most early drug discovery is performed under in vitro conditions in cell-based models that poorly represent the disease they are intended to represent. To enable in vivo analysis of anti-cancer agent efficacy at earlier stages of drug development, and to potentially enable toxicity-sparing assessment of novel agents in the oncology clinic, we have developed a technology platform called CIVOTM . CIVOTM allows for simultaneous assessment of up to eight drugs or drug combinations in a single solid tumor. Controlled microinjection-based delivery of doxorubicin, docetaxel, mafosfamide, and gemcitabine both as single agents and combinations was tested in the canine sarcoma clinic. Drugs were co-injected in a columnar array with UV fluorescent beads resulting in easy-to-identify bands of drug, each at a distinct position of the patient’s tumor. Tumors were resected 72 h following microinjection and were examined for responses using standard histology methods. The CIVO introduced drug microdoses induced spatially defined, graded, and mechanism-specific cellular changes around sites of drug exposure in a patient-specific manner. Consistent with the use of doxorubicin as first line therapy in the soft tissue sarcoma clinic, the frequency and extent of response of localized responses to doxorubicin exceeded those of all other agents tested. This preclinical data, along with early responses observed in the human clinic, set the stage for clinical application of this technology to identify which novel agents are likely to succeed or fail in subsequent clinical trials.

Immunotherapy (Immunecheckpoints, Vaccination, Oncolytic viruses, Cytokines) 106 POSTER (Board P100) Beta-3 integrin inhibition reduces inflammatory cytokine release but not anti-cancer activity of oncolytic adenovirus in ovarian cancer A.K. Browne1 , L.A. Tookman1 , C.K. Ingemarsdotter1 , R. Bouwman1 , K. Pirlo1 , Y. Wang1 , K.M. Hodivala-Dilke2 , I.A. McNeish3 , M. Lockley1 . 1 Barts Cancer Institute, Centre For Molecular Oncology, London, United Kingdom; 2 Barts Cancer Institute, Centre For Tumour Biology, London, United Kingdom; 3 University of Glasgow, Institute of Cancer Services, Glasgow, United Kingdom Background/Introduction: The potential of oncolytic adenoviruses as anti-cancer therapy has repeatedly been demonstrated. A consistent and worrying feature of adenoviral gene therapy is the rapid cytokine release that occurs after viral administration. These cytokines give rise to dose limiting inflammatory toxicities, which can be severe and have hindered further investigation and clinical development of these promising anti-cancer therapies. Using primary ovarian cancer cells and genetically modified mice, we show that the E1A-CR2 deleted replicating oncolytic adenovirus, dl922–947, induces cytokines via beta-3 integrin in macrophage-rich tissues. We present new evidence that co-administration of a Cilengitidelike integrin inhibitor controls the inflammatory cytokines and hepatic toxicity

Poster Session – Immunotherapy induced by dl922–947 in tumour bearing mice. Importantly, although alphav/beta-3integrin functions as a secondary adenoviral receptor, we found no evidence that beta-3 inhibition compromised viral infectivity and oncolysis in vitro or anti-cancer efficacy in vivo. Material and Methods: We quantified production of cytokine mRNA (qRTPCR) and protein (Mesoscale Discovery System) following dl922–947 in ovarian cancer cells lines, tumour cells harvested from the ascites of women with ovarian cancer and in murine tissues, peritoneal cells and serum. Murine models included nude mice bearing intraperitoneal ovarian cancer xenografts and non-tumour bearing, immunocompetent beta-3 null mice. Pharmacological inhibition of beta-3 integrin was achieved using H2574, a cyclic RGD mimetic inhibitor of alpha-v/beta-3andalpha-v/beta-5 integrins. Results: Primary and established ovarian cancer cell lines in vitro did not release inflammatory cytokines in response to adenovirus. In contrast, intraperitoneal delivery of dl922–947 caused rapid, systemic cytokine induction in ovarian cancer xenografts. Cytokines originated predominantly in macrophage-rich murine tissues (liver, spleen and peritoneal macrophages), rather than the injected malignant cells, and was independent of viral replication. Adenoviruses are known to induce cytokine release via beta-3 integrin-expressing macrophages. We found that co-administration of the integrin inhibitor, H2574, controlled production of inflammatory cytokines in the circulation of tumour-bearing mice. Cotreatment also reduced pathological features of viral hepatic toxicity such as eosinophilic degeneration and liver enzyme elevation. Importantly, combining dl922–947 with H2574 did not compromise anti-cancer activity in vitro or in vivo. Conclusions: Combining oncolytic adenoviruses with pharmacological inhibition of beta-3 integrin enables safe systemic delivery of replicating adenoviruses, without compromising anti-cancer activity. This novel approach could have a major impact on the future development of these effective anti-cancer agents. 107 POSTER (Board P101) CIGB-247: Anti-VEGF therapeutic vaccine in patients with advanced solid tumors 1 2 ´ F. Hernandez-Bernal ´ , J.V. Gavilondo2 , M. Ayala Avila , A.V. de la Torre3 , J. de la Torre4 , K.H. Selman-Housein4 , Y. Morera2 , M. Bequet-Romero2 , C.M. Valenzuela1 , Y. Martin5 . 1 Center for Genetic Engineering and Biotechnology (CIGB), Clinical Trials, Havana City, Cuba; 2 Center for Genetic Engineering and Biotechnology (CIGB), Pharmaceuticals, Havana ´ City, Cuba; 3 “Celestino Hernandez” Hospital, Oncology, Santa Clara, Cuba; 4 Medical and Surgical Research (CIMEQ), Oncology, Havana City, ´ Hospital, Oncology, Havana City, Cuba Cuba; 5 “Celestino Hernandez”

Background: The CIGB-247 is a vaccine preparation for the therapy of cancer that combines the recombinant antigen p64K-hVEGFKDR− , produced in Escherichia coli and the VSSP adjuvant. This antigen is a representative molecule of the human Vascular Endothelium Growth Factor (VEGF-A), in its isoform 121. Vaccination with CIGB-247 in experimental animal models has been safe, with anti-tumor and anti-metastatic effects. Material and Methods: A non-controlled, dose up scaling phase I clinical trial was performed in patients with advanced solid tumors, which previously received the best onco-specific treatment available without response. The general objective of the trial was to study the safety profile of the vaccine at three antigen dose levels. A maximum of 10 patients per group were planned. Individuals were subcutaneously immunized for 8 consecutive weeks with 50, 100 or 400 mg of antigen (all in the same amount of VSSP adjuvant), and re-immunized on week 12. On week 16, evaluations of safety, tolerance, clinical status, and immunogenicity (seroconversion for anti-VEGF IgG, serum VEGF/KDR-Fc blocking ability, and gamma-IFN ELISPOT with blood cells stimulated in vitro with mutated VEGF) were done. Results: Vaccination was shown to be safe at the three dose levels, with only grade 1−2 adverse events. CIGB-247 was immunogenic and higher numbers of individuals positive to the three immune response tests were seen with increasing antigen dose. Conclusions: This is the first clinical testing report of a cancer therapeutic vaccine based on a human VEGF related molecule as antigen. The CIGB247 vaccine is safe, immunogenic, and merits further clinical development.

Poster Session – Immunotherapy 108 POSTER (Board P102) CD70 (TNFSF7), a receptor involved in acute immune modulation of viral infection, is frequently overexpressed in solid and hematological malignancies J. Jacobs1 , K. Zwaenepoel1 , P. Aftimos2 , C. Rolfo3 , S. Rottey4 , L. Ysebrant de Lendonck2 , K. Silence5 , A. Awada2 , A. Thibault5 , P. Pauwels1 . 1 Universitair Ziekenhuis Antwerpen, Pathology, Antwerp, Belgium; 2 Institut Jules Bordet, Oncology, Brussels, Belgium; 3 Universitair Ziekenhuis Antwerpen, Oncology, Antwerp, Belgium; 4 Ghent University Hospital, Oncology, Ghent, Belgium; 5 arGEN-X BVBA, Research, Zwijnaarde, Belgium Tissue expression of CD70 (TNFSF7), a receptor of the TNF superfamily normally involved in transient, rapid-onset immune response, is highly restricted to small subsets of T-, B-, and dendritic cells. It is often chronically over-expressed in patients with hematological and solid malignancies in whom it mediates tumor cell growth and immune escape. To date, the incidence of CD70 positivity has not been systematically characterized in patients with advanced malignancies. We developed an immunohistochemistry (IHC) method to detect CD70 on paraffin-embedded tumor biopsies in support of a Phase 1 trial of ARGX110, a monoclonal IgG1 SIMPLE Antibody™ endowed with enhanced ADCC properties (POTELLIGENT® ). A total of 182 samples (120 solid tumors and 62 hematological malignancies) were selected at random from a university hospital tissue bank and tested for CD70 expression. Samples were considered positive for CD70 (cut-off: 10% cells staining) in 33% and 58% of cases, respectively. As of March 2014, CD70 positivity has also been documented in 147 of 296 patients (50%) who underwent IHC screening as an eligibility requirement for study participation (ClinicalTrials.gov Identifier: NCT01813539). EBV-induced malignancies (e.g.: Hodgkin’s and Burkitt’s lymphoma, nasopharyngeal carcinoma) consistently overexpressed CD70. A high incidence of CD70 positivity was observed in T- and B-cell lymphomas (64%), as well as renal cell carcinoma (90%) and esophageal cancer (64%). Consistent with the intracellular trafficking of CD70, staining patterns in malignant cells included membranous, cytoplasmic and perinuclear distribution. In conclusion, the expression of CD70 is commonly increased in a wide variety of solid and hematological malignancies. 109 POSTER (Board P103) Functional activity, but not PD-1 expression level, differentiates primary CLL from healthy PD1+ T cells using SCNP S. Liang1 , L. Leung1 , S. Putta1 , D. Hotson1 , D. Rosen1 , R.E. Hawtin1 . Nodality Inc., Research, So San Francisco CA, USA

1

Background: Drugs inhibiting PD-1 signaling have demonstrated clinical efficacy in the treatment of cancer. To increase understanding of PD-1+ T cell biology, PD-1 expression was profiled across CD4/CD8 T cell subsets in peripheral blood mononuclear cells (PBMC) of CLL and healthy donors (HD), and the functional responsiveness of these PD-1+/− subsets to cytokine modulation or T cell receptor (TCR) modulation +/− PD-1 blockade was interrogated. Materials and Methods: Single Cell Network Profiling (SCNP) is a multiparametric flow cytometry based technology that enables simultaneous analysis of signaling networks in multiple immune cell subsets. CLL (n = 9) and HD (n = 8) PBMC were profiled by SCNP to interrogate CD4/CD8 +/− T cell subsets for PD-1 expression, cell subset specific signaling following modulation with IL-2, IL-7, IL-15, IL-21 or TCR (a-CD3/a-CD28), and proliferation following TCR modulation +/− PD-1 blockade. CLL and HD data were compared to identify dysfunctional signaling associated with disease and PD-1 expression. Results: CLL and HD did not differ significantly in median PD-1 expression, although CLL samples were more heterogeneous. Significant differences were detected in signaling capacity in CLL vs HD, particularly in PD-1+ CD8 T cells. Unmodulated p-STAT5 was elevated in CLL in PD-1+ and PD-1− CD8 T cells and in PD-1+ CD4−/CD8− T cells. IL-2, IL-7 and IL-15 induced lower p-STAT5 levels in PD-1+ CD8 T cells and PD-1+ CD4−/CD8− T cells of CLL donors, indicating dysfunctional signaling through these common g-chain cytokines in these PD-1+ subsets. In contrast to reduced cytokine responsiveness, increased TCR modulation of p-ERK was observed in PD-1+ CD8 T cells in CLL vs HD. Conversely, CLL T cells demonstrated decreased proliferation in response to TCR modulation, which was partially reversed with PD-1 blockade. Conclusions: The dysregulated cytokine signaling, elevated TCR responsiveness and reduced proliferation observed in CD8, but not CD4, PD-1+ T cell subsets in CLL is consistent with the reported ‘pseudo-exhausted state’. These data establish the application of SCNP to interrogate PD-1+ T cell subset signaling and the functional consequences of tumor–

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microenvironment interactions. Building upon and verifying these data, SCNP will be applied to both developing predictive and prognostic markers of response in hematologic and solid tumor indications, and interrogating the efficacy of therapeutics to restore T cell function. 110 POSTER (Board P104) IL-6/STAT3/Fra-1 signaling axis promotes colorectal cancer aggressiveness through epithelial–mesenchymal transition J. Shao1 , H. Liu1 . 1 Zhejiang University School of Medicine, Pathology and Pathophysiology, Hangzhou City, China Fos-related antigen-1 (Fra-1), a member of the Fos family, is aberrantly expressed in several types of human cancer, but its functional roles in cancer development and the regulatory mechanisms are still not well understood. In this study, we investigated the significance of Fra-1 expression in human colorectal cancer (CRC). Fra-1 levels were positively correlated with the local invasion depth as well as lymph node and liver metastasis in a total of 229 CRC patients. Intense immunohistochemical staining of Fra-1 was observed at the tumor invasive front adjacent to inflammatory cells and in parallel with secretion of the pro-inflammatory cytokine interleukin-6 (IL-6) in the CRC tissues. Exogenous IL-6 administration to CRC cell lines significantly upregulated Fra-1 expression in a manner dependent on signal transducer and activator of transcription 3 (STAT3), during which both phosphorylated and acetylated post-translational modifications were required for STAT3 activation to promote the transcription of Fra-1 gene by directly binding to its promoter. Importantly, Fra-1 upregulated by IL-6/STAT3 signaling endowed the CRC cells with properties of epithelial– mesenchymal transition (EMT), an essential step for tumor progression and metastasis. RNA interference-based attenuation of STAT3 or Fra-1 prevented IL-6-induced EMT, cell migration and invasion, whereas ectopic expression of Fra-1 markedly reversed the inhibitory effect of STAT3knockdown on the EMT process. Collectively, this study uncovered the existence of an aberrant IL-6/STAT3/Fra-1 signaling axis leading to EMT and aggressiveness in colorectal cancer and suggested novel therapeutic opportunities for the treatment of malignant disease. 111 POSTER (Board P105) Identification of peptides which could block PD-1 checkpoint for NSCLC immunotherapy Y. Zhu1 , C. Li1 . 1 Suzhou Institute of Nano-tech and Nano-bionics CAS, Nanobiomedicine, Suzhou, China Background: Non-small cell lung cancer (NSCLC) has been reported responding to programmed death-1 (PD-1) immune checkpoint blockade, thus inhibitory peptides blocking PD-1/programmed death ligand-1 (PDL-1) pathway may contribute the effective therapy to NSCLC patients. Materials and Methods: X15 bacteria peptide library was used to screening and selecting binding peptides to PDL-1 and PD-1. Focused library was designed and constructed according to the peptide screening results from X15 random bacteria peptide library. Affinity and specificity of binding peptides for PDL-1 and PD-1 were examined with methods of SPR and competition experiments. In vitro assessment of peptides for blocking the PD-1/PDL-1 pathway was performed with MDA-MB-231 cell lines. Metastatic mice model of A549 cells were constructed with nude mice after 2 months since A549 cells were injected into the lung position of mice. The evaluation of peptides for their anti-tumor effect was done via measuring and comparing tumor sizes and life spans of mice injected either with binding peptides or scramble peptides. Immunohistology were performed with anti-CD31 antibody and blood of mice was collected for circulating tumor cells detection. Results: 1. Mice model with metastatic lung cancer was established; 2. Peptides binding with PDL-1 were obtained from random X15 library; 3. Conserved sequences for peptides binding with PDL-1 were identified and their physiochemical properties were examined roughly; 4. Focused library was established according to the conserved binding peptide sequences; 5. Peptides interacted with PDL-1 could block proliferation of MDA-MB-231; 6. Life span of tumor mice got elongated and tumor size was decreased after mice injected with binding peptides; 7. The amount of CTC was alleviated and CD-31 positive signals got decreased in binding peptides-treated mice group. Conclusion: Peptides binding to PDL-1 could behave as potential drugs for metastatic lung cancer therapy since they can block signal pathway initiated by PD-1/PDL-1 interaction, inhibit angiogenesis and metastasis of tumor cells.

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Poster Session – Immunotherapy

112 POSTER (Board P106) A novel primary human tumor explant platform provides a preclinical translational link from tissue culture to the clinic G. Juan1 , K. Paweletz1 , E. Trueblood2 , J. Rossi1 , M. Damore1 , A. Anderson1 , R. Loberg1 . 1 Amgen, Medical Sciences, Thousand Oaks CA, USA; 2 Amgen, Pathology, Seattle WA, USA Background: To explore a candidate immunotherapeutic (BiTE® ), a translational explant platform was developed consisting of cultures of thinly sectioned tissues from freshly resected solid tumors. The tumor explants are being used to study treatment responses in a model system that preserves the tumor architecture and microenvironment, including immune effector populations. Material and Methods: 18 freshly resected epithelial tumors were sectioned and treated in culture for 48 or 72 h with increasing concentrations (0, 5, 50 and 500 pM) of an EGFR-BiTE® or a MEC14-BiTE® (negative control). The culture supernatant was then collected to quantitate cytokines released as a measure of T-cell activation using Myriad’s CytokineMAP® A v1.0 panel. CD25 and CD3 enumeration to demonstrate in situ T-cell activation was performed by IHC in FFPE tissues derived from the treated explants. In addition, the parental tumors were profiled by IHC to characterize the EGFR target levels together with the initial tumor T-cell infiltrate. Finally, gene expression profiles of the parental tumors were generated to explore potential resistance mechanisms. Results: Tumor resident T-cells are activated ex vivo by BiTE® . A dose dependent increase of several cytokines including IFN-g, IL-2 and TNF-a was observed in conditioned media harvested from EGFR-BiTE® treated but not in MEC14-BiTE® treated explants. In addition, a dose dependent increase of granzyme B into media as measured by ELISA was observed. Importantly, a dose dependent increase of total CD25 counts and the %CD3CD25 is observed in the EGFR-BiTE® explants vs. those treated with the control BiTE® . EGFR-BiTE® (N = 18)

0 pM

IFN-g (pg/mL) IL-2 (pg/mL) TNF-a (pg/mL) %CD3CD25

5.6±0.9 6.8±0.7 24.4±4.2 28.5±15

5 pM 28.9±9.8 55.9±23 43.6±7.3 41.2±17.7

50 pM 101±37 114.4±43.5 88±23.6 57.9±26.2

mode of action, its activity in selected pre-clinical models, and to identify pharmacodynamic biomarkers for response monitoring. In vitro functional tests using lymphocytes from various species were conducted. A series of in vivo syngenic mouse tumor models were selected based on immune checkpoint ligand expression. Immune-related biomarkers and drug concentrations were assessed from circulating blood and tumor samples from those models. Results: A first-in-class therapeutic peptide, designated W014A (formerly AUR-012), was characterized. W014A is a 29-amino acid branched peptide designed as a PD-1 decoy using selected portions of the human PD-1 receptor. W014A displayed equipotent antagonism towards PD-L1 and PDL2-mediated T cell exhaustion. Robust activity in rescue of proliferation and effector functions (IFN-g secretion) from mouse, non-human primate and human lymphocytes was demonstrated. W014A also prevented the interaction of PD-L1 with B7.1. A complete rescue of CD4+ and CD8+ T cells and suppression of regulatory T cells were observed using proliferation assays of immune cells stimulated with anti-CD3/anti-CD-28. Different in vivo models, including melanoma, colon, breast and kidney cancers (B16, CT26, 4T1 and Renca) demonstrated the potent effects of W014A on both primary tumor growth and metastasis. Active drug concentration levels in tumour (Renca model), as well as intratumoral recruitment (CT26 model) of CD4+ and CD8+ T cells, and a reduction in PD-1+ T cells (both CD4+ & CD8+ ) were observed concomitantly with antitumoral activity. Conclusions: Altogether, the unique mechanism of action and preclinical activity of W014A support the set-up of clinical trials in the near future and suggest potential pharmacodynamic biomarkers for use in the clinic. The synergy with other treatments that enhance endogenous antitumor immunity will also be investigated. 114 POSTER (Board P108) Toll-like receptor 5 agonist entolimod as a potential anticancer immunotherapeutic agent L. Burdelya1 , C. Brackett1 , B. Kojouharov1 , J. Veith1 , A. Gudkov1 . 1 Roswell Park Cancer Institute, Cell Stress Biology, Buffalo NY, USA

500 pM 455.3±118.8 605.8±207.7 272.9±61.8 94.9±51.3

Conclusions: Infiltrating T cells can be activated by BiTE® molecules, as demonstrated by cytokine secretion and T-cell activation in the explant culture system. Using a novel preclinical ex vivo model of human primary tumors, the explants provide mechanistic insights into the modulation of the tumor immunoresponse upon administration of a BiTE® . 113 POSTER (Board P107) Preclinical activity and pharmacodynamic biomarkers of W014A, a PD-1 decoy peptide blocking both PD-1 immune checkpoint ligands, PD-L1 and PD-L2 C. Bailly1 , M. Broussas2 , M. Ramachandra3 , P.G. Sasikumar3 , K. Shrimali3 , S. Adurthi3 , M. Ramachandra3 , L.K. Satyam3 , A.A. Dhudashia3 , S. Dhodheri3 , K.B. Sunilkumar3 , N. Corva¨ıa4 , P. Ferre5 . 1 Institut de Recherche Pierre Fabre, Head of Research IRPF, Toulouse Cedex 1, France; 2 Institut de Recherche Pierre Fabre, Experimental Oncology Department, Saint-Julien en Genevios, France; 3 Aurigene Discovery Technologies Ltd, Bangalore, India; 4 Institut de Recherche Pierre Fabre, Saint-Julien en Genevois, France; 5 Institut de Recherche Pierre Fabre, Toulouse, France Background: Cancer immunotherapy is starting to change the way cancer is treated. The goal is to re-activate patient’s immune system to specifically destroy tumor cells. Recent clinical trials in various settings have confirmed that several monoclonal antibodies blocking either the programmed cell death-1 (PD-1) receptor on infiltrating T cells, or its ligand PD-L1 re-activate tumor-specific T cell cytotoxicity, and induce impressive and durable clinical responses. However, the immune evasion of tumor cells cannot be restricted to the sole interaction between PD-1 and PD-L1. PD-1 also interacts with a higher binding affinity with PD-L2, and PD-L1 is capable of binding to B7.1 (CD80) on the T cell surface. Similarly as PD-1/PD-L1, both PD-1/PD-L2 and PD-L1/B7.1 interactions result in deactivation of T cells. Antagonizing those 3 interactions might induce a more robust anti-tumor activity. Materials and Methods: A novel drug candidate was designed to antagonize both PD-L1 and PD-L2. Our goal was to characterize its

Mobilization of innate immunity by Toll-like receptor (TLR) agonists and the subsequent development of adaptive immunity have been considered as an attractive approach to cancer immunotherapy. Unfortunately, the majority of TLR agonists induce prohibitive acute inflammatory responses limiting their clinical use. In this regard, TLR5 stands alone since the TLR5 agonist, bacterial flagellin, induces much less severe systemic inflammation than agonists of other TLRs. Entolimod is a flagellin derivative and a clinical stage experimental drug initially developed as a candidate radiation antidote capable of protection from and mitigation of radiation injury in mice and non-human primates. We found that entolimod, in addition to its radioprotective properties, had anticancer effects in a variety of TLR5-positive tumors in mice. We hypothesized that entolimod can induce antitumor defense if tumors are located in a TLR5-responsive microenvironment such as the liver independently of the TLR5 status of tumor cells. This hypothesis was tested in three syngeneic spontaneous and experimental liver metastasis models: CT26 and MC38 colorectal and 4T1 breast adenocarcinomas. The results demonstrate that entolimod treatment was efficient in suppressing metastatic disease when injected systemically either before or after surgical removal of primary tumors in all tested mouse models of metastatic colon, breast, and melanoma cancers. This effect was mediated by mobilization of immune effectors to the liver, the major primary target organ for TLR5 agonists, and did not depend on the TLR5 status of the tumors. Rapid and massive recruitment of neutrophils, NK, CD4/CD8 T cells and other types of immune cells to the liver was followed by eradication of dormant tumor cells and micrometastases and development of protective T cell memory. Importantly, entolimod has passed through two Phase I clinical trials in 150 healthy volunteers and is currently undergoing a Phase I trial in cancer patients with advanced solid tumor liver metastases with the expectation to be used in adjuvant or neoadjuvant settings for prevention of metastatic disease. 115 POSTER (Board P109) Anti-metastatic activity via co-blockade of PD-1 and adenosine A2A receptor D. Mittal1 , A. Young1 , K. Stannard1 , M.W. Teng1 , B. Allard2 , J. Stagg2 , M.J. Smyth3 . 1 QIMR Berghofer Medical Research Institute, Immunology in Cancer and Infection, Brisabane, Australia; 2 Faculte´ de Pharmacie ´ Montreal ´ Quebec ´ et Institut du Cancer de Montreal Canada, Centre de ´ Recherche du Centre Hospitalier de l’Universite´ de Montreal, Montreal, Canada; 3 QIMR Berghofer Medical Research Institute, Immunology in Cancer and Infection, Brisbane, Australia Adenosine targeting is an attractive new approach to cancer treatment, but no clinical study has yet examined adenosine inhibition in oncology despite

Poster Session – Immunotherapy the safe clinical profile of adenosine A2A receptor inhibitors (A2ARi) in Parkinson’s disease. Monoclonal antibodies (mAb) that block programmed death (PD)-1 or cytotoxic T lymphocyte antigen (CTLA-4) receptors have been associated with durable clinical responses against a variety of cancer types and hold great potential as novel cancer therapeutics. Metastasis is the main cause of cancer related deaths worldwide, and therefore we have studied experimental and spontaneous mouse models of melanoma and breast cancer metastasis to demonstrate the efficacy and mechanism of a combination of A2A receptor inhibitor in combination with various immune check point inhibitors. The combination of anti-PD-1 and A2A receptor inhibitor significantly reduces metastatic burden and prolongs the life of mice compared with either monotherapy alone. Importantly, the combination was only effective when the tumor expressed high levels of CD73, suggesting a tumor biomarker that at a minimum could be used to stratify patients that might receive this combination. The mechanism of the combination therapy was critically dependent on NK cells and interferon gamma, and to a lesser extent, CD8+ T cells and the effector molecule, perforin. Consistent with the anti-metastatic role of NK cells, we observed significantly high number of NK cells in the lungs of tumor bearing mice after combination immunotherapy. Overall, our preclinical data provide a strong rationale to use A2ARi with anti-PD-1 mAb for the treatment of minimal residual and metastatic disease. 116 POSTER (Board P110) Novel targets for antibody–drug conjugate therapy A.G. Grandi1 , S.C. Campagnoli1 , M.P. Parri1 , E.D.C. De Camilli2 , B.J. Jin3 , P.S. Sarmientos1 , G.G. Grandi4 , L.T. Terracciano5 , P.P. Pileri1 , G.V. Viale2 , R. Grifantini6 . 1 Externautics, R&D, Siena, Italy; 2 European Institute of Oncology, Pathology, Milan, Italy; 3 Fourth Military University, Immunology, XI’an, China; 4 Novartis Vaccines, R&D, Siena, Italy; 5 Basel Medical University, Pathology, Basel, Switzerland; 6 Externautics, Siena, Italy The study focuses on two novel potential therapeutic targets identified by a systematic immune-histochemistry (IHC) screening with a large collection of polyclonal antibodies (approximately 1600) raised against marginally characterized human proteins. Here we describe the molecular characterization of two surface-associated proteins (EXN36 and EXN91) associated to different cancer types. EXN36 is mainly over-expressed in ovary and breast cancers (frequency of approximately 30−40%). Interestingly, it is also over-expressed in triple negative breast cancer. The protein is involved in cell proliferation, migration and invasiveness. Concerning EXN91, it is an adhesion molecule and it acts as a signaling receptor, likely to be important in developmental processes and cell communication. The protein is mainly detected in colon cancer with high frequency (more than 80%), both in early and advanced stages, in high and low grade cancers. Interestingly, EXN91 is over-expressed in KRAS and BRAF mutant colon cancers with significant frequency (approximately 50%). Finally, it is also detected in esophagous SCC and ccRCC (10−20%). Murine monoclonal antibodies able to recognize EXN36 and EXN91 on the surface of cancer cells have been selected and characterized to assess their potential for specific therapeutic indications. In particular, five antiEXN36 mAbs are able to recognize the target protein in breast (Her2+ ,Er+ and triple negative cells) and ovary cell lines. A murine monoclonal antibody is able to recognize EXN91 on the surface of colon cancer cells. This antibody also shows the ability to inhibit growth of colon cancer in xenograft mouse models. Finally, it specifically binds cancer tissues by IHC, suggesting that it could be also developed as companion diagnostic tool for EXN91-based therapies. EXN36 and EXN91 monoclonal antibodies show a high number of binding sites on the cancer cell surface, ranging from 10,000 to 100,000 sites per cells. The antibody specificity has been confirmed in different immunoassays (Western blot, FACS, IHC) by gene silencing experiments and/or competition with peptides containing the antibody epitopes. Thes eantibodies have high affinity for their target epitopes (KD : 10−9 ,10−10 nM). Moreover, they show limited IHC reactivity in normal tissues (FDA tissue panel). Some antibodies are efficiently internalized by cancer cells, suggesting that they can be exploited for the development of Antibody–Drug Conjugate (ADC). The analysis of their potential for ADC is ongoing. Initial results from in vitro studies show that these antibodies, indirectly linked to commercially available drugs (e.g. auristatin-based, DM1, and duocarmycin), show significant anti-tumor activity with specific drugs and linker chemistry. Overall, the results indicate that EXN36, EXN91 and their specific monoclonal antibodies could be developed for the targeted therapy of cancer indications at high medical need, either alone or in combinatorial strategies.

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117 POSTER (Board P111) “Arming” the chimeric oncolytic adenovirus enadenotucirev to deliver checkpoint inhibitors and other therapeutics directly to tumours B. Champion1 , P. Kodialbail1 , S. Illingworth1 , N. Rasiah1 , D. Cochrane1 , J. Beadle1 , K. Fisher1 , A.C.N. Brown1 . 1 PsiOxus Therapeutics Ltd, Abingdon Oxford, United Kingdom Enadenotucirev (EnAd; formerly called ColoAd1) is a potent, chimeric Ad11p/Ad3 adenovirus active against a range of epithelial cancer cells, with a shorter time-to-lysis than either wild type Ad11p, Ad3 or Ad5. In normal cells, EnAd is attenuated and shows little or no activity by either cytotoxicity or by qPCR. In vivo, EnAd shows efficacy in a range of xenograft human tumour models following intra-tumoural, intravenous and intra-peritoneal injection, and is currently being evaluated clinically for treatment of several different epithelial cancers. Data from an ongoing clinical mechanism of action study have shown that i.v. dosed EnAd infects and replicates in tumour cells, producing significant amounts of viral protein (hexon), indicating that transgene encoded proteins will also be made in significant amounts by tumours following i.v. delivery of an armed EnAd virus. To develop ‘armed’ variants for delivery of therapeutic agents that enhance EnAd’s anti-tumour activity, we have developed a system for rapid generation of modified viruses that can be dosed systemically to deliver immunomodulatory antibodies into tumours. We chose to first encode antiVEGF antibodies since, unlike immunomodulators, they could be readily evaluated in vivo in immunodeficient mouse human tumour xenograft models. We have successfully produced EnAd variants encoding fulllength (NG-135) and ScFv (NG-76) forms of anti-human VEGF antibodies which have similar virus activity profiles to EnAd in cancer cell lines in vitro (virus replication, gene expression and oncolytic action), but also express and release the respective anti-VEGF antibody forms into the culture supernatant. Using either HCT-116 or DLD human colon carcinoma xenograft models we have shown that the virus infection profile following intra-tumoural injection is similar to the parental EnAd virus (virus replication and Hexon gene expression). Anti-VEGF antibody expression by these tumours could be detected in the tissue as both mRNA and functional antibody. Antibodies were detectable early (within 3 days of infection) and expression was sustained over several weeks. Furthermore, low levels of anti-VEGF antibody were detectable in the blood. Production and evaluation of viruses similarly expressing checkpoint inhibitor antibodies is now in progress, together with evaluation of anti-VEGF armed oncolytic viruses for their impact on the growth and microenvironment of tumour xenografts. 118 POSTER (Board P112) Major synergy between Coxsackievirus A21 (CAVATAK™) and radiotherapy or chemotherapy in bladder cancer G.R. Simpson1 , N. Annels1 , M. Ajaz1 , F. Launchbury1 , G. Bolton1 , A.A. Melcher2 , K.J. Harrington3 , G. Au4 , D. Shafren4 , H. Pandha1 . 1 The University of Surrey, Faculty of Health and Medical Sciences, Guildford, United Kingdom; 2 St James’s University Hospital Leeds, Institute of Molecular Medicine, Leeds, United Kingdom; 3 Institute of Cancer Research, Targeted Therapy Team, London, United Kingdom; 4 Viralytics Ltd & The University of Newcastle, Viralytics Ltd & The University of Newcastle, Newcastle, Australia Introduction: There are still no treatments for superficial bladder cancer (SBC) which alter its natural progression, where 20% of patients develop metastatic disease. SBC is often multifocal, has high recurrences after surgical resection and recurs after intravesical live BCG.As this is a clinical setting in which local live biological therapy is already well established, it presents intriguing opportunities for oncolytic virotherapy. Coxsackievirus A21 (CVA21) has recently been shown to be an efficient oncolytic agent that specifically targets and rapidly lyses human malignant melanoma, multiple myeloma, prostate and breast tumours, which express high levels of the CVA21 cellular uptake receptors both in vitro and in vivo. In addition, a Phase I clinical trial in late stage melanoma patients has recently been completed, and has demonstrated that intratumorally administered CVA21 is well tolerated in humans, and that 55.55% of patients experienced stabilization or reduction in injected tumour volumes. Materials and Methods: Infection Radio- & Chemotherapy Synergy assays, Combination index analyses, QPCR and IHC for ICAM-1/DAF, Culture of SBC. Results: Characterization of CVA21 cytotoxicity in a panel of cell lines yielded a range of sensitivities. CVA21 cytotoxicity seems to correlate expression of viral receptors ICAM-1 & DAF. The addition of radiotherapy or chemotherapy resulted in significantly increased cytotoxicity over CVA21 alone. When 5637 or T24 cells were irradiated (4−10 Gy) then 24 hours

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later exposed to CVA21 (multiplicities of infection 0.961–12.6), clear synergy was seen. Dose matrix analysis showed that combination indices reached minima of approximately 0.4. Similar analyses showed synergy between Mitomycin C (1.4–0.021ug/ml) and CVA21 at multiplicities of infection between 0.78 and 50 on 5637 cells, which was confirmed on T24 cells. Crucially exposure to Mitomycin C or radiotherapy up-regulates the expression of viral receptors ICAM-1 & DAF in bladder cancer cell lines at both the RNA and protein level. Ru19−19 cells showed low ICAM-1 expression and almost no cytotoxicity when infected with CVA21. Exposure to Mitomycin C increased Ru19−19 ICAM-1/DAF expression, resulting in enhanced cytotoxicity killing, higher than each agent alone. Changing the sequence of treatment with CVA21 and Mitomycin C on bladder cancer cell lines does not appear to change the cytotoxicity killing. Patient derived primary bladder cancer cell lines appear to be highly susceptible to CVA21 infection. Conclusions: Combining CVA21 with either radiotherapy or chemotherapy synergistically enhances cytotoxicity in bladder cancer cell lines. Radiation and chemotherapy enhanced CVA21 oncolysis, likely by increased viral receptors ICAM-1 and DAF expression. These results offer strong support for translational clinical trials of CVA21 plus chemotherapy or radiotherapy that have been initiated in the clinic 119 POSTER (Board P113) 3-Bromopyruvate as an inducer of immunogenic cell death in colon cancer cells K.H. Jung1 , J.H. Lee1 , J.W. Park1 , C. Quach1 , K.H. Lee1 . 1 Samsung Medical Center Sungkyunkwan University School of Medicine, Nuclear Medicine, Seoul, South Korea Background: As cancer cells tend to resist cell death and evade immune surveillance, there is a need to develop therapeutic strategies that induce apoptosis and immune-mediated clearance of cancer cells. 3-Bromopyruvate (3-BrPA) is a halogenated alkylating analog of pyruvic acid that has been shown to inhibit tumor proliferation. The anti-cancer effect is presumed to occur by compromised ATP synthesis through inhibition of glycolysis and mitochondrial complex II activity. In this study, we evaluated the ability of 3-BrPA to induce immunogenic cell death (ICD) in colon cancer cells. Materials and Methods: CT26 murine colon cancer cells were treated with 10 to 20 mM of 3-BrPA for 24 h. Well-known ICD inducers including doxorubicin (DX; 20 mM) and combined mitomycinC/tautomycin (MitoC; 30 mM/TTM; 150 nM) were used as positive controls. Cell surface exposed calreticulin (CRT) was detected by confocal microscopy and fluorescenceactivated cell sorting (FACS) analysis using alexa488 anti-CRT antibody. The magnitude of CRT exposure was measured by binding of 125 I labeled anti-CRT antibody prepared by the iodogen-method. Apoptotic and necrotic cells were identified by FACS using FITC-annexinV and PI staining. Results: Confocal microscopy showed increased binding of alexa488 antiCRT antibodies to the surface of CT26 cells treated with 3-BrPA, in magnitudes comparable to that by DX and MitoC/TTM. FACS analysis confirmed a significant right-shift of fluorescent signals in cells treated with 3-BrPA, in a fashion similar to that by DX and MitoC/TTM. Hence, 3-BrPA treatment increased fluorescent signal-positive cells from 2.7±0.8% to 16.9±0.9%. The amount of cell bound 125 I-labeled anti-CRT antibody increased to 246.1±109.4 % of control level by 3-BrPA treatment. Furthermore, FITC-annexinV and PI staining of 3-BrPA-treated cells showed that CRT was exposed on early apoptotic (AnnexinV+ PI− ) but not secondary necrotic (AnnexinV+ PI+ ) cells. Conclusion: This study provides evidence that, in addition to its glycolysis inhibiting property, 3-BrPA induces early apoptotic CRT exposure on the surface of cancer cells, indicating a potential role of ICD in its anti-cancer effects. 120 POSTER (Board P114) Density of CD 8 +ve T cells & CD 56 +ve NK cells in follicular adenoma & papillary carcinoma of thyroid in Pakistani population J. Varda1 , N. Naseem1 , A.H. Nagi1 . 1 University of Health Sciences Lahore, Morbid Anatomy & Histopathology, Lahore, Pakistan Background: In Pakistan, papillary thyroid carcinoma (PTC) is the commonest (69−71%) of all thyroid malignancies, while follicular adenoma is the most common variety of benign lesions accounting for 60% of all thyroid nodules. In tumour microenvironment, CD 8 +ve cytotoxic T lymphocytes (CTLs) & CD 56 +ve natural killer (NK) cells play a key role in progression from benign to malignant neoplasm as well as in the metastatic potential of tumour. Objectives: To determine the density of CD 8 +ve CTLs & CD 56 +ve NK cells in biopsies of patients with follicular adenoma and papillary thyroid carcinoma

Poster Session – Immunotherapy Material and Methods: A total of 50 patients presenting with various histological subtypes of follicular adenoma and different grades of PTC were recruited through non-probability convenient sampling during study period of January–December 2013 from different clinical centers of Lahore, Pakistan. Relevant clinical data was recorded and morphological findings including subtypes of follicular adenoma and histological grades of PTC was ascertained microscopically. Density of CD 8 +ve CTLs & CD 56 +ve NK cells was determined immunohistochemically and related with other histological parameters. Results: The mean age of the patients with thyroid lesions was 36.72±14.04 years (Age range: 14−84 years) with 74% females and 26% males and a female to male ratio of 2.8: 1. Among n = 25 cases each of follicular adenoma and PTC, 76.0% and 72.0% were females and 24.0% and 28.0% were males with a female to male ratio being 3.1: 1 and 2.1:1 respectively (p = 1.0). The ages of patients were divided into three age groups; Group 1 (10−34 years), Group 2 (35−59 years) & Group 3 (60−84 years). Among n = 25 cases each of follicular adenoma and PTC 64.0% and 56.0% cases were present in Group 1, 32.0% and 16.0% cases were present in Group 2 while 4.0% and 28.0% cases were seen in Group 3 respectively (p = 0.046). The density of CD 8 +ve CTLs was scored from 1−5 where increased frequency of score 3 (56%) and score 5 (40%) were observed in cases of follicular adenoma and PTC respectively (p = 0.01). Whereas when the density of CD 56 +ve NK cells was related to the total number of follicular adenoma and PTC, increased frequency of score 3 (96%) was observed in cases of follicular adenoma as compared to PTC (52%) (p = 0.000). Score 3 for CD 8 +ve CTLs and CD 56 +ve NK cells was most frequent among the microfollicular variety of follicular adenoma while this density increased with the increasing histological grades of PTC. Conclusion: The density of CD 8 +ve CTLs and CD 56 +ve NK cells was related strongly with the microfollicular subtype of follicular adenoma and grade II of PTC. Hence, the lymphocytic infiltrate in tumour microenvironment might be an independent prognostic factor if studied on a comparatively larger sample size with follow up of the patients. 121 POSTER (Board P115) A modified double-deleted vaccinia virus combining viral oncolysis and potential gene therapy as a novel therapeutic for atypical teratoid/rhabdoid tumors Y. Ruan1 , A. Narendran2 . 1 Alberta Children’s Hospital, Calgary Alberta, Canada; 2 Alberta Children’s Hospital, Pediatrics and Oncology, Calgary Alberta, Canada Background: AT/RT is a highly malignant brain tumour of infancy that has a poor outcome. The tumor is characterized by the loss of tumor suppressor gene SNF5, a core subunit of the SWI/SNF chromatin remodeling complex, which consequently leads to substantial epigenetic changes and tumorigenesis. Due to the unacceptable toxicity of current chemotherapy on children, safe and effective novel therapies are urgently needed. Oncolytic virotherapy has emerged as a promising approach that uses replication-competent virus to provide anti-neoplastic activity, through the action of direct killing via cell lysis and the additional effect via the activation of the host anti-tumour immune response. We have previously demonstrated the efficacy of double-deleted vaccinia virus (vvDD), a virus with improved safety and tumor specificity, in the ability of homing to and killing AT/RT xenografts. Vaccinia virus, in particular, has the additional advantage of having non-essential genes on its genome that can be replaced with foreign DNA to enhance cytotoxicity. We therefore generated a modified vvDD that expresses wild-type SNF5 gene and tested whether this virus possesses better efficacy against AT/RT cells. Methods: Modified virus, vvDD-hSNF5, was generated by cloning wild type human SNF5 gene into shuttle vector pSC65-mCherry. This construct was transfected and co-infected with vvDD-GFP to HEK293 cells to generate recombined viral particles. Successfully recombined viruses, in which the mCherry and hSNF5 genes replaced GFP in the TK locus, were selected by FACS (sorting of mCherry+ GFP− cells). Viral titre was determined by plaque assay and cytotoxicity against AT/RT cells was determined by crystal violet staining. The ability of vvDD-hSNF5 to express SNF5 gene in AT/RT cells was determined by western blotting. Results: We successfully generated the modified vvDD-hSNF5 that expresses high levels of SNF5 protein in AT/RT cells. Arming of vvDDhSNF5 does not affect the replication of the virus. SNF5 protein is primarily localized to the cell nucleus but is also present in the cytoplasm. vvDDhSNF5 is capable of decrease cell cycle progression in AT/RT cells, in contrast to control virus (vvDD). The ability to cause effective cytotoxicity remains unchanged in vvDD-hSNF5 virus. Conclusions: We generated a modified oncolytic virus that can potentially combine oncolytic virotherapy with gene therapy. As a proof-of-concept,

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we demonstrated that vvDD-hSNF5 possesses similar replication potency and in vitro cytotoxicity. The expression of SNF5 can induce cycle arrest in AT/RT cells. These preliminary observations grant further investigation of vvDD-hSNF5 in in vivo testing. 122 POSTER (Board P116) Analysis of immune-response markers in resectable NSCLC M. Uso´ 1 , E. Jantus-Lewintre1 , R. Sirera2 , S. Calabuig-Farinas ˜ 1, S. Gallach1 , E. Escorihuela1 , A. Blasco3 , R. Guijarro4 , C. Camps5 . 1 ´ de Investigacion ´ Hospital General de Valencia, Molecular Fundacion ` ` Oncology, Valencia, Spain; 2 Universitat Politecnica de Valencia, Biotechnology, Valencia, Spain; 3 Hospital General de Valencia, Oncology, 4 Valencia, Spain; Hospital General de Valencia, Thoracic Surgery, ` Medicine, Valencia, Spain Valencia, Spain; 5 Universitat de Valencia, Background: Several markers have been indentified to be involved in pro- and anti-tumor immune responses, and some of them may have a prognostic impact. In this study we have analyzed gene expression of a set of markers related to different immune responses in samples from resectable NSCLC patients. Material and Methods: RNA was isolated from fresh-frozen lung specimens (tumor and normal lung) from resectable NSCLC patients (n = 178). RTqPCR was performed to analyze the expression of CCL2, CCL22, CD1C, CD209, CTLA4, IL10, IL23A, LGALS1, LGALS2, and TGFB1 by the use of hydrolysis probes. Relative gene expression was assessed by Pfaffl formula and normalized by the use of CDKN1B, GUS and ACTB as endogenous genes (selected by GeNorm algorithm). Statistical analyses were considered significant at p < 0.05. Results: Patient’s median age was of 65 [26−85], 86.5% were male, 47.2% with squamous (SCC) histology and 11.2% presented a mutation in KRAS gene. Mann-Whitney test revealed significant differences between histology and TGFB1 expression (p = 0.020), being higher in patients with SCC. Moreover, higher levels of LGALS2 (p = 0.023) were found to be associated with absence of lymph node involvement and higher IL23A expression was associated with KRAS WT status (p = 0.026). Survival analyses revealed that higher levels of IL23A and LGALS2 were associated with longer overall survival (OS) (p < 0.001 and 0.007, respectively) and progression free survival (PFS) (p = 0.003 and p = 0.002, respectively) (Table 1). Furthermore, survival analyses restricted to adenocarcinoma (ADC) patients revealed that higher levels of IL10 and CTLA4 were both correlated with longer OS (p = 0.017 and 0.028, respectively) and PFS (p = 0.015 and p = 0.006), as well as IL23A and LGALS2 (Table 1). Table 1. Marker

NSCLC (n = 178) IL23A Low High LGALS2 Low High ADC (n = 74) IL10 Low High CLTA4 Low High IL23A Low High LGALS2 Low High

OS Median (months)

p

PFS Median (months)

0.001* 42.6 NR

0.003* 23.4 81.23

0.007* 46.6 NR

0.002* 26.2 NR

0.017* 37 81.2

0.015* 17.8 49.3

0.028* 37.5 NR

0.006* 18.8 81.2

0.025* 42.9 NR

0.015* 19.2 81.2

0.001* 37.4 NR

p

0.005* 19.2 NR

Gene expression levels were dichotomized as high (median or higher) and low (less than the median). Conclusions: Our data indicated that the expression of certain immuneresponse markers could be associated with better outcome in resectable NSCLC patients. Supported by grants PS09/01149, RD06/0020/1024 and RD12/0036/0025 from RTICC, and Instituto de Salud Carlos III.

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123 POSTER (Board P117) Targeting tryptophan metabolism in human lung cancer J. Deshane1 , C. Schafer1 , Y. Wang1 , A. Sawant2 , T.H. Jin1 , D. Zhi3 , S. Ponnazhagan2 , S. Grant4 . 1 UAB (University of Alabama Birmingham) Cancer Center, Medicine/Pulmonary, Birmingham AL, USA; 2 UAB (University of Alabama Birmingham) Cancer Center, Pathology, Birmingham AL, USA; 3 UAB (University of Alabama Birmingham) Cancer Center, Biostatistics, Birmingham AL, USA; 4 UAB (University of Alabama Birmingham) Cancer Center, Medicine-Hematology Oncology, Birmingham AL, USA Background: The purpose of the study is to determine whether combination chemotherapy-mediated targeting of tryptophan (Trp) metabolism by inhibition of indoleamine 2,3-dioxygenase (IDO) enzyme activity in immunosuppressive myeloid-derived suppressor cells (MDSCs) predicts improved clinical outcome in lung cancer patients. Methods: Percentages of circulating MDSCs were determined using CD33+ myeloid cells enriched from peripheral blood samples collected from Stage III−IV Non-Small Cell Lung Cancer (NSCLC) patients and their normal healthy relatives. Quantitation of L-Kynurenine (L-kyn), a read out for IDO enzymatic activity, was performed in serum and purified circulating MDSCs. Results: Serum IDO activity (levels of L-kyn) was found to be significantly correlated (P = 3.0×10−5 ) with disease status of patients (n = 11) and their normal relatives (n = 8) using a linear mixed model with family ID as random effect. The percentages of circulating granulocytic (G-MDSCs) and monocytic-MDSCs (Mo-MDSCs) immunosuppressive subsets are significantly associated with disease status. Using linear mixed model, we found G-MDSCs (P = 0.004) and Mo-MDSCs (P = 3.7×10−5 ) are both higher in cancer patients compared to normal healthy subjects. Following the second cycle of treatment with combination chemotherapies, serum L-Kyn was reduced significantly (n = 5, p = 0.026, paired t test). Mean effect of chemo is −55.0 nmol kynurenine/hour, suggesting response to combination chemotherapy strategies. Percentages of subsets of MDSCs are also significantly reduced after chemotherapy treatments (n = 5, p = 0.030 for G-MDSCs, p = 0.026 for Mo-MDSCs, paired t test). Mean effects of chemotherapy treatments are −2.9% for G-MDSCs and −12.4% for Mo-MDSCs. Ongoing studies will determine correlations of change in IDO activity in MDSCs with reduction in tumor burden and overall survival of lung cancer patients. Conclusions: These preliminary studies suggest that the IDO enzymatic pathway and tryptophan metabolism may serve as important therapeutic targets and diagnostic markers in predicting clinical outcomes of combination chemotherapies in the treatment of NSCLC in humans. Supported by ACS-IRG-60–001−53. 124 POSTER (Board P118) A WT1-derived peptide protects against metastatic melanoma in a syngeneic model by in vivo immunomodulatory effects on dendritic cells M.H. Massaoka1 , C.R. Figueiredo1 , N. Girola1 , R.A. Azevedo2 , L.R. Travassos1 . 1 Experimental Oncology Unit, Department of Microbiology ˜ Paulo SP, Brazil; 2 Butantan Institute, Immunology and Parasitology, Sao ˜ Paulo SP, Brazil Department of Biochemistry and Biophysics, Sao Background: The cell-penetrating peptide, WT1-pTj, derived from a zinc finger domain of the Wilms Tumor Protein 1 (WT1), displays antimelanoma activity in vitro by suppression of cell growth and induction of cellular senescence. We now report that WT1-pTj-mediated antitumor effects in vivo rather depend on the immune system primarily involving dendritic cells (DCs). Materials and Methods: In vivo protection experiments with WT1-pTj were made in mice challenged endovenously with B16F10-Nex2 melanoma cells. Syngeneic C57Bl/6 or immune deficient animals were used. The peptide significantly reduced lung metastatic nodules in syngeneic animals when injected i.p. In the present work: (a) The same protocol was used in immune deficient NOD/Scid/IL-2rgnull mice; (b) To investigate the role of DCs, the syngeneic melanoma model was used. Mice were injected e.v. with B16F10-Nex2 melanoma cells to induce lung metastasis, and seven days after tumor inoculation animals received a single dose of syngeneic bone-marrow DCs primed with melanoma lysate and treated ex vivo with WT1-pTj for two days. Lung nodules were counted and compared with the untreated control; c) CD11c+ DCs from the cervical lymph nodes of WT1-pTj-treated metastatic melanoma-bearing mice were examined for the expression of co-stimulatory molecules by FACS and production of cytokines by ELISA. Additionally, the levels of CD8+ and CD4+ T lymphocytes and NK1.1+ cells in the lung and spleen of treated and untreated mice with metastatic disease were determined.

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Results: Systemic WT1-pTj treatment protected against metastatic melanoma in immune competent but not in immune deficient animals. Remarkably, peptide-activated syngeneic DCs ex vivo protected against metastatic melanoma, even in animals with established pulmonary nodules. By culturing CD11c+ cells isolated from cervical lymph nodes of WT1pTj-treated animals developing metastatic melanoma, we found that the peptide induced phenotypic maturation of DCs (e.g. upregulation of CD40, MHC-II and CD86) and enhanced production of type-1 cytokines, such as IL-6 and IL-12. Simultaneously, a marked decrease in TGF-b1 production was observed in DCs retrieved from WT1-pTj-treated mice. Moreover, the WT1-pTj-mediated antitumor activity was associated with significantly augmented frequency of tumor-infiltrating lymphocytes (CD8+ and CD4+) and NK1.1+ cells, and down-regulation of splenic CD4+ FoxP3+ regulatory T cells. Conclusions: The present results show the immunomodulatory role of WT1-pTj and indicate that the WT1-derived peptide may act as a potent adjuvant in cancer immunotherapy. Supported by FAPESP no. 2010/51423-0 and the Brazilian National Research Council (CNPq). 125 POSTER (Board P119) Critical issues in the clinical development of oncolytic viruses − A regulatory perspective M. Schuessler-Lenz1 . 1 Paul-Ehrlich-Institute, Langen, Germany Oncolytic viruses are claimed to replicate selectively and preferentially in tumor tissue, without causing excessive damage to normal tissues. In Europe there has been a steady increase in the number of clinical trials performed with oncolytic viruses, and some development candidates have reached the pivotal stage of clinical development. As oncolytic viruses are replication competent there is a need to balance the therapeutic promises linked to this new treatment concept against the risks that may be associated with replication competent viruses. Most of the oncolytic viruses are classified as gene therapy medicinal products, based on genetic modifications to change the viral tissue tropism and to increase tumor specificity. From a European regulatory perspective, gene therapy medicinal products are in the scope of the Advanced Therapy Medicinal Product (ATMP) regulation. This implies that for the development of oncolytic viruses towards marketing authorisation, both national and European regulatory and legal requirements apply, based on the different key players for clinical trial authorisation and central marketing authorisation. At the European Medicines Agency the Committee for Advanced Therapies formulates the draft opinion on quality, safety and efficacy of oncolytic viruses for final approval by the Committee for Human Medicinal Products. In Germany clinical trial authorisations with oncolytic viruses are approved by the Paul-Ehrlich Institute. A review of clinical trials shows that oncolytic viruses are at different stages of clinical development. They constitute one of the main innovative treatment concepts in cancer therapy, as can be seen from the number of clinical trials submitted to the Paul-Ehrlich Institute. We review the main issues in early clinical development, based on our experience with clinical trial assessment and national advice. We also review the interaction between the national competent authority and the Committee for Advanced Therapies. 126 POSTER (Board P120) IMCgp100: A novel bi-specific biologic for the treatment of malignant melanoma W. Shingler1 , J. Harper2 , G. Bossi2 , D. Barker2 , J. Dukes2 , N. Liddy3 , S. Paston2 , T. Mahon3 , P. Molloy3 , M. Sami1 , E. Baston3 , B. Cameron3 , A. Johnson1 , A. Vuidepot3 , N. Hassan1 , Y. McGrath1 , B. Jakobsen1 . 1 Immunocore Ltd, Development, Abingdon Oxford, United Kingdom; 2 Immunocore Ltd, Cell Biology, Abingdon Oxford, United Kingdom; 3 Immunocore Ltd, Protein Science, Abingdon Oxford, United Kingdom Background: Despite significant advances in the treatment of metastatic melanoma, long-term remission for the majority of patients remains elusive. Kinase inhibitors provide potent but short-term responses for a significant proportion of patients and immunotherapy elicits long-term responses with the prospect of cure, but only in a minority. IMCgp100 comprises an affinity-enhanced T cell receptor (TCR) specific for the HLAA2 restricted melanoma gp100 peptide (YLEPGPVTA) fused to an anti-CD3 antibody fragment. Binding of IMCgp100 to melanoma cells redirects T cell cytotoxicity allowing them to kill even HLA down-regulated melanoma cells otherwise invisible to cancer specific T cells. Materials and Methods: Preclinical efficacy and safety testing of IMCgp100 has been performed using a range of in vitro assays of immunological activity against human tissue, including ELISpot, cytotoxicity

Poster Session – Immunotherapy assay and Incucyte. A Phase I clinical study is currently underway to determine the safety and tolerability of IMCgp100 in patients with metastatic melanoma, with extensive laboratory testing accompanying this first in human study to try to identify pharmacodynamic markers. Results: Here, we present data which provides the foundation for the clinical observations. In vitro, IMCgp100 is demonstrated to potently redirect T cells from late stage cancer patients to target melanoma tumours exhibiting HLA down-regulation, even in the presence of high numbers of regulatory T cells. Target cell killing is observed within hours and is specific for gp100. In addition killing is associated with the release of various proinflammatory cytokines and chemokines as well as cross-presentation of gp100 and other melanoma-associated antigens by dendritic cells. Thus, IMCgp100 demonstrates the potential to elicit potent short-term responses and trigger longer-term anti melanoma activity in vivo. Maximum tolerated dose has been established for weekly dosing with the drug being well tolerated and showing evidence of tumour shrinkage. Analyses of trial patient samples provide evidence for drug mediated T cell mobilisation and transient cytokine and chemokine release, including those reported to play a key role in anti-melanoma responses. Conclusion: These data support the potential of IMCgp100 as a new immunotherapy against advanced melanoma. In-vitro data demonstrate the drug is specific for melanoma cells, and preliminary early phase clinical data suggest acceptable toxicity and promising durable efficacy. 127 POSTER (Board P121) Correlation of clinical activity of pembrolizumab (MK-3475) with immunohistochemical staining for programmed death-1 ligand (PD-L1) in 50% of tumor cells in a prospective non-small cell lung cancer (NSCLC) validation population E.G. Garon1 , N.A. Rizvi2 , N.B. Leighl3 , R. Hui4 , J.P. Eder5 , A. Patnaik6 , C. Aggarwal7 , L. Horn8 , A.S. Balmanoukian9 , M.A. Gubens10 , E. Felip-Font11 , E. Carceny Costa12 , J.C. Soria13 , M.J. Ahn14 , H.T. Arkenau15 , J.S. Lee16 , G. Robinet17 , G.M. Lubiniecki18 , J. Zhang19 , K. Emancipator21 , R. Rutledge22 , M. Dolled-Filhart23 , L. Gandhi20 . 1 David Geffen School of Medicine at UCLA, Dicision of Hematology/Oncology, Los Angeles, USA; 2 Memorial Sloan Kettering Cancer Cente, Department of medicine, New York, USA; 3 Princess Margaret Cancer Centre, Department of Medicine, Toronto, Canada; 4 Westmead Hospital University of Sydney, Department of Medical oncology, Sydney, Australia; 5 Yale University, Department of medical oncology, New Haven, USA; 6 South Texas Accelerated Research Therapeutics, Clinical research, San Antonio, USA; 7 Abramson Cancer Center of the University of Pennsylvania, Department of medicine, Division of Hematology/Oncology, Philadelphia, USA; 8 Vanderbilt Ingram Cancer Center, Department of Medicine, Nashville, USA; 9 The Angeles Clinic and Research Institute, Department Lung and Thoracic cancer, Los Angeles, USA; 10 University of California San Francisco, Thoracic Tumours Group, San Francisco, USA; 11 Hospital General Universitari Vall d’Hebron, Department of medical oncology, Barcelona, Spain; 12 Catalan Institut of Oncology − Badalona, Badalona, Spain; 13 Gustave Roussy, Drug Development Department (DITEP), Villejuif, France; 14 Samsung Medical Center, Department of Internal Medicine, Samsung, South Korea; 15 Sarah Cannon Research UK, Oncology, London, United Kingdom; 16 Seoul National University Bundang Hospital, Department of Hematology/Oncology, Seongnam-si, South Korea; 17 Hopital Morvan, Institute de Cancerologie, Brest, France; 18 Merck & Co. Inc., Clinical Research, North Wales, USA; 19 Merck & Co. Inc., BARDS, North Wales, USA; 20 Dana-Farber Cancer Institute, Medical Oncology, Boston, USA; 21 Merck & Co., Inc., Molecular Biomarkers and Diagnostics, Whitehouse Station, NJ, USA; 22 Merck & Co., Inc., Oncology Clinical Researchs, Whitehouse Station, NJ, USA; 23 Merck & Co., Inc., Molecular Biomarkers and Diagnostics, Whitehouse Station, NJ, USA Background: The phase I KEYNOTE-001 study evaluated the safety, tolerability, and clinical activity of pembrolizumab, a selective anti-PD-1 antibody that blocks the interaction between programmed death-1 (PD-1) on T cells and its ligands PD-L1 and PD-L2 on tumor cells. Of those patients (pts) with tumor tissue evaluable for PD-L1 status by immunohistochemistry (IHC) in the initial 38-pt NSCLC cohort, all pts with clinical response by immune-related response criteria (irRC) were in the group with the highest PD-L1 expression. Tissue from these pts and an additional 119 pts were evaluated to generate a formalized training set. Among the 129 pts with tumor evaluable for PD-L1 expression and measurable disease at baseline per RECIST v1.1 as assessed by independent central review, a highly significant correlation between strong PD-L1 expression (i.e., staining in 50% of tumor cells) and the overall response rate (ORR) and progressionfree survival (PFS) was observed. Materials and Methods: 92 treatment-na¨ıve and 216 previously treated pts with measurable NSCLC whose tumors expressed detectable PD-L1

Poster Session – Immunotherapy (1% cutoff) using a prototype IHC assay using the 22C3 antibody were randomized to receive pembrolizumab at 10 mg/kg every 2 or 3 weeks; 2 pts were never treated. A new tumor biopsy 60 days prior to or, with Amendment 8, any time after the most recent therapy before the first pembrolizumab dose was required. Tumor response was assessed every 9 weeks by RECIST v1.1 by independent central review. All pts will have a minimum of 6 mo of follow-up. PD-L1 expression was subsequently evaluated in the study eligibility tumor samples by a clinical trial IHC assay using the 22C3 antibody for assessment of the relationship with outcomes with a goal of validating the utility of IHC staining in 50% of tumor cells as a biomarker to predict the efficacy of pembrolizumab. Results: After selection of the training set population, over 500 additional pts signed consent between 20 May 2013 and 12 May 2014 and provided tissue for PD-L1 assessment. Based on PD-L1 staining using the prototype IHC assay and other study eligibility criteria, 306 pts received pembrolizumab. Conclusions: At the meeting, we will present data evaluating whether the cutoff of PD-L1 staining by IHC in 50% of tumor cells that was selected based on our training set correctly predicts ORR and PFS in the validation set of 306 pts with advanced NSCLC treated with pembrolizumab. 128 POSTER (Board P122) T cell-mediated cancer immunotherapy through OX40 agonism M. Huseni1 , C. Du2 , J. Zhu2 , P. Pacheco-Sanchez3 , M. Moskalenko3 , H. Chiu4 , K. Dalpozzo1 , K. Totpal3 , L. Damico-Beyer5 , J. Kim2 . 1 Genentech, Oncology Biomarkers, South San Francisco CA, USA; 2 Genentech, Cancer Immunology, South San Francisco CA, USA; 3 Genentech, Translational Oncology, South San Francisco CA, USA; 4 Genentech, Biochemical and Cellular Pharmacology, South San Francisco CA, USA; 5 Genentech, Portfolio Management and Operations, South San Francisco CA, USA Background: Productive immune responses to tumors are hypothesized to require T cell costimulation. OX40 is a costimulatory molecule that is expressed on activated effector (Teff) and regulatory T cells (Treg). Agonistic antibodies targeting OX40 are predicted to counteract the immunosuppressive tumor microenvironment and promote T cell dependent anti-tumor immunity via two distinct mechanisms − activation and expansion of antigen experienced T cells and inhibition of T cell suppression. An anti-human OX40 antibody MOXR0916, and a surrogate anti-mouse OX40 antibody were tested for their ability to costimulate effector T cells and inhibit Treg function in vitro and in vivo, respectively. Materials and Methods: MOXR0916 is a humanized agonist OX40 antibody, which binds to human OX40 with subnanomolar affinity. Due to sequence divergence between human and mouse OX40, a murine agonist OX40 antibody was employed to assess anti-tumor efficacy, Teff costimulation and Treg dynamics in murine syngeneic tumor models. The role of Fc effector function was determined utilizing Fc receptor knockout mice and isotype variants of anti-mouse OX40. In vitro analysis of MOXR0916 and isotype variants of MOXR0916 in Teff costimulation and Treg suppression assays were conducted with sorted peripheral blood T cell populations from healthy donors. Results: In syngeneic mouse tumor models, anti-mouse OX40 treatment induced durable tumor regression which were associated with intratumoral Treg depletion and CD8+ T cell infiltration, and enhancement of interferon gamma (IFN-g) production. Tumor regression and pharmacodynamic modulation of Teff and Tregs required antibody crosslinking via Fc receptors. MOXR0916 costimulated CD4+ memory T cell proliferation and IFN-g production following T cell receptor engagement in a dose dependent manner. Additionally, MOXR0916 impaired the suppressive function of Tregs in na¨ıve T cell coculture assays. Memory CD4+ T cell costimulation was dependent on MOXR0916 effector function. In in vitro T cell activation assays, MOXR0916 costimulated Teff production of IFN-g at comparable levels as anti-mouse OX40. Conclusions: Anti-mouse OX40 antibody induced durable anti-tumor immunity in multiple syngeneic tumor models. Potent single agent activity correlated with Teff costimulation and modulation of Treg dynamics. MOXR0916 and anti-mouse OX40 induced comparable levels of IFN-g production, supporting the clinical investigation of MOXR0916 as a T cell directed therapy for advanced malignancies. 129 POSTER (Board P123) Characterization of PD-L1 expression in circulating tumor cells (CTCs) of non-small cell lung cancer (NSCLC) patients R. Krupa1 , D. Lu1 , M. Harvey1 , J. Louw1 , A. Jendrisak1 , D. Marrinucci1 , R. Dittamore1 . 1 Epic Sciences Inc., San Diego CA, USA Background: Novel PD-1 and PD-L1 targeting immunotherapies are demonstrating efficacy in multiple tumor types. Recent data have suggested

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that increased progression free survival (PFS) is observed in patients whith higher expression of the PD-L1 protein in tumor tissue. However, many NSCLC patients have insufficient tumor sample or have high co-morbidities preventing access to tissue IHC to determine PD-L1 expression and potential benefit to novel PD-1/PD-L1 immunotherapies. We developed a protein assay for PD-L1 protein and examined CTCs and CTC subpopulation incidence and molecular characterization in blood samples from newly diagnosed NSCLC patients. Material and Methods: 16 samples from newly diagnosed NSCLC patients prior to therapy were recruited and blood specimens were collected and shipped to Epic Sciences. All nucleated cells were plated onto glass slides and subjected to immunofluorescent (IF) staining and CTC identification by fluorescent scanners and algorithmic analysis. CTCs, defined as traditional (CK+, CD45− with intact and morphologically distinct DAPI+ nuclei), apoptotic (CK+, CD45−, non-intact nuclei) and CK− (CK−, CD45−, intact and distinct nuclei). CTC subtypes were characterized with PD-L1 IF to assess expression. Results: Assays for the PD-L1 protein were developed and specificity confirmed utilizing Colo205, SU-DHL-1 and A549 cells spiked into donor blood and run through the Epic Assay. Additionally, expression analysis of Colo205, SU-DHL-1 and A549 cell lines show increased differential PD-L1 expression when cells were exposed to interferon gamma. PD-L1 positive CTCs were detected in 8/16 (50%) of NSCLC patients. PD-L1 positivity was seen in traditional and CK− CTCs, as well as apoptotic and CTC clusters. Conclusion: PD-L1 protein assessment of CTCs and CTC subpopulations from NSCLC patients at diagnosis is feasible on the Epic CTC platform. This test demonstrates sensitivity and specificity and may aid in the identification of patients suitable for clinical trial studies with novel PD-1 or PD-L1 therapies. The identification of PD-L1 positive CTC subpopulations identifies unique tumor cell morphology and suggests evidence of epithelial plasticity in some patients. Further analysis of pharmacodynamics of CTCs and PD-L1 expression on CTCs in the setting of PD-1/PD-L1 therapies is warranted. 130 POSTER (Board P124) Preclinical results of ProCervix, a first in class, first in indication therapeutic vaccine targeting HPV16/18 infected women M. Esquerre1 , M. Bouillette-Marussig1 , A. Goubier1 , M. Momot1 , H. Keller1 , M. Bissery1 . 1 Genticel, Labege, France With the availability of HPV diagnostic tests, it is now possible to identify HPV infected women and to develop treatment in order to clear the infection and prevent the onset of neoplasia and cancer. ProCervix is a bivalent therapeutic vaccine consisting of two adenylate cyclase (CyaA) recombinant proteins originating from Bordetella pertussis into which HPV16 E7 and HPV18 E7 have been inserted. The CyaA targets CD11b expressed by Antigen Presenting Cells (APC). It delivers the vaccine antigens to both MHC-I and MHC-II pathways in the APC for presentation to CD4+ and CD8+ T cells. We report here on the preclinical properties of ProCervix therapeutic vaccine designed to treat women infected with HPV16 and/or 18 before they develop high grade cervical lesions. ProCervix binding to CD11b was performed using a cell line expressing human CD11b in a competition assay using biotinylated-CyaA HPV16 E7. The immunogenicity was evaluated in C57BL/6 mice vaccinated i.d. with ProCervix adjuvanted with Imiquimod 5% cream. E7-specific T cell responses were measured using IFN-g ELISpot and TH 1/TH 2 cytokines, using Cytometric Bead Array and FACS analysis. Cytotoxic T lymphocytes (CTL) were evaluated by in vivo killing assays. Therapeutic efficacy was evaluated in mice bearing 106 HPV16 E7-expressing tumour cells implanted s.c. and vaccinated on day 11 and 39. Tumour growth was measured with a caliper twice a week. Results obtained with an in vitro functional cell-based assay demonstrated that ProCervix binds to human CD11b-expressing cells. In vivo, using C57BL/6 mice, intradermal administration of ProCervix in combination with a TLR7 agonist, resulted in the development of both HPV16 E7-specific and HPV18 E7-specific T cell responses with a clear TH 1 cytokine profile. Using an in vivo killing assay, we demonstrated that ProCervix-induced CD8+ T cells are functional CTLs. Using syngeneic TC-1 tumour cells, our results demonstrated that these CTLs were able to eradicate HPV16-E7expressing cells. Finally, using this tumour-rejection model, we showed that even a low dose of E7 antigens lead to strong therapy when it delivered via ProCervix. These preclinical data prompted a phase I clinical study in HPV16/18 infected women with normal cytology which indicated that ProCervix was well tolerated, and exhibited a higher frequency of peripheral HPV-specific interferon-gamma associated T-cells and a higher viral clearance in the treated group compared to the placebo group.

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131 POSTER (Board P125) Zfra activates novel Hyal2+ CD3− CD19− memory spleen cells to block cancer growth, stemness, and metastasis in vivo N. Chang1 . 1 National Cheng Kung University, Tainan City, Taiwan Zfra is a 31-amino-acid zinc finger-like protein, which participates in tumor necrosis factor signaling. Here, we determined that synthetic full-length Zfra1−31 peptide selfpolymerized in degassed buffers without catalytic enzymes. When nude mice and BALB/c mice were pre-injected with micromolar levels of Zfra1−31 or truncated Zfra4−10 via tail veins, these mice became resistant to the growth, metastasis and stemness of prostate, breast, and lung cancer cells, melanoma cells, and many malignant cancer cells. Alteration of the Ser8 phosphorylation site to Gly8 abolished Zfra polymerization and cancer suppression in vivo. Injected Zfra was deposited mainly in the spleen. Transfer of Zfra-stimulated spleen cells to na¨ıve mice conferred resistance to cancer growth. Mechanistically, Zfra bound membrane hyaluronidase Hyal-2 and suppressed the TGF-b/Hyal-2/WWOX/Smad4 signaling, via down-regulation of Hyal-2 and activated WWOX (with Y33 phosphorylation), in the spleen for generating novel non-T/non-B memory cells, designated Hyal2+ CD3− CD19− cells. Transfer of these cells to na¨ıve mice also induced anticancer response. Similarly, agonist anti-Hyal-2 antibody mimicked the effect of Zfra in causing cancer suppression. In conclusion, Zfra self-polymerizes in the spleen to activate Hyal2+ CD3− CD19− cells for blocking cancer growth, stemness and metastasis in vivo. Supported in part by NSC and NHRI, Taiwan, and DoD, USA 132 POSTER (Board P126) A novel anti-PDL1 antibody-based bifunctional protein with enhanced immunological activity Y. Wu1 , S. Martomo1 , Z. Zhong1 , D. Lu1 , Z. Polonskaya1 , X. Luna1 , Z. Zhang1 , H. Zhang1 , L. Witte1 , S. Waksal1 , Z. Zhu1 . 1 Kadmon Corporation LLC, NYC, USA Preclinical and clinical evidence suggest that immunotherapies based on immunomodulating agents that inhibit immunosuppressive pathways (e.g., PD-1/PD-L1), or that stimulate the immunity of T-cells and NK cells (e.g., by cytokines such as IL-12, IL-15, IL-21), hold great potential for the effective treatment of cancer. The anti-PD1/PDL1 approach, via attenuation of inhibitory checkpoints, has shown success in certain clinical settings across multiple cancer types. Its full potential may be limited however by impaired overall immunity within the tumor micro-environment. The broad clinical application of immune-stimulating cytokines has been greatly hindered by their limited bioavailability, short half-life and systemic toxicities due to their lack of target specificity. It is therefore desirable to develop biotherapeutic agents that are capable of both inhibiting immunosuppressive pathways and simultaneously stimulating and expanding immune effector cells thereby increasing both innate and adaptive immune responses within the tumor environment for greater antitumor activity. To this end, we engineered a panel of recombinant bifunctional proteins that combine an anti-PD1 antibody with an immune-stimulating cytokine. These bifunctional proteins were genetically constructed and produced by mammalian cell cultures. One of the bifunctional proteins, KD-033, was efficiently expressed in mammalian cells and was fully stable when incubated in serum samples. The bifunctional protein retained binding and functional activity of both its parental components as tested by ELISA and cell-based assays.

Poster Session – Immunotherapy Further, KD-033 showed strong activity in relieving PD1/PDL1-meditated suppressive effect on CD4 T cell proliferation and cytokine (IL-2, IFN) production, and simultaneously showed significant enhancement of the killing activity of CD8 T cells and NK cells. 133 POSTER (Board P127) Analysis of immune microenvironment in resectable NSCLC: Prognostic value of regulatory and conventional T cell markers proportion M. Uso´ 1 , R. Sirera2 , S. Calabuig-Farinas ˜ 1 , A. Blasco3 , E. Pastor4 , ´ R. Guijarro4 , E. Jantus-Lewintre1 , J. Forteza5 , C. Camps6 . 1 Fundacion ´ Hospital General de Valencia, Molecular Oncology, de Investigacion ` ` Valencia, Spain; 2 Universitat Politecnica de Valencia, Biotechnology, Valencia, Spain; 3 Hospital General de Valencia, Oncology, Valencia, Spain; 4 Hospital General de Valencia, Thoracic Surgery, Valencia, Spain; 5 Instituto Valenciano de Patolog´ıa, Valencia, Spain; 6 Universitat de ` Valencia, Medicine, Valencia, Spain Background: Immune cells present in tumor microenvironment have been demonstrated to play an important role in tumor progression, and therefore, in patient’s outcome. Methods: FFPE samples from 117 early-stage NSCLC patients of primary tumor tissue were used. We assessed the mRNA expression levels of 11 genes (CD127, CD25, FOXP3, CTLA-4, IL-10, TGFB-1, LAG-3, GITR and TNFA as well as CD4 and CD8) in microdisected tumor and tumor stroma areas by RTqPCR. The presence of CD4+, CD8+ and FOXP3+ (+ = positive) lymphocytes was also assessed in 84 of these FFPE samples by immunohistochemistry (IHC). All statistical analysis were considered significant at p < 0.05. Results: Survival analyses revealed that patients with lower expression levels of CD8 in tumor areas have worse overall survival (OS) and progression free survival (PFS). Lower expression levels were also associated with worse OS for CD4 and LAG-3 in tumor. We also found that those patients with higher levels of FOXP3 stroma/FOXP3 tumor ratio had worse OS and the same was observed for FOXP3 stroma/CD4 tumor ratio and for FOXP3 stroma/CD8 tumor ratio. Furthermore, the presence of FOXP3+ and CD8+ lymphocytes assessed by IHC was associated with prognosis (Table 1). Gene expression levels were dichotomized according to the median in high and low. Infiltration of FOXP3+ lymphocytes was defined as high (10% of total lymphocytes) and low (<10%). Infiltration of CD8+ lymphocytes was dichotomized in high and low according to the median of the absolute number of CD8+ lymphocytes (X400). Conclusion: The presence of immune biomarkers in tumor microenvironment have a prognostic impact in resectable NSCLC patients. Supported by grants PS09–01149 and RD12/0036/0025 from ISCIII.

Table 1 (abstract 133). Survival analysis (log-rank test).

Analyzed by RTqPCR CD8 in tumor, low vs high CD4 in tumor, low vs high LAG3 in tumor, low vs high FOXP3 stroma/FOXP3 tumor ratio, low vs high FOXP3 stroma/CD4 tumor ratio, low vs high FOXP3 stroma/CD8 tumor ratio, low vs high Analyzed by IHC CD8+ lymphocytes in tumor, low vs high High FOXP3+ lymphocytes stroma/Low FOXP3+ lymphocytes tumor, vs others High FOXP3+ lymphocytes stroma/Low CD8+ lymphocytes tumor, vs others

OS Median (months)

p

PFS Median (months)

p

37.2 vs 81.23 42.9 vs 81.23 36.2 vs 69 NR vs 42.9 81.23 vs 46.66 74.33 vs 46.43

<0.001* 0.026* 0.017* 0.002* 0.025* 0.017*

19.43 vs 81.23 23 vs 37.8 22.1 vs 30.1 NR vs 19.43 37.8 vs 22.13 37.8 vs 22.13

0.002* 0.058 0.085 <0.001* 0.027* 0.022*

40.4 vs 73.98 36.2 vs 69 17.43 vs 68.8

0.021* 0.020* 0.011*

23 vs 58.83 17.49 vs 35 15.3 vs 35.9

0.026* 0.16 0.035*

Poster Session – Immunotherapy 134 POSTER (Board P128) Epigenetic immunomodulation by SGI-110 combined with immune checkpoint blockade for new therapeutic strategies A. Covre1 , C. Fazio1 , H.J.M.G. Nicolay2 , P.G. Natali3 , P. Taverna4 , M. Azab4 , S. Coral2 , M. Maio1 . 1 University Hospital of Siena, Medical Oncology and Immunotherapy, Siena, Italy; 2 Epigen Therapeutics s.r.l., Pordenone, Italy; 3 University of Chieti, Lab CINBO, Chieti, Italy; 4 ASTEX Pharmaceuticals Inc., Dublin, USA Background: SGI-110 is a dinucleotide of decitabine (DAC) and deoxyguanosine formulated as a low volume SQ injection that extends DAC exposure compared to DAC IV. Our in vitro and in vivo evidences identified a strong immunomodulatory activity of SGI-110 on human cancer cells of different histotype and on human melanoma xenografts, and showed a remarkable anti-tumor effect once combined with anti-CTLA-4 mAb in a syngeneic mouse model. In this study we evaluated the contribution of anti-tumor immune responses in the reduction of tumor growth achieved by this therapeutic combination. Materials and Methods: The mammary carcinoma cells TS/A (2×105 ) were implanted SQ in Balb/c mice. Animals bearing palpable tumors were treated with 3 mg/kg of SGI-110 (days 1−5), alone or combined with 100 mg of anti-murine CTLA-4 mAb (days 8, 11 and 14). The immunomodulatory effects of treatment were studied on tumor and normal tissues by RTPCR and by quantitative RT-PCR analysis of murine CTA expression. Immunohistochemical evaluation of tumor infiltrating immune cells was also performed. P1A-promoter methylation was tested by quantitative Methylation-Specific PCR (qMSP) on genomic DNA from tumor tissues. Results: The expression of P1A and Mage-a family members was induced in tumor tissues from animals treated with SGI-110, either alone or in combination with anti-CTLA-4 mAb, but not from mice treated with antiCTLA-4 mAb alone. Levels of P1A-specific mRNA were similar in tumors from mice treated with SGI-110 alone (3.18×10−04 P1A/b-actin molecules) or combined with anti-CTLA-4 mAb (1.18×10−04 P1A/b-actin molecules). The DNA hypomethylating effect of SGI-110 was sustained by the reduction of P1A promoter methylation in cancer tissues from SGI-110- (16%) and combination- (7%) treated mice vs control. Epigenetic remodelling was restricted to tumor tissue leaving almost unaltered normal ones. The contribution of immune cells in the therapeutic effectiveness of treatment was supported by the increased frequency of tumor infiltrating CD3+ cells in the combination arm (11±1.9) vs control (3.7±1.4) or single agent, antiCTLA-4 mAb (3±1.1) and SGI-110 (4.1±1.7), treated mice. Conclusion: These data highlight the involvement of the immune system in the anti-tumor effect of SGI-110 combined with CTLA-4 blockade. Based on these experimental evidences, an exploratory phase I trial to evaluate safety and immunobiologic activities of the combination is being activated in advanced melanoma patients. 135 POSTER (Board P129) AD-O64.3: IFN-g–TRAIL fusion protein. Use of two independent signaling pathways for a strong synergistic antitumor effect B. Zerek1 , J.S. Pieczykolan1 , S.D. Pawlak1 , P.K. Rozga1 , A. Pieczykolan1 , M. Szymanik1 , A. Jaworski1 , M. Galazka1 , K. Bukato1 , K. Poleszak1 , M. Teska-Kaminska1 , W. Strozek1 . 1 Adamed Sp. z.o.o., Drug Discovery, Warsaw, Poland Background: Interferon gamma (IFN-g) is a dimeric soluble cytokine, an representative of Type II interferons group. Secreted by NK, NKT, Th1, Tc and dendritic cells IFN-g ligand binds to two types of receptors and acts by activation the JAK-STAT pathway. Generally IFN-g antitumor action is mediated through the immunomodulatory function which results from the expression and action of many different classes of IFN-stimulated genes (ISGs) responsible for antigen processing, leukocyte trafficking and tumor cytotoxicity. IFN-g stimulates antibody-dependent toxicity and potentiates the process of connecting the cells with tumor cells. Additionally, it is able to induce an anti-proliferative effects and activates caspases, thereby inducing apoptosis in many cancer cell lines. IFN-g may cause Bak upregulation, cytochrome C secrection and Bax translocation. In many tumor lines showing resistance to TRAILstimulated apoptosis, interferon gamma acted synergistically, contributing to their sensitivity to TRAIL. Additionally, one of IFN-g effects is an intense stimulation of human monocytes to produce TRAIL protein. We developed the novel fusion protein AD-O64.3 consists of artificial dimer of IFN-g, recombinant variant of human TRAIL fragment and sequences recognized by tumor-specific proteases (MMP’s, uPa) in between. Materials and Methods: AD-O64.3 protein was expressed in E. coli and purified by IEC. Obtained protein was characterized biochemically and biophysically using CD spectroscopy, SEC-HPLC, protease cleavage and MTT cell cytotoxicity assays. Interaction with the respective receptors was

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confirmed with SPR method. The proapoptotic effect was tested using active caspase 3 staining. For in vivo potential we examined the efficacy on mice xenograft models of human renal cell carcinoma (ACHN), human colon adenocarcinoma (SW620) and human hapatoma (PLC/PRF/5) cell lines. Results: Obtained protein has well-defined secondary and quarternary structure and partially verified mechanism of action. The molecule showed in vitro specific cytotoxic effect on various cancer cell lines (IC50 below 0.1 ng/ml). New protein showed very low activity on normal cells. In vivo scINFTR3.0 showed promising effect displaying significant tumor volume inhibition. Conclusions: We developed very promising molecule with high antitumor activity showing synergistic effect with TRAIL. 136 POSTER (Board P130) Tumor-infiltrating lymphocytes (TILs) following intratumoral administration of ONCOS-102 are associated with prolonged overall survival in last line solid tumor patients S. Pesonen1 , T. Joensuu2 , E. Jager3 , J. Karbach3 , C. Wahle4 , K. Kairemo2 , K. Partanen2 , R. Turkki5 , A. Hemminki6 , C. Backman1 , M. von Euler1 , T. Hakonen1 , T. Ranki1 , L. Vassilev1 , A. Vuolanto1 , M. Jaderberg1 . 1 Oncos Therapeutics Ltd., Helsinki, Finland; 2 Docrates Cancer Center, Helsinki, Finland; 3 Krankenhaus Nordwest, ¨ Hamatologie-Onkologie, Frankfurt, Germany; 4 Krankenhaus Nordwest, ¨ Hamatologie-Onkologie, Helsinki, Finland; 5 Institute for Molecular Medicine Finland, Helsinki, Finland; 6 University of Helsinki, Cancer Gene Therapy Group Haartman Institute, Helsinki, Finland Intratumoral administration of adenovirus activates Toll-like receptor signalling leading to production of pro-inflammatory cytokines and activation of the innate immune system. Adenovirus causes immunogenic cancer cell death and the subsequent release of tumor antigens in the presence of danger signal results in the priming of a potent adaptive anti-tumor immune response. This effect may be further enhanced by immune-stimulating transgenes expressed by the virus. We present immunomonitoring results from a phase I study with ONCOS-102, a chimeric oncolytic adenovirus coding for GMCSF, in 12 patients with refractory injectable solid tumors. A total of 9 intratumoral injections were given at three different dose levels. Peripheral blood mononuclear cells (PBMCs) were collected at baseline and during the study to assess the antigen specificity of CD8+ T cells by IFN-gamma ELISPOT (analysis ongoing). The presence of tumor-infiltrating lymphocytes (TILs) were analyzed at baseline and 1 and 2 months after treatment initiation by immunohistochemistry (IHC). A computer assisted quantification of IHC samples was performed by calculating the fraction of a positively stained cellular region in the whole biopsy. In an exploratory analysis, a correlation between the post-treatment increase in different subpopulations of TILs and overall survival (OS) was assessed by Spearman’s rank correlation analysis. Intratumoral ONCOS-102 triggered an innate immune response in every patient as measured by a transient increase in systemic pro-inflammatory cytokines and induction of fever within 6 to 10 hours post administration. Infiltration of innate immune cells into tumors post-treatment was detected in 11 out of 12 patients. Concomitant infiltration of T cells was detected in 11 out of 12 patients with the most prominent increase seen in CD8+ T cells. Two patients showing the most robust increase in tumor-infiltrating CD8+ T cells post-treatment showed a prominent induction of tumor specific CD8+ T cells in peripheral blood. Biopsies from a non-injected distant tumor were obtained from one patient and a 2.5-fold increase in CD8+ T cells following ONCOS-102 treatment was detected. The correlation between OS and increase in TILs was analyzed. Among all sub-populations, CD8+ T cells (correlation coefficient (r) 0.74, p = 0.008) and CD68+ cells (r = 0.84, p = 0.001) showed the most significant correlation with OS. A correlation between post-treatment changes in CD8+ and CD68+ cells in tumors was also detected (r = 0.59, p = 0.046). Concomitant trafficking of innate and adaptive immune cells to the tumors and the induction of tumor specific CD8+ T cells suggest that ONCOS102 is able to induce de novo anti-tumor immune responses in advanced cancer patients. Correlation between TILs following treatment and OS, as well as CD8+ T cell infiltration into a non-injected distant lesion, suggest an involvement of a systemic immune activation.

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137 POSTER (Board P131) Immune checkpoint blockade enhances measles virotherapy C.E. Engeland1 , R. Veinalde1 , C. Grossardt1 , S. Bossow1 , 4 I. Shevchenko2 , V. Umansky2 , D.M. Nettelbeck3 , D. Jager ¨ , C. von Kalle1 , G. Ungerechts5 . 1 National Center for Tumor Diseases (NCT), Translational Oncology, Heidelberg, Germany; 2 German Cancer Research Center (DKFZ), Clincal Cooperation Unit Dermato-Oncology, Heidelberg, Germany; 3 German Cancer Research Center (DKFZ), Heidelberg, Germany; 4 National Center for Tumor Diseases (NCT), Medical Oncology, Heidelberg, Germany; 5 National Center for Tumor Diseases (NCT), Translational Oncology and Medical Oncology, Heidelberg, Germany

Poster Session – Immunotherapy 139 POSTER (Board P133) GBR1302: a BEAT® bispecific antibody for the treatment of HER2 positive cancers A. Croset1 , J. Macoin1 , R. Ollier1 , M. Pluess1 , C. Delon1 , D. Skegro1 , S. Blein1 , S. Hou1 , J. Back1 . 1 Glenmark Pharmaceuticals S.A., R&D, La Chaux de Fonds, Switzerland

Background: We hypothesized that combining oncolytic Measles virus (MV) with immune checkpoint blockade using antibodies against CTLA-4 and PD-L1 can provide synergistic anti-tumor effects. Material and Methods: Targeted immunomodulatory MV vectors encoding anti-CTLA-4 and anti-PD-L1 antibodies were cloned and characterized in vitro. Oncolytic efficacy was addressed in human melanoma xenografts. Immunotherapeutic efficacy was evaluated in a fully immunocompetent murine model of subcutaneous syngeneic malignant melanoma (B16CD20) and compared to parental MV with or without systemic administration of anti-CTLA-4 and anti-PD-L1. Results: Viral replication and oncolysis of MV anti-CTLA-4 and MV antiPD-L1 were not impaired. In the B16-CD20 model, treatment with MV antiCTLA-4 and MV anti-PD-L1 led to an increase in tumor-infiltrating cytotoxic T cells as well as a decrease in regulatory T cells. Therapeutic benefits in terms of delayed tumor growth and prolonged median overall survival were observed for animals treated with MV anti-CTLA-4 and MV anti-PD-L1, respectively. Further investigations in preparation of a Phase I trial are currently ongoing. Conclusions: Combining oncolytic MV with immune checkpoint modulation is a promising approach with direct implications for future clinical application.

Background: Human epidermal growth factor receptor 2 (HER2) is highly expressed in a significant proportion of breast, ovarian and gastric cancers. Since the discovery of its role in tumorigenesis and metastasis, HER2 has received great attention in cancer research over the past two decades. HER targeting therapies (such as Herceptin) have demonstrated efficacy but are limited by several resistance mechanisms. Targeting, recruiting, redirecting or bridging the cytotoxic T cells to HER2 tumor cells has the potency to trigger a more efficient elimination of tumor cells. Using the Glenmark BEAT® platform, we have produced a bispecific antibody, GBR1302, targeting human CD3e and HER2 which is designed to recruit cytotoxic T lymphocytes against HER2 positive cancer cells. Material and Methods: Re-directed lysis (RDL) in vitro killing assays using non-activated human peripheral blood mononuclear cells (PBMC) as effector cells and multiple carcinoma cell lines over-expressing HER2 at different levels were performed to determine the potency of GBR1302. In vivo, the potency of GBR1302 was evaluated using tumor models, in which a mixture of human HER2 expressing tumor cells and human PBMCs were xenografted subcutaneously in immuno-deficient mice (NOD-SCID). Results: GBR1302 was able to bridge cytotoxic T lymphocytes to HER2 tumor cells and trigger killing of the tumor cells at a very low concentration. A clear correlation between GBR1302 killing and HER2 expression levels on target cells was demonstrated. In in vivo xenograft experiments, GBR1302 induced a strong antitumor effect which was also dependent on HER2 expression levels. Conclusion: These data highlight a very good potency of GBR1302 for HER2 overexpressing cancers and warrant further clinical studies with this antibody.

138 POSTER (Board P132) Preclinical activity and safety of MGD006, a CD123xCD3 Bispecific DART® molecule for the treatment of hematological malignancies

140 POSTER (Board P134) Relationship between programmed death ligand 1 (PD-L1) expression and clinical outcome in patients (pts) with melanoma (MEL) treated with pembrolizumab (pembro; MK-3475)

P. Moore1 , G.R. Chichili1 , L. Huang1 , H. Li1 , S. Burke1 , F. Chen2 , L. He1 , Q. Tang1 , L. Jin1 , S. Gorlatov1 , V. Ciccarone1 , S. Koenig1 , M. Shannon1 , R. Alderson1 , S. Johnson1 , E. Bonvini1 . 1 MacroGenics, Rockville MD, USA; 2 MacroGenics, South San Francisco CA, USA T-cell directed killing of tumor cells using bispecific molecules may hold promise for cancer treatment. MGD006 (also encoded as S80880 and known as CD3xCD123-DART) is a bispecific diabody-based molecule consisting of two polypeptides, each composed of the VH of one antibody in tandem with the VL of the second antibody and covalently linked at the COOH termini via a disulfide bridge. MGD006 simultaneously binds T lymphocytes and cells expressing CD123(IL3RA), an antigen up-regulated in several hematological malignancies and differentially expressed in AML blasts compared to normal hematopoietic stem and progenitor cells. MGD006 mediates dose-dependent T-cell redirected killing of CD123-positive cell lines as well as primary AML blasts. Furthermore, MGD006 demonstrates potent activity in tumor-bearing mice engrafted with human peripheral blood mononuclear cells or when tumors were coinoculated with human T lymphocytes. MGD006 binds to both human and cynomolgus monkey antigens and can redirect T cells from either species to kill CD123-expressing cells. Preclinical safety and pharmacology was therefore assessed in monkeys that were infused with escalating doses of MGD006 on continuous or intermittent schedules over a period of 4 weeks. Monkeys infused with MGD006 showed depletion of circulating CD123positive cells, an activity biomarker, as early as 72 h after treatment initiation and persisting throughout the 4-week treatment period. Furthermore, T cells from treated monkeys exhibited efficient ex vivo redirected target cell lysis, indicating no functional exhaustion. Transient release of cytokines, particularly IL-6, was observed following the first MGD006 infusion, but not after subsequent administrations even when the dose was escalated. A reversible, decrease in red cell mass with concomitant reduction in CD123-positive progenitors in the bone marrow was also observed. No significant changes in circulating platelets or neutrophil levels were observed. The preclinical safety and efficacy profile of MGD006 supports advancement to clinical evaluation. A phase 1 safety study of MGD006 in relapse/refractory AML is currently recruiting patients (http://clinicaltrials.gov/ ct2/show/NCT02152956?term=mgd006&rank=1). Note: Studies involving animal subjects were performed following institutional IACUC approval.

A. Daud1 , O. Hamid2 , C. Robert3 , F.S. Hodi4 , J.D. Wolchok5 , W.J. Hwu6 , J.S. Weber7 , R. Kefford8 , P. Hersey9 , A.M. Joshua10 , R. Joseph11 , T.C. Gangadhar12 , R. Dronca13 , A. Patnaik14 , H. Zarour15 , K. Gerigich16 , J. Lunceford16 , K. Emancipator16 , M. Dolled-Filhart16 , X. Li16 , P. Kang16 , S. Ebbinghaus16 , A. Ribas17 . 1 University of California San Francisco, Medicine Division of Hematology and Medical Oncology, San Francisco CA, USA; 2 The Angeles Clinic and Research Institute, Melanoma Center, Los Angeles CA, USA; 3 Gustave Roussy and INSERM U 981, Medical Oncology, Villejuif Paris-Sud, France; 4 Dana-Farber Cancer Institute, Medicine, Boston MA, USA; 5 Memorial Sloan-Kettering Cancer Center, Medicine, New York NY, USA; 6 The University of Texas MD Anderson Cancer Center, Melanoma Medical Oncology, Houston TX, USA; 7 H. Lee Moffitt Cancer Center, Cutaneous Oncology, Tampa FL, USA; 8 Crown Princess Mary Cancer Centre Westmead Hospital and Melanoma Institute Australia University of Sydney, Medicine, Sydney, Australia; 9 University of Sydney, Medicine, Sydney, Australia; 10 Princess Margaret Cancer Centre, Medical Oncology, Toronto, Canada; 11 Mayo Clinic, Medical Oncology, Jacksonville FL, USA; 12 Abramson Cancer Center of the University of Pennsylvania, Medicine, Philadelphia PA, USA; 13 Mayo Clinic, Oncology, Rochester MN, USA; 14 South Texas Accelerated Research Therapeutics, Phase I Research, San Antonio TX, USA; 15 University of Pittsburgh, Medicine, Pittsburgh PA, USA; 16 Merck & Co. Inc., Molecular Biomarkers and Diagnostics, Whitehouse Station NJ, USA; 17 University of California Los Angeles, Hematology/Oncology, Los Angeles CA, USA Background: In an initial analysis of the relationship between PD-L1 expression and efficacy of the anti-PD-1 monoclonal antibody pembro (training set; n = 125), the preliminary cutpoint for PD-L1 positivity was an Allred proportion score (APS) 2 (ie, staining in 1% of tumor cells). Using this cutpoint, the PD-L1+ pts (71%) had a significantly higher ORR (P = 0.0007) and longer PFS (P = 0.0051) than PD-L1− pts. We report the results of an independent validation set to confirm the cutpoint and provide pooled results of the training and validation sets. Methods: The validation set included 150 PD-L1-evaluable pts with ipilimumab na¨ıve and treated MEL treated with pembro 10 mg/kg Q3W or 2 mg/kg Q3W in KEYNOTE-001. Response was assessed every 12 wk by RECIST 1.1 by independent central review. PD-L1 expression was assessed in pretreatment tumor biopsies by IHC using the 22C3

Poster Session – Preclinical Models

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Table (abstract 140): Relationship between tumor PD-L1 expression and clinical activity Measurable disease and interpretable PD-L1 IHC N

Validation Set PD-L1+ (APS 2) PD-L1− (APS <2) HR (95% CI), PD-L1+ vs PD-L1− 2-sided P c Pooled Set PD-L1+ (APS 2) PD-L1− (APS <2) HR (95% CI), PD-L1+ vs PD-L1− 2-sided P a b c

ORRa , n (%)

Interpretable PD-L1 IHC N

PFSa 24-wk, %

Median (range), wk

OSb 12-mo, %

Median (range), mo

111 24 − −

40 (36) 1 (4) − 0.002

123 27 − −

45 19 − −

24 12 0.43 (0.27–0.69) 0.0002

67 43 − −

NR 8 0.43 (0.24–0.78) 0.0042

194 54 − −

81 (42) 5 (9) − <0.0001

212 63 − −

51 29 − −

24 12 0.50 (0.36–0.69) <0.0001

72 53 − −

NR 13 0.49 (0.33–0.74) 0.0005

Analysis cutoff: Oct 18, 2013. Analysis cutoff: May 6, 2014. PD-L1 association test.

antibody. APS included staining in tumor cells. Response rate, PFS, and OS were compared between PD-L1+ and PD-L1− pts using Miettinen and Nurminen’s method (ORR) and Cox regression (PFS, OS). Results: 82% of validation set pts were PD-L1+ (APS2). There was a significantly higher response rate and significantly longer PFS and OS for PD-L1+ pts in the validation set (Table). The positive (PPV) and negative (NPV) predictive values for ORR in the validation set were 36% and 96%. 77% of pooled set pts were PD-L1+ (Table). PPV and NPV for the pooled set were 42% and 91%. Conclusions: Tumor PD-L1 expression was associated with higher ORR and prolonged PFS and OS in advanced MEL pts treated with pembro. Although NPV is >90%, the high prevalence of PD-L1 positivity, durable responses in PD-L1− pts, and unknown prognostic value and dynamic nature of tumor PD-L1 expression in MEL suggest PD-L1 expression may not be useful for selecting MEL pts for pembro treatment. 141 POSTER (Board P135) Phase I study of ipilimumab with stereotactic radiosurgery for melanoma patients with brain metastases W. Shi1 , E. Wuthrick2 , K. Feeney3 , M. Werner-Wasik4 , D.W. Andrews5 , J.J. Evans5 , K. Judy5 , C. Farrell5 , A.P. Dicker4 . 1 Thomas Jefferson University Hospital, Radiation Oncology, Philadelphia PA, USA; 2 Ohio State University, Radiation Oncology, Philadelphia PA, USA; 3 Thomas Jefferson University, Medical Oncology, Philadelphia PA, USA; 4 Thomas Jefferson University, Radiation Oncology, Philadelphia PA, USA; 5 Thomas Jefferson University, Neurological surgery, Philadelphia PA, USA Background: Brain metastases (BM) occur in more than half of patients with advanced melanoma. Stereotactic radiosurgery (SRS) is an effective treatment for symptom palliation and provides a benefit in local control for patients with limited BM. Ipilimumab (Bristol-Myers Squibb) is a human monoclonal antibody targeted to cytotoxic T-lymphocyte Antigen 4 antibody (CTLA-4) receptor with survival benefit in patients with metastatic melanoma. We hypothesize that combining SRS with Ipilimumab will yield a synergistic effect. However, the toxicities of this combined therapy are unknown. This is a phase I study is to assess the safety profile of combining escalating doses of Ipilimumab with standard dose SRS. Methods: Eligible patients had  4 BM with the largest lesion 4 cm, or resection cavity 4 cm. Ipilimumab starting dose was 3 mg/kg once on weeks 1, 4, 7, and 10. SRS was 15−24 Gy, based on the size, delivered on day 1 of week 1. The primary endpoint is maximum tolerated dose (MTD) assessed 30 days following SRS. Secondary endpoints include the rate of new BM, extra-cranial disease response, overall survival (OS), progression free survival (PFS), and adverse events. Results: Nine patients with a median 1 BM (range, 1−3) were enrolled from November 2011 to March 2014. Eight (89%) patients had extra-cranial metastases. Five received ipilimumab 3 mg/kg and four patients received 10 mg/kg. The median follow up was 4 months (range, 2−19) and no doselimiting toxicities have been observed. All patients have local control of treated index lesins, 80% of assessable patients have developed new BM, and 80% of assessable patients have stable extra-cranial disease. Median OS was not reached. Intracranial PFS at 4 months was 38.9%. PFS of any site was 22% at 4 months. Adverse events were mild, grade 1−2 and grade 3 in six (66%) and one (11%) patient, respectively. Conclusions: Concurrent ipilimumab and SRS is well tolerated in patients with BM from melanoma. This phase I study will continue to assess the MTD of ipilimumab for future phase II trials.

142 POSTER (Board P136) IL-17 as a novel molecular target for prostate cancer prevention A. De Angulo1 , R. Faris2 , C. Jolly1 , L. deGraffenried1 . 1 University of Texas at Austin, Nutritional Sciences, Austin, USA; 2 Texas A&M University Health Science, Microbial Pathogenesis and Immunology, Bryan, USA Background: Prostate cancer represents 14% of all new cancer cases in the US and is the fifth leading cause of cancer death. Incidence is closely associated with aging, but the mechanisms by which aging promotes prostate cancer development are unclear, limiting the development of effective preventive interventions. Aging is accompanied by immune dysfunction and a progressive increase in pro-inflammatory cytokines, including interleukin 17 (IL-17). Aging-associated increases in circulating IL-17 promotes pro-inflammatory signaling in prostate epithelial cells, possibly contributing to tumorigenesis. Methods: Serum and splenic CD4+ T-lymphocytes from young T cell aging-mimic mice as well as young and aged wild-type mice were collected. Surface markers and intracellular levels of IL-17, IFN-g and IL-4 in isolated CD4+ T-cells were measured using flow-cytometry. shRNA was used to knock down the IL-17 receptor in LNCaP prostate cancer cells and RWPE-1 non-transformed prostate epithelial cells, which were then exposed to mouse sera or conditioned media from stimulated T-lymphocytes. NFkB and STAT3 activation, NF-kB and STAT3 target gene expression, and epithelial cell transformation were all measured in prostate epithelial cells. Results: T cells from aging and aging-mimic mice secrete elevated levels of IL-17, possibly due to an imbalance in the TH17/TH1 and TH17/TH2 cell ratios. T-lymphocyte-secreted IL-17 from aging-mimic mice induced NF-kB and STAT3 activity and target gene expression in LNCaP and RWPE-1 cells. Importantly, IL-17 also promoted pro-tumorigenic changes in RWPE-1 cells, leading to a pre-transformed phenotype. Inhibition of IL-17 signaling blocked age-induced changes in both the cancer and non-transformed prostate epithelial cells. Conclusion: Aging of the immune system is associated with increased levels of IL-17, which stimulates the pro-inflammatory NF-kB and STAT3 pathways in prostate epithelial cells and activates programming associated with cellular transformation. Inhibition of IL-17 signaling blocks the protumorigenic activity induced by aging T cells. These findings provide evidence that the dysregulation of IL-17 production in aged T-cells may directly contribute to the increased risk for prostate cancer. Future novel immunotherapies for prostate cancer could target the increase production of IL-17 associated with aging.

Preclinical Models 143 POSTER (Board P137) Regional bias of intratumoral genetic heterogeneity of nucleotide repeats in colon cancers with microsatellite instability C. An1 , Y. Choi2 , M. Kim2 , J. Lee1 , N. Yoo2 , S. Lee2 . 1 Uijongbu St. Mary’s Hospital, Surgery, Uijongbu City, Korea; 2 Catholic University of Korea, Pathology, Seoul, Korea Background: Intratumoral heterogeneity (ITH) may produce regional biases in genotype and phenotype evaluation in a single tumor and may impede proper cancer diagnosis.

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Material and Methods: To evaluate the extent of ITH in colorectal cancer (CRC) with microsatellite instability (MSI), we obtained 4−7 biopsies from 39 CRCs followed by MSI analysis either using the Bethesda MSI evaluation system or Promega system with 5 mononucleotide markers. Results: We found decreased prevalence of MSI (+) by the Promega system compared to the Bethesda system. The overall discordance between the two systems was 54%. In contrast to the previous studies that had shown discordance only in low MSI (MSI-L), our results showed the discordance not only in MSI-L, but also in high MSI (MSI-H) cases. Among the MSI (+) CRCs, ITH of MSI status was identified in 41.7% of CRC by the Bethesda system and 22.2% by the Promega system. In terms of MSI markers, the ITH originated from dinucleotide markers in most cases (69%), but it originated from mononucleotide markers (31%) as well. Pooling of DNA from a regional biopsy with MSI (+) with additional biopsies from stable MSI (MSS) showed that this approach was beneficial to increase the sensitivity of MSI detection. Conclusions: Our results indicate that ITH of MSI phenotype by the Bethesda system is more overestimated than previously identified. However, because there was considerable ITH of MSI subtypes and markers even by the Promega system, our data suggest that analysis of MSI status in multiple regional biopsies is needed for a better evaluation of MSI status in CRC. 144 POSTER (Board P138) Association between interleukin 17/interleukin 17 receptor gene polymorphism and papillary thyroid cancer Y. Eun1 , H. Chung1 , Y.C. Lee1 . 1 Kyung Hee University, Otolaryngology, Seoul, South Korea Background: Interleukin 17 (IL17) is an important cytokine for inflammatory and autoimmune disease. Recently, although numerous studies have been suggested the role of IL17 in tumor development, the mechanism remains to be unknown. The aim of this study is to determine whether single nucleotide polymorphisms (SNPs) in IL17 and IL17R contribute the development of papillary thyroid cancer (PTC) and assess the relationship between IL17 and IL17R SNPs and the clinicopathologic characteristics of PTC. Material and Methods: Eight SNPs located within the three genes of IL17A, IL17RA and IL17RB were genotyped using direct sequencing in 94 patients with PTC and 213 patients without PTC (controls). Genetic data were analyzed using commercially available software. And, the statistical analyses were performed according to clinicopathologic characteristics of PTC. Results: Genotyping analysis demonstrated that the SNP rs4819554 of IL17RA (codominant model 1, OR = 0.39, P = 0.001; and dominant model, OR = 0.45, 95% CI, P = 0.002) and the SNP rs1025689 of IL17RB (dominant model, OR = 0.59, P = 0.043) were significantly associated with the development of PTC. The SNP rs2275913 of IL17A (codominant model 2, OR = 0.19, P = 0.034; dominant model, OR = 0.34, P = 0.033) was significantly associated with multifocality. Furthermore, IL17RA SNP rs4819554 (dominant model, OR = 0.25, P = 0.010) was significantly associated with bilaterality of cancers. Conclusion: In our case–control study of SNPs in the IL17 and IL17R gene in patients with PTC, we demonstrated that IL17RA polymorphism have the possibilities of developing PTC and can influence the bilaterality of PTC. 145 POSTER (Board P139) ARQ 087, a novel pan FGFR-inhibitor crosses the BBB (blood–brain barrier) and distributes to the brain of rats R. Savage1 , T. Hall1 , B. Schwartz1 . 1 ArQule Inc., Woburn, USA Background: ARQ 087 is a potent multi-kinase inhibitor with pan-FGFR activity against FGFR1, FGFR2, FGFR3 and FGFR4 kinases. Preclinical data from human cell lines and xenograft models support the exploration of its anti-tumor activity across a broad range of human solid and hematological malignancies. Material and Methods: QWBA (Quantitative Whole Body Autoradiography) and metabolite profiling studies were conducted with 14 C-ARQ 087 in rats. Free concentrations of 14 C-ARQ 087 related radioactivity were determined in rat brain via microdialysis. The brain regions sampled by microdialysis were the right striatum and left ventricle. The concentrations sampled in the brain were compared to those obtained in peripheral blood sampled from the right jugular vein. Results: From QWBA studies, 14 C-ARQ 087 derived radioactivity was found to distribute to the cerebellum, cerebrum, choroid plexus, corpus callosum, lateral ventricle, meninges and spinal cord (as well as broadly distributing to the rest of the body) after 5-Day repeat dosing in SpragueDawley rats. At 24 hours post dose on Day 5, 14 C-ARQ087 derived

Poster Session – Preclinical Models radioactivity ranged from 0.540 mg equiv./g (cerebellum) to 4.12 mg equiv./g (choroid plexus) versus 0.756 mg equiv./g in plasma. In brain tissues AUC(0−24 h) ranged from 12.4 (cerebellum) to 408 (choroid plexus) mg equiv.·hr/g versus 34.2 mg equiv.·hr/g in plasma. Metabolite profiling data in rat brain showed that ARQ 087 is present primarily as parent drug. Preliminary microdialysis data in rat brain showed that free unbound ARQ 087 (based on AUC) is present in the striatum (17.45%) and in the left ventricle (16.30%) compared to that in systemic circulation. Conclusions: ARQ 087 distributes to the brain of rats as well as broadly to the rest of the body. Metabolite profiling and preliminary microdialysis studies confirmed that in brain primarily parent ARQ 087 is present and available in part as free ARQ 087. 146 POSTER (Board P140) Establishment of patient-derived xenografts (PDX) models for triple negative breast cancer (TNBC) as a pre-clinical platform for drug development J. Thatte1 , M. Meza1 , J. Ricono1 , T. Broudy1 , C. Mirsaidi1 , P. Nair1 . 1 Molecular Response, San Diego California, USA TNBC comprises 15−20% of breast cancers in the United States. TNBC lacks expression of estrogen receptor, progesterone receptor and receptor tyrosine protein kinase ERBB2 or Her-2/neu oncogene amplification. As a result, this type of breast cancer is difficult to treat as most of the chemotherapies target these 3 receptors. TNBC is an aggressive form of cancer associated with high morbidity, mortality and shorter diseasefree survival. Prognosis and management of TNBC is complicated due to its heterogeneous clinical presentation, histology and response to therapy. PDX models in immune compromised mice have become a valuable tool for preclinical drug development in recent years. Here, we report development and characterization of the TNBC PDX model. We used patient derived TNBC tissues to generate 5 new TNBC models in NOD-SCID mice. Patient tumors were pre-screened for their ER, PR and Her-2/Neu expression by IHC, prior to inoculation in mice. We report efficacy of two standard of care drugs, cisplatin and vinorelbine, in the TNBC PDX model which demonstrates the potential utility of the TNBC model in drug discovery effort in oncology for treatment of TNBC. 147 POSTER (Board P141) An integrated approach for identifying E-cadherin synthetic lethality networks I. Bajrami1 , S.J. Pettitt1 , R. Brough1 , H. Pemberton1 , D. Kastrev1 , Y. Fontebasso1 , J. Frankum1 , J. Campbell1 , A. Ashworth1 , C.J. Lord1 . 1 Institute of Cancer Research, Division of Breast Cancer, London, United Kingdom The E-cadherin (CDH1) tumour suppressor gene encodes a calciumdependent cell–cell adhesion glycoprotein, which has roles in maintaining cell polarity, differentiation, cell migration and survival. E-cadherin dysfunction is a feature common to many epithelial tumours, with the highest incidence occurring in diffuse gastric cancer (50%) and lobular breast cancer (56%) and can occur via CDH1 mutation, deletion or epigenetic silencing. Although E-cadherin dysfunction is relatively common, approaches to target this pathogenic alteration do not as yet exist. We have taken an integrated functional genomics approach to identifying E-cadherin synthetic lethality effects that exploits siRNA. Using a combination of Achilles’ Heel siRNA and small molecule inhibitor screens in histologically and genetically diverse tumour cell line panels, we have identified a compendium of genes whose disruption selectively targets E-cadherin deficient cells. Alongside the Achilles’ Heel screens in tumour cell models, complementary screens in isogenic systems with shRNA and CRISPR-engineered E-Cadherin defects will also be presented. As an orthogonal approach, we have also exploited piggyBac transposonbased mutagenesis in haploid ES cells to generate a diverse mutant library consisting of 100,000 mutants which are now being used in E-cadherin synthetic lethal screens. Together, these systems provide a framework for identifying candidate synthetic lethal effects suitable for mechanistic dissection and subsequent validation. The results from these complementary approaches and subsequent validation of selected effects will be presented.

Poster Session – Preclinical Models 148 POSTER (Board P142) RAS synthetic lethal interactions from yeast to human cells S. van Wageningen1 , A. Prahallad1 , G. Heynen1 , R. Rothstein2 , R. Bernards1 . 1 Netherlands Cancer Institute Antoni van Leeuwenhoek hospital, Molecular Carcinogenesis, Amsterdam, Netherlands; 2 Columbia University, Genetics Department, New York, USA Background: Synthetic lethal (SL) interactions are used to develop targeted cancer therapy. However, novel SL interactions discovered in mammalian cell cultures are often cell type specific and are therefore only relevant to a small, or difficult to define, subset of patients. We developed a strategy in which we prioritize potential SL drug targets using the genetically tractable model system Saccharomyces cerevisiae. Material and Methods: Weperformed a SL screen by expressing a constitutively active RAS allele, RAS2(V19), in ~4800 S. cerevisiae strains in which each individual gene is deleted. Next we tested if SL interactions were conserved in human cancer cell lines. Results: The yeast screen yielded a hit list highly enriched for mutants with a defect in ‘endoplasmic reticulum (ER)-to-Golgi-to-vacuole’ transport. Moreover, we found that this list had a significant overlap with strains sensitive to b-mercaptoethanol, DTT and tunicamycin. We hypothesized that ER homeostasis was disturbed in these cells. The two gene deletion mutants most sensitive to ER stress are IRE1 and HAC1. These genes make up the unfolded protein response (UPR) in yeast; the signaling pathway that restores ER homeostasis. Both UPR genes were SL with RAS2(V19). Next we asked if we could detect a SL interaction between oncogenic RAS and the UPR in human cells. We find that a SL interaction between oncogenic RAS and the UPR is dependent on specific RAS effector pathways in human cell cultures. Conclusions: The UPR is conserved in evolution. However, signaling pathways downstream of RAS have diverged over time. We will present how the interaction between oncogenic RAS and the UPR has evolved in human cells and how this interaction can be exploited for therapeutic intervention. 149 POSTER (Board P143) Quantitative mutational assessment of circulating tumor DNA using massively parallel deep sequencing in plasma and urine from advanced colorectal cancer patients J.C. Poole1 , C.R.T. Vibat1 , L. Benesova2 , B. Belsanova2 , S. Hancock1 , T.L. Lu1 , M.G. Erlander1 , M. Minarik2 . 1 Trovagene Inc., R&D, San Diego, USA; 2 Genomac Research Institute, Center for Applied Genomics of Solid Tumors, Prague, Czech Republic Background: Technologies enabling the assessment of circulating tumor DNA (ctDNA) in biofluids expand the clinical utility to detect and monitor cancer patient oncogenic mutations by minimally invasive and non-invasive liquid biopsy methods. Mutational tumor load quantification with high clinical sensitivity is vital for robust individualized assessment of systemic therapeutic responsiveness and resistance. A quantitative ctDNA assay using a massively parallel deep sequencing approach was developed to determine patient ctDNA mutational status. Material and Methods: Initial assay development was for the simultaneous detection of 13 known (7 reported) oncogenic mutations in KRAS codons 12/13. An ultrashort 31bp region encompassing KRAS codons 12/13 was PCR amplified; G12A/C/D/R/S/V, and G13D mutations were enriched by suppressing wild-type (WT) sequence amplification with a WT blocking oligo. Barcoded adaptor primers were added for compatibility with massively parallel deep sequencing. Limits of detection (LOD) were independently determined for each of the 7 KRAS mutations by spiking 5–500 copies of each mutant into 60 ng of a WT genomic background. Limits of quantitation (LOQ) were confirmed with 7 copies of each mutation in an increasing WT genomic DNA background of 60–360 ng. Archived, matched plasma and urine samples (stored between 3−5 years prior to ctDNA extraction) from 20 treatment na¨ıve, advanced cancer patients with known tumor tissue KRAS mutations determined by an accredited clinical laboratory, were used in a retrospective setting for a blinded pilot study. These samples were used to compare KRAS status in urine and plasma to tumor tissue, and assess clinical sensitivity of the ctDNA assay. Results: LOD data for 5–500 KRAS G12A/C/D/R/S/V, and G13D mutant copies in 60 ng WT DNA showed a highly correlative response with an average R2 of 0.90 for the 7 mutations evaluated. LOQ assessed for each mutation in an increasing WT DNA background, revealed a robust signal for each mutation versus WT alone; an estimated analytical LOD of 7 copies per ~100,000 genome equivalents (0.007%) was observed. Of 20 blinded retrospective plasma ctDNA samples evaluated, 19 (95%) displayed the KRAS mutation concordant with tumor tissue. Of 20 matched urine samples tested, 16 were deemed evaluable; 15 (94%) had a significant sequence call consistent to tumor and to plasma.

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Conclusion: The developed method for quantitative massively parallel deep sequencing of ctDNA for KRAS mutational assessment has reported high clinical sensitivity in plasma and urine. This technical approach is scalable and has the potential for detecting and quantifying a multitude of genomic alterations indicative of therapeutic responsiveness and resistance. Expansion of retrospective colorectal patient cohort described herein will be reported. Partially supported by the Czech Ministry of Health grant NT 13660. 150 POSTER (Board P144) Pirin downregulates E-cadherin gene expression and contributes to EMT K. Komai1 , Y. Niwa1 , Y. Sasazawa1 , S. Simizu1 . 1 Keio University, Faculty of Science and Technology, Yokohama, Japan Background: Downregulation of E-cadherin, a cell–cell adhesion protein, induces epithelial–mesenchymal transition (EMT), which plays crucial roles in metastatic progression. A nuclear protein pirin enhances NF-úB related transcription by binding to Bcl3-p50 complex, which is important for the SLUG expression and melanoma migration. Additionally, proteomics analysis indicated that pirin expression was decreased in metastatic adenoid cystic carcinoma cells. Although these reports suggest that pirin may be involved in tumor metastasis, there are no reports which directly demonstrate the contribution of pirin to metastasis. Here, we investigated the effects of pirin on EMT which associates with metastasis, and its mechanisms. Material and Methods: Pirin was overexpressed or silenced in HeLa cells, and then EMT-related genes and protein expressions were detected. The morphological changes of pirin stably-expressing HeLa cells were measured by employing parameter of ‘circularity’, [4p(area)/(perimeter)2 ]×100, which decreases by morphological changes from cobblestone-like epithelial cells to spindle-like mesenchymal cells. The effect of pirin on cell migration and anticancer drug resistance was measured by wound healing assay and MTT assay, respectively. The binding of wild-type pirin or its mutant to Bcl3 was confirmed by GST pull-down assay using recombinant GST-Bcl3. Results: Knockdown of pirin increased E-cadherin gene expression whereas overexpression of pirin decreased its level. Pirin stably-expressing HeLa cells exhibited spindle-like morphology and loss of cell–cell adhesion, which are reminiscent of EMT. From the result of MTT assay, we demonstrated that pirin contributed to acquire anticancer drug resistance. Furthermore, RNAi experiment revealed that pirin positively regulated A549 cell migration. Next, we examined whether Bcl3, a binding partner of pirin, is involved in EMT induction by pirin overexpression. GST pull-down analysis indicated that Pirin/E103A mutant was decreased its binding ability to Bcl3; however, as with wild-type pirin, this mutant also downregulated E-cadherin gene expression, suggesting that pirin decreases E-cadherin expression in Bcl3-SLUG-axis-independent manner. Conclusions: Pirin downregulates E-cadherin gene expression in Bcl3 independent manner, and contributes to EMT and cancer malignancy. These data provide evidence that pirin may be a potent target toward cancer therapy. 151 POSTER (Board P145) Impact of EGFR amplification pattern on the expression of miRNA-200c in primary glioblastoma multiforme ´ Gines2 , E. Serna3 , D. Monleon1 , L. Munoz ˜ Hidalgo1 , C. Lopez R. Callaghan2 , R. Gil Benso2 , H. Martinetto4 , A. Gregori Romero2 , J. Gonzalez Darder5 , M. Cerda Nicolas2 . 1 Fundation HCU-INCLIVA, Pathology, Valencia, Spain; 2 University of Valencia, Pathology, Valencia, Spain; 3 University of Valencia, UCIM, Valencia, Spain; 4 Institute FLENI, Neurological, Buenos Aires, Argentina; 5 Clinical Hospital, Neurosurgery, Valencia, Spain Glioblastoma Multiforme (GBM) is the most common tumor in the primary tumors of the central nervous system, accounting for 60% of neoplasms in this location. It is a highly aggressive tumor with a median survival of twelve months. Heterogeneity in the biological behavior of this neoplasm of astrocytic glial origin is expressed in the infiltrative nature, this is a critical feature in glioblastoma. Several miRNAs have been related with different types of cancer, some of them related with ability of modulation behavior neoplastic cells expressed epithelial/mesenchymal changes. In this regard, miRNA expression is deregulated in most, if not all, types of cancer. Based on the literature the most common dysregulation of miRNAs in GBM is over-expression; more than two hundred miRNAs have been found to be significantly overexpressed. We have evaluated whole-genome miRNA expression profiling associated with different EGFR amplification patterns in primary glioblastoma multiforme, whose clinicopathological and immunohistochemical features have

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also been analyzed. All samples used for histopathological examination were fixed in neutral-buffered formalin, embedded in paraffin, sectioned and stained with hematoxylin-eosin. The immunohistochemical study was performed on paraffin-embedded sections using the avidin–biotin peroxidase method. To evaluate EGFR gene status, dual-color FISH analysis was performed on paraffin tissue arrays from 30 samples. Epigenetic analysis study of miRNAs and mRNA in 30 samples was performed using miRNA and mRNA Genechip Array (Affymetrix, Santa Clara, CA, USA). Expression levels of the selected miRNA (mir-200c) and mRNAs (CDH1, EGFR and ZEB1) were quantified using real-time reverse transcription-PCR (RT-PCR) analysis. MirRNA-200c showed a very significant difference between tumors having or not EGFR amplification. With respect to EGFR status our cases were categorized into three groups: high level EGFR amplification, low level EGFR amplification, and no EGFR amplification. Our results showed that microRNA-200c and E-cadherin expression are down-regulated, while ZEB1 is up-regulated, when tumors showed a high level of EGFR amplification. Conversely, ZEB1 mRNA expression levels were significantly lower in the group of tumors without EGFR amplification. Tumors with a low level of EGFR amplification showed ZEB1 expression levels comparable to those detected in the group with a high level of amplification. In this study we provide what is to our knowledge the first report of association between mirRNA-200c and EGFR amplification in glioblastomas. The mirRNA-200c plays an important role in epithelial–mesenchymal transition, but its implication in the behavior of glioblastoma is largely unknown, and we suggest that microRNA-200c may act as a potential regulator of glioblastoma migration and invasion by targeting ZEB1 mRNA. 152 POSTER (Board P146) Preclinical characterization of MM-151, an oligoclonal antibody therapeutic that targets EGFR by three distinct mechanisms of action A. King1 , M. Sevecka1 , N. Gerami-Moayed1 , O. Burenkova1 , J. Kearns1 , G. Tan1 , C. Sloss1 , R. Bukhalid1 , U. Nielsen1 , B. Wolf1 . 1 Merrimack Pharmaceuticals, Cambridge, USA Background: EGFR is a well-documented driver of solid tumor growth and a validated therapeutic target. However, currently approved EGFRtargeting monoclonal antibodies have limited clinical benefit, indicating the need for improved antibody therapeutics. We hypothesized that sub-optimal inhibition of the EGFR network and engagement of the immune system may limit the activity of current monoclonal antibody therapeutics, such as cetuximab and panitumumab. MM-151, an oligoclonal combination of three fully human IgG1s with subnanomolar affinities and non-overlapping epitopes, was developed using a systems biology approach to address these unmet needs. Methods: Preclinical characterization studies were performed, including signaling, proliferation, and immune effector assays to elucidate the mechanisms of action of MM-151 and to differentiate it from current monoclonal antibody therapeutics. Results: Our characterization assays demonstrate that MM-151 has three distinct mechanisms of action: ligand antagonism, receptor downregulation, and the pathway extrinsic activities of complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. MM-151 displayed an advantage over cetuximab for all three mechanisms in the preclinical assays. Seven redundant ligands with a wide range of affinities can activate EGFR. We therefore examined the ability of MM-151 to antagonize ligand-driven signaling and proliferation in detail. Initially, we compared MM-151 with cetuximab and observed similar inhibition of signaling and proliferation driven by each of the three low-affinity ligands, but MM-151 showed increased inhibition against each of the four high-affinity ligands. A bioinformatics analysis using primary tumor data from The Cancer Genome Atlas revealed expression of distinct ligand mixtures comprised of both low- and high-affinity ligands across indications. MM-151 provided superior inhibition of proliferation with indication-specific ligand mixtures. MM-151 was also active against a cetuximab-resistant cancer cell line with upregulated expression of high-affinity ligands. Conclusions: MM-151 is a EGFR-targeting antibody combination designed using systems biology to overcome ligand redundancy and signal amplification within the EGFR network. MM-151’s novel oligoclonal composition enables enhanced antagonism of EGFR ligands, EGFR downregulation and immune effector function. These preclinical studies provide a rationale for clinical study of MM-151.

Poster Session – Preclinical Models 153 POSTER (Board P147) Moving beyond in vitro models and addressing the challenges of pooled RNAi screens in mouse xenografts D. Tedesco1 , K. Bonneau1 , M. Makhanov1 , D. Deng1 , P. Sun2 , A. Chenchik1 . 1 Cellecta Inc., Mountain View CA, USA; 2 The Scripps Research Institute, La Jolla CA, USA RNAi loss-of-function screening with pooled shRNA expression libraries has proven to be a useful tool to identify essential cancer genes and drug resistance mechanisms in numerous cell lines in vitro. The effects of knocking down large numbers of genes on cancer cell growth and viability can be measured simultaneously using this approach. Now there is considerable interest to adapt RNAi viability screens to ex vivo xenograft mouse models. However, small take rates, growth rate variance of cells, and differing cell microenvironments in these more complex tumor models impose significant challenges to this sort of assay. We have developed a novel approach based on the use of pooled shRNA libraries with clonal barcodes that enables proliferation measurements of clonal populations produced by the individual shRNA-expressing founder cells that produce the tumor. This RNAi clonal analysis provides a basis to separate, across large cell populations, the external parameters that strongly influence cell growth rates from shRNA-induced growth inhibition in these systems. We will present in vivo RNAi screening validation data for several cancer models. 154 POSTER (Board P148) Drug response database with PDX tumor models in biomarker-driven multi-drug multi-arm clinical trial settings J. Jiang1 , T.F. Yu1 , Y. Yan1 , W. Du1 , T.T. Tan1 , L. Hua1 , J.L. Gu1 , X.Q. Yang1 , Z.H. Liu1 , X.K. Ye1 , Z. Gu1 . 1 GenenDesign, Oncology, Shanghai, China Recent advances in affordable genome sequencing and molecular profiling provide opportunities to better understand the complexity and heterogeneity of cancer, and perform multi-biomarker diagnosis in a single comprehensive test for personalized clinical treatment. A new biomarker-driven multi-drug multi-arm clinical trial in lung squamous cell carcinoma (Lung-MAP) uses cutting-edge genomic profiling to match patients with investigational treatments targeting their unique tumor mutations. However, there are still difficulties in enrolling enough patients with specific parameters, comparing different regimes on same clinical tumor and validating predictive biomarkers, especially those for drugs in early development stage. Patient derived xenograft (PDX) tumor models have been proved to recapitulate the complexity and heterogeneity of their corresponding human tumors by phenotypic and genomic characterization, and thus become to be widely used in recent years in preclinical setting to facilitate drug discovery, translational studies and clinical trials support. To further meet the increasing needs and complement the current clinical practice for precision medicine, better characterized PDX models with genomic profiles and drug response information to multiple chemo/targeted therapies are in demand. GenenDesign has established over 700 PDX tumor models and derived around 100 resistance models to drugs of interest. Through our in-house efforts, PDX models of different tumor types were tested with related SOCs and clinical candidates in biomarker-driven multi-drug multi-arm clinical trial settings. So far, more than 800 data sets have been generated, including responses to chemotherapy drugs, targeted inhibitors against HER2, EGFR, FGFRs, c-Met, MEK and Ras/Raf pathway, as well as PI3K/Akt pathway etc. Moreover, genomic profiling data of many PDX model have also been acquired at hot-spot mutation, gene expression, gene copy number and RNA/Exome sequence levels. The combination of functional and molecular information will help to design and support clinical trials, discover predictive biomarkers and unravel underlying drug resistance mechanisms. 155 POSTER (Board P149) SNIPER(TACC3) degrades TACC3 protein via the ubiquitin– proteasome pathway and induces apoptosis in cancer cells expressing a large amount of TACC3 N. Ohoka1 , K. Nagai2 , K. Okuhira1 , N. Shibata1 , T. Hattori1 , N. Cho2 , M. Naito1 . 1 National Institute of Health Sciences, Division of Biochemistry and Molecular Biology, Tokyo, Japan; 2 Takeda Pharmaceutical Co. Ltd., Medicinal Chemistry Research Laboratories Pharmaceutical Research Division, Fujisawa, Japan Microtubule inhibitors are widely used as anti-cancer drugs. They arrest cancer cells and often induce mitotic catastrophe and cell death. However, they also affect microtubule function in non-dividing cells, which limit their

Poster Session – Preclinical Models utility. Recently, inhibitors of spindle-regulatory proteins have attracted considerable attention, and transforming acidic coiled-coil-3 (TACC3) is a spindle-regulatory protein overexpressed in many human cancers. We have developed a protein knockdown system to induce degradation of target proteins via the ubiquitin–proteasome system in cells with hybrid molecules named SNIPER (Specific and Non-genetic IAP-dependent Protein ERaser). In this study, we designed and synthesized novel SNIPER(TACC3)s that target TACC3 for degradation, and evaluated their activity in vitro. SNIPER(TACC3)s induce poly-ubiquitylation and proteasomal degradation of TACC3, and reduce the TACC3 protein level in cells. Mechanistic analysis indicated that the ubiquitin ligase APC/CCDH1 mediates the SNIPER(TACC3)-induced degradation of TACC3. Cancer cells express larger amount of TACC3 than do normal fibroblasts, and SNIPER(TACC3) selectively induced cell death in cancer cells. These results suggest protein knockdown of TACC3 by SNIPER(TACC3) is a potential strategy to treat cancers overexpressing the TACC3 protein. 156 POSTER (Board P150) Behaviour of platinum(IV) complexes with prodrug function in different models of hypoxia E. Brynzak1 , P. Heffeter2 , V. Pichler1 , M.A. Jakupec1 , B.K. Keppler1 . 1 University of Vienna, Institute of Inorganic Chemistry, Wien, Austria; 2 Medical University of Vienna, Institute of Cancer Research, Wien, Austria Background: Solid tumours frequently contain regions of low oxygen concentration, which have been identified as important component for drug modification, therapy resistance and metastasis formation. Selective targeting of this tumour hypoxia might be a proper strategy to overcome current therapeutic limits. Therefore platinum(IV) compounds with prodrug function, which require the reductive environment of tumor hypoxia for their biological activation, can be highly selective therapeutics. Material and Methods: To investigate the cytotoxicity of platinum(IV) compounds we used the AlamarBlue assay in hypoxic spheroid models of CH1 (ovarian carcinoma), HT1080 (fibrosarcoma) and HCT116 (colon carcinoma). Evidence of hypoxia has been provided by antibody staining of HIF-1alpha, and propidium iodide staining revealed necrotic regions in spheroid centres. Distribution of the drug within the spheroids has being determined by LA-ICP-MS. Further investigations of compound activity in an in vivo mouse model are ongoing. Results: Two test compounds were found to be at least 2−6 times more potent in hypoxic CH1 and HT1080 spheroid models than in monolayer culture, while satraplatin, another platinum(IV) compound, was 2 times less active. Furthermore platinum(II) complexes such as cis- and oxaliplatin were found to be 2−11 times less active in hypoxic models. Distribution studies showed adequate spheroid penetration for all tested compounds and give evidence of platinum(IV) accumulation within hypoxic regions. Conclusions: In this study we showed an advantage of platinum(IV) prodrugs over platinum(II) therapeutics currently used in the clinic and pointed out relevance of spheroid based in vitro models for drug screenings. 157 POSTER (Board P151) Sensitive and specific detection of 1p/19q codeletion in gliomas by next generation sequencing E. Dubbink1 , P.N. Atmodimedjo1 , R.M. van Marion1 , J.M. Kros1 , M.J. van den Bent2 , W.N.M. Dinjens1 . 1 Erasmus MC, Pathology, Rotterdam, Netherlands; 2 Erasmus MC Cancer Insitute, Neuro-oncology, Rotterdam, Netherlands Molecular subtyping of malignancies from the central nervous system becomes increasingly important to establish histological diagnosis and to predict differential treatment outcome of histologically similar tumors. One of these molecular markers is deletion of chromosomal arms 1p and 19q. Current methods to detect 1p/19q codeletion involve fluorescent in situ hybridisation (FISH) and loss of heterozygosity (LOH) analysis using polymorphic microsatellite markers. We will present a novel method to detect these aberrations based on single nucleotide polymorphism (SNP) analysis using next generation sequencing (NGS) on an Ion Torrent platform. We show that targeted NGS analysis of multiple heterozygous SNPs spread over the entire chromosomal arms 1p and 19q allows sensitive (down to 40% of tumor cells) and reliable detection of 1p and/or 19q deletion in gliomas. The strength of this method is that it accurately detects allelic imbalance in small amounts of suboptimal quality DNA derived from routine formalin-fixed, paraffin-embedded (FFPE) tissue in a fast turnaround time and that it can be easily adapted to perform additional mutational analysis of genes of interest. The test is therefore perfectly suited to become standard practice for routine glioma diagnostics.

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158 POSTER (Board P152) The development of a series of orthotopic solid tumour models of prostate, lung and ovarian cancer using optical and X ray imaging M. Batey1 , M. Brown1 , E. Bowden1 . 1 Epistem Ltd, Manchester, United Kingdom Background: One of the most important aspects in anti cancer drug development is the availability of robust preclinical models which allow the efficacy of novel therapies to be examined. Several orthotopic models have been described where cancer cells are grown at clinically relevant sites. The objective is that these models, compared to more conventional subcutaneous models, will provide a more realistic environment, and will be superior models for therapeutic evaluation. A major difficulty with such techniques however is assessment of disease burden, with deep lying or metastatic tumours not being amenable to the usual methods of measurement. The introduction of non invasive small animal imaging has opened up the possibility of using such models in preclinical drug development programs, and in time the prospect of lower drug attrition rates in human trials. Here we discuss the development of three preclinical imaging models of solid human cancers, using the prostate line LnCAP, the ovarian line A2780, and the lung line A549. Materials and Methods: Following stable lentiviral transduction of these cell lines with a vector expressing firefly luciferase and GFP, LnCAP cells were implanted directly into the testis of male SCID mice, A2780 cells were implanted into the intraperitoneal space of female CD-1 nude mice, and A549 cells were implanted into the left lung of female CD-1 nude mice. Using the Bruker In Vivo Xtreme System, animals were monitored following initial transplantation, and disease burden quantified longitudinally over several weeks using co-registered 2D and 3D bioluminescent and X-ray detection. Results: Establishment of disease can be measured in each individual animal prior to commencement of therapy, and can be tracked in each animal throughout the duration of the study, with sites of metastasis readily visualised. In untreated animals the A2780 model runs over approximately 8 weeks, with multiple sites of solid tumour formation and frequent associated tumour ascites. Control mice in the A549 model are viable for approximately 6 weeks, with metastatic tumour development common in the model and a third of mice developing a pleural effusion. The LnCAP model runs over approximately 10 weeks, with the local development of dense, highly vascularised tumours, which show some invasion into the surrounding tissues. In all models visualisation of engrafted disease by imaging is possible at an early stage, and all show consistently high take rates. Conclusion: The ability to quickly identify engrafted animals, and the predictable development of disease demonstrated allows for improved randomisation based on disease signal, early therapeutic intervention, and extension of the treatment window. Efficacy of treatment can be easily followed by optical imaging, and as such, these models provide an attractive, clinically relevant environment for the preclinical assessment of novel therapeutics and combination treatment regimens. 159 POSTER (Board P153) Neoadjuvant chemotherapy in breast cancer patients induces expression of miR-34a and miR-122 P. Freres1 , C. Josse1 , N. Bovy2 , M. Boukerroucha3 , I. Struman2 , V. Bours3 , G. Jerusalem1 . 1 C.H.U. Liege, Laboratory of Medical ` Oncology, Liege, Belgium; 2 GIGA-Research, Unit of Molecular Biology ` and Genetic Engineering, Liege, Belgium; 3 GIGA-Research, Human ` Genetics, Liege, Belgium Background: Circulating microRNAs (miRNAs) are extensively studied in cancer as biomarkers but little is known about the influence of anti-cancer drugs on their expression. In this article, we describe the modifications of circulating miRNAs profile under neoadjuvant chemotherapy (NAC) for breast cancer. Methods: The expression of 188 circulating miRNAs was assessed by RT-qPCR in plasma of 25 patients before and after NAC. 2 miRNAs significantly increased under NAC, miR-34a and miR-122, were measured in the tumor tissue before and after the chemotherapy for 7 patients with pathological partial response (pPR) to NAC. These 2 chemotherapyinduced miRNAs were further studied in the plasma of 22 patients with adjuvant chemotherapy (AC) as well as in 12 patients who did not receive any chemotherapy and 20 healthy women. Results: 25 plasma miRNAs are found significantly modified by NAC. Tumor suppressor miR-34a and miR-122 are highly upregulated at the end of the NAC, notably in pPR patients with aggressive breast cancers. Furthermore, miR-34a level is elevated in remaining tumor tissue after NAC treatment. Studying the kinetic of circulating miR-34a and miR-122 expression during NAC reveals that their levels are especially increased

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after anthracycline-based chemotherapy. AC induces lower levels of plasma miR-34a and doesn’t modify miR-122. The tumorectomy alone doesn’t deregulate miR-34a and miR-122. Circulating miR-34a and miR-122 are downregulated in NAC treated breast cancer patients compare to controls and normalized after treatments. Conclusion: This study demonstrates for the first time that NAC specifically induces expression of tumor suppressor miRNAs in plasma and tumor tissue that might be involved in the anti-tumor effect of the chemotherapy. 160 POSTER (Board P154) Identification of fusion genes through kinome-centered RNA sequencing in different types of solid tumors L. Mittempergher1 , C. Sun1 , F.H. Groenendijk1 , A.J. Bosma1 , S.M. Willems1 , T. Sustic1 , I.J. Majewski1 , W. Grernrum1 , N.M. Davidson2 , I. de Rink3 , H.M. Horlings1 , W. Theelen-Engelsman4 , S.F. Chin5 , A. Oshlack6 , B.W. van Rhijn7 , M. van den Heuvel4 , M.S. van der Heijden1 , C. Caldas5 , R. Bernards1 . 1 Antoni van Leeuwenhoek − Netherlands Cancer Institute, Molecular Carcinogenesis, Amsterdam, Netherlands; 2 Murdoch Childrens Research Institute Royal Children’s Hospital, Bioinformatics Division, Victoria, Australia; 3 Antoni van Leeuwenhoek − Netherlands Cancer Institute, Genomics Core Facility, Amsterdam, Netherlands; 4 Antoni van Leeuwenhoek − Netherlands Cancer Institute, Thoracic Oncology, Amsterdam, Netherlands; 5 University of Cambridge, Oncology, Cambridge, United Kingdom; 6 The Walter and Eliza Hall Institute, Cancer and Haematology, Victoria, Australia; 7 Antoni van Leeuwenhoek − Netherlands Cancer Institute, Urology, Amsterdam, Netherlands Background: Oncogenic fusion genes involving kinases are effective therapeutic targets in different tumor types. However, diagnostic screening for kinase fusion genes in solid tumors is particularly challenging, as many occur with a low frequency. To overcome this, we developed a capture enrichment strategy to enable high throughput transcript sequencing of the human kinome. Using this approach, we recently identified novel translocation events involving the FGFR3 and ALK genes in lung squamous cell and adenocarcinoma (Majewski IJ et al, 2013). Methods: To date we screened with this approach 192 non-small cell lung carcinomas, 480 breast cancers of different subtypes from the METABRIC cohort (Curtis C et al, 2012), 80 head and neck carcinomas and 80 muscle-invasive urothelial carcinomas. Patient material was available from frozen or formalin-fixed paraffin-embedded tissues. Sequencing libraries were constructed with a TruSeq mRNA library preparation kit using poly-A enriched RNA (Illumina). Capture enrichment was performed with the human kinome DNA capture baits (Agilent). Captured libraries were sequenced on an Illumina HiSeq2000 platform with a paired-end 51 base protocol. Sequences were aligned to the human genome (Hg19) with TopHat (Trapnell C et al, 2009). Two pipelines were used to identify and rank candidate fusion genes: TopHat-fusion (Kim D et al, 2012) and de novo transcript assembly with Trinity (Grabherr MG et al, 2011). In order to validate the fusion events, PCR primers were designed to amplify across the fusion breakpoints and fusion breakpoints were confirmed by PCR amplification from cDNA and subsequently by capillary sequencing. Results: About 90% of the fusion events predicted with TopHat and de novo assembly algorithm were validated by capillary sequencing. Intriguingly, a gene was found recurrently fused at the exact same breakpoint in the breast cancer set. Fusion genes identified in the discovery set will be screened in independent sets of samples in order to determine their frequency. In addition, association of genomic rearrangements with clinico-pathological parameters has been assessed. Detailed results and functional validations will be presented at the meeting. Conclusion: Our kinome capture enrichment strategy for systematically profiling kinase fusion genes across different cancer types can be reliably used to identify novel translocation events in any expressed kinase. 161 POSTER (Board P155) Leveraging a novel DNA barcoding platform for integrated profiling and pharmacodynamic readouts C. Castro1 , V. Peterson2 , A. Ullal2 , S. Agasti2 , S. Tuang2 , N. Miller2 , M. Birrer3 , R. Weissleder4 . 1 Massachusetts General Hospital Cancer Center, Medicine/Center for Systems Biology, Boston, USA; 2 Massachusetts General Hospital, Center for Systems Biology, Boston, USA; 3 Massachusetts General Hospital Cancer Center, Medicine, Boston, USA; 4 Massachusetts General Hospital, Center for Systems Biology/Radiology, Boston, USA Background: Studies cite modest overlap between DNA, RNA, and protein levels within tumor cells. Integrated testing could generate valuable and complementary mechanistic insight. We recently developed a DNA

Poster Session – Preclinical Models barcoding platform to quantify ~100 protein markers using scant clinical trial samples down to a single cell. Here, we expand this tool to predict drug testing by enabling simultaneous testing of tumor DNA, RNA, and protein. Materials and Methods: We highlight two ovarian cancer cases with contrasting clinical characteristics (low and high grade). Besides profiling, biopsies were also treated ex-vivo with cytotoxic (carboplatin, paclitaxel) or targeted drugs (PKI-587 [PI3K/mTOR], selumetinib [MEK]). Antibodies conjugated to unique DNA barcodes were used for multiplexed proteomic measurements using one fine needle aspirate (FNA) pass. After labeling with Ab-DNA conjugates, cells were lysed and unique barcodes cleaved from antibodies. Lysates contained cellular DNA and mRNA along with cleaved DNA barcodes measurable by automated detection platforms with attendant high femtomolar sensitivity. Results: Integrative analyses and expanded drug testing were feasible on clinical specimens. Profiling: Subject 1 (low grade) had increased KRAS (~3 fold) and MYC (~5 fold) copy number suggesting MAPK pathway dysregulation. In contrast, Subject 2 (high grade) had increased copy number of PI3KCA (~3 fold) and AKT2 (~11 fold) genes suggesting PI3K pathway dysregulation. Concordant with DNA results, Subject 1 had multiple unregulated proteins in the MAPK pathway (p38 MAPK, MET) along with MAPK1 and MET mRNA levels. Subject 2 had high AKT expression, pS6, and MTOR at both protein and mRNA levels. Drug testing: Both subjects downregulated EpCAM, S6RP, and p44/42 MAPK protein levels following carboplatin, selumetinib, or paclitaxel. Subject 1 was very sensitive to selumetinib (IC50: ~20 nM), supporting our profile testing. While Subject 1 showed limited in vitro sensitivity to PKI-587, as predicted, we noted acquired sensitivity with HM30181 (MDR1 inhibitor) use; results were validated with cell line testing. Conclusion: Our novel platform’s potential for expanded ex-vivo target modulation testing using scant specimens and various drug dosages or combinations could render it a feasible preclinical drug screening tactic. Our data support recent ovarian cancer MEK inhibitor trial evidence and uncover potential synergism with MDR1, poised for further clinical exploration. 162 POSTER (Board P156) Drug–drug interaction predictions for MLN2480, an investigational pan-RAF inhibitor, based on nonclinical data S.K. Balani1 , A. Bulychev1 , L. Cohen1 , M. Liao1 , C.Q. Xia1 , F. Wang2 , P. Li3 , B. LeClair3 , T. Bohnert3 , L. Gan3 , X. Zhou4 , V. Bozon5 , S. Prakash6 . 1 Takeda Pharmaceuticals International Company, DMPK, Cambridge, USA; 2 Takeda Pharmaceuticals International Company, Cancer Pharmacology, Cambridge, USA; 3 Biogen Idec Inc, DMPK, Cambridge, USA; 4 Takeda Pharmaceuticals International Co., Clinical Pharmacology, Cambridge, USA; 5 Takeda Pharmaceuticals International Co., Oncology Clinical Research, Cambridge, USA; 6 Takeda Pharmaceuticals International Co., DMPK, Cambridge, USA Background: MLN2480 is an investigational small molecule pan-RAF inhibitor in clinical trials in patients with metastatic melanoma and other solid tumors. Non-clinical assessments were made to predict the potential for drug–drug interactions (DDIs), and pH and formulation effects in the clinic. Material and Methods: In vitro assessments of enzymes involved in metabolism, their quantitative phenotyping, and CYP inhibitions were carried out using human liver microsomes. Freshly isolated human hepatocytes were used to assess CYP induction and mRNA expression. Caco-2 cells were used to assess membrane permeability and transporters’ inhibition potential. Pharmacokinetic (PK) studies used mice to assess the effect of pH and cremophor on PK of MLN2480. Results: Studies with human liver microsomes indicated that MLN2480 is metabolized by multiple enzymes, with aldehyde oxidase and CYP2C8 playing predominant roles. Due to multiple metabolic enzymes involved in MLN2480 clearance, CYP2C8 inhibitor and inducer drugs are expected to have a low potential to alter exposure to MLN2480. Additionally, the compound is a significant inhibitor of CYP2C8, hence a potential to affect PK of CYP2C8 drugs. MLN2480 did not exhibit time-dependent inhibition of CYP3A, or induction of CYPs. The compound exhibits high membrane permeability in Caco-2 model and does not appear to be an efflux pump substrate, reducing any concerns of DDIs with efflux pump inhibitor drugs. Additionally, it is not an inhibitor of Pgp, but of BCRP. Thus, a DDI potential exists with BCRP substrate drugs. Physicochemical-based interactions also have been assessed in mice. Consistent with the fact that the solubility of MLN2480 is not pH dependent, there was no effect on exposure to MLN2480 in an alkaline formulation in rodents. Additionally, since paclitaxel would be tested as a combination agent, and Cremophor® EL (BASF SE) in Taxol is known to affect PK of some drugs, the effect of cremophor on MLN2480 PK was assessed

Poster Session – Preclinical Models in mice. The results showed that with Cremophor concentration similar to that at the planned Taxol dose of 80 mg/m2 in the clinic, the increase in MLN2480 exposure (AUC) in mice was only 14%. Conclusions: Overall, based on nonclinical data, there is a DDI potential for MLN2480 with CYP2C8 substrates/inhibitors/inducers and BCRP substrate drugs in the clinic. The possible alteration of MLN2480 PK by Cremophor in Taxol (80 mg/m2 ) is likely to be minimal. 163 POSTER (Board P157) Interrogation of pharmacogenes in cancer patients using targeted DNA sequencing F. Innocenti1 , N. Gillis1 , J. Parker1 , N. Hayes1 , D. Eberhard1 , K. Richards1 , J.T. Auman1 , E. Seiser1 . 1 University of North Carolina at Chapel Hill, Chapel Hill NC, USA Background: Germline sequence variation and somatic alterations in genes associated with drug processing can generate individualized pharmacogenetic data for cancer patients that may aid in understanding therapeutic response. Material and Methods: Targeted sequencing of over 200 genes, including known druggable targets and genes relevant to cancer pathogenesis, was performed for matched germline and tumor DNA from over 200 patients representing many common cancer types. Aligned sequence data was used for genotype variant calling in both germline and tumor DNA and for mutation and copy number identification within tumors. Genotype data was used to determine genetic ancestry and to provide summary statistics for known pharmacogenetic variants in germline DNA. Additionally, somatic alterations within genes relevant to the pharmacology of anticancer agents were examined in the represented cancer types: brain/central nervous system, breast, gastrointestinal, genitourinary, gynecologic, head and neck, and hematologic. Results: The use of genetic markers to determine ancestry identified patients that were mainly Caucasian (78%) and African American (16%), and provided a means to correct for errors and ambiguity in self reporting for nine individuals. The most common cancer types included gastrointestinal (19%), genitourinary (18%), and breast (13%). A total of 38 pharmacogenetic variants in 18 genes, including cytochrome P450 genes, demonstrated similar minor allele frequencies when compared to 1000 Genomes data and were predominantly in Hardy-Weinberg equilibrium. Observed genotype concordance of these variants in matched germline and tumor DNA was greater than 95% for the majority of loci. Analysis of 16 genes associated with anticancer agents identified mutations 15 genes (22% of patients exhibited at least one mutation in one or more genes), including predicted non-synonymous coding changes and gains of stop codons in ABCB1 and DPYD. Aberrant tumor copy numbers were sporadically observed in most of these genes, although recurrent deletions of DPYD and SULT1A1 were present across tumor types. Conclusions: Next generation sequencing of pharmacogenes in both nonmalignant and tumor tissue from an individual provides a comprehensive catalog of germline variants and both known and novel somatic alterations. The integration of genetic data from germline and tumor may aid in the elucidating the pharmacology underlying therapeutic effect. Analysis of the effect of these variants and molecular alterations on drug response for a subset of individuals is ongoing and will be presented at the meeting. 164 POSTER (Board P158) Augmentation of NAD+ by NQO1 activation attenuates cisplatinmediated hearing impairment S. Yang1 , G.S. Oh2 , K.B. Kwon2 , R.K. Park2 , S.Y. Lee3 , S.R. Moon4 , H.S. So2 . 1 Wonkwang University School of Medicine, Department of Internal Medicine, Iksan-city Jeonbuk, South Korea; 2 Wonkwang University School of Medicine, Center for Metabolic Function Regulation, Iksan-city Jeonbuk, South Korea; 3 Wonkwang University School of Medicine, Department of Thoracic Surgery, Iksan-city Jeonbuk, South Korea; 4 Wonkwang University School of Medicine, Department of Radiation Oncology, Iksan-city Jeonbuk, South Korea Introduction: Cisplatin [cis-diaminedichloroplatinum-II] is an extensively used chemotherapeutic agent, and one of its most adverse effects is ototoxicity. A number of studies have demonstrated that these effects are related to oxidative stress and DNA damage. However, the precise mechanism underlying cisplatin-associated ototoxicity is still unclear. The cofactor nicotinamide adenine dinucleotide (NAD+ ) has emerged as a key regulator of cellular energy metabolism and homeostasis. Although a link between NAD+ -dependent molecular events and cellular metabolism is evident, it remains unclear whether modulation of NAD+ levels has an impact on cisplatin-induced hearing impairment.

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Material and Methods: To investigate whether augmentation of NAD+ by NQO1 activation using b-Lapachone (b-Lap) attenuates cisplatinmediated hearing impairment, male C57BL/6mice and NQO1 knockout mice on a C57BL/6 background were used. For analysis of the auditory threshold, auditory brainstem response (ABR) was recorded. For biochemical analysis, we measured the enzymatic activity of SIRT1, PARP1, ROS production, NAD+/NADH ratio, mRNA levels of miR-34a and pro-inflammatory cytokines. Immunohistochemistry and western blot analysis were also performed. Results and Discussion: We have demonstrated for the first time that both the protein expression level and the activity of SIRT1 were suppressed by the reduction of intracellular NAD+ levels in cisplatin-treated cochlear tissue. We also found that the decrease in SIRT1 protein expression and its activity after cisplatin exposure were mediated by the increase in transcriptional activity of p53 for miR-34a expression and PARP-1 activation causing NAD+ -depletion, respectively. However, the increase in cellular NAD+ levels by NQO1 activation using b-Lap prevented mice from cisplatininduced cochlear damage and hearing impairment through the modulation of PARP-1, SIRT1, p53, and NF-kB. Conclusion: Considering that b-Lap itself did not attenuate the tumoricidal effect of cisplatin (Kidney International, 2014, in press), these results suggest that the direct modulation of the cellular NAD+ level by pharmacological agents could be a promising therapeutic strategy for enhancing the efficacy of cisplatin chemotherapy without its adverse effects. 165 POSTER (Board P159) Effect of zoledronic acid on the post-translational modification of activated leukocyte cell adhesion molecule (ALCAM) in cancer cells ` R. Toth1 , G. Trombino1 , V. Castronovo1 , A. Bellahcene1 . 1 C.H.U. Liege, ` Metastasis Research Laboratory, Liege, Belgium Background: Activated leukocyte adhesion molecule (ALCAM/CD166) is a 105kDa protein that is a member of the immunoglobulin superfamily. This family consists of proteins that are involved in recognition binding and adhesion processes of the cells. Zoledronic acid (ZA) is a bisphosphonate that has been extensively studied as an osteoclast inhibitor. ZA decreases bone turnover by inhibiting the mevalonate pathway in cells. This pathway has been notably implicated in post-translational protein modifications such as prenylation and N-linked glycosylation. Methods: MDA-MDA-231 cells have been treated with different concentrations of ZA for 48 h. Enzymatic digestion was performed with PNGaseF on protein extracts from treated and non-treated cells. Western blot analysis and cell aggregation assays have been performed upon ZA treatment. Results: We observed the appearance of a 95kDa form of ALCAM after 48 h of treatment with ZA in a dose dependent manner. MDA-MB-231 cytosolic/membrane protein fractionation showed that the 95kDa form of ALCAM was detectable in the membrane fraction. PNGaseF treatment of MDA-MB-231 cell extracts resulted in a single 68kDa band in both treated and non-treated conditions, indicating that the 95kDa form potentially represents an intermediate glycosylation form of ALCAM. We showed that ALCAM is crucial for MDA-MB-231 cell aggregation. Next, we observed that ZA treated-cells presented with a decreased aggregation capacity potentially linked to their expression of the 95kDa ALCAM form. Conclusion: Our results suggest for the first time that ZA affects the glycosylation of ALCAM in MDA-MB-231 cells in vitro, which will be further investigated in breast cancer tumor models in vivo. Reduced N-linked glycosylation of ALCAM was associated with a decreased aggregation potential in ZA-treated cancer cells. Cell aggregation is associated with anchorage independent growth and proliferation in MDA-MB-231 cells. Ongoing experiments will help to explore the potential implication of the 95kDa ALCAM form in these processes. 166 POSTER (Board P160) Pre-clinical and clinical activity of Anti-DLL4 (demcizumab) in combination with gemcitabine plus nab-paclitaxel in pancreatic cancer M. Hidalgo1 , A. Cubillo2 , R. Stagg3 , J. Dupont3 , Y. Wan-Ching3 , T. Hoey3 . 1 CNIO, Madrid, Spain; 2 CIOCC, Madrid, Spain; 3 Oncomed Pharmaceutical, California, USA DLL4-Notch signaling plays a key role in cancer stem cell (CSC) biology in numerous tumor types including pancreatic cancer. We have developed an anti-DLL4 antibody (demcizumab) that blocks Notch signaling and inhibits tumor growth though multiple mechanisms including a reduction of CSC frequency and the inhibition of productive angiogenesis. Previous studies have shown broad spectrum activity of anti-DLL4 & gemcitabine in a panel of patient derived pancreatic cancer xenografts. Data from a Phase 1b clinical study of demicizumb & gemcitabine in PDAC resulted in a 25%

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partial response rate in 16 evaluable patients. Based on the emergence of nab-paclitaxel & gemcitabine as a standard of care regimen for the treatment of pancreatic cancer, we have tested anti-DLL4, gemcitabine & nab-paclitaxel in PDAC PDX models, and observed enhanced antitumor activity in all seven pancreatic cancer xenografts tested. Based on serial transplantation experiments, the triple combination of anti-DLL4, gemcitabine, & nab-paclitaxel reduced CSC frequency whereas treatment with the chemothearpy agents alone did not. Interestingly, the combination activity of anti-DLL4 appeared to be greater with gemcitabine & nabpaclitaxel than with gemcitabine alone. In a co-clinical study, a PDX model derived from a Phase 1b patient who progressed rapidly on treatment with demcizumab & gemcitabine was, as expected, insensitive to gemcitabine or the combination of anti-DLL4 & gemcitabine, correlating well with the lack of clinical response. Notably, this tumor was more sensitive to nabpaclitaxel, gemcitabine & anti-DLL4 treatment. As a result of these data and the emergence of nab-paclitaxel & gemcitabine as a new standard of care for pancreatic cancer, the Phase 1b clinical study mentioned above was amended to test the triple combination of demcizumab, gemcitabine & nab-paclitaxel in patients with 1st line pancreatic cancer. Early data from the 1st 14 patients treated with this combination revealed a partial response in 6 (43%) patients and stable disease in another 6 (43% pts). Related AEs observed in 20% in the 38 patients in the Phase 1b study were nausea (37%), fatigue (34%), vomiting (32%), decreased appetite (24%) & hypertension (21%). Additional cohorts of patients are being treated with demcizumab, gemcitabine & nab-paclitaxel to further assess the preliminary safety, efficacy, pharmacokinetics and impact on biomarkers of this combination. 167 POSTER (Board P161) Genomic characterisation of 1003 cancer cell-lines G. Bignall1 , F. Iorio2 , P.A. Futreal3 , M.R. Stratton1 , P. Campbell1 , U. McDermott1 . 1 Wellcome Trust Sanger Institute, Cambridge, United Kingdom; 2 EMBL-EBI, Cambridge, United Kingdom; 3 MD Anderson Cancer Centre, Houston, USA Over the last twenty years there has been a paradigm shift in cancer treatment, moving away from the cytotoxic effects of chemicals targeting all cells going through cell division to a more targeted approach aimed at inactivating specific cellular components upon which the cancers rely to drive cell growth; a process termed oncogene addiction. The ‘poster child’ for such therapies is ‘imatinib’ a small molecule inhibitor of ABL kinase activity targeting the BCR−ABL fusion protein in CML. Here we present the genomic characterisation of 1003 cancer cell-lines currently being used as reagents in a high-throughput screen of anti-cancer agents. This screen is aimed at identifying novel biomarkers with which to stratify patients, identifying those most likely to respond to specific cancer therapies. To characterise the cell-lines we performed exome sequencing, copy number, expression and methylation analysis. Since most cell-lines are unmatched we screened out germline variants by comparison to ~8000 normal exomes. To further reduce the ‘genetic space’ for the subsequent downstream correlation to drug response data we included only ‘clinically relevant’ variants. The list of clinically relevant variants was identified by looking for recurrence across the ‘systematic screen data’ in COSMIC and by reducing the gene set to those genes identified as ‘frequently mutated’ across a series of >7000 clinical exomes/genomes from 29 cancer types using IntOGen, MutSigCV and dN/dS analysis. Tissue specific and pan cancer gene sets were identified for downstream analysis. In total we identified ~500,000 putative somatic non-synonymous variants across the set of 1003 cancer cell-lines, of which ~6000 were classed as clinically relevant. Analysis is currently on-going correlating this ‘clinically relevant’ variant data to the drug response. Comparison of the mutation frequencies between the clinical samples and the filtered variant data from the cancer cell-lines indicate that the cell-lines are likely to represent good model systems for downstream correlation to drug response. The variant data for the cancer cell-lines is available via the COSMIC web portal. 168 POSTER (Board P162) CDCP1 as a new marker of aggressiveness in triple-negative breast cancers M. Campiglio1 , F. Turdo1 , F. Bianchi1 , M. Sasso1 , L. De Cecco1 , P. Casalini1 , P. Gasparini1 , L. Forte1 , R. Agresti1 , I. Maugeri1 , G. Sozzi1 , E. Tagliabue1 . 1 Fondazione IRCCS Istituto Nazionale Tumori, Dept. Experimental Oncology and Mol. Med., Milano, Italy Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype showing high recurrences and mortality rate, for which no therapies besides chemotherapy are available to date. Lacking specific markers for an effective targeted therapy, TNBCs still represent the most

Poster Session – Preclinical Models important challenge for clinical oncologists. In an effort to identify specific markers responsible for aggressiveness of TNBCs that may serve as target(s) of therapy, we used post-surgery wound-healing fluids (WHFs) from breast cancer patients, known to be extremely enriched in growth factors and cytokines, as a tool mimicking the pro-tumorigenic post-surgery host microenvironment to stimulate TNBC cells lines. Material and Methods: Cell lines knock-down for CDCP1 by specific siRNA were analyzed for migration, invasion and proliferation in vitro. Immunohistochemistry on FFPE TNBC specimens were performed using a polyclonal antibody. Cases were classified as positive when plasmamembranes were stained in at least 10% of tumor cells. FISH analysis was performed using a pool of 3 BAC (bacterial artificial chromosome) clones that cover the CDCP1 gene at chromosome 3p21.31 and a commercial chromosome 3 enumeration probe. Kaplan–Meier methods were used to calculate the disease free survival (DFS) and distant DFS (DDFS) and differences in survival curves were evaluated with the log-rank test. Results: Gene expression profiling (GEP) on the Illumina platform using TNBC cell lines derived after stimulation with WHFs identified among the surface receptors the non-catalytic receptor CDCP1 (CUB domaincontaining protein 1) as the most significantly up-modulated gene. CDCP1 protein was found basally highly expressed in 6 of 8 TNBC cell lines. Its silencing in the most highly expressing TNBC lines (MDA-MB-231 and BT549) strongly impaired both their migration (~70%) and invasion (~50%) ability but did not affect their in vitro proliferation, suggesting a role for CDCP1 in TNBC dissemination. IHC analysis of CDCP1 in 126 human primary TNBC FFPE specimens revealed intense membrane staining in 60% of cases. CDCP1 expression was found to be a risk factor that significantly reduces both DFS (log-rank p = 0.0115) and DDFS (log-rank p = 0.0063) of TNBC patients. To evaluate whether the high expression level of CDCP1 in human TNBC specimens can depend on a genetic gain, FISH analysis of FFPE sections from 30 human TNBC cases (20 CDCP1positve and 10 CDCP1-negative in IHC) was performed, revealing that in more than 50% of CDCP1-positive cases CDCP1 gene was polysomic. Additionally, polysomy strongly correlated with CDCP1 protein expression levels. Conclusions: Our data identify CDCP1 as a marker of extremely aggressive TNBCs, suggesting its candidacy as a target of novel therapeutic strategies against this disease. Supported by AIRC 169 POSTER (Board P163) BRAF mutation testing in cell-free DNA from plasma of patients with advanced cancers using a novel, rapid, automated molecular diagnostics prototype platform (Idylla™) F. Janku1 , H.J. Huang1 , B. Claes2 , G.S. Falchook1 , A. Naing1 , S. Piha-Paul1 , A.M. Tsimberidou1 , R.G. Zinner1 , D.D. Karp1 , S. Fu1 , V. Subbiah1 , D.S. Hong1 , J.J. Wheler1 , R.G. Luthra3 , S.P. Patel4 , E.S. Kopetz5 , E. Sablon2 , G. Maertens2 , R. Kurzrock6 , F. Meric-Bernstam1 . 1 The University of Texas MD Anderson Cancer Center, Investigational Cancer Therapeutics (Phase I Clinical Trials Program), Houston, USA; 2 Biocartis, Mechelen, Belgium; 3 The University of Texas MD Anderson Cancer Center, Molecular Diagnostic Laboratory, Houston, USA; 4 The University of Texas MD Anderson Cancer Center, Melanoma Medical Oncology, Houston, USA; 5 The University of Texas MD Anderson Cancer Center, Gastrointestinal Medical Oncology, Houston, USA; 6 The University of California San Diego, Moores Cancer Center, La Jolla, USA Background: Cell-free (cf) DNA from the plasma of cancer patients offers an easily obtainable, low-risk, inexpensive and repeatedly applicable source of biologic material for mutation analysis of druggable targets and monitoring molecular changes in tumor(s) during and after therapeutic interventions. Novel, fast, and accurate diagnostic systems are needed for further development of plasma cfDNA testing in personalized therapy. Methods: cfDNA from plasma samples of patients with advanced cancers who progressed on systemic therapy was purified and 50– 100 ng DNA was used for testing for V600 BRAF mutations using the prototype molecular diagnostics (IdyllaTM ) fully integrated real-time PCRbased platform (Biocartis, Mechelen, Belgium) and BRAF V600 mutation prototype cartridges with a quick turnaround time (<60 minutes for cfDNA). The IdyllaTM platform and the BRAF V600 mutation prototype assay were used for research purposes only. Results were compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care from a CLIA-certified laboratory if available. Results: cfDNA was extracted from plasma samples of 127 patients with advanced cancers (colorectal, n = 54; melanoma, n = 28; non-small cell lung, n = 12; breast, n = 6, Erdheim-Chester disease, n = 6; thyroid, n = 7; appendiceal, n = 3; ovarian, n = 3; endometrial, n = 3; other cancers, n = 5). BRAF mutations were detected in 29% (36/127) of plasma samples and in 43% (50/117) of available archival tumor samples, resulting in concordance

Poster Session – Preclinical Models for 102 (87%) of 117 patients who had both plasma and tissue tested cases (kappa = 0.73, 95% confidence interval 0.61–0.85) with sensitivity 72%, specificity 99%, positive and negative predictive value 97% and 83%, respectively. In all 15 discrepant cases identical plasma cfDNA samples were tested using an alternative cfDNA BRAF mutation PCRbased method (BEAMing, Sysmex Inostics, Baltimore, MD), which yielded 100% agreement with the IdyllaTM platform. Longitudinally collected plasma samples were available in 13 patients (appendiceal, n = 2; colorectal, n = 2; melanoma, n = 4; papillary thyroid cancer, n = 2; other, n = 3) with plasma BRAF V600 mutations treated with predominantly BRAF targeting combinations and changes in the amount of BRAF-mutant cfDNA tracked changes in tumor markers and disease burden visualized via imaging. Conclusions: Detecting V600 BRAF mutations in cfDNA from plasma using the IdyllaTM platform and BRAF V600 mutation prototype cartridges is a fast and noninvasive alternative to mutation testing of tumor tissue with an acceptable level of concordance and sensitivity, and should be investigated further for testing and monitoring of BRAF mutation status in patients with cancer.

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170 POSTER (Board P164) Novel, ultra-deep next-generation sequencing for BRAF mutation testing using small amount of cell-free DNA from plasma of patients with advanced cancers

associated with sensitivity or resistance, and other cell line characteristics. EGR1 was identified as one of the top genes for which drug-induced changes in expression were associated with sensitivity to gemcitabine (gem; 32 cell lines up regulated >2-fold) and to erlotinib (17 cell lines down regulated >2-fold) in at least two treatment conditions. In the clinic, combinations of gem and erlotinib have been tested with mixed results. We hypothesized that divergent response of EGR1 to these two drugs might form the basis for antagonism. Treatment with the gem/erlotinib combination in 6 cell lines generated a range of antagonism in vitro, which depended on sensitivity to either the gem or erlotinib. Cell lines that were more sensitive to erlotinib, with greater EGR1 down regulation, demonstrated greater antagonism in response to the drug combination. In support of a role for EGR1, we found that transient down regulation of EGR1 in some gem-responsive cell lines led to decreased sensitivity to gem, while overexpression of the gene led to increased gem sensitivity. The measured antagonism was cell line dependent, but independent of drug scheduling. Sensitivity to gem appears mediated by EGR1 and can be antagonized by other pharmacologic effects on EGR1 as evidenced by the antagonism of growth inhibition between gem and erlotinib. These data may provide a rationale for the poor clinical response to this combination in some tumor types. Funded by NCI Contract No. HHSN261200800001E and supported in part by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis, NCI.

F. Janku1 , H.J. Huang1 , N.M. Ramzanali1 , X. Cai2 , R. Klausner2 , F. Meric-Bernstam1 , J.B. Fan2 . 1 MD Anderson Cancer Center, Investigational Cancer Therapeutics (Phase I Clinical Trials Program), Houston, USA; 2 Illumina, San Diego, USA

172 POSTER (Board P166) Modulation of estrogen-dependent transcription by cohesin in MCF7 human breast adenocarcinoma cells

Background: Plasma cell-free (cf) DNA of cancer patients offers an easily obtainable and repeatedly applicable source of DNA for mutation analysis, which provides attractive alternative to tumor tissue testing. Novel ultrasensitive technologies using small amounts of DNA are needed for further development of plasma cfDNA testing in personalized therapy. Methods: We have developed a next-generation sequencing method for somatic BRAF mutation detection in cfDNA with high sensitivity and specificity. The protocol was designed specifically to utilize small DNA fragments, using a very low DNA input of 5 ng. Each cfDNA fragment was uniquely barcoded and amplified prior to Illumina target enrichment workflow, followed by ultra-deep sequencing (>10,000×). Proprietary data processing and analysis tools were developed to enable sensitive detection of rare mutant molecules over high wild-type background (i.e. detection of 1 in 1000 molecules). Results were compared to mutation analysis of archival primary or metastatic tumor tissue obtained at different points of clinical care from a CLIA-certified laboratory Results: cfDNA was extracted from plasma samples of 24 patients with advanced cancers (melanoma, n = 9; colorectal, n = 5; non-small cell lung, n = 2; papillary thyroid, n = 2; other cancers, n = 6) and 5 ng were used for BRAF mutation analysis. BRAF mutations were detected in 71% (17/24) of plasma samples and in 88% (21/24) of archival tumor samples, resulting in concordance in 87% (20/24) of cases. Conclusions: Detecting V600 BRAF mutations using ultra-deep sequencing of small amounts of cfDNA (5 ng) from plasma is feasible with an acceptable level of concordance with BRAF testing of tumor tissue obtained at different time points and should be investigated further for testing and monitoring of BRAF mutation status in patients with cancer. 171 POSTER (Board P165) Antagonistic interaction between gemcitabine and erlotinib is influenced by EGR1 (early growth response 1) transcription factor expression C.D. Hose1 , Y. Zhao2 , E.C. Polley2 , J. Fang2 , N.D. Fer1 , A. Rapisarda1 , B.A. Teicher2 , R.M. Simon2 , J.D. Doroshow2 , A. Monks1 . 1 Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc., Frederick MD, USA; 2 National Cancer Institute, DCTD, Frederick MD, USA Drug-induced transcriptional changes may help to ascertain mechanisms of action and possible bystander effects, and can suggest potentially favorable or adverse drug combinations. We developed a database of gene expression changes that were observed following treatment with 15 commonly used anticancer agents in 60 human tumor cell lines, across 2 drug concentrations and 3 incubation times. Gene expression profiles were measured using Affymetrix HTA array plates 133A and 133B, and drug-induced fold-changes were calculated and compared to timematched controls. These dynamic transcriptional response data will be provided on a public website that can be facilely accessed and explored using the Transcriptional Pharmacology Workbench, a powerful web-based tool-set designed by the NCI for finding drug-modulated genes that are

T. Dasgupta1 , J. Antony1 , J. Rhodes1 , M. McEwan1 , M. Eccles1 , J. Horsfield1 . 1 University of Otago, Pathology, Dunedin, New Zealand Background: RAD21, a component of the evolutionary conserved chromosome cohesion protein cohesin, is amplified in breast cancer. Amplification of RAD21 is correlated with poor prognosis and endocrine resistance in luminal breast cancers. However, the reasons for this correlation are not understood. Our lab discovered that depletion of RAD21 reduces estrogen induction of the proto-oncogene c-MYC, and impairs binding of estrogen receptor a (ERa) to its enhancers. Chromosome binding of RAD21 coincides with ERa in estrogen-induced breast cancer cells suggesting cohesin could modulate estrogen-dependent gene transcription. This study aimed to identify estrogen-sensitive genes that are dysregulated upon loss of cohesin, and determine the mechanisms of regulation mediated by cohesin. Material and Methods: To map global changes in gene expression, a microarray analysis was carried out in RAD21-depleted MCF7 cells in the presence/absence of estrogen. Chromatin immunoprecipitation (ChIP) and quantitative PCR (qPCR) was used to determine if RAD21 depletion influenced binding of ERa at selected altered genes (IL20 and SOX4). Results: Microarray analysis revealed that cohesin depletion affected transcription of not all, but a subset of estrogen-sensitive genes. Loss of cohesin resulted in bidirectional (up or down) regulation of ERa target genes. The most significantly regulated genes were co-bound by both ERa and cohesin and were over-represented in oncogenic pathways such as ErbB and PI3K/mTOR. Preliminary ChIP analysis suggests that depletion of RAD21 alters binding of ERa at sites previously identified3 for genes, SOX4 and IL20. Conclusion: Cohesin can promote or inhibit expression of estrogenresponsive genes in a context-specific manner. Ongoing experiments are aimed at confirming the ChIP findings, to further elucidate cohesin’s role in regulation of the ER transcription program. 173 POSTER (Board P167) Ras-mediated activation of mitogen-activated protein kinase pathway unleashes basement membrane damaging activity of serine protease hepsin T. Tervonen1 , S. Pant1 , D. Belitskin1 , J. Englund1 , K. Narhi ¨ 2, E. Verschuren2 , P. Kovanen3 , J. Klefstrom ¨ 1 . 1 University of Helsinki, Translational Cancer Biology Research Program, Helsinki, Finland; 2 University of Helsinki, Institute for Molecular Medicine Finland, Helsinki, Finland; 3 University of Helsinki, Haartman Institute and Department of Immunology and Pathology, Helsinki, Finland Membrane-anchored serine proteases have emerged as novel players in invasion and metastasis of epithelial cancers. Among these, type II transmembrane serine protease hepsin is prominently upregulated in prostate and breast cancer both in mRNA and protein level. In agreement what was previously observed in ovarian cancer, normal human mammary gland expresses low levels of pericellularly and basally localized hepsin

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contrasting to diffuse, cytosolic expression observed in more than 80% (n = 49) of breast cancer specimens representing all major subtypes. Mutations in hepsin gene don’t explain widespread deregulation of hepsin protein in breast cancer, suggesting a critical role for upstream factors as culprits for hepsin deregulation. Hepsin expression in breast cancer was studied in paraffin sections and fresh tissue lysates from breast cancer patients. We also generated 2D and 3D human and mouse non-malignant mammary epithelial and cancer cell lines stably transduced with recombinant retro- or lentivirus vectors endowing cells with constitutive or inducible hepsin expression. Hepsin activity was probed with cell-based peptide substrate cleavage assay. We used lox-stop-lox (LSL)-KrasD12; p53−/− mice where sporadic lung tumors are induced with inhaled adeno-cre virus. Furthermore, mammary epithelial cells from LSL-KrasD12 mice were isolated and Kras activated ex vivo with adeno-cre virus to investigate pattern of 3D structure formation in Matrigel. To elucidate factors upstream of hepsin, we explored the effects of oncogenic Ras proteins, c-Myc, E2F-1, dominant negative p53 and Lkb1 silencing on hepsin protein expression levels in non-malignant mammary epithelial cells. We demonstrate that HrasV12, KrasV12, ectopic wildtype Kras, endogenous mutant LSL-KrasD12 and E2F-1 deregulate and enhance the proteolytic activity of hepsin. Oncogenic Ras proteins also reduced expression levels of HAI-1, the negative regulator of hepsin. Concomitantly, oncogenic Ras expression led to disappearance of desmoplakin and desmoglein from desmosomal junctions, which correlated with mislocalization of hepsin from its predominant localization in desmosomes to cytosol. shRNA-mediated silencing or function blocking Ab25 antibody for hepsin partially rescued HrasV12 induced transformation in 2D and 3D cultures of MCF10A cells and preserved the basal lamina integrity. Furthermore, MEK and Erk1/2 inhibitors prevented HrasV12dependent hepsin alterations as well as desmosomal and basement membrane defects. Our results couple Ras-MAPK pathway to deregulation of hepsin, revealing a possible role for hepsin in mediating Ras transformation of epithelial structures. 174 POSTER (Board P168) Combination of molecular and drug response data in patient-derived xenografts to assist patient stratification S. Cairo1 , O. Deas ´ 1 , A. Beurdeley1 , V. Yvonnet1 , M.F. Poupon1 , J.G. Judde1 . 1 Xentech, R&D, Evry, France Patient-derived xenografts (PDXs) are the preclinical models that most closely resemble tumors in patients. PDX and the tumor of origin show strikingly similar histological and molecular features, and when tested they possess very similar drug-response profile. Thanks to these characteristics, PDXs are potent surrogates of human tumor to investigate the impact of the genetic background of transformed cells on tumor response to treatment. Tumor heterogeneity is likely the reason why anticancer treatments fail to display the same efficacy in different patients. This diversity is probably due to the variability observed in tumor genetics, and in particular to the combination of genetic alterations in each tumor. In patients, the relationship between the degree of response to treatment and tumor genetic features is often loose. One of the difficulties in identifying genetic markers associated with anticancer treatment efficacy is that as each patient is subjected to only one therapeutic strategy, chosen as the best adapted based on the clinical parameters, it is impossible to know how this same patient would have responded to a different treatment. XenTech holds a large PDX collection, with more than 100 validated PDXs models characterized so far. These models represent tumors occurring in different tissues such as breast, lung, colon, prostate, liver and brain among others. Most of these modes have been subjected to standardof-care (SOC) treatments to characterize their response profile. As the studies are performed on the same tumor model, the genetic background heterogeneity that can bias molecular comparison of patient cohorts subjected to different therapeutic options is eliminated. The use of PDXs for this type of studies enables to investigate the response of the same tumor to different treatments by generating as many preclinical arms of xenografted mice as desired. These studies, besides providing a personalized profiling of tumor sensitivity to treatment, allow the investigation of the associated molecular features. In parallel, extensive molecular characterization has been performed, including gene expression array-CGH microarrays, and mutational profile of 73 genes most frequently mutated in the tumor types available in our PDX collection by microarray-based exon trapping coupled to high-throughput sequencing. For each model, mutational, gene expression and SNP/aCGH data of each PDX have been associated with treatment response profile in order to check for biomarkers associated with tumor sensitivity or resistance to treatment. The results obtained have been crossed with genomic data from patient cohorts with annotated response to SOC, and the results of these analyses will be discussed.

Poster Session – Preclinical Models The use of each PDX as representative of a patient subset carrying the same overall genomic alteration could be the good compromise between the panel-driven and personalized use of PDXs to stratify patients. 175 POSTER (Board P169) Functional analysis of [methyl-3 H]choline uptake in glioblastoma cells: Influence of anti-cancer and central nervous system drugs M. Inazu1 , C. Taguchi2 , T. Yamanaka3 , H. Uchino2 . 1 Tokyo Medical University, Institute of Medical Science, Tokyo, Japan; 2 Tokyo Medical University, Department of Anesthesiology, Tokyo, Japan; 3 Tokyo Medical University, Department of Molecular Preventive Medicine, Tokyo, Japan Positron emission tomography (PET) and PET/computed tomography (PETCT) studies with 11 C- or 18 F-labeled choline derivatives are used to differentiate between malignant and benign lesions in various regions of the body, including the brain, head, bone, and soft tissue. Concerning glioma, choline PET may make it possible to differentiate between low-grade and high-grade gliomas. Moreover, untreated and recurrent high-grade gliomas can be visualized with sharp delineation, without any influence by reactive inflammatory changes caused by choline PET. Further, a recent in vitro study suggested that choline PET may be a useful indicator of the response to chemotherapy agents that act by inhibiting signal transduction. However, the nature of the choline transport system in glioblastoma is poorly understood. In this study, we performed a functional characterization of [methyl-3 H]choline uptake and sought to identify the transporters that mediate choline uptake in the human glioblastoma cell lines A-172 and U-251MG. In addition, we examined the influence of anti-cancer drugs and central nervous system drugs on the transport of [methyl-3 H]choline. High- and low-affinity choline transport systems were present in A-172 cells, U-251MG cells and astrocytes, and these were Na+ -independent and pH-dependent. Cell viability in A-172 cells was not affected by choline deficiency. However, cell viability in U-251MG cells was significantly inhibited by choline deficiency. Both A-172 and U-251MG cells have two different choline transporters, choline transporter-like protein 1 (CTL1) and CTL2. In A-172 cells, CTL1 is predominantly expressed, whereas in U-251MG cells, CTL2 is predominantly expressed. Treatment with anti-cancer drugs such as cisplatin, etoposide and vincristine influenced [methyl-3 H]choline uptake in U-251MG cells, but not A-172 cells. Central nervous system drugs such as imipramine, fluvoxamine, paroxetine, reboxetine, citalopram and donepezil did not affect cell viability or [methyl3 H]choline uptake. The data presented here suggest that CTL1 and CTL2 are functionally expressed in A-172 and U-251MG cells and are responsible for [methyl-3 H]choline uptake that relies on a directed H+ gradient as a driving force. Furthermore, while anti-cancer drugs altered [methyl3 H]choline uptake, central nervous system drugs did not affect [methyl3 H]choline uptake. 176 POSTER (Board P170) Resolvin D2 has mitogenic activity in estrogen receptor positive breast cancer cell lines via activation of estrogen receptor N. Al-Zaubai1 , C. Johnstone2 , M. Rizzacasa3 , A. Stewart1 . 1 University of Melbourne, Pharmacology and Therapeutics, Melbourne Victoria, Australia; 2 Peter Maccallum Cancer Centre, Research Division, Melbourne Victoria, Australia; 3 University of Melbourne, School of Chemistry, Melbourne Victoria, Australia Background: Inflammation has been implicated in tumour initiation, angiogenesis and metastasis and linked to the development of more aggressive, therapy-resistant estrogen receptor positive breast cancer. Resolvin D2 (RvD2) is a potent anti-inflammatory lipid mediator. As RvD2 is present in plasma at bioactive concentrations and may be synthesized within breast tumours by both tumour and stromal cells, we have characterized the impact of RvD2 on cell processes underlying breast tumour growth and spread. Materials and Methods: Viable cells were enumerated by Trypan-blue exclusion. Transactivation of estrogen response element was assessed by transient transfection with estrogen response element (ERE) reporter and pGL3 vector constructs. RT-qPCR was used to examine gene expression. Binding to the estrogen receptor was investigated by competitive radioligand binding assays. Western blotting and immunofluorescence were the techniques used to ascertain estrogen receptor a nuclear localization. Results: Unexpectedly, whilst RvD2 (10–1000 nM) supported the proliferation of the ER-positive breast tumour, MCF-7, cells, it did not affect the ER-negative, MDA-MB-231 cell number. The proliferative effect of RvD2 in MCF-7 cells was attenuated by the estrogen receptor antagonist ICI 182,780 (fulvestrant). Furthermore, RvD2 increased ERE transcriptional activity in a number of ER positive breast and ovarian tumour cell lines.

Poster Session – Preclinical Models This activation was also inhibited by ICI 182,780. RvD2 altered the expression of a subset of estrogen-responsive genes. Prior exposure of MCF-7 cells to RvD2 resulted in a significant reduction in the apparent cytosolic ER density. However, binding experiments showed that RvD2 did not directly compete with 3 [H]-17b-estradiol (E2) for ER binding. Confocal immunocytochemistry and western blotting studies showed that RvD2 promoted nuclear localization of ERa with a corresponding decrease in cytosol ER density. Conclusions: These observations indicate that RvD2 displays significant but indirect estrogenic activities and that it has the potential to play a role in estrogen-dependent breast cancer progression. 177 POSTER (Board P171) Validation of 3D primary organoid cultures of colorectal carcinoma as discovery and validation platform for personalized cancer therapy P. Halonen1 , A. Kuijpers2 , B. Morris1 , B. Diosdado1 , S. Mainardi1 , R. Bernards1 , V. Verwaal2 , R. Beijersbergen1 . 1 Netherlands Cancer Institute − Antoni van Leeuwenhoek Hospital, Molecular Carcinogenesis, Amsterdam, Netherlands; 2 Netherlands Cancer Institute − Antoni van Leeuwenhoek Hospital, Colorectal Surgery, Amsterdam, Netherlands Background: Primary organoid cultures derived from patients’ tumors hold promise for the improvement of cancer therapy. For this, organoid cultures should accurately represent primary tumors with respect to their morphological and genomic characteristics. We have developed a 3D organoid culturing platform for primary and metastatic colorectal carcinoma derived from human tissues to conduct comparative studies of the primary tumors and the organoid culture counterparts. Material and Methods: Surgical specimens of primary and metastatic colorectal carcinoma were used to derive the primary tissue for culturing. 3D cultures were established based on the protocol by Sato et al. and adjusted for human colorectal cancer cells. Phenotypic comparison of primary tissue and organoids was done by immunohistochemistry. Genomic analysis consisted of copy number variation analysis by low-resolution full genome sequencing and exome sequencing by kinome capture; RNAseq was used to analyze gene expression. Signaling pathway status and drug responses were quantified using western blot analysis and Luminex technology combined with viability read-outs and fluorescence microscopy. Results: A high degree of similarity between the 3D cultures and original patient tissues was observed by immunohistochemistry using general and differentially expressed colorectal cancer markers. Genomic alterations identified in the primary tumor were also present in the organoid cultures derived thereof. Although some mutations were enriched in the organoids, we did not observe the appearance of specific mutations within the organoid panel. Comparison of short and long term organoid cultures indicated the preservation of the genomic alterations with some additional mutations indicative of the genomic instability of tumors. Gene expression analysis by RNAseq revealed clustering of organoids with the primary tissue and a concordance with subtype classification. Conclusions: This study shows that organoid cultures are highly representative of primary tumors and maintain these characteristics upon prolonged culturing. This establishes patient derived organoids as a discovery and validation platform for the prediction of drug response and a platform for the discovery of treatment options for personalized therapy. 178 POSTER (Board P172) Establishment of patient-derived xenografts (PDX) models for small cell lung (SCL) as a pre-clinical platform for drug development T. Broudy1 , J. Ricono1 , C. Mullins1 , C. Mirsaidi1 , P. Nair1 . 1 Molecular Response, San Diego, USA With over 150,000 deaths in 2012, lung cancer is the deadliest carcinoma in the United Sates. Small cell lung cancer, encompassing about 15−20% of all lung cancer cases, is more invasive and has a higher rate of proliferation with respect to non-small cell lung cancer, leading to a higher mortality rate. Most cases are responsive to chemotherapy, however there is a high rate of recurrence with treated patients and those in advanced stage of the disease often have a refractory response to treatment. Due to the fast growing nature of this cancer, the long term survival rate is low whilst advanced stage cancers typically will lead to expiry within 1 year of diagnosis. Preclinical PDX models have become valuable tools for novel drug development in recent years. Here, we used patient derived tissue to generate 8 new SCLC models in NOD-SCID mice. Patient tumors were pre-screened for their myc amplification status, before inoculating into immunocompromised animals. Once established as PDX models, cells were also grown in 3D cultures to compare morphology and to perform drug treatments to assess anti-proliferative potential of various compounds. Further characterization of the models were done by H&E, immunohistochemistry, and mutation analysis by next-generation

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sequencing. Efficacy of standard of care drugs irinotecan, and cisplatin– etoposide combination was demonstrated in these models. Current studies are underway to derive correlations between in vivo drug response and mutational status of these models. Our data strongly suggests the potential utility of these unique PDX models in drug development efforts in oncology. 179 POSTER (Board P173) Effects of human breast cancer cells secreted factors on macrophage differentiation 1 S. Coimbra de Sousa1 , R. Brion2 , J. Monkk ¨ onen ¨ , H. Joensuu3 , ¨ a¨ 1 . 1 University of Eastern Finland, School of D. Heymann2 , J. Ma¨ att Pharmacy, Kuopio, Finland; 2 INSERM, UMR957, Nantes, France; 3 Helsinki University Central Hospital, Department of Oncology, Helsinki, Finland

Background: Tumour associated macrophages (TAM) are a macrophage (Mf) population recruited and educated by tumour cells. Therefore, TAM exposed to IL-10, TGF-b, M-CSF and other immunosuppressive stimuli are more closely related to the M2 than M1 type macrophages. TAMs perform preferentially trophic tasks promoting epithelial outgrowth and invasion, common to the Mf role in development, rather than immune roles. The current study focuses on the in vitro differentiation of human Mfs in the presence of breast cancer conditioned medium (CM) and the differential expression of M1 and M2c markers by TAMs in tissue samples from breast cancer patients. Materials and Methods: CD14+ freshly isolated human monocytes were in vitro differentiated to M1 (IFN-g), M2a (IL-4) and M2c (IL-10) Mfs in the presence or absence of human breast cancer cell line CM (MCF-7, T47D and MDA-MB231). Differentiated cells were analysed by flow cytometry for the expression of CD14, CD16, CD64, CD86, CD200R and CD163. CD68, HLA-DRIIa and CD163 were analysed by immunohistochemistry in tissue microarray (TMA) samples from a large cohort of human breast cancer patients. Results: All the breast cancer CM enhanced M2a differentiation, by increasing the percentage of cells in the population of CD200Rhigh CD86med that normally results from IL-4 stimulus. Further, MDA-MB231 CM was sufficient to increase CD163 expression levels to the same extent as IL-10, the M2c inducing cytokine. In conjugation IL-10 and MDA-MB231 CM further increased CD163 expression. From the ongoing TMA analysis we expect to find out the effect of breast tumour Mf infiltration (assessed by CD68), M1 (HLA-DRIIa) and M2c differentiation (CD163) on recurrencefree survival of breast cancer patients treated in a randomised clinical trial (the FinXX trial). Conclusions: Breast cancer cell lines affect alternative Mf differentiation, inducing more matrix remodelling and immune-suppressive status, respectively M2a and M2c. Clinically this effect is relevant as human breast cancer TAM differentiation status may influence metastases formation, therapy efficacy and ultimately patient outcome. 180 POSTER (Board P174) Identifying and monitoring somatic mutations in cell free DNA of patients with metastatic melanoma J. Wisell1 , C.M. Amato2 , W.A. Robinson2 . 1 University of Colorado, Pathology, Aurora CO, USA; 2 University of Colorado, Medical Oncology, Aurora CO, USA Background: Within the last decade, significant advances have been made in understanding the molecular pathogenesis of melanoma, and several mutations such as those found in KIT, a tyrosine kinase receptor, and BRAF proto-oncogene, play a significant role in the disease. Patients with melanoma benefit from somatic mutation screening, particularly when it comes to predicting for therapeutic response. Such mutational analysis is performed on tumor samples, and with recent advancements in molecular biology, it is now possible to detect genetic changes, i.e. somatic mutations, in patient blood. In this study, our laboratory explored possible candidates for monitoring disease progression in patient blood samples. Material and Methods: The University of Colorado Skin Cancer Biorepository hosts an extensive collection of clinically annotated blood and tumor samples. We screened 175 metastatic melanoma tissue samples for 37 separate mutations in 10 different genes to identify somatic mutations suitable for monitoring in matched patient blood samples. Cell free DNA (cfDNA) was isolated from patient plasma samples, and somatic mutations were measured using digital PCR (dPCR). Results: In our tissue screen, approximately 65% of the samples contained at least one mutation, BRAF being the most commonly mutated oncogene, while RAS (KRAS and NRAS) and KIT mutations were second and third. In our cfDNA analysis, BRAFV600E levels in patient plasma correlated with disease burden. Conclusion: We demonstrate that somatic mutations are detectable in cfDNA isolated from patient blood samples. Our results show that

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BRAFV600E mutation levels correlate with tumor burden and suggest a possible role as a prognostic indicator. Studies are currently underway to examine RAS mutations in patient blood as a possible marker for acquired resistance to BRAF inhibitors, and to monitor disease progression in patients with BRAF wildtype tumors. 181 POSTER (Board P175) Aberrant Wnt signaling activation in human cancers: In vitro and in vivo models to facilitate Wnt targeted drug development 1

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G. Liu , C. Dong , R. Zhang , L. Zhang , S. Qian , J. Cai , J. Zhang , J. Ning1 . 1 Crown Bioscience Inc., Molecular and Cellular Biology, Taicang, China Activation of Wnt signaling has emerged as one of the major oncogenic aberrations in human cancers, which has been demonstrated to play critical roles in the maintenance of the undifferentiated cancer stem/progenitor cell phenotype, as well as to directly stimulate the malignant growth of tumors. Mutations in the downstream signaling components, including APC, AXIN, and b-catenin, have been well described in several cancer types, and recent studies have further extended the Wnt activation mechanisms beyond these downstream mutations to upstream signaling molecules in this pathway. By screening primary cancer cells established from the patient derived xenograft models, we identified multiple cancer types that secrete Wnt stimulating ligands. We also successfully established primary cancer cell line harboring recurrent R-spondin fusion mutations. Inhibition of the activated Wnt signaling in these cancer cells results in reduced cancer cell growth, indicating the critical dependence of cancer cells on the autocrine Wnt signaling. Thus, these in vitro and in vivo models provide a valuable resource for the high throughput screening of Wnt antagonists, efficacy assessment of candidate Wnt inhibitors, biomarker analysis, as well as the preclinical development of Wnt targeted therapeutics. 182 POSTER (Board P176) TRAP1 represents a key mediator of stemness and glycolytic metabolism in colorectal cancer cells G. Lettini1 , F. Maddalena1 , L. Sisinni1 , V. Condelli1 , L. Del Vecchio2 , M. Gemei2 , T. Notarangelo1 , M. Landriscina3 . 1 CROB − IRCCS, Laboratory of Pre-Clinical and Translational Research, Rionero in Vulture (PZ), Italy; 2 CEINGE, Biotecnologie Avanzate, Napoli, Italy; 3 University of Foggia, Clinical Oncology Unit Department of Medical and Surgical Sciences, Foggia, Italy Background: Tumor cells undergo a metabolic shift from mitochondrial oxidative phosphorylation to a preferential glycolytic metabolism, known as Warburg effect. Furthermore, cancer stem cells (CSCs), known for being responsible for tumor initiation and growth, are located in a hypoxic microenvironment, heavily relying on anaerobic glycolysis. Recently, tumor necrosis factor receptor-associated protein 1 (TRAP1), a HSP90 mitochondrial molecular chaperone up-regulated in colorectal, prostate, breast and lung carcinomas, has been identified as a key regulator of tumor cell metabolism, being responsible for suppression of mitochondrial oxidative phosphorylation and post-translational stabilization of HIF1a. This study was designed to address the hypothesis that TRAP1 is relevant in determining specific features of the CSC phenotype and, more specifically, the reprogramming of their metabolism. Methods: TRAP1 levels were investigated in CD166-positive CSC fractions of HCT116 and HT29 colorectal carcinoma cells (CRCs) and primary stem cells derived from dental pulp by flow cytometry, cell sorting and upon colony formation assay. Interfering strategies were used to evaluate TRAP1 role in regulating glucose uptake and glycolytic metabolism. Results: TRAP1-interfered CRC HCT116 cells showed lower clonogenic potential than scramble cells in soft agar assay. Furthermore, CD166positive CSCs derived from CRC cell lines exhibited higher TRAP1 levels compared to non-CSC subpopulations and, consistently, TRAP1 stable interference resulted in the down-regulation of CD166 expression in HCT116 cells. In parallel experiment, TRAP1 expression was downregulated upon differentiation of primary stem cells derived from dental pulp. Finally, TRAP1 interference resulted in increased O2 consumption and in reduced glucose uptake, and the parallel inhibition of the expression of glucose transporters 1 and 2 and lactate production in CRC cells. Conclusion: These preliminary data suggest a potential role of TRAP1 in the maintenance of the CSC phenotype, likely due to its capacity to enhance glucose uptake and glycolytic metabolism, thus favoring the adaptation to a hypoxic microenvironment.

183 POSTER (Board P177) Spatio-temporal characterization of tumor growth and invasion A.M. Jimenez1 , O. Yogurtcu2 , M. Horn-Lee1 , P. Rao1 , S.X. Sun2 , D. Wirtz1 . 1 Johns Hopkins University, Chemical and Biomolecular Eng, Baltimore MD, USA; 2 Johns Hopkins University, Mechanical Engineering, Baltimore MD, USA Background: Tumors are a complex arrangement of tissues made up of several components, including dense masses of cancer cells and extracellular matrix (ECM). Recent studies have revealed the crucial role extracellular matrix components have on single cancer cell behavior, but how the interaction of ECM components affect the growth dynamics of an entire tumor is not well understood. Materials and Methods: We designed a novel three-dimensional (3D) in vitro system and used human derived fibrosarcoma cell (HT1080) aggregates embedded in 3D collagen matrices in combination with live cell imaging, cryo-stat sectioning, and immunostaining to characterize tumor growth and invasion. Results: Multicellular aggregates grow linearly with a rate that decreases with increasing collagen concentration. The spatio-temporal cell density distribution of the aggregates differs with differing collagen concentrations. Both cell migration and proliferation are highly dependent on collagen concentration and local cell density. Conclusion: We identified cell migration as a key contributor to multicellular aggregate size and demonstrate that different cell phenotypes correlate with different invasion patterns of the cell aggregates at various collagen concentrations. 184 POSTER (Board P178) Molecular profiling of heterogeneous tumor cells A. Chenchik1 , D. Deng1 , K. Bonneau1 , M. Makhanov1 , M. Coram2 , G. Dolganov2 , S.S. Jeffrey2 . 1 Cellecta Inc., Mountain View CA, USA; 2 Stanford University, Palo Alto CA, USA Molecular profiling of heterogeneous circulating tumor cells (CTCs) and tumor biopsy samples at the single-cell level is critical for identifying different cancer cell subpopulations and understanding their value in predicting metastatic cancer progression and responses to treatment. Unfortunately, the isolation and comprehensive characterization of hundreds to thousands of individual cancer cells present formidable analytical and technical challenges. To this end, we developed a high-throughput (HT) HiCellex technology to obtain the genome-wide expression and mutation profiles of hundreds to thousands of epithelial cancer cells at the single cell level. Importantly, HiCellex allows selective molecular profiling of a complete set of viable cells at the single-cell level in the presence of contaminating leukocytes without physical separation of individual cells. The genetic profiling technology uses unique cell-specific molecular barcodes to label mRNAs in individual viable cancer cells by a spliceosome-mediated trans-splicing mechanism. Barcoded trans-splicing constructs, necessary for the labeling of the entire population of pre-mRNAs, are delivered and expressed in a pool of target cancer cells with lentiviral vectors. Digital expression data are generated by HT sequencing of barcoded amplified cDNAs and easily cluster to each cell in silico using cell-specific barcodes. This presentation will provide results of development and validation of genome-wide and multiplex RT-PCR profiling assay of the 500 most informative subtyping and metastatic signature genes (MGS) in model cancer cell lines, CTC and tumor samples. The developed HiCellex 500 MGS profiling assay is a cost-effective approach to effectively characterize, subtype and identify prognostic and diagnostic biomarkers in heterogeneous cancer cells at the single-cell level. 185 POSTER (Board P179) Treatment of patient-derived NSCLC xenograft preclinical models using image-guided small animal irradiation N. Papadopoulou1 , A. McKenzie1 , J. King1 , M. Page1 , R. Kumari1 . 1 Precos Ltd, Crown Bioscience, Loughborough Leicestershire, United Kingdom Background: Radiotherapy is a primary, adjuvant or neoadjuvant treatment for a number of different cancers such as glioblastoma, breast, lung and prostate. Image-guided micro-irradiation (IGMI) is widely used to treat cancer patients providing more accurate treatment plans and reduced side effects. However in the preclinical setting the use of IGMI is less common with traditional irradiation studies utilising whole body irradiation with lead shielding to focus the radiation to a specific area on the animal or simple single beam techniques. The development of the image-guided small animal radiation research platform (SARRP) allows the treatment of animal models of cancer more accurately and with planned protocols

Poster Session – Preclinical Models similar to those utilised in the clinic. We have established a panel of Caucasian NSCLC patient-derived xenograft (PDX), which are sensitive or have acquired resistance to standard of care treatments. Methods: NSCLC samples obtained from untreated patients undergoing surgery were collected with ethical consent, disaggregated and implanted subcutaneously in MF-1 nude mice (Harlan, UK) admixed with a human stromal cell component to generate patient-derived xenograft (PDX) models. Resistant models were generated in vivo through repeated cycling of treatment for up to 10 weeks with standard of care or targeted agents. Patient-derived xenografts were maintained in vivo and monitored by calliper measurements three times weekly and mice recruited to the study when mean tumour volume was approximately 100–200 mm3 . Irradiation protocols and dose calculation were designed to deliver appropriate irradiation dose (2−3 Gy/day/mouse) to the tumours either alone or in combination with a targeted agent/chemotherapeutic, whilst sparing the surrounding normal tissue. Response to treatment was evaluated by tumour growth inhibition and clinical condition of mice monitored daily. Results: Mice treated with IGMI using the SARRP tolerated irradiation doses either alone or in combinations. Response and resistance to irradiation across a panel of Caucasian NSCLC PDX and resistant models will be reported along with any outcome of the combination treatments to re-sensitize resistant cancer to radiotherapy. Conclusions: The SARRP platform allows the evaluation of irradiation alone or in combination with anti-cancer agents in small animals with reduced side effects and improved safety outcome, allowing these novel preclinical models to be used effectively for drug discovery programmes and to derive irradiation schedules and regimens suitable for testing subsequently in clinical trials. 186 POSTER (Board P180) The use of Quantitative Textural Analysis imaging biomarkers to predict response to temsirolimus treatment in advanced HCC subjects R. Korn1 , R. Osarogiagbon2 , R. Newbold3 , D. Burkett4 , J. Sachdev5 . 1 Scottsdale Healthcare Research Institute TGEN, Imaging Endpoints, Scottsdale, USA; 2 Baptist Cancer Center, Oncology, Memphis, USA; 3 Scottsdale Medical Imaging, Radiology, Scottsdale, USA; 4 Imaging Endpoints Core Lab, Radiology, Scottsdale, USA; 5 Scottsdale Healthcare Research Institute-TGEN, Virginia G Piper Cancer Center, Scottsdale, USA Background: The treatment of advanced HCC can be challenging. Being able to predict who will respond to therapy would represent a major step forward in disease control. We have developed an exploratory CT imaging biomarker that may predict treatment response to temsirolimus (T) after sorafenib (S) failure using quantitative textural analysis (QTA) of hepatic tissue and tumors on CT scans. Material and Methods: Pre and Post venous phase contrast CT scans through the abdomen were obtained in 10 subjects enrolled in a IIT from a single institution prior to treatment with T after S failure. QTA was performed on the axial slice of the liver containing both uninvolved hepatic tissue and tumor in the same slice. QTA parameters were generated at a fine filter level (SSF 3) and displayed as histogram derived features of mean, SD, mean positive pixel (MPP), entropy, skewness and kurtosis. Statistical Correlation between QTA parameters and subsequent CT tumor responses by mRECIST criteria, Barcelona score, tumor markers, OS, PFS were performed using spearman correlations, regression analysis and ROC analysis. P values <0.05 were considered significant. Results: A single arm Simon two-stage phase II trial was conducted to test the activity of T in previously S treated HCC patients. Of the 26 subjects enrolled 10 subjects qualified for QTA analysis as having both pre and post contrast venous phase CT scans available for evaluation. Of the 10 subjects, 7 had PR and 3 had SD as best response by mRECIST. The mean change in tumor size was −26.7% (median −34.4%, range −52.5% to 11.3%) for the 10 subjects. Using QTA there was a significant correlation between baseline tissue-tumor texture and best mRECIST response as measured by MPP (rho = 0.7551, p = 0.0011). ROC analysis showed that a MPP cutoff value of 7.95 had the best separation of predicting PRs with a 85% sensitivity and 100% specificity. No significant correlation was noted between QTA and PFS, OS, serum tumor markers, Barcelona scores. Conclusion: This limited analysis of responding HCC patients indicates that textural analysis from CT scans may provide an imaging biomarker for predicting response to T therapy following S failure. Although preliminary, these findings merit further investigation.

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187 POSTER (Board P181) Targeted genomic profiling of penile squamous cell carcinoma using the Oncomine cancer research panel A.S. McDaniel1 , D. Hovelson1 , A. Cani1 , C.J. Liu1 , Y. Zhang1 , S. Sadis2 , S. Bandla2 , P. Williams2 , D. Rhodes2 , S.A. Tomlins1 . 1 University of Michigan, Pathology, Ann Arbor MI, USA; 2 ThermoFisher Scientific, Ann Arbor MI, USA Background: Penile squamous cell carcinoma (PeSCC) is a rare cancer notable for significant morbidity and mortality as well as an incomplete understanding of the underlying molecular alterations and lack of effective non-surgical therapeutic approaches. Material and Methods: A retrospective cohort of 60 formalin fixed, paraffin embedded (FFPE) tumor samples from 44 PeSCC cases (including 14 matched primary/metastasis pairs) was subjected to targeted next generation sequencing (NGS) using the Oncomine Cancer Research Panel encompassing actionable recurrent somatic alterations in ~125 oncogenes and tumor suppressors identified by analysis across multiple cancer types. Sequencing of multiplexed templates was performed using the IonTorrent Proton system. Sequence analysis was performed in Torrent Suite 4.0, with sequence alignment by TMAP, and variant calling using the Torrent Variant Caller plugin. HPV infection status for each sample was assessed using additional genomic DNA for PCR with the GP5/6 and My09/11 consensus primer sets for viral detection and typing. Results: Highly multiplexed targeted NGS yielded an average of 1,136,032 mapped reads per sample with high coverage (mean >450×) over targeted bases using 20 ng of input genomic DNA. High risk HPV was detected in five cases (Four with HPV 16, one with HPV 33). All classes of genomic alterations were evaluated, including single nucleotide variants (SNVs), insertions, deletions, stopgains, and copy number variants (CNVs). Frequently altered genes included CDKN2A (20 of 44), TP53 (19 of 44), NOTCH1 (9 of 44), FBXW7 (8 of 44), PIK3CA (7 of 44), NFE2L2 (6 of 44), and HRAS (6 of 44). Notably, tumor specimens from four cases harbored amplifications of EGFR and one case demonstrated CDK4 amplification; genes for which approved and investigational targeted therapies are available. Importantly, cases with multiple tumor samples profiled showed significant differences in 4 of 14 paired samples, indicating heterogeneity for actionable mutations such as EGFR amplification between primary tumors and metastases. Conclusions: We evaluated a cohort of PeSCC FFPE specimens using an NGS panel of recurrently altered cancer-associated somatic variants, providing detailed molecular analysis of this disease for the first time. The scope of mutations identified was similar to squamous cell carcinomas from other locations such as the lung and head and neck region. We identified a subset of cases harboring mutations with immediate therapeutic potential, including EGFR amplifications. This research suggests that NGS profiling of PeSCC may have utility as part of a precision medicine approach to aid clinical decision making. 188 POSTER (Board P182) RANBP2 knock-down is synthetic lethal with BRAF V600E in colon cancer L. Vecchione1 , V. Gambino1 , G. d’Ario2 , S. Tian3 , A. Schlicker1 , S. Mainardi1 , B. Diosdado1 , I. Simon3 , M. Delorenzi2 , C. Lieftink1 , R. Beijersbergen1 , S. Tejpar4 , R. Bernards1 . 1 NKI/AVL, Division of Molecular Carcinogenesis, Amsterdam, Netherlands; 2 Swiss Institute for Bioinformatics, Bioinformatics Core Facility, Lausanne, Switzerland; 3 Agendia BV, Agendia BV, Amsterdam, Netherlands; 4 KULeuven, Laboratory of Digestive Oncology, Leuven, Belgium Background: Approximately 8−15% of colon (CC) patients carry an activating mutation in BRAF. This CC subtype is associated with poor outcome and with resistance to chemotherapeutic treatments. We recently showed that BRAF (V600E) colon cancers have a characteristic gene expression signature, which is found also in subsets of KRAS mutant and KRAS-BRAF wild type (WT2) tumors. Tumors having this gene signature, referred to as ‘BRAF-like’, have a similar poor prognosis irrespective of the presence of the BRAF (V600E) mutation. By using a shRNA-based genetic screen in BRAF mutant CC cell lines we aimed to identify genes necessary for survival and growth of BRAF mutant CC in order to reveal novel targets for therapy. Method: We selected 363 genes that are specifically overexpressed in 89 BRAF mutant tumors as compared to 608 WT2 type tumors, based on gene expression profiles generated in two independent datasets The 363 genes list was used to generate a shRNA library consisting of 1815 hairpins targeting these genes (BRAF library) selected from the TRC human genome-wide shRNA collection (TRC-Hs1.0). Vaco432 and WIDR (BRAFV600E) CC cell lines and Lim1215 (WT2) CC cell line were infected with the BRAF library and screened for shRNAs that are selectively synthetic lethal with BRAFV600E mutation. Cells stably expressing the shRNA

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library were cultured for 13 days, after which shRNAs were recovered by PCR. Deep sequencing was applied to determine the relative abundance of each shRNA in BRAF (V600E) cells as compared to WT2 CC cells. Results and Conclusions: Based on the results of the pooled shRNA screen, we were able to identify six candidate synthetic lethal genes in BRAF mutant CC cell lines. In particular, further validation showed RANBP2 gene knock-down to be synthetic lethal with BRAFV600E and BRAFlikeness in CC. Experiments addressing the identification and the biological characterization of RANBP2 will be presented. 189 POSTER (Board P183) Molecular and pharmacological characterization of primary mesothelioma tumor cell lines orthotopically xenografted in nude mice C. Pisano1 , A. Cole1 , A. Barbarino1 , E. Bianchino1 , M. Guglielmi1 , C. Melito1 , G. Mercadante1 , A. Porciello1 , A. Riccio1 , I. La Porta1 , S. Orecchia2 , R. Libener2 , L. Mazzucco2 , S. Licandro3 , M. Ceccarelli4 , F. D’Angelo4 , P. De Luca1 . 1 Biogem, Preclinical Research&Development Service, Ariano Irpino (AV), Italy; 2 Azienda Ospedaliera, Hematology, Alessandria (AL), Italy; 3 IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Oncology, Milano (MI), Italy; 4 Biogem, Bioinformatic, Ariano Irpino (AV), Italy Background: Mesothelioma is estimated at less than 1% of all cancers, however its incidence is increasing, with an expected peak in the next 10−20 years. In order to setup in vivo models of human mesothelioma, putatively relevant to studies of the human pathology in terms of histology, antigen expression, and pharmacological response to chemotherapy, we have established in vivo models of mesothelioma, using primary tumors, having different histotypes. Material and Methods: primary cells derived from nine mesothelioma patients were xenografted orthotopically in immunodeficient nude mice. Cells derived from three of nine xenografted mice (MM432: sarcomatoid, MM473: epithelioid and MM487: biphasic) were stably transfected with luciferase expression vector. These cells and the nine original cell lines were used for further molecular and biochemical characterization. Results: The selected MM473 and MM487 high luc-expressing clones were intrapleurally reinoculated in immunodeficient nude mice. They successfully invaded and proliferated within the murine host. In particular, the epithelioid histotype presented tumour growth in 100% of mice, after a short latency period. The biphasic histotype showed tumour growth in 30% of the cases, although it was far more aggressive than the epithelial histotype in engrafted mice. Immunohystochemistry evaluation of resultant tumour masses confirmed the histotype from the parental patient tumours (i.e. for CEA, EMA, Mesothelin, Podoplanin, calretinin). Interestingly, biochemical characterization (i.e. EGFR, VEGF, Top1, TKs pathways) of the three histotypes, revealed histotype-related differences. The exposure of the nine cell lines to a panel of pharmaceutical drugs (i.e. Doxorubicin, Topotecan, Cyclophosphamide, Dacarbazine, Gemcitabine, Temozolomide, Bortezomib, 5-Azacitidine, Paclitaxel, Etoposide, 5-FU, Cisplatin, and AZD-2281) revealed a different not histotypes-related sensitivity. Western blot and RT-PCR analysis revealed that mesothelioma cell lines express elevated levels of EGFR (RNA and protein) not correlate with downstream signaling pathways. Thus, the sensitivity to gefitinib of these cells was not correlated with the EGFR expression. Finally, gene expression analysis between the three histotypes (MM432, MM473 and MM487) revealed that more than 1000 genes are differentially expressed. Conclusions: Taken together, these results have led to expand our knowledge on the molecular and biochemical features of various type of mesotheliomas. In addition, our in vivo models, being established from primary cells from patients, enlarged the available models of mesothelioma that, with the limitations intrinsic to the experimental models, can be predictive about the efficacy of new drugs or new therapeutic approaches for this incurable disease. 190 POSTER (Board P184) Differential chemosensitivity between CETCs and tumour spheroids in cancer patients with solid tumors D. Zimon1 , M. Pizon2 , E.L. Stein2 , U. Pachmann2 , K. Pachmann2 . 1 Simfo GmbH, Bayreuth, Germany; 2 Simfo GmbH, Research, Bayreuth, Germany Background: In vitro chemosensitivity testing of circulating epithelial tumor cells (CETCs) provides real-time information about the sensitivity of the tumor cells present in the patient and correlates with treatment success. Nevertheless, a fraction of CETCs can survive after conventional chemotherapy and grow into distant metastasis. A subpopulation of CETCs with proliferation activity has the ability to form spheroids in suspension

Poster Session – Preclinical Models culture. Spheroids exhibit stem cell-like properties and may be responsible for chemo therapeutic resistance. Therefore, the aim of our study was the comparison of the efficacy of chemotherapeutics on CETCs and on spheroids originated from the same individuals. Methods: The enumeration of CETCs collected from patients with solid tumors in clinical stage 1−4 were performed using the maintrac® method. Subsequently, viable CETCs were cultured in suspension culture system allowing for spheroid formation. To evaluate the cytotoxic effect CETCs and spheroids we exposed to anticancer drugs in short time culture in different concentrations and for different periods of time. Results: The response to chemotherapeutics was different between CETCs and spheroids. In contrast to CETCs, spheroids from the same patients were significantly more chemoresistant. Whereas active drugs led to membrane permeability in single CETCs with subsequent staining of the nuclei with propidium iodide, the same drugs led to disintegration of tumorspheres with destruction of part of the cells but often part of the cells in the spheres were able to survive. Epirubicin and, interestingly, and especially salinomycin, a polyether ionophore antibiotic isolated from Streptomyces albus, showed the best effects. Docetaxel, cyclophosphamide and 5-fluoruracil showed almost no cytotoxic effects onto the cells in the spheres. Conclusion: Our results show, for the first time, that stem cells circulating in peripheral blood, capable of forming spheroids are way more resistant to anticancer drugs than the remnant circulating tumor cells. We, furthermore, demonstrate that salinomycin efficiently destroy spheroids cultured from CETCs, strengthening its role as promising anti-cancer therapeutic. 191 POSTER (Board P185) Homotypic and heterotypic cell signaling transduction using a dielectrophoresis microfluidic device M. Tellez Gabriel1 , D. Heymann1 . 1 Universite´ de Nantes, UMR 957, Nantes, France Studying cell signaling transduction in homotypic and heterotypic cell interactions can have a great impact in clinics as could be useful to determine which cells are able to establish communication (e.g. tumoral niche). In this study, taking the advantage of a dielectrophoresis based microfluidics device (the DEPArray), we show the possibility of observing homotypic (Human osteosarcoma cells) and heterotypic (Human osteosarcoma cells vs. Mesenchymal Stem Cells) cell signaling transduction either single cells or clusters. We prepared two different cell populations − acceptors and donors − stained with two different fluorescent markers, one with DiI − a red non transferable molecule − and the other one with Calcein-AM − a green fluorochrom, able to be transferred through GAP junctions- and mixed them. We predetermined spatial coordinates by software-guided routing for establishing cell interactions − at different ratios of populations − within the chip, and we programmed a time-lapse for taking images of the interacting cells. The analysis of the images showed a decreasing in time of the green fluorescence intensity in donor cells, concomitant with an increasing of green fluorescence in acceptor cells, indicating the existence of cell signaling transduction between cells in both homotypic and heterotypic interactions. These findings are promising to study which specific cell–cell communications are established between different populations present in a microenvironment. 192 POSTER (Board P186) Extensive characterization of patient derived colon cancer xenografts for preclinical biomarker identification V. Vuaroqueaux1 , F. Kiefer1 , P. Bronsert2 , A.L. Peille1 , B. Zeitouni1 , F. Foucault1 , T. Kees1 , J. Guo3 , J. Schuler ¨ 3 , H.H. Fiebig4 . 1 Oncotest GmbH, Biomarker Development and Bioinformatics, Freiburg, Germany; 2 Institute of Pathology University Hospital, Freiburg, Germany; 3 Oncotest GmbH, In vivo, Freiburg, Germany; 4 Oncotest GmbH, Freiburg, Germany The use of large collections of preclinical models with extensive molecular characterization is essential for improving preclinical drug sensitivity testing and biomarker identification. We established a collection of 70 patient derived xenografts (PDX) of colorectal cancer and analyzed them for morphological features, genomic alterations (Whole Exome Sequencing & Affymetrix SNP6), transcriptomic profiles (affymetrix U133 2 plus) and sensitivity to various anticancer agents. Furthermore, we set up an ‘R’ based bioinformatics process for integrative analyses and automated biomarker identification. Here, we used an integrative OMICS approach to characterize our colon cancer PDX collection and we evaluated their relevance for biomarker identification. At the histological level, colon PDX tumor tissues closely resembled the patient tumors and retained similar differentiation grades. Furthermore, we observed heterogeneity in between the different colon PDX regarding

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stroma content and vascularization. At the transcriptomic level, we identified several PDX clusters similar to Goblet-like, Enterocyte, Transit-Amplifying, Inflammatory and Stem-like subtypes recently reported in CRC patients. In addition, well and moderately differentiated PDX showed a different gene expression profile than poorly and undifferentiated models. At the genomic level, the colon PDX models exhibited alteration profiles comparable to those reported for patient tumors regarding signatures of mutational processes (patient-age related or associated to mismatch repair gene deficiencies), chromosomal rearrangement and mutated genes (e.g. APC, TP53, KRAS and TP53). Moreover, we identified 6 PDX models with microsatellite instability (MSI), 5 of them being hyper-mutant with a loss of MLH1 expression. Interestingly, colon PDX displaying the MSI phenotype showed gene expression patterns of the Globlet-like or Inflammatory subtypes. To demonstrate the potential of our approach, we searched for molecular determinants of cetuximab sensitivity in colon PDX by combining in vivo drug sensitivity and molecular data. The study identified the Transit Amplifying subtype, AREG/EREG expression and KRAS mutation among the best predictors of response. Similarly, predictive biomarkers for oxaliplatin and irinotecan were found, and their associations with particular PDX subtypes are under investigation. The accuracy in predicting patient outcome following treatment with FOLFOX and FOLFIRI will be evaluated by assessing publicly available datasets. The comprehensive characterization of our colon PDX models confirmed their similarities with patient tumors and their inter-tumoral diversity. The combined use of PDX molecular and drug sensitivity data represents a promising approach for biomarker identification and tumor response prediction at a preclinical stage.

with advanced PDA have improved their overall survival, but the disease still inexorably progresses. The most commonly used system for drug discovery/screening and development are PDA monolayer cell lines coupled to preclinical studies in mice. However, this approach has not been successful and new ones are needed. Material and Methods: In the last year our lab has developed an organotypic three-dimensional culture system to study PDA. Murine organoids derived from normal ductal, preneoplastic or neoplastic pancreatic cells are cultured in semisolid media. In the case of human, normal and tumor are also grown in this semisolid media. Following orthotopic transplantation, these cultures form the normal, premalignant or malignant ductal structures from which they were derived and they are termed ‘pancreatic ductal organoids’. Using different methods that assess cell viability (ATP based luminescence and fluorescence), we have measured the effect of single drugs and combinatorial strategies. Results: This platform allows us to compare the IC50 of different drugs and search for those with a higher therapeutic index by comparing normal vs tumor. We have evaluated several chemotherapeutic drugs (Gemcitabine, 5-Flurouracil, paclitaxel, irinotecan and oxaliplatin) as well as some targeted agents (eg. Selumetenib, Neratinib, and MK-2206). We have also included tumor microenvironment cell types in our cultures to evaluate their role in drug responsiveness. Furthermore, organoid cultures have been transplanted orthotopically into immune-competent mice allowing for the study of therapeutic responses in vivo. Conclusions: We present this versatile ex vivo system as a promising experimental platform for achieving further insight into biologically and clinically important questions regarding cancer therapeutics in PDA.

193 POSTER (Board P187) The identification of new therapies for ependymoma subgroups

195 POSTER (Board P189) A humanized mouse model for preclinical testing of molecules targeting immune checkpoints

N. Boulos1 , J.D. Dapper1 , Y.T. Patel2 , M. DeCuypere1 , B. Bianski3 , K.M. Mohankumar1 , M.O. Jacus2 , B. Freeman III4 , K.D. Wright5 , A. Gajjar5 , A.A. Shelat6 , C.F. Stewart2 , R.K. Guy6 , R.J. Gilbertson1 . 1 St Jude Children’s Research Hospital, Developmental Neurobiology, Memphis TN, USA; 2 St Jude Children’s Research Hospital, Pharmaceutical Sciences, Memphis TN, USA; 3 St Jude Children’s Research Hospital, Radiologic Sciences, Memphis TN, USA; 4 St Jude Children’s Research Hospital, Preclinical Pharmacokinetics Shared Resources, Memphis TN, USA; 5 St Jude Children’s Research Hospital, Oncology, Memphis TN, USA; 6 St Jude Children’s Research Hospital, Chemical Biology and Therapeutics, Memphis TN, USA Current treatment for ependymoma is limited to surgery and radiation; existing chemotherapies are generally ineffective. We aimed to identify new therapies for ependymoma subgroups using accurate mouse models driven by ependymoma oncogenes, including the highly recurrent C11orf95RELA fusion (RELA+ ). First, we performed unbiased, high throughput drug screening against tumor cells generated from a RTBDN-driven ependymoma (mEPRTBDN ) mouse model. Initial screens performed at a single concentration and dose response screen of 6500 compounds, comprising FDA-approved drugs and developmental candidates, identified 377 compounds with 50% activity. Of these, 30 potent compounds (with EC50 1mM) were selected for rigorous preclinical pharmacokinetics (PK) and efficacy studies that included mouse tumor neurosurgery and cranial irradiation. Our lead compound gemcitabine proved highly active in vitro against RELA fusion-negative (RELA− ) ependymoma (mEPRTBDN and mEPEPHB2 ) and RELA+ ependymoma. Applying stringent preclinical PK with consideration of pediatric clinical dosing and toxicity, we identified the optimal route of administration, dose and schedule for gemcitabine (fixed dose rate infusion of 0.805 mg/kg/min over 3 hrs). Treatment of mice harboring murine RELA− and human RELA+ ependymoma with this PK guided regimen significantly prolonged survival. Moreover, combination of gemcitabine infusions with surgery further improved survival that we are now testing in a triple treatment regimen in mice that includes surgery, radiation and gemcitabine. We currently are developing gemcitabine as a new treatment for children with both RELA+ and RELA− ependymoma. 194 POSTER (Board P188) Pancreatic ductal organoids as a new platform for drug discovery 1 ¨ M. Ponz-Sarvise1 , V. Corbo1 , D. Ohlund , T. Oni1 , A. Handly-Santana1 , D. Engle1 , H. Tiriac1 , C. Chio1 , M. Feigin1 , L. Baker1 , C. Ardito-Abraham1 , Y. Park1 , C. Hwang1 , E. Elyada1 , K. Yu1 , H. Clevers2 , D.A. Tuveson1 . 1 Cold Spring Harbor Laboratory, Cancer Center, Cold Spring Harbor, USA; 2 Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, Netherlands

Background: Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with a 5-year survival rate of 6%. Newer cytotoxic regimens for patients

G. Baia1 , D. Vasquez1 , D. Ciznadija1 , D. Sidransky2 , A. Katz1 , K. Paz1 . 1 Champions Oncology, Baltimore MD, USA; 2 Johns Hopkins University, Baltimore MD, USA Background: The blockade of immune checkpoints is a promising therapeutic avenue for cancer therapy, with durable objective responses observed in patients with a variety of solid tumors. Despite these successes, current animal models do not reliably identify targets with the greatest clinical potential due in part to differences between human and murine immune systems. Hence, robust preclinical tools to test these drugs directly against human cancers in the context of a human immune system are required. To circumvent this limitation, Champions Oncology has developed the ImmunoGraft, whereby two innovative technologies, the Champions TumorGraft (patient-derived xenograft) and humanized mice (immunodeficient mice reconstituted with a human immune system), are combined in a single platform. Materials and Methods: Thirty-nine melanoma, colorectal, breast, and lung TumorGrafts were characterized with respect to HLA expression, mutation status, and expression of PD-L1, a pathway targeted by several therapeutics currently in clinical trial. Immune-compromised NOG (Prkdcscid Il2rgtm1Sug ) mice were reconstituted with human CD34+ cells and the animals monitored for cell engraftment and expansion. Humanized and non-humanized NOG animals were subcutaneously implanted with tumor fragments and growth rates were compared between the two groups. Results: As early as 6 weeks after humanization was initiated, mature human CD45+cells could be detected in the circulation of humanized animals. We found at least 70% of reconstituted animals had >15% hCD45+ cells in the peripheral blood 10 weeks post-hCD34+ reconstitution. Of the hCD45+ cells present in the peripheral circulation of humanized animals, 19% were hCD3+ (T cells), of which 67% were hCD4+ helper T cells, and 25% were hCD8+. Although still early in the growth phase, tumor volumes in humanized animals are comparable to those in nonhumanized animals. Assessing ImmunoGraft responses to checkpoint inhibitors is underway. Conclusion: Our study has demonstrated the potential of combining humanized mice with Champions TumorGrafts to generate a novel and unique preclinical model. The ImmunoGraft will allow direct assessment of immuno-modulatory agents on tumor growth and progression in a platform more reflective of the complete human microenvironment. It will also facilitate research examining the critical interplay between tumors and the immune system, potentially leading to identification of the most active drugs, and perhaps additional drug targets.

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196 POSTER (Board P190) Neuropilin 2 (NRP2) modifies CXCL12/CXCR4 signaling and promotes lymph node metastases in colon cancer 1 H. Schneider1 , P. Honscheid ¨ , S. Scholch ¨ 2 , C. Jakob1 , M. Muders1 . 1 ¨ Universitatsklinikum Carl Gustav Carus, Institute of Pathology, Dresden, ¨ Germany; 2 Universitatsklinikum Carl Gustav Carus, Department of Surgery, Dresden, Germany

Aims: Dissemination of cancer cells to regional lymph nodes is one of the most important prognostic factors for colorectal cancer. To be able to form lymph node metastases tumor cells need a supportive microenvironment. One important part of this niche are lymph vessels that are located inside the lymph node. This lymphovascular niche has been shown to be an independent prognostic factor in rectal cancer patients after neoadjuvant radiochemotherapy (Jakob et al., Plos One 2011). These intranodal lymph vessels produce chemoattractant factors like CXCL12, that can bind to CXCR4-positive tumor cells and mediate cell migration. We investigated if the receptor Neuropilin-2 (NRP2) mediates lymph node metastasis by modulating the CXCR4/CXCL12 signaling pathway. Methods: In a cohort of 113 node negative colon cancer patients the lymph vessel density in regional lymph nodes was quantified by standard immunohistochemistry staining against D2−40, which labels lymphatic endothelium. In vitro experiments were performed with standard cell lines. Protein and mRNA levels were assessed by Western Blot and quantitative real time PCR. After stimulation with human recombinant CXCL12, RNAi mediated depletion of NRP2 and CXCR4 as well as after addition of recombinant Semaphorin 3F (Sema3F) Boyden Chamber Migration Assays were performed. To confirm our in vitro results a cohort of 78 patients with locally advanced node positive colon cancer was analyzed for NRP2expression in primary tumors and lymph node metastases by standard immunohistochemistry. Parallely, a nude mouse model with orthotopically implanted colorectal cancer cells was assessed. Results: In node negative colon cancer patients, intranodal lymphangiogenesis scorrelated with disease-free survival (P < 0.05). siRNA mediated depletion of NRP2 decreased CXCR4-mRNA and protein expression, while CXCR4 had no influence on NRP2 expression. Blocking NRP2 abrogated CXCL12-induced AKT phosphorylation. Increase of CXCR4 mRNA after stimulation with CXCL12 (‘feed forward loop’) was inhibited after NRP2 depletion. Tumor cell migration towards a CXCL12-gradient was reduced when NRP2 function was blocked by siRNA or Sema3F (P < 0.001). Our in vitro results were confirmed by an up-regulation of NRP2 in lymph node metastases of advanced colon cancer patients’ tissue (P < 0.001) as well as an increased expression of NRP2 in metastasis after orthotopic implantation of colorectal cancer cells in nude mice. Conclusion: NRP2 modulates the CXCL4/CXCL12 signaling axis in colorectal cancer cells and influences cell migration. Therefore, NRP2 might have an impact on the metastatic potential of colorectal cancer cells. Additionally, the detection of intranodal lymh vessels in node negative colon cancer patients by standard immunohistochemistry against D2−40 might offer a new tool for oncologists to decide whether an adjuvant treatment is recommended. 197 POSTER (Board P191) Statistical aspects of kinetic analysis of gliomas with FDG-PET D. Hawe1 , F.R. Hernandez1 , S. Murphy1 , E. Wolsztynski1 , J. Huang1 , J. O’Sullivan1 , M. Muzi2 , J. Eary3 , K. Krohn2 , F. O’Sullivan1 . 1 University College Cork, Statistics, Cork, Ireland; 2 University of Washington, Radiology, Seattle, USA; 3 University of Alabama, Radiology, Birmingham, USA Background: Positron emission tomography (PET) is used to examine the distribution of radio-labelled molecules in vivo, which allows the status of healthy and pathological tissue to be examined by studying how it processes substances of interest. Material and Methods: The interpretation of dynamic PET time-course data is complicated because the measured signal is a combination of vascular delivery and tissue retention effects. The tissue time-course can be expressed as a convolution between the arterial time-course (directly measurable) and the tissue residue. In light of this, estimating the residue is essentially a survival analysis problem, which has been examined in great detail in the literature. Kinetic analysis of PET data is concerned with estimation of the residue function and parameters such as flow, flux and volume of distribution. This is generally done using compartmental models, however as these models assume that the area being studied is homogenous, and that there is instant mixing within compartments, its validity has been questioned. Accordingly, we examine both a nonparametric model, and a mixture model approach, which do not require the same assumptions of the compartmental model and compare

Poster Session – Preclinical Models these alternatives with the classic compartment model approach to the estimation of the residue function. Results: We evaluate the improvement in the mixture and nonparametric models relative to compartment models using simulations and crossvalidation. Estimates of key functionals including flow, flux and volume of distribution for the three models are compared. This is illustrated with data from ongoing 18 F-fluorodeoxyglucose-PET studies in a series of 46 brain tumour subjects. Conclusions: Significant statistical evidence against the compartment model in favour of both the nonparametric and mixture models has been found. Supported by Science Foundation Ireland under SFI-PI 11/27 and by the National Institute of Health (NCI) under PO1-CA-42045. 198 POSTER (Board P192) Genetic and pharmacological inhibition of PIM-1 reduces tumor development in a K-Ras-driven mouse model of non-small cell lung cancer E. Aguirre1 , O. Renner1 , M.C. Rodriguez de Miguel1 , M.I. Albarran1 , A. Cebria1 , D. Cebrian1 , F. Ramos-Lima1 , J. Pastor1 , C. Blanco-Aparicio1 . 1 Spanish National Cancer Research Centre (Cnio), Experimental Therapeutics Program, Madrid, Spain Lung cancer is the leading cause of cancer deaths worldwide, and 40% of these cases are diagnosed as adenocarcinomas, a subtype of nonsmall cell lung cancer (NSCLC). Activating mutations in the K-Ras protooncogene have been found in 30% of human NSCLC, and these tumors are still an unmet medical need as in most of the cases the current treatments failed. K-Ras G12V is one of the commonest mutations observed in NSCLC. Several mouse models have been created to understand the role of K-Ras in tumor initiation and progression. In this study, we have evaluated the effect of inhibiting PIM proteins in tumor progression in a mouse model in which K-Ras (G12V) mutation is conditionally activated resembling human NSCLC. The PIM proteins are a family of highly homologous serine/threonine kinases that are implicated in the regulation of apoptosis, metabolism, the cell cycle, and homing and migration, which makes these proteins interesting targets for anti-cancer drug discovery. To evaluate Pim1 inhibition as a therapeutic target for NSCLC, a conditional knock-in mouse model had been used in which Pim1 kinase activity was ablated, controlling its expression using a Cre/loxP recombination system. This kinase-dead (KD) mutant was crossed with K-Ras (G12V) mice, and 7 months after intra-tracheal administration of Cre-recombinase adenovirus, tumor development was assessed by computer tomography. The images showed that K-Ras (G12V) Pim1-KD mice developed later and less lung tumors than K-Ras (G12V) mice, significantly increasing their survival rate. Besides, histological examination of these tumors revealed that only 14% of K-Ras (G12V) Pim1-KD mice developed adenocarcinomas, whereas 73% of K-Ras (G12V) mice presented this malignant type of lung cancer. Having genetically validated PIM1 kinase as a molecular target for NSCLC treatment, we reinforced these results by pharmacological inhibition of the PIM proteins activity. For this purpose, a selective and potent proprietary panPIM inhibitor was generated in our Programme. In the PK/PD assay, this compound revealed that 24 hours after oral single dose administration (100 mg/kg), a 60% specific inhibition of phospho-4E-BP1 (Thr37/46) was detected in K-Ras (G12V) lung tumors samples. After that, the compound efficacy was evaluated by orally administrating a daily dose of 50 mg/kg for three weeks to K-Ras (G12V) mice. Pre- and post-treatment computer tomography images revealed that tumor growth inhibition rate was 61% in treated mice when compared to control mice. Besides, when the administered dose was of 80 mg/kg, tumor growth inhibition reached the value of 70%. In summary, in this study we have confirmed that genetic or pharmacological inhibition of Pim-1 kinase is an effective strategy to be considered in the treatment of NSCLC using a transgenic mouse model. In the future, further studies will be needed to establish the role of Pim inhibitors in the treatment of human NSCLC.

Poster Session – Radiation Interactive Agents 199 POSTER (Board P193) Inhibitor of differentiation 1 (Id1) expression in lung cancer cells and liver microenvironment is required for liver metastasis (LM) development from non-small cell lung cancer (NSCLC) by regulating EMT-related and proliferation-related proteins 3 E. Castanon1 , A. Soltermann2 , I. Lopez ´ , M. Ecay4 , M. Collantes4 , J.M. Lopez Picazo1 , M. Ponz5 , C. Rolfo6 , A. Calvo7 , I. Gil-Bazo1 . 1 Clinica Universidad de Navarra, Department of Oncology, Pamplona, Spain; 2 University Hospital Zurich, Institute of Surgical Pathology, Zurich, ´ Medica ´ Switzerland; 3 Centro de Investigacion Aplicada, Laboratorio ´ de Nuevas Dianas Terapeuticas, Pamplona, Spain; 4 Clinica Universidad de Navarra, Nuclear Medicine, Pamplona, Spain; 5 Cold Spring Harbour, Cold Spring Harbour Laboratory, New York, USA; 6 University of Amberes, ´ Phase I Clinical Trials Unit, Antwerp, Belgium; 7 Centro de Investigacion ´ Medica Aplicada, Department of Oncology, Pamplona, Spain

Introduction: Id1 is involved in carcinogenesis. We previously showed Id1 as an independent prognostic factor in NSCLC. Id1 and Id3 are required for breast cancer metastasizing to the lungs. We studied the potential role of Id1 in LM in an in vivo model of NSCLC and its correlation with EMT-related proteins. Methods: We selected 40 Id1 wild-type mice (Id1+/+) and 40 Id1 knockout mice (Id1−/−). We intrasplenically injected 5×105 Id1-silenced (Id1s), Id1/Id3-silenced (Id1/Id3s) or wild-type (Id1Id3wt) Lewis Lung Carcinoma (LLC) cells. Mice were followed by weekly microPET for 4 weeks or until death. Microscopic LMs were studied in all necropsies. A gene expression study (Affymetrix) was performed to assess differences in LM from Id1+/+ and Id1−/− mice. Three tissue microarrays (TMA) containing 538 NSCLC human samples were stained with against Sox10, Betacatenin, E-cadherin, Vimentin, Slug, Podoplanin, S6, ERCC1, Id1 and Id3. An H-score was calculated. Multivariable analysis investigated potential predictive factors for LM colonization in mice. Spearman’s Rho test for measuring the possible correlation between Id1, Id3 and the proteins studied in the TMA was used. Results: Among Id1+/+ mice the LM rate was: 57.1% when Id1/Id3wt cells were injected, 33.3% for animals injected with Id1s cells and 0% in those in which Id1/Id3s cells were used (p < 0.001). Nonetheless, no significant differences in LM were found regarding the cell type injected among Id1−/− mice (p > 0.05). In the logistic regression model, Id1 silencing in tumor cells reduced the risk of LM in an 84.9% independently of the mice strain used (p = 0.03). We also found an 84.5% reduction in the probability of developing LM among Id1−/− mice compared to Id1+/+ animals regardless the expression of Id1 and Id3 in the tumor cells injected (p = 0.006). In the LM gene expression profile, the loss of Id1 expression in mice produced a significant downregulation of different genes involved in proliferation such as FGFGR1 (p < 0.001), Myc (p < 0.001), Akt (p = 0.03) and Hoxd10 (p = 0.02). EMT-related genes such as Versican (p = 0.003), Vimentin (p = 0.007), Snail (p = 0.04), Podoplanin (p = 0.02) and IntegrinBeta 1 (p = 0.04) were also downregulated by Id1 loss in the tumor microenvironment compared to Id1+/+ mice (p < 0.001). Coincidentally in the NSCLC samples studied, Vimentin, Podoplanin and Akt expression was positively correlated with Id1 levels. In human NSCLC samples a positive and significant correlation between Beta-catenin, E-cadherin, Slug, ERCC1 and S6 with Id1 expression was found too. Conclusions: Id1 expression in lung cancer cells and liver microenvironment may be required for LM. Id1 expression would favor the migration and colonization capacity of lung cancer cells by regulating EMT-related and proliferation-related proteins in mice and humans. If proven in further mechanistic experiments, Id1 and Id3 targeting might help prevent and treat liver metastasis from lung cancer. 200 POSTER (Board P194) The influence of different cMET and EGFR backgrounds on the cytotoxicity of cMET and EGFR small molecule inhibitors in vitro N. Van Der Steen1 , K. Zwaenepoel2 , C. Rolfo3 , E. Giovannetti4 , M. Castiglia2 , V. Deschoolmeester1 , A.P. Carreca2 , P. Germonpre5 , P. Pauwels2 . 1 University Antwerp, Center for Oncological Research, Antwerp, Belgium; 2 Antwerp University Hospital, Molecular Pathology Unit Department of Pathology, Antwerp, Belgium; 3 Antwerp University Hospital, Phase I − Early Clinical Trials Unit Oncology Department, Antwerp, Belgium; 4 VU Amsterdam, Pharmacology Lab Cancer Center, Amsterdam, Netherlands; 5 AZ Maria Middelares, Department of Respiratory Medicine, Ghent, Belgium Background: Despite recent advances lung cancer shows poor prognosis and high incidence of recurrence. Today, targeted therapies against the epidermal growth factor receptor (EGFR) are frequently used in the clinic. Besides EGFR, the cMET receptor and its ligand hepatocyte growth factor

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(HGF) play a role in cancer growth, as cMET amplification is a known resistance mechanism against these EGFR-targeted therapies. In vitro, the combination of anti c-Met synergistically enhanced gefitinib-induced growth inhibition in all NSCLC cMET-expressing cell lines. The current study determines the influence of cMET amplification, expression and autocrine stimulation, and EGFR mutational status and expression on the cytotoxicity of small molecule inhibitors. Materials and Methods: Therefore 4 non small cell lung cancer (NSCLC) and 1 myeloma cell line were selected: HCC827 (EGFR exon 19 deletion), H1975 (EGFR L858R and T790M substitutions), EBC-1 (cMET amplification), LUDLU-1 (EGFR and cMET wild-type) and the JJN3 myeloma cell line (HGF secreting). Furthermore, 4 tyrosine kinase inhibitors (TKI) were selected: erlotinib (EGFR-TKI), PHA665752 (cMETTKI), crizotinib (dual ALK and cMET-TKI) and 17-AAG [Heat shock protein 90 (Hsp90) inhibitor]. To determine the cytotoxic effect of these inhibitors, a sulforhodamine B assay was performed. Each cell line was treated using monotherapy for 72 h under normoxic conditions. Results: The cMET amplified EBC-1 cell line seemed to be very sensitive to the Hsp90 inhibitor (IC50 = 90 nM), possibly because Hsp90 folds the cMET-protein. The JJN3 cell line showed moderate sensitivity to cMETTKIs (IC50 = 3000–4000 nM) and very low sensitivity to Hsp90 inhibition (IC50 = 941 nM) compared to the other cell lines. The EGFR mutant cell lines (sensitizing and resistance mutations) are relatively sensitive to the Hsp90 inhibitor (IC50 = 200–250 nM) in comparison with the wild-type LUDLU cell line (IC50 = 780 nM), which showed low sensitivity to the whole panel of inhibitors. Conclusions: These observations can possibly open the door for combination therapy that targets both EGFR and cMET. Further research will be needed to investigate the sensibility of this cMET amplified cell line to Hsp90 inhibition. 201 POSTER (Board P195) Transcriptome analysis of CD133-positive stem cells and prognostic value of survivin in colorectal cancer S. Kim1 , S.H. Park2 , J. Lee2 , Y.S. Park2 , H.C. Kim3 . 1 Korea University Hospital, Seoul, Korea; 2 Samsung Medical Center, Medical Oncology, Seoul, Korea; 3 Samsung Medical Center, Surgery, Seoul, Korea CD133 is an important, but not exclusive, biomarker of colorectal cancer (CRC) stem cells. In order to identify other CRC stem cell-specific genes, we performed a comparative expression profiling of CD133+ and CD133− cell populations in primary and metastatic tumors from four patients with CRC. CD133+ and CD133− CRC cells were isolated using MagSweeper and were used for whole transcriptome analysis with RNA-Seq. We found thatin CD133+ cells, 17 genes (RNASE2, PRB2, IL4, MGC27382, CLEC4C, SALL3, GIMAP1, ISG15, LOC728875, ZIK1, ICAM2, CCDC7, CDYL2, LRRC2, ZEB1, OSTF1, and CCDC144B) were significantly upregulated compared to CD133− CRC cells. Among them, IL4 has been known as an inducer of survivin implicated in the survival and proliferation of cancer cells. However, the prognostic value of survivin in CRC is controversial. We evaluated survivin expression in formalin-fixed paraffin-embedded tumor samples of 188 patients with CRC by immunohistochemistry. Survivin overexpression was detected in 85 patients (45.2%) and was significantly associated with primary tumor sites (P = 0.028), lymph node metastasis (P = 0.029), and advanced III/IV CRC stages (AJCC 7; P = 0.001). Furthermore, survivin upregulation correlated with reduced disease-free survival (DFS; P = 0.021) and overall survival (OS; P < 0.000) and was proved to be an independent prognostic factor for both DFS and OS in multivariate analysis. Collectively, our data suggest that CD133+ CRC stem cells have a distinct expression pattern and that survivin, upregulated by differentially expressed IL-4, is a candidate biomarker for the prediction of recurrence and survival in CRC.

Radiation Interactive Agents 202 POSTER (Board P196) The novel microtubule-destabilizing drug BAL101553 (prodrug of BAL27862) sensitizes a treatment refractory tumor model to ionizing radiation A. Broggini-Tenzer1 , F. Bachmann2 , V. Vuong1 , A. Messikommer1 , K. Nytko-Karouzakis1 , T. O’Reilly2 , H.A. Lane2 , M.N. Pruschy1 . 1 University Hospital Zurich (USZ), Radiation Oncology, Zurich, ¨ Switzerland; 2 Basilea Pharmaceutica International Ltd, Basel, Switzerland Background: Microtubule-targeting agents (MTAs) are widely used for cancer treatment, both alone and in combination. BAL27862 is a novel

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microtubule-destabilizing drug with a dual action against human tumors; targeting tumor cells refractory to standard MTAs as well as tumor blood supply. The water soluble prodrug BAL101553 has completed Phase I clinical evaluation administered i.v. but is also orally bioavailable. We have investigated the effect of ionizing radiation (IR) in combination with BAL27862 in vitro in human cancer lines resistant to standard MTAs, and with BAL101553 in a genetically defined paclitaxel-, epothilone- and IRrefractory xenograft model. Different routes of BAL101553 administration were compared. Methods: Alamar blue/crystal violet proliferation, clonogenic survival and apoptotic death assays were performed in human lung carcinoma A459, b-tubulin-mutated A549EpoB40 and Pgp-overexpressing colon carcinoma SW480 cells. Antitumor response was determined in SW480- and A549EpoB40-derived tumor xenografts with BAL101553 administered i.v. (21.3 mg/kg/wk) or p.o. (15 mg/kg QD×5 or 38 mg/kg/wk) and IR fractionated over a week (3×5 Gy). Results: BAL27862 reduced the proliferation/survival of wild type A549 cells, paclitaxel/epothilone-resistant A549EpoB40 cells and paclitaxelresistant SW480 cells with similar potency (anti-proliferative IC50s: 28nM, 22nM & 9nM, resp). BAL27862 combined with IR resulted in additive cytotoxicity with no apoptosis induction. In SW480 xenografts, BAL101553 exhibited antitumor activity when administered i.v. or p.o., with daily and weekly oral administration eliciting equivalent antitumor responses (final D%T/C: 36 & 41, resp). Equipotent single i.v. bolus and daily p.o. BAL101553 regimens were identified which partially inhibited tumor growth (%T/C: 66 & 52, resp) in microtubule stabilizing agent-resistant A549EpoB40 tumor xenografts, as did IR (%T/C: 35). Interestingly, both BAL101553 regimens induced a profound antitumor effect when combined with IR, associated with almost complete tumor stabilization (%T/C: 4 & 7, resp) over 5 weeks. Based on the in vitro data, involvement of tumor microenvironment in antitumor response to this combination is likely. Conclusions: BAL101553 single bolus (i.v.) or daily (p.o.) treatment regimens positively interact with IR in a human cancer model refractory to clinically relevant MTAs and IR, demonstrating the potential of this combination therapy for the treatment of cancer patients. 203 POSTER (Board P197) Ionizing radiation induced phosphatidylserine externalization on endothelial cell surface − a potential target for vascular targeting Z. Zhao1 , M. Johnson2 , B. Chen1 , M. Grace3 , J. Ukath3 , V. Lee1 , M. Stoodley1 . 1 Macquarie University, Australian School of Advanced Medicine, Sydney NSW, Australia; 2 University of Technology Sydney, Faculty of Science, Sydney NSW, Australia; 3 Genesis Cancer Care, Macquarie University Hospital, Sydney NSW, Australia Background: Vascular targeting agents can deliver anti-cancer drugs specifically to tumor sites by binding to unique markers/targets on endothelial surface of tumors. The discriminating power of the markers/targets relative to normal tissues determines the specificity of the technique. Stereotactic RadioSurgery (SRS) can precisely deliver focused ionizing radiation to a target tumor site. Radiation-induced molecular changes should be restricted within the tumor tissue, and be good targets for vascular targeting. This study investigated radiation-induced externalization of phosphatidylserine (PS) in endothelial cells, a potential target for vascular targeting. Materials and Methods: An immortalized cell line generated from mouse brain endothelium, bEnd3 cells, were cultured and irradiated at different radiation doses using a linear accelerator (LINAC) Elekta Synergy. Then PS externalization in the cells was visualized using pSIVA-IANBD, a polarity sensitive probe for PS. Live cell imaging was used to monitor the PS externalization in real time. Results: Ionizing radiation has remarkable effects on the cells and the effects are found to be dose dependent. The cell proliferation rate decreased after exposure to 5 Gy radiation whereas higher radiation doses (15 Gy and 25 Gy) totally inhibited proliferation. In comparison with shamradiation treated cells, the irradiated cells showed distinct pseudopodial elongation with little or no spreading of the cell body. The percentages of pSIVA positive cells were significantly higher in the cells that received 25 Gy and 15 Gy radiation 24 hours after treatment. This effect sustained until the end of the experiment (3 days). Radiation at 5 Gy did not induce significant PS externalization compared with the sham-radiation control at any time points. Conclusions: Ionizing radiation can cause remarkable cellular changes in the endothelial cells. Significant PS externalization can be induced by the radiation at dose levels 15 Gy and above. Given the precise focusing of the radiation beams, the radiation-induced markers/targets may have high discriminating power to be harnessed in vascular targeting for cancer treatment.

Poster Session – Radiation Interactive Agents 204 POSTER (Board P198) High-throughput functional screening identifies the flavoreductase POR as a principal determinant of sensitivity to the hypoxia-targeting prodrug SN30000 F.W. Hunter1 , Z. Shalev2 , J. Wang1 , J. Moffat3 , T. Katella4 , M. Koritzinsky3 , W.R. Wilson1 , B.G. Wouters3 . 1 University of Auckland, Auckland Cancer Society Research Centre, Auckland, New Zealand; 2 University Health Network, Princess Margaret Cancer Centre, Toronto, Canada; 3 University of Toronto, Department of Radiation Oncology, Toronto, Canada; 4 University of Toronto, Donnelly Centre and Banting and Best Department of Medical Research, Toronto, Canada Background: Hypoxia constitutes an attractive therapeutic target owing to its prevalence in tumours and its contribution to disease progression and treatment resistance. Several hypoxia-selective prodrugs have been developed to address this target, including the preclinical agent SN30000 which is an optimised analogue of the well-studied prodrug tirapazamine. These prodrugs are enzymatically activated under hypoxic conditions and thus selectively toxic to viable hypoxic cells. Biomarkers that are predictive of prodrug activation and tumour sensitivity are urgently needed to support their clinical development.

Figure: SN30000. Materials and Methods: We deployed whole-genome lentiviral shRNA screens interrogating 82,017 unique hairpins targeted to 16,019 human genes, and a high-representation focused library enriched for oxidoreductases (1,821 hairpins targeting 359 genes), in tandem with massively parallel sequencing, to identify determinants of sensitivity to SN30000 in HCT116, HT-29 and PANC-1 cells under hypoxic conditions. Potential determinants were evaluated by RNAi-mediated knockdown and zinc finger nuclease-mediated genetic knockout in cell lines and xenografts. Results: Our functional screens identified the flavoprotein P450 (cytochrome) oxidoreductase (POR) as the predominant determinant of sensitivity to SN30000 in all cell lines evaluated, except a POR knockout clone derived from HCT116, with no other genes consistently selected. Specific hairpins against POR were validated by qPCR and immunoblotting, where POR suppression directly correlated with the degree of enrichment in the high-throughput screens. Knockdown or knockout of POR inhibited metabolic activation of SN30000 and enhanced clonogenic survival of cells exposed under hypoxic conditions. We are currently assessing effects of POR knockout on the activity of SN30000 against hypoxic cells in xenografts. Gene

shRNA clone

Z-score HT-29

PANC-1

HCT116

POR POR POR

TRCN0000046524 TRCN0000046526 TRCN0000046527

3.3 3.2 2.6

3.5 3.1 2.2

5.0 3.3 2.3

Conclusions: POR acts as a critical determinant of tumour cell sensitivity to SN30000 by catalysing reductive activation of the prodrug. As a single gene apparently responsible for a major component of SN30000 activity, tumour expression of POR may be a clinically tractable predictive marker of response to SN30000. The methodology established in this study may be repurposed to study determinants of sensitivity to additional hypoxiaactivated prodrugs such as the clinical agents TH-302 and PR-104.

Poster Session – Radiation Interactive Agents 205 POSTER (Board P199) Metformin to modulate AMP-kinase and enhance chemotherapy and radiotherapy in non-small cell lung cancer M.J. Troncone1 , S.M. Cargnelli1 , G. Pond2 , E. Tsiani3 , J. Wright2 , G. Steinberg4 , H. Skinner5 , L. Bo6 , J. Bradley7 . 1 McMaster University, Medicine, Hamilton Ontario, Canada; 2 McMaster University, Oncology, Hamilton Ontario, Canada; 3 Brock University, Community Health Science, Hamilton Ontario, Canada; 4 McMaster University, Biochemistry, Hamilton Ontario, Canada; 5 M.D. Anderson, Radiation Oncology, Houston Texas, USA; 6 Jefferson University, Radiation Oncology, Philadelphia, USA; 7 Washington University, Radiation Oncology, St. Louis, USA Background: In the past few years our group investigated the response of AMP-activated kinase (AMPK) to clinical doses of radiation therapy (RT). We found that AMPK is not only a metabolic but also a genomic stress sensor that is activated in epithelial tumour cells downstream of Ataxia Telengiectasia Mutated (ATM) and leads to induction of p53 and cyclin dependent kinase inhibitors such as p21cip1 and p27kip1 . We observed that AMPK is involved in mediation of the G2-M cycle arrest and the cytotoxicity of RT. Knock down of AMPK in cancer cells and knockout models of AMPK show dramatic activation of the Akt-mTOR pathway. In tumours RT induced growth inhibition and chronic suppression of angiogenesis that was associated with sustained activation of ATM and AMPK and inhibition of Akt and mTOR axis. We used the anti-diabetic drug metformin (MET) to activate AMPK in lung tumours. MET is a safe and well-tolerated agent that has no reported toxicity when combined with RT or chemotherapy. Materials and Methods: We performed pre-clinical evaluation of MET in combination with RT in in-vitro and in-vivo models of lung prostate cancer. Further, we performed a retrospective analysis of clinical outcomes in stage III NSCLC treated with chemotherapy and radiotherapy at the Juravinski Cancer Center over the past 14 years. Results: MET at low microM concentrations, that are safely achieved in the serum of diabetic patients, activated the AMPK pathway, inhibited growth and enhanced RT responses in cancer cells and tumours. The antiproliferative and radio-sensitizing activity of MET was depended on AMPK and led to effective inhibition of the Akt-mTOR pathway. MET increased the pro-apoptotic and anti-angiogenic action of RT. Retrospective analyses of clinical outcomes show that diabetic patients with locally advanced (LA) non-small cell lung cancer (NSCLC) treated with RT and chemotherapy have improved survival after chemotherapy or RT treatment if they receive metformin for the treatment of diabetes. Conclusions: Based on our pre-clinical and retrospective clinical data we propose prospective clinical trials in LA-NSCLC. Two phase II studies of MET in combination of chemo-RT will open to accrual soon. The Canadian Ontario Clinical Oncology Group (OCOG) ALMERA and US NRG LU001 studies will investigate MET in combination with concurrent chemo-RT in LA-NSCLC. LU001 will examine the pure chemo-radio-sensitizing action of MET using the drug only concurrently with chemo-RT while ALMERA will examine the benefits of MET when used both concurrently with chemoRT and adjuvant for 1 year. Tumour bio-specimens collected in NRGLU001 and OCOG-ALMERA will help show whether expression or mutation status of tumour (LKB1, K-Ras, p53, EGFR and Alk) and microenvironment (HIF1a and micro-vessel density) markers could serve as biomarkers of MET response in NSCLC. 206 POSTER (Board P200) Identification of novel targets for radiosensitisation of non-small cell lung cancer by secretome analysis A. Sharma1 , S. Bender1 , O. Riesterer1 , A. Broggini-Tenzer1 , M. Pruschy1 . 1 University Hospital Zurich, Department of Radiation Oncology, Zurich, Switzerland Background: The therapeutic response of ionizing radiation (IR) is imparted by genomic instability and DNA damage. However, IR also triggers multiple intracellular signaling processes as part of IR-induced stress responses that lead to the secretion of various para- and autocrine factors into the tumor microenvironment. Here we investigated treatmentdependent secretion of auto- or paracrine factors, which drive acquired rescue mechanisms and determine the overall radiation sensitivity of the tumor. Material and Methods: Exhaustive large scale secretome analysis was performed using antibody arrays for a wide range of secretory factors. Secretion kinetics of selected factors were determined using ELISA across different established tumor cells and in murine blood serum, derived from irradiated tumor xenograft-carrying mice. Clonogenic survival and xenograft tumor growth delay assays were performed in response to IR in siRNAtargeted tumor cell lines or in combination with small molecular agents. Results: We performed an exhaustive IR-dependent secretome analysis (>300 factors) in lung carcinoma cells and investigated IR-induced

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expression and tumor cell secretion of the top hits, including amphiregulin, transforming growth factor-a and ALCAM. All these factors were secreted in a similar IR-induced time- and dose-dependent way from several nonsmall cell lung cancer (NSCLC) cell lines, indicative of a common upstream mechanism. No changes were observed at the transcriptional level implying potential modulation at the posttranslational level. Interestingly, irradiation induced a dose-dependent increase in cleavage of the proform of ADAM17 (A Disintegrin and metalloprotease domain 17), which resulted in enhanced ADAM17 activity and correlated with subsequent substrate shedding. IRinduced ADAM17 activation required both p38 mitogen-activated protein kinases (MAPKs) and Furins. siRNA mediated silencing of ADAM17 or targeting of ADAM17 with the small molecular inhibitor TMI-005 suppressed IR-induced shedding of these factors, down regulated ErbB-signaling in target cells and enhanced IR-induced cytotoxicity in vitro and in vivo. Ex vivo substrate analysis of murine blood serum derived from irradiated tumor xenograft-carrying mice correlate with our in vitro results. Conclusions: Our findings demonstrate that IR significantly activates ADAM17, which results in shedding of multiple survival factors, growth factor pathway activation and contributes to treatment resistance in NSCLC cells. We provide a sound rationale for positioning ADAM17 inhibitors as radiosensitizers to improve the treatment of NSCLC. 207 POSTER (Board P201) The enhancement of radiotherapy efficacy with docetaxel-titanate nanotubes as a new nanohybrid for localized high risk prostate cancer C. Mirjolet1 , J. Boudon2 , A. Loiseau2 , S. Chevrier1 , T. Gautier2 , R. Boidot3 , J. Paris2 , N. Millot2 , G. Crehange1 . 1 Georges-Francois Leclerc Cancer Centre, Radiotherapy, Dijon, France; 2 UMR 6303 CNRS U Bourgogne, NanoScience Department MaNaPI, Dijon, France; 3 Georges-Francois Leclerc Cancer Centre, Molecular Biology, Dijon, France Background: From 30% to 50% of high risk prostate cancer patients who undergo radiation therapy (RT) will have a biochemical failure. These failures are either due to a poor local control or to distant disease which may also be related to a local failure. Taxane-based chemotherapy has proved to be useful in prostate cancer. Combining chemotherapy, such as docetaxel (DXL), with RT can enhance its efficiency, however systemic injection of the classical formulation leads to 95% uptake by healthy tissues whereas 2−5% only reach tumors; adverse side effects are a crucial problem. Moreover, multidrug resistance mechanisms often limit drug efficacy by decreasing tumor cell intracellular concentration of drugs. There is interest to develop nanocarrier of DXL to maintain drug inside cancer cells by improving its efficacy. In a previous in vitro study, we have highlighted that titanate nanotubes (TiONTs) which have a needle shape can enter and stay inside cancer cells until 10 days without cytotoxic effects induction. We suggest in this study to develop TiONt-DXL nanocarrier and to evaluate its in vivo biodistribution as well as its efficacy in association with RT on a prostate cancer model. Materials and Methods: TiONts are obtained from hydrothermal synthesis. DXL molecules were grafted on TiONts using PEG-3000 molecules to generate the nanohybrid. In vitro cytotoxic activity of nanohybrid was evaluated on PC-3 cell line using MTS assay. BALB/c nude mice bearing subcutaneous PC-3 human prostate tumors were used to evaluate nanohybrid biodistribution and efficiency after intratumoral injection. Biodistribution analysis was performed by SPEC-CT imaging using 111Indium. To evaluate the benefit of TiONt-DXL and RT association, tumors were irradiated using three daily fractions of 4 Gy administrated the day after nanohybrids injection. Mice behavior, health status and tumor volume were monitored twice a week until tumor growth recovery. Result: TiONt-DXL in vitro cytotoxic activity was showed on PC-3 cells. Biodistribution kinetics showed that more than 70% of nanohybrids were localized into the tumor 96 hours after injection. Tumor growth was stopped for mice receiving RT combined or not to TiONt or TiONt-DXL. Moreover, mice receiving TiONT-DXL alone exhibited a decrease in tumor growth when compared to mice receiving free DXL. Conclusions: These results highlight that our nanocarrier improve DXL efficacy. Local control might be improved by injecting TiONT-DXL in the tumor during prostate brachytherapy as a boost in high risk localized prostate cancer. A longer follow-up period will be needed to determine if TiONt-DXL combined to RT will improve outcome. This work was supported by the ‘Ligue Contre le Cancer du Grand Est’ (Comites ´ Doubs and Cote ˆ d’Or).

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208 POSTER (Board P202) Combined treatment of a DNA-PKcs inhibitor (NU7441) and ionizing radiation causes a differential mode of cell death in a panel of NSCLC cell lines and exhibits robust radiosensitisation D. Saha1 , Y. Lan1 , F. Hsu2 , V. Tumati1 , Z. Zhang1 , Y. Lin1 , B. Chen1 . 1 UT Southwestern Medical Center, Radiation Oncology, Dallas TX, USA; 2 National Taiwan University College of Medicine, Urology, Taipei, Taiwan Background: Lung cancer is still the leading cause of cancer death worldwide. The current standard of care for lung cancer consists of concurrent chemotherapy and radiation. Several studies have shown direct evidence that a novel DNA-PKcs inhibitor NU7441 is highly potent radiosensitizer in different tumor models. However, the mechanism of NU7441 induced radiosensitization has not been fully elucidated. In this study, the combined effect of NU7441 and ionizing radiation (IR) in a panel of non-small cell lung cancer cell lines (A549, H460 and H1299) has been thoroughly investigated. The purpose of the study is to enhance the effect of the radiation using a DNA-PKcs inhibitor in radio-resistant NSCLC lines representing different genetic background. Material and Methods: Clonogenic surviving fraction analysis, DNA double strand break repair kinetics, cell cycle analysis, immunofluorescence, immunoblot, apoptosis and autophagy assays were used in this study. Results: We found that NU7441 significantly enhances the effect of IR in all cell lines tested. The notable findings in response to this combined treatment are (i) prolonged delay in IR induced DNADSB repair, (ii) induced robust G2/M checkpoint in these cell lines, (iii) increase of aberrant mitosis followed by mitotic catastrophe specifically in H1299 cells, (iv) dramatically induced autophagy in A549 cells and (v) IR-induced senescence specifically in H460 cells. We observed that H1299 cells show greater G2 checkpoint adaptation after combined treatment which can be attributed to the higher expression level of Plk1 compared to A549 and H460. The enhanced autophagy after NU7441 treatment in A549 cells is possibly due to the higher endogenous expression of pS6K compared to H1299 and H460 cells. Conclusion: Differential mode of death was noticed and that leads to the extensive radio-sensitization in all NSCLC lines tested. The choice of cell death pathway, in response to the combined treatment with NU7441 and IR, can be attributed to mutation status of the cells treated.

Poster Session – Radiation Interactive Agents

th

26 EORTC–NCI–AACR Symposium on Molecular Targets and Cancer Therapeutics

Thursday 20 November 2014

Plenary Session 5

Thursday 20 November 2014

Thursday 20 November 2014

08:00–10:00

PLENARY SESSION 4

Antibody-Based Therapies (ADC and others) 209 ORAL PRESENTATION Pre-clinical and translational pharmacology, pharmacokinetics and pharmacodynamics for a humanized anti-OX40 antibody MOXR0916, a T-cell agonist in the treatment of solid tumors S. Sukumaran1 , J.M. Kim1 , M. Huseni1 , J. Ruppel1 , H. Taylor1 , K. Totpal1 , J. Zhu1 , C. Zhang1 , H. Chiu1 , E.G. Stefanich1 . 1 Genentech, gRED, So San Francisco CA, USA Background: Targeting T cell surface proteins to restore and enhance the function of tumor-reactive T cells have been found to be an effective way to combat cancer. OX40 is a costimulatory receptor in the TNFreceptor family that is transiently expressed on antigen-experienced T-cells. OX40 signaling in the context of antigen recognition by the T-cell receptor enhances the proliferation and survival of CD4+ and CD8+ effector T cells and inhibits the suppressive function of regulatory T cells. MOXR0916 is a humanized effector-competent IgG1 agonistic antibody against human OX40 that is being developed as treatment for refractory solid tumors. Methods: In vitro characterization of the effects of MOXR0916 on T cell proliferation and interferon g (IFNg) production was performed in human primary CD4+ memory T cells stimulated with anti-CD3. In vivo studies in mouse syngeneic tumor models were performed with the surrogate antimouse OX40 antibody PRO307205, as MOXR0916 does not bind mouse OX40. Dose ranging efficacy studies were performed with the EMT6 model and relevant pharmacodynamic (PD) markers including CD4 and CD8 T cell proliferation, IFNg production, and FOXP3 and CD8B gene expression were measured in blood and/or tumor. Pharmacokinetics (PK) of MOXR0916 and PRO307205 were assessed in SCID mice and PK of MOXR0916 was assessed in cynomolgus monkeys. Results: Treatment of CD4+ memory T cells with varied concentrations of MOXR0916 and a fixed concentration of anti-CD3 produced a concentration-dependent increase in cell proliferation and IFNg production with EC50 values estimated as 10 ng/mL. Both single and multiple dosing of 0.1 to 10 mg/kg of PRO307205 treatment were found to be efficacious in the EMT6 model, with more animals responding to treatment with higher and more frequent dose administration. No efficacy was observed with a 0.01 mg/kg dose. Efficacious doses were associated with evidence of peripheral and intratumoral increase in IFNg, proliferation of effector T cells, and reduction in number of regulatory T cells. MOXR0916 showed linear PK with typical IgG1 clearance and distribution parameters in both mouse and cyno. Human PK was projected based on allometric scaling of cyno PK. Conclusion: Based on in vitro and in vivo studies of anti-OX40 pharmacology, MOXR0916 is predicted to counteract the immunosuppressive tumor microenvironment and promote T cell-dependent, anti-tumor immunity via two distinct mechanisms: (1) enhancing proliferation and cytokine production of antigen-experienced effector T cells and (2) reducing the number and activity of intra tumoral regulatory T cells. Quantitative data from pharmacology, PK and PD were used for predicting relevant activity and doses of MOXR0916 for testing in clinical trials. 210 ORAL PRESENTATION A phase 1 study of KTN3379, a human anti-ErbB3 monoclonal antibody in patients with advanced cancers P. LoRusso1 , T. LaVallee2 , L. Kimmel2 , C. Lubeski2 , R. Gedrich2 , C. Sidor2 . 1 Wayne State University, Detroit Michigan, USA; 2 Koltan Pharmaceuticals, New Haven CT, USA Purpose: ErbB3 is an important dimerization partner for other ErbB family members and is emerging as a key component in the development of resistance. KTN3379 is a human monoclonal antibody against human epidermal growth factor receptor-3 (ErbB3 or HER3) that blocks both ligand-dependent and independent activation of ErbB3 and is engineered to extend half-life. The Phase 1 trial (KTN3379-CL-001) is an open label, dose escalation and expansion study to assess the safety and pharmacokinetics of KTN3379 in patients with advanced cancers to determine a recommended dose for efficacy studies and to evaluate potential biomarkers related to KTN3379 activity. Methods: The study is being conducted in two Parts. In Part I, using a 3 + 3 design, patients received escalating doses of KTN3379 ranging from 5 to 20 mg/kg every 3 weeks until disease progression or intolerable

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toxicity. In Part II, KTN3379 will be administered along with a Standard of Care (SOC) regimen in disease specific cohorts of 6 patients. Adverse events are assessed according to the National Cancer Institute Common Terminology Criteria for Adverse Events and dose limiting toxicities (DLTs) were evaluated during the initial cycle. RECIST tumor measurements were obtained every 3rd cycle and pharmacokinetics (PK) and pharmacodynamic (PD) assessments were evaluated during each cycle. Archival tumor tissue was obtained if available. Results: In Part I, 12 patients with advanced cancer (CRC, ovarian, pancreatic, endometrial, pancreatic, HCC and germ cell cancers), median age of 62 with ECOG performance status of 0 or 1 and multiple prior therapies (median of 4) received at least one dose of KTN3379. There were no DLTs or Grade 3 related events reported to date and the highest dose administered was 20 mg/kg. The most frequent treatment related AEs were Grade 1 and 2 diarrhea (one Grade 3), dry mouth and skin, and anemia. PK parameters were consistent with humanized monoclonal antibodies and could support q3 week dosing. Early PD assessments indicated elevation of sErbB3 levels in all patients following treatment. Additional assessments are ongoing. Conclusions: Results suggest that doses of 20 mg/kg are safe and well tolerated and PK supports 3-week dosing. Expansion is ongoing to assess the safety and tolerability of KTN3379 in combination with other targeted agents in specific cancers and to evaluate the potential for biomarkers to identify sensitive patients.

Thursday 20 November 2014

10:30–12:30

PLENARY SESSION 5

Epigenetic Targets 211 ORAL PRESENTATION A novel synthetic lethal interaction between the histone mark H3K36me3 and checkpoint kinases S.X. Pfister1 , E. Markkanen1 , Y. Jiang1 , S. Sarkar1 , V. D’Angiolella1 , G. Dianov1 , A.J. Ryan1 , T.C. Humphrey1 . 1 CRUK MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, United Kingdom Background: Synthetic lethal interactions between cellular pathways can be exploited to selectively kill cancer cells that carry a mutation in a gene of one pathway by chemically inhibiting the function of a gene in a second pathway. SETD2, a histone H3K36me3 methyltransferase, has recently been recognised as a tumour suppressor in several cancer types, thereby identifying SETD2 loss of function mutation as potential target for cancer therapy. Here, we report a novel synthetic lethal interaction between SETD2 loss and inhibition of DNA replication checkpoint proteins, and provide insights on the underlying molecular mechanisms. Material and Methods: Growth of p53 wild type SETD2-deficient (A498, LB996) or -proficient (RCC4, U2OS) human cancer cell lines were measured after treatment using WEE1 (MK1775), CHK1 (LY2603618, AZD7762, Go6976) ¨ or ATR (VE821) kinase inhibitors. DNA replication was analysed by DNA fibre assay and by iPOND (isolation of proteins on nascent DNA). dNTP pools were measured by polymerase-catalyzed incorporation of radioactive dNTP. We confirmed the in vitro efficacy of MK1775 in vivo in SETD2-deficient A498 xenografts. Results: Compared to wild type, SETD2-deficienct cells were more sensitive to growth inhibition by VE821 (IC50 = 8.5 vs 3.8mM), LY2603618 (IC50 = 750 vs 374nM) or MK1775 (IC50 = 327 vs 109nM) (p < 0.0001). Increased cell death in SETD2-deficient cells was due to DNA damage arising from replication stress, and not premature mitosis as has been previously reported in TP53 mutant tumour cell lines. SETD2independent methods to reduce H3K36me3 levels (overexpression KDM4A demethylase, or expression of the dominant-negative K36M histone H3.3 variant) also significantly inhibited cell growth and increased apoptosis on treatment with ATR, CHK1, or WEE1 inhibitors (p < 0.0001). SETD2 and WEE1 acted on two independent pathways regulating dNTP production during DNA replication. An in vivo experiment in established SETD2deficient A498 xenografts grown in nude mice showed that treatment with MK1775 (60 mg/kg, po, bid, d1−12) regressed tumours, producing a marked reduction in tumour size compared with vehicle-treated control animals (mean tumour size = 291.2±40.0 vs 50.2±4.7 mm3 , p < 0.0001). In addition, MK1777-treated tumours had significantly greater levels of DNA damage as measured by gH2AX foci (p < 0.0001). Conclusions: The findings suggest that targeted inhibition of ATR, CHK1 or WEE1 may be exploited to create novel treatments for cancers with reduced levels of H3K36me3.

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Thursday 20 November 2014

Plenary Session 6

212 ORAL PRESENTATION Novel anti-tumor activity of targeted LSD1 inhibition by GSK2879552 H. Mohammad1 , K. Smitheman1 , G. Van Aller2 , M. Cusan3 , S. Kamat4 , Y. Liu2 , N. Johnson2 , C. Hann4 , S. Armstrong3 , R. Kruger2 . 1 GlaxoSmithKline, Cancer Epigenetics, Collegeville PA, USA; 2 GlaxoSmithKline, Cancer Epigenetics, Collegeville, USA; 3 MSKCC, Pediatrics, New York, USA; 4 Johns Hopkins University, Oncology, Baltimore, USA Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. LSD1 mediated H3K4 demethylation can result in repressive chromatin environment that silences gene expression and has been shown to play a role in development and hematopoietic differentiation. LSD1 is overexpressed in multiple tumor types, including acute myeloid leukemia (AML). Together, these studies suggest LSD1 is an important regulator of the epigenome that modulates gene expression through modification of histones and its presence in transcriptional complexes. The current study describes the anti-tumor effects of a novel, potent, irreversible, GSK LSD1 inhibitor (GSK2879552) in AML and small cell lung cancer (SCLC). Screening of over 150 cancer cell lines revealed that SCLC and AML cells have a unique requirement for LSD1. While LSD1 inhibition did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of putative LSD1 target genes. This increase in the transcriptionally activating histone modification correlates with increased gene expression. Treatment of AML cells with GSK2879552 promotes the expression of cell surface markers associated with a differentiated immunophenotype, including CD11b and CD86. In an MV-4−11 engraftment model, increases in CD86 and CD11b were observed as early as 8 hours post dosing. GSK2879552 treatment resulted in a potent anti-proliferative growth effect in a subset of SCLC cell lines tested and all AML cell lines tested. Potent growth inhibition was also observed on AML blast colony forming ability of bone marrow samples derived from primary AML patient samples. The effects of LSD1 inhibition were further characterized in vivo using a mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into secondary recipient mice that were treated with an LSD1 tool molecule inhibitor for 17 days. Control mice succumbed to AML by 45 days post transplant, while treated mice showed prolonged survival. GSK2879552 treatment of mice engrafted with SCLC cell lines resulted in greater than 80% tumor growth inhibition. Studies using patient derived primary SCLC showed similar efficacy demonstrating the growth inhibition of SCLC with an LSD1 inhibitor extended beyond cell lines. Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML and SCLC. A Phase I clinical trial using GSK2879552 was initiated in March, 2014. All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed.

Thursday 20 November 2014

13:30–15:35

PLENARY SESSION 6

Proffered Paper Session 213 ORAL PRESENTATION A phase I dose-finding study of BI 853520, a potent and selective inhibitor of focal adhesion kinase (FAK), in Japanese and Taiwanese patients with advanced or metastatic solid tumors T. Doi1 , C.C. Lin2 , A. Ohtsu3 , J.C.H. Yang2 , K. Shitara3 , L.C. Pronk4 , A. Sarashina5 , A.L. Cheng2 . 1 National Cancer Center Hospital East, Gasrtointestinal Oncology, Chiba, Japan; 2 National Taiwan University Hospital, Department of Oncology, Taipei, Taiwan; 3 National Cancer Center Hospital East, Department of Experimental Therapeutics, ˜ S.A., Clinical Development Chiba, Japan; 4 Boehringer Ingelheim Espana Oncology, Barcelona, Spain; 5 Nippon Boehringer Ingelheim Co. Ltd, Clinical PK/PD department, Kobe, Japan Background: Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that contributes to activation of multiple downstream-signaling pathways involved in tumor cell survival, proliferation, invasion, and metastasis. BI 853520 is a potent and highly selective inhibitor of FAK with an IC50 of 1nM. It has shown efficacy in multiple xenograft models of human cancer.

Material and Methods: This is a phase I, dose-finding study of BI 853520 given as a continuous oral dosing regimen in 28-day cycles in a mixed population of Japanese and Taiwanese patients (pts) with progressive, advanced or metastatic solid tumors. Endpoints include safety, determination of maximum tolerated dose (MTD), pharmacokinetics (PK), pharmacodynamics, and efficacy, determined according to RECIST v1.1 criteria. Results: To date, 18 pts have received doses of 50, 100 and 200 mg qd of which 14 pts are evaluable for dose limiting toxicity (DLT). Male/female ratio was 12/6, median age 65 years (range, 35−77 years), ECOG PS 0/1:10/8. Primary tumor types: 4 gastric cancer, 2 colorectal cancer; 2 oesophageal cancer, and 10 others. Median number of prior systemic treatments: 3. Drug-related adverse events (AEs) in >10% of pts included proteinuria (44.4%), diarrhea (38.9%), nausea (22.2%), vomiting (22.2%), inflammatory fibrous thickening in the palm of the hand (11.1%), maculopapular rash (11.1%) and decreased appetite (11.1%), all of NCI Common Terminology Criteria for AEs (v4.03) grade 1−2 except for one patient who had proteinuria grade 3 that occurred during cycle 2 at the 200 mg dose and was considered DLT. So far, no DLTs have been observed during cycle 1 and no drug-related serious AEs have been reported. The MTD was determined as 200 mg qd and the 200 mg cohort is expanded to a total of 12 patients to confirm the MTD. PK:Preliminary data suggest that plasma exposure increased with increasing doses. Based on the observed accumulation and half-life, a once-daily dosing scheme is supported. Preliminary efficacy: Of 12 evaluable pts, 1 pt with gastric cancer achieved a partial response (100 mg qd), 2 pts presented stable disease: 1 pt with oesophageal cancer and 1 with urachal cancer lasting 7 and 8 cycles, respectively (both 50 mg qd), and 9 pts progressed. Conclusion: The safety profile of BI 853520 is favourable. PK analysis supports the once-daily dosing schedule. Recruitment at the 200 mg dose is ongoing to confirm the MTD. An update will be provided at the meeting.

214 ORAL PRESENTATION Homologous recombination deficiency (HRD) score and niraparib efficacy in high grade ovarian cancer P. Haluska1 , K.M. Timms2 , M. AlHilli1 , Y. Wang3 , A.M. Hartman2 , J. Jones2 , A. Gutin2 , Z. Sangale2 , C. Neff2 , J. Lynchbury2 , L. Rudolph-Owen3 , M.A. Becker1 , S. Agarwal3 , K.M. Wilcoxen3 . 1 Mayo Clinic, Rochester, USA; 2 Myriad Genetics, Salt Lake City, USA; 3 Tesaro Inc., Waltham, USA Purpose: The therapeutic potential of PARP inhibitors is predicted to extend beyond BRCA mutant (BRCAmut ) phenotypes to homologous recombination deficient (HRD) cancers. An HRD assay amenable for clinical testing was applied to treatment na¨ıve, high grade ovarian primary tumor samples. Niraparib treated patient-derived tumorgraft models selected from these primary ovarian tumors were utilized to evaluate the correlation between HRD score, BRCA deficiency, platinum sensitivity and niraparib anti-tumor response. Methods: Utilizing patient-derived ovarian xenografts we previously demonstrated response to niraparibin both BRCAmut and BRCA wild type (BRCAwt ) tumors. BRCAmut status alone was neither necessary nor sufficient to predict response to niraparib. To understand the selectivity observed, samples from a collection of >100 high grade ovarian tumors were subjected to HRD analysis. The HRD analysis is a DNA-based assay that is capable of detecting homologous recombination deficiency independent of its etiology. Genome-wide SNP data was generated from a custom Agilent SureSelect XT2 capture followed by sequencing on an Illumina HiSeq2500. SNP data was analyzed using all three algorithms; Loss of Heterozygosity (LOH), Telomeric Allelic Imbalance (TAI) and Large-scale State Transistions (LST). The final HRD score is the sum of the LOH+TAI+LST scores with numerical outputs ranging from 0– 100. RNAseq analysis was also conducted on a subset of the ovarian tumors. Niraparib was then evaluated as a monotherapy in a series of high grade ovarian cancer tumorgrafts with a diverse range of HRD scores. Intraperitoneal tumorgrafts were monitored for tumor growth with twiceweekly transabdominal ultrasound imaging. In-vivo response to niraparib was correlated to the HRD score, BRCA status, patient’s platinum response and RNAseq data. Results: One hundred and six high grade ovarian tumors were evaluated for HRD, BRCA mutation, and RNAseq analysis. Twenty one tumors (20%) had a deleterious somatic or germline BRCAmut . BRCA1 hypermethylation was evaluated in 98 tumors and found in 6, representing approximately 6% of this cohort. Collectively, there was 26% BRCA deficiency in this primary tumor collection. HRD scores ranged from 1−86 with a median score of 32 and an average score of 39. All BRCAmut tumors and 83% (5/6) of the BRCA1 hypermethylated tumors had an HRD score of 31 or greater. The BRCA1 hypermethylated tumor with a low HRD score does not have loss of heterozygosity (LOH) at BRCA1, and likely retains functional BRCA1. Invivo response to niraparib monotherapy was demonstrated in BRCAmut and

Plenary Session 6 BRCAwt models with high HRD scores. In addition, niraparib resistance or lack of in vivo efficacy was demonstrated in tumors with low HRD scores. Conclusion: HRD testing of ovarian cancer was predictive of BRCA deficiency. Response to niraparibwas seen in both BRCAmut and BRCAwt tumors. Our data are supportive of the use of HRD testing to select high grade ovarian cancer patients for the clinical evaluation of niraparib treatment. 215 ORAL PRESENTATION Updated clinical and preliminary correlative results of ARIEL2, a Phase 2 study to identify ovarian cancer patients likely to respond to rucaparib E. Swisher1 , J. Brenton2 , S. Kaufmann3 , A. Oza4 , R.L. Coleman5 , D. O’Malley6 , G. Konecny7 , L. Ma8 , M. Harrell9 , D. Visscher3 , A.W. Hendrickson3 , K. Lin10 , M. Raponi10 , E. Mann10 , H. Giordano10 , L. Maloney11 , L. Rolfe12 , I. McNeish13 . 1 University of Washington School of Medicine, Seattle WA, USA; 2 Cancer Research UK Cambridge Institute, Oncology, Cambridge, United Kingdom; 3 Mayo Clinic, Oncology, Rochester, USA; 4 Princess Margaret Cancer Centre, Oncology, Toronto, Canada; 5 The University of Texas MD Anderson Cancer Center, Oncology, Houston, USA; 6 The Ohio State University James Cancer Center, Oncology, Columbus, USA; 7 University of California Los Angeles (UCLA), Oncology, Los Angeles, USA; 8 Rocky Mountain Cancer Center, Oncology, Lakewood, USA; 9 University of Washington School of Medicine, Oncology, Seattle WA, USA; 10 Clovis Oncology, San Francisco, USA; 11 Clovis Oncology, Boulder, USA; 12 Clovis Oncology, Cambridge, United Kingdom; 13 Institute of Cancer Sciences University of Glasgow, Glasgow, United Kingdom This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

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Methods: Patients with recurrent gliomas (WHO grade II-IV) were eligible for this trial. FSRT was prescribed to 30−35 Gy delivered in 10 daily fractions. Panobinostat was administrated orally once daily every Monday, Wednesday, and Friday concurrently with radiation treatment. The panobinostat dose was escalated from 10 to 30 mg using a 3+3 trial design. Results: A total of 16 patients were enrolled on the trial. Four patients were excluded from the analysis due to 1 patient failing to receive study treatment due to rapid progression, and 3 patients having increased QTc exceeding the protocol limit and were unable to finish protocol treatment. Of the 12 evaluable patients, 8 had recurrent GBM, and 4 had recurrent anaplastic astrocytoma. There were 3 patients in the 10 mg panobinostat cohort, 3 patients in the 20 mg cohort, and 6 patients in the 30 mg final cohort. There were no treatment related grade 3 or higher toxicities in the 10 mg or 20 mg cohort. In the 30 mg panobinostat cohort, one patient developed grade 3 leukopenia and grade 4 neutropenia which was possibly related to the treatment. There are 5 living patients in the study. The minimum follow up of the living patients is 8.5 m. The 4-m progression survival (PFS) of all the evaluable patients is 87%, and 6-m PFS of 56%. The medial overall survival is 9.5 m. There is a suggestion of dose response to the panobinostat. The 4-m PFS is 100%, 100%, and 67% for 30 mg, 20 mg, and 10 mg cohort respectively. The 6-m PFS is 83%, 33%, and 67% for 30 mg, 20 mg, and 10 mg cohort respectively. The overall survival for the 30 mg cohort is not reached with a median follow up of 11.5 m. Conclusions: Panobinostat administrated with FSRT is well tolerated at a 30 mg dose, which is the recommended dose for phase II trial. There is a suggestion of a dose–response relationship with panobinostat dose. The median overall survival is not reached with a median follow up of 11.5 m for the 30 mg cohort. A phase II trial is warranted to confirm the efficacy of combining panobinostat with FSRT for recurrent gliomas. 217 ORAL PRESENTATION Results of a phase I, open-label, multicentre study to assess the safety, tolerability, pharmacokinetics and preliminary antitumour activity of AZD9150 in patients with advanced/metastatic hepatocellular carcinoma Y.K. Kang1 , B.Y. Ryoo1 , T.Y. Kim2 , K.H. Lee2 , H.Y. Lim3 , S.J. Lee3 , M. Ikeda4 , T. Okusaka5 , S. Nadano6 , C.C. Lin7 , T.P. Poon8 , C.J. Yen9 , P. McCoon10 , F. Neumann10 , K. Vishwantahan10 , R. DuPont10 , P. Lyne10 . 1 Asan Medical Center, Oncology, Seoul, Korea; 2 Seoul National University Hospital, Oncology, Seoul, Korea; 3 Samsung Medical Center, Oncology, Seoul, Korea; 4 National Cancer Center Hospital East, Chiba, Japan; 5 National Cancer Center Hospital, Tokyo, Japan; 6 National Hospital Organization, Ehime, Japan; 7 National Taiwan University Hospital, Oncology, Taipei, Taiwan; 8 Queen Mary Hospital, Surgery, Hong Kong, Hong Kong; 9 National Cheng Kung University Hospital, Tainan, Taiwan; 10 AstraZeneca Pharmaceuticals, Oncology IMED, Waltham, USA

216 ORAL PRESENTATION Phase I study of panobinostat and fractionated stereotactic re-irradiation therapy (FSRT) for recurrent high grade gliomas W. Shi1 , Y.R. Lawrence1 , M. Werner-Wasik1 , D.W. Andrews2 , J.J. Evans2 , J. Glass2 , L. Kim1 , V. Bar Ad1 , Y. Moshel1 , K. Judy1 , C. Farrell1 , N.L. Simon1 , A.P. Dicker1 . 1 Thomas Jefferson University Hospital, Radiation Oncology, Philadelphia PA, USA; 2 Thomas Jefferson University Hospital, Neurological Surgery, Philadelphia PA, USA Background: This is a phase I study to evaluate the safety, and tolerability of oral panobinostat, an HDAC inhibitor, when combined with fractionated stereotactic re-irradiation therapy (FSRT) for recurrent gliomas.

Background: STAT3 plays a role in tumour biology by modulating critical functions of both tumour and stromal/immune cells. AZD9150 is a STAT3 antisense oligonucleotide (ASO) in Phase I dose expansion in two clinical trials. We present safety, preliminary response, pharmacokinetic (PK) and pharmacodynamic (PD) data for the dose escalation portion of a Phase I clinical, open-label, multi-center study in advanced hepatocellular carcinoma (HCC) patients. Methods: Patients with advanced sorafenib relapsed/refractory HCC and Child-Pugh A liver function were enrolled in a 3+3 dose escalation study to determine dose limiting toxicities (DLTs), maximum tolerated dose (MTD), safety, tolerability, PK, PD, and preliminary efficacy. AZD9150 was administered intravenously with 3 loading doses of AZD9150 on days 1, 3, and 5 and weekly thereafter in 28 day cycles. STAT3 RNA and protein expression in circulating leukocytes and serum CRP were evaluated on day 8 of treatment and the first day of subsequent cycles vs. pre-treatment. Results: Twenty-four patients were dosed in 1, 1.5, 2 and 3 mg/kg cohorts between May 2013 and May 2014. One partial response was observed in the 2 mg/kg cohort with 61% reduction in lung metastases and 87% decrease in AFP. Adverse events reported in 20% patients included AST elevation (G1/2/3 46, 8, 4%), ALT elevation (G1/2/3 35, 15, 4%), thromobocytopenia (G1/2/3 31, 19, 4%) neutropenia (G1/2/3 0, 23, 0%) hypertension (G1/2/3 0, 19, 4%). Transaminase elevations were mostly mild and were reversible after interruption of AZD9150. Thrombocytopenia was mostly mild/moderate, typically occurred after ~10 weeks of dosing, and did not require dose modification or intervention. One patient had G3 thrombocytopenia and transient dosing interruption. DLTs of G3 transaminase elevations occurred in 3 patients, one each in the 1.5, 2 and 3 mg cohort. 3 mg/kg was declared the MTD and the recommended dose for subsequent studies. Maximum AZD9150 plasma concentration (Cmax) was observed at the end of the 3 hr infusion and was dose proportional between 1 and 3 mg/kg dose levels. There was no accumulation in mean plasma Cmax or AUC0−24 hr after repeated 3-hr infusions. Knockdown of

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Plenary Session 7

STAT3 RNA and protein (up to 80%) in all circulating leukocytes of patients by day 8 of treatment was detected in the majority of samples analyzed so far and confirms activity of the drug at the target level. Decreased serum CRP in the majority of patients treated in the 1 and 1.5 mg/kg cohorts and all patients in the 2 and 3 mg/kg cohorts is consistent with functional inhibition of STAT3. Updated data will be presented. Conclusion: Preliminary data suggest activity of AZD9150 in patients with advanced HCC. A tolerated dose with clinical and pharmacodynamic activity has been identified and is being explored further in dose expansion. 218 ORAL PRESENTATION Genomic analysis identifies novel drivers and targetable pathways in inflammatory breast cancer patient samples D.M. Moran1 , K. Rao1 , P. Bacon-Trusk1 , K. Pry1 , V. Weigman2 , V. Velculescu3 , M. Cristofanilli4 , S. Bacus1 . 1 Quintiles, Translational R&D Oncology, Westmont, USA; 2 Quintiles/Expression Analysis, Translational Genomics, Durham, USA; 3 Johns Hopkins University, Oncology, Baltimore, USA; 4 Thomas Jefferson University, Medical Oncology, Philadelphia, USA Background: Inflammatory breast cancer (IBC) is a rare aggressive breast cancer in which cancer cells block the lymph vessels in the skin of the breast. IBC tumors are typically hormone receptor negative but have shown a high rate of HER2 (human epidermal growth factor receptor 2) positivity and response to HER2 targeted therapies such as lapatinib. Genomic drivers associated with progression and drug response of IBC are not well established. This study interrogates the mutational background of IBC to understand drug responses in breast cancer. Materials and Methods: A targeted NGS panel that covers whole coding regions of 208 of the most common cancer related genes (copy numbers and somatic mutations) and rearrangements in 17 well characterized cancer genes was used to analyze 20 IBC patient tumor and matched normal samples. Pathway analysis was performed on genomic variants identified. Cell line studies were performed to understand the impact of genomic variants on drug treatment. Results: Intra- and inter-tumor heterogeneity was observed across the IBC samples studied, however, common pathway motifs were also identified among cases. Multiple variants in the HER signaling pathway were observed including HER2 amplification (54% of samples) and a high rate of ERBB3 mutations (26% of samples). ERBB3 point mutations were discovered in hotspot regions in both the extracellular and kinase domains and occurred at higher rates than previously observed in other cancers. Genomic alterations were also identified among many genes of the PI3K-mTOR pathway in the majority of IBC cases. Activity of the PI3K-mTOR pathway was further confirmed by immunohistochemistry for phosphorylated S6, a target of mTOR kinase activity. Cell studies demonstrated potent effects of lapatinib on proliferation of IBC cells harboring ERBB3 mutations in conjunction with HER2 amplification. Breast cancer cells harboring PI3K mutations and HER2 amplification were less sensitive to lapatinib but were synergistically responsive to a combination of PI3KCA inhibitors and lapatinib. Other frequent genomic alterations were also detected in pathways related to chromatin modification, DNA repair, APC, JAK-STAT, KIT and Notch signaling which may also be novel drug targets in IBC. Notably, hotspot and/or kinase domain mutations were discovered in JAK (1 & 2) and KIT genes in multiple IBC samples. Conclusions: Genomic and protein analysis of IBC identified multiple pathways that may be targetable using single and/or combination targeted therapies in all cases studied. This study also highlighted that drugs such as PI3K/mTOR and novel ERBB3 targeted therapies, used alone or in combination with HER2 inhibitors, may be important in the treatment of IBC. Co-occurrence of ERBB3 mutations and HER2 amplification/overexpression likely sensitize cancer cells to HER2 targeted therapies and should be further explored in other HER2 positive cancers. 219 ORAL PRESENTATION Phase I trial evaluating the antiviral agent Cidofovir in combination with chemoradiation in cervical cancer patients: A novel approach to treat HPV related malignancies? E. Deutsch1 , A. Levy2 , R. Mazeron2 , A. Gazzah2 , E.A. Angevin3 , V. Ribrag3 , R. Balheda3 , A. Varga3 , C. Lhomme4 , C. Haie-Meder2 , J.C. Soria3 . 1 Institut Gustave Roussy, Villejuif, France; 2 Institut Gustave Roussy, radiation oncology, Villejuif, France; 3 Institut Gustave Roussy, DITEP, Villejuif, France; 4 Institut Gustave Roussy, Medicine, Villejuif, France This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

Thursday 20 November 2014

16:00–17:50

PLENARY SESSION 7

Novel Mechanisms for Drug Resistance 220 ORAL PRESENTATION Overcoming drug-resistance in multiple myeloma by XPO1 inhibitor combination therapy J. Turner1 , J. Dawson1 , S. Grant2 , K. Shain3 , C. Cubitt4 , Y. Dai2 , L. Zhoui2 , M. Kauffman5 , S. Shacham5 , D. Sullivan1 . 1 Moffitt Cancer Center, Chemical Biology and Molecular Medicine Program, Tampa Florida, USA; 2 Massey Cancer Center, Virginia Commonwealth University, Richmond Virginia, USA; 3 Moffitt Cancer Center, Department of Malignant Hematology, Tampa Florida, USA; 4 Moffitt Cancer Center, Translational Research Laboratory, Tampa Florida, USA; 5 Karyopharm Theraeutics, Natick Massachusetts, USA Background: The purpose of this study was to investigate the use of XPO1 (exportin 1, CRM1) inhibitors (XPO1i) to sensitize de novo and acquired drug resistant multiple myeloma (MM) cells to the proteosome inhibitors (PI) bortezomib (BZ) and carfilzomib (CZ), the topoisomerase II (topo II) inhibitor doxorubicin (DX) and the alkylating agent melphalan (ML). Materials and Methods: Cells were treated in vitro with XPO1i (KPT-330 or KOS-2464) +/− BZ, CZ, DX or ML. Sensitivity was measured by cell viability assay (CellTiter-Blue). Proximity ligation assays (PLA) were performed to assess XPO1-topoIIa binding in the presence of an XPO1i. Western blot of proteins related to PI and XPO1i were used to determine the potential mechanism of XPO1i-PI synergy. Comet assay for DNA damage was performed in XPO1i/DX treated cells. Drug resistant U266 and 8226 MM cell lines were developed by incremental exposure to BZ. Resistant and parental MM cells were treated in vitro with XPO1i +/− MM drugs. Sensitivity was measured by apoptosis (caspase 3). U266 resistant MM cells were also used to challenge NOD/SCID-g mice treated with XPO1i +/− BZ or pegylated liposomal doxorubicin (PLD). MM cells isolated from patients with newly diagnosed, relapsed or refractory MM were treated with XPO1i +/− BZ, CZ, DX or ML and CD138+/light chain+ MM cells assayed for apoptosis. Results: MM cell viability was decreased synergistically by XPO1i when used in combination with BZ, CZ, DX or ML (CI values 0.502, 0.482, 0.092 and 0.687). XPO1i prevented binding of XPO1 to topoIIa as shown by PLA, and XPO1i increased DNA fragmentation when combined with DX (Comet assay). Western blot showed that the XPO1i/PI combination increased IkB and decreased NFkB in MM cells. BZ selected MM cells were found to be resistant (>10-fold) to BZ, CZ, DX and ML when compared to parental cell lines. Resistant MM cell lines were sensitized by the XPO1i to drugs as shown by apoptosis assay (3 to10-fold). Drug resistant MM challenged mice treated with XPO1i +/− BZ or PLD had increased survival when compared to BZ or PLD alone (p0.027). CD138+/light chain+ MM cells derived from

Poster Session – Chemoprevention newly diagnosed, relapsed and refractory MM patients were sensitized by XPO1 inhibitors to BZ, CZ, DX and ML as shown by apoptosis. Conclusions: XPO1i greatly improved the response of de novo and acquired drug resistant MM to BZ, CZ, DX and ML in vitro, in vivo and ex vivo. Combination therapies using XPO1i may significantly improve the treatment of myeloma.

Thursday 20 November 2014

Poster Sessions Chemoprevention 221 POSTER (Board P001) Docosahexaenoic acid along with modulation of actin binding proteins reduces cancer cell migration M. Ali1 , L.K. Rogers1 . 1 Nationwide Children’s Hospital The Ohio State University College of Medicine, The Research Institute at Nationwide Children’s Hospital and Department of Pediatrics, Columbus Ohio, USA Background: Cancer is the leading cause of death worldwide and all age groups, including children, are at risk for cancer associated death. Disease progression and metastasis are major contributors to cancer-associated morbidity and mortality. Increased cell migration rate is characteristic of tumor progression and metastasis. Actin binding proteins regulate cytoskeletal remodeling at the leading edges of cancer cells, facilitate invasive organelle (invadopodia) formation, and promote higher migration rates. Docosahexaenoic acid (DHA), a poly unsaturated fatty acid, has been shown to inhibit the cancer cell metastatic phenotype. Here, we test the hypothesis that changes in actin binding proteins regulate cancer cell migration and that supplementing cells with DHA will prevent these changes. Materials and Methods: Fatty acid profile of Non-cancer (MLE12) and cancer (A549) done with mass spectrophotometry. Further cells were treated with 8-Br-cAMP and/or DHA. F-actin content was measured using confocal microscopy. Cell migration was estimated by wound assay and transwell apparatus. Actin binding proteins, profilin, cofilin, vimentin and gelsolin, were identified and quantified using confocal microscopy and western blot to evaluate wound edges and actin co-immunoprecipitate, respectively. Results: F-actin content and cell migration were increased by cAMP in association with changes in profilin, cofilin, vimentin and gelsolin levels. DHA treatment suppressed the increase in actin content and cell migration in cancer cells but not in non-cancer cells in association with altered levels of actin binding proteins. Conclusion: We postulate that DHA specifically inhibits cancer cell migration via alterations in actin binding proteins indicating a therapeutic potential against cancer cell metastasis. The changes in actin binding proteins could serve as biomarkers for cancer progression and as innovative therapeutic targets. 222 POSTER (Board P002) Estimating predictive values of short-term morphologic assays of cancer chemoprevention for efficacy in animal tumor assays B. Dunn1 , V.E. Steele1 , R.M. Fagerstrom1 , C.F. Topp2 , D. Ransohoff3 , C. Cunningham4 , R. Lubet5 , L.G. Ford5 , B.S. Kramer5 . 1 National Cancer Institute NIH, Division of Cancer Prevention, Bethesda Maryland, USA; 2 CCS Associates, Preclinical Research and Development Resources, McLean Virginia, USA; 3 University of North Carolina at Chapel Hill, Department of Medicine, Chapel Hill NC, USA; 4 Information Management Services, Rockville MD, USA; 5 National Cancer Institute, Division of Cancer Prevention, Bethesda MD, USA Background: The predictive value of chemopreventive agent efficacy in morphologic (in vitro/in vivo) assays for efficacy in in vivo tumor assays is not well characterized. Over a 25-year period, the Chemopreventive Agent Development Research Group in the U.S. NCI’s Division of Cancer Prevention has tested approximately 800 agents for potential chemopreventive activity. The current project focuses on agents tested in both morphologic and tumor assays in order to gain a deeper understanding of the relevant predictive values. Materials and Methods: The early stages of the testing pathway involve two critical steps: (1) in vitro/in vivo morphologic assays and, for agents

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successful in these, (2) testing for tumor prevention (measured in terms of tumor incidence and multiplicity reduction) in animal tumor assays. The ultimate goal is to test agents that successfully decrease tumor incidence and multiplicity in animal tumor assays in humans. In the current project we evaluated the predictive values of our preclinical models by determining how well the earlier-stage (morphologic) assays predict efficacy in the laterstage (animal tumor) assays. The 210 agents that were tested in both morphologic and animal tumor assays in our program were included in our analysis. Statistical modeling to determine how well the six most commonly used morphologic assays predicted efficacy of the 210 tested agents in animal tumor assays was carried out by multimodel inference applied to ordinal logistic regression. Results: The ability of these six morphologic assays to predict tumor outcomes was evaluated in three statistical models, one for each animal tumor setting: (1) all tumor types (general model), (2) colon cancer only, and (3) mammary gland cancer only. Based on this statistical modeling, each morphologic assay was assigned a value describing how strongly it predicted outcomes in each of the three animal tumor assay settings. Conclusions: These predictive models can be used to guide our future decision-making with respect to agent selection as well as morphologic and animal tumor assay use. The overall goal is to improve the efficiency of the process of chemopreventive agent development. 223 POSTER (Board P003) Preclinical assessment of nintedanib for chemoprevention in hepatocellular carcinoma V. Tovar1 , A. Moeini1 , S. Torrecilla1 , M. Higuera1 , J. Peix1 , I.M. Quetglas1 , L. Rodriguez-Carunchio1 , H. Cornella1 , M. Sole1 , J.M. Llovet1 . 1 Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS), HCC Translational Research Laboratory, BCLC group, Hospital Cl´ınic, Liver Unit − CIBEREHD, University of Barcelona, Barcelona, Spain Background: Hepatocellular carcinoma (HCC) is the 2nd cause of cancerrelated mortality. Cirrhosis is the most common risk factor in 80−90% of HCC patients. Angiogenesis has a pivotal role in both chronic liver disease and hepatocarcinogenesis. Thus, early interference with anti-angiogenic agents at cirrhotic stages may prevent HCC development. In this study, we evaluate the chemopreventive efficacy of the angiokinase inhibitor nintedanib (VEGFR1−3, FGFR1−3, PDGFR-a/b; Boehringer Ingelheim) in an animal model of HCC. Material and Methods: HCC was induced in male C57Bl/6 mice (n = 55) by a single intraperitoneal (i.p.) injection of DEN (25 mg/kg) at day 15 postpartum followed by weekly i.p. injections of CCl4 (0.5 ml/kg) starting at 4 weeks of age. At 12 weeks-old, mice were randomized to receive nintedanib (50 mg/kg) (n = 29) or vehicle (n = 26). To assess chemopreventive efficacy, mice were sacrificed at 15, 17 and 18 weeks of age. Liver samples were collected for immunohistochemical and molecular analysis (western blot and qRT-PCR). The primary study end-points included HCC incidence, number and size of macroscopic tumors. Drug tolerance was evaluated by body weight losses and plasma ALT/AST levels. Results: Nintedanib reduced HCC incidence in treated animals compared to placebo group at 15 weeks (13% vs 43%), 17 weeks (30% vs 89%, p = 0.02), and 18 weeks (90% vs 100%). The number of tumor/mouse significantly decreased at 17 weeks (0.9 vs 4.7, p = 0.003) and 18 weeks (3.5 vs 11.4, p = 0.003). Nintedanib also prevent the development of large number of tumors (>4 tumors) at 17 weeks (p = 0.01) and 18 weeks (p = 0.008). Moreover, tumor size was significantly reduced at 17 weeks (0.7 mm vs 6.4 mm, p = 0.01) and 18 weeks (4.8 mm vs 16.2 mm, p = 0.001). Nintedanib was well tolerated and no significant toxicity was reported. Histological analysis and up-regulation of collagen levels confirmed the development of HCC in a context of well-established fibrosis. Assessment of nintedanibmechanism of action showed a trend to reduce VEGFR-2 activation while significantly decreased downstream activation of AKT and ERK. Conclusion: Nintedanib decreases overall HCC incidence, number of tumors and tumor size in a liver fibrosis-based mouse model. Nintedanib acts by blocking the MAPK/PI3K pathway downstream of the proangiogenic receptors (VEGFR and PDGFR). Our data provides rationale for testing nintedanib as a potential chemopreventive agent for the development of HCC in cirrhotic patients.

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Poster Session – Clinical Methodology

224 POSTER (Board P004) Synergic tumor growth suppression with carbohydrate-restriction diet and natural AMP-dependent protein kinase activators

226 POSTER (Board P006) Efficacy of cancer preventing drugs administered by intermittent dosing regimens

M. Choi1 , J. Lee2 . 1 Yonsei Cancer Center Yonsei University College of Medicine, Department of Nuclear Medicine, Seoul, South Korea; 2 Yonsei Cancer Center Yonsei University College of Medicine, Department of Nuclear Medicine Brain Korea 21 Project for medical Sciences, Seoul, South Korea

V.E. Steele1 , C. Grubbs2 , C.V. Rao3 , R.A. Lubet1 . 1 National Institutes of Health/National Cancer Institute, Division of Cancer Prevention, Bethesda MD, USA; 2 University of Alabama at Birmingham, Department of Surgery, Birmingham AL, USA; 3 Oklahoma University Health Sciences Center, Center for Cancer Prevention and Drug Development, Oklahoma City OK, USA

Background: Calorie restriction (CR) or low-carbohydrate diet (LCD) can increase life-span in normal cells but inhibit carcinogenesis. Various AMPdependent protein kinase (AMPK) activating phytochemicals also have CR-mimetic anticancer properties. We investigated whether carbohydraterestriction diet and phytochemical supplementation induce synergic tumor suppression. Methods: We used a mixture of curcumin, quercetin, catechins and resveratrol extracts for natural AMPK-activating agent. Survival analysis was performed in B16F10 melanoma model after fed control diet, control diet with phytochemicals (MP formula), LCD, LCD with phytochemicals (LCDmp), moderate-carbohydrate diet (MCD), and MCD with phytochemicals (MCDmp). Tumor suppression mechanisms, especially alterations in energy-dependent signaling pathways, histone methylation, and global gene expression were investigated. We evaluated the role of SIRT1 under glucose-restriction condition with MTT assay. Diet-induced cancer prevention effects were analyzed in a transgenic liver cancer model. Results: In melanoma model, MP, LCD, or MCD intervention did not produce survival benefit but LCDmp and MCDmp interventions increased median survival time significantly (p < 0.05). Suppression of the IGF-1R/PI3K/Akt/mTOR signaling, activation of the AMPK/SIRT1/LKB1pathway and NF-kB suppression were the major tumor suppression mechanisms. MTT assay showed suppressed proliferation of the B16F10 and A375SM cells after treatment of SIRT1 activator under low-glucose condition. Alterations in histone methylation within Pten and FoxO3a were demonstrated in tumor tissues. In transgenic liver cancer model, the MCDmp and LCDmp groups showed fewer tumor nodules. Microarray analysis revealed increased PPARa with decreased IL-6 and NF-kB after MCDmp intervention, which could be the major cancer prevention mechanism. Conclusion: AMPK-activating phytochemicals exert synergic anti-cancer activities under low-carbohydrate condition, and SIRT1 induces tumor suppression. 225 POSTER (Board P005) Molecular targets of interest to the NCI PREVENT cancer preclinical drug development program R.H. Shoemaker1 , B.K. Dunn1 , C. Suen1 , R.A. Lubet1 , D.L. Boring1 , B.D. Klein1 , M.S. Miller1 , V.E. Steele1 . 1 National Cancer Institute Division of Cancer Prevention, Chemopreventive Agent Development Research Group, Bethesda MD, USA Chemoprevention research in the Division of Cancer Prevention was re-structured in 2011 creating the PREVENT Cancer Preclinical Drug Development Program. This Program provides for peer-review of proposals from the general research community for development of small molecules or biologicals, including vaccines, for cancer prevention or biomarkers to facilitate clinical evaluation of prevention strategies. The process for applying to the Prevent Program is described at: http://prevention.cancer.gov/ programs-resources/programs/prevent. Applications are submitted twice yearly and reviewed by a panel of experts and scored for scientific merit, feasibility, etc. Top scoring applications undergo secondary review and prioritization by a panel of NIH scientists. Applications with potential for near-term clinical translation are given highest priority. Approved projects are implemented as Task Orders via a system of contracts with academic and nonprofit, independent research institutions that provide the full range of preclinical studies, including GLP toxicology, needed to support Investigational New Drug Applications. Twenty-five Task Orders addressing a range of molecular targets have been awarded in the first two years of the Program. Small molecules targeting classic aspects of inflammation as well as newer molecular-targeted agents (n = 16) are under study. Pharmacodynamic assays are coupled to efficacy studies. Immunoprevention strategies targeting tumor-associated antigens as well as antigens from cancer-associated infectious agents (n = 6) are being pursued. Preliminary data from current projects will be presented. The PREVENT Cancer Program has engaged a broad cross-section of investigators, supporting preclinical development of agents addressing a wide variety of cancer prevention targets.

Background: Many potential chemopreventive drugs have adverse effects on human subjects when given daily. One strategy to alleviate these adverse events is to give the drugs on an intermittent dosing schedule. Such intermittent dosing has proven effective in other scenarios to reduce toxicity with limited effect on efficacy. Two animal models were used: the rat methylnitrosourea (MNU)-induced mammary cancer model and the rat hydroxy-butyl(butyl)-nitrosamine (OH-BBN)-induced urinary bladder cancer model. Materials and Methods: For the mammary study female Sprague-Dawley rats were given 75 mg of MNU/kg body weight at 50 days of age and five days later the EGFR inhibitors, erlotinib, gefitinib or lapatinib were administered either daily or once a week at two dose levels. Multiplicity of ER-positive mammary cancers was followed weekly for four months. For the urinary bladder study female Fischer 344 rats were given 150 mg OHBBN/gavage twice weekly beginning at 56 days of age and continuing for 8 weeks. Two weeks after the last OH-BBN treatment the rats were given naproxen either daily, one week on/one week off, or 3 weeks on/3 weeks off. Bladders were excised after 8 months of naproxen treatment and bladders with tumors were excised and weighed. Results: EGFR inhibitors and the NSAID, naproxen, have similar efficacy when given intermittently compared to daily. Data will be presented with two organ specific chemoprevention animal models: ER-positive mammary and invasive urinary bladder cancer. The EGFR antagonists, erlotinib, gefitinib and lapatinib, decreased mammary cancer multiplicity by 90% given daily and 75% given weekly. The NSAID, naproxen, decreased large bladder cancers by about 65−80% given either daily, one week on/one week off or 3 weeks on/3 weeks off. Conclusions: Intermittent dosing can be used for agents in two different mechanistic classes in animal mammary and bladder cancer models to lower toxicity yet show little reduction in chemoprevention efficacy compared to daily dosing. Intermittent dosing schedules should reduce EGFR mediated rash and NSAID induced gastric toxicity clinically while retaining efficacy.

Clinical Methodology 227 POSTER (Board P007) Multiplexed ICE COLD-PCR: A mutation detection methodology for achieving sensitivities of <0.01% using either Sanger or NGS G. Wu1 , B. Legendre2 , S. Cherubin1 , C. Cubrich1 , A. Dowers2 , S. Jensen1 , J. Gniffke3 , A. Kruempel2 , P. Krzycki2 , E. McCutchen2 , E. Montagne1 , S. Peterson4 , J. Pope2 , K. Scott1 , K. Richardson5 . 1 Transgenomic, R&D, Omaha, USA; 2 Transgenomic, Biomarker Identification, Omaha, USA; 3 Transgenomic, IT, Omaha, USA; 4 Transgenomic, CLIA, Omaha, USA; 5 Transgenomic, Omaha, USA Background: Blood-based mutation analysis from circulating free DNA (cfDNA) is becoming very important for molecular demographics and diagnostics where no tumor is available as well as in the pharmacodynamic monitoring of the patient during therapy. ICE COLD-PCR technology is capable of high sensitivity detection for both point mutations and insertion/deletions through unbiased enrichment of relevant gene regions. This method preferentially amplifies low levels of mutant DNA in a sample containing a vast excess of wild-type DNA. Materials and Methods: In order to increase throughput as well as address the limited amounts of DNA present from cfDNA sources, a multiplex approach for ICE COLD-PCR has been developed. Horizon Cell line DNA with digital PCR verified mutation percentages was first amplified using singleplex PCR for a single region of interest or multiplex PCR for several regions of interest. The advantage of the multiplex PCR is to allow simultaneous amplification of all targets with the same input DNA. The digitally-verified DNA was used for the LOD dilutions where the starting mutation percentage was below 1%. The amplified DNA can then be used in multiple singleplex or multiplex ICE COLD-PCR reactions. A constraint of all ICE COLD-PCR reactions is the optimal thermal cycling parameters needed for mutation enrichment and this critical temperature

Poster Session – Clinical Methodology (Tc) is dependent on the sequence context. A Veriti thermal cycler was used for ICE COLD-PCR analysis of EGFR Exons 19, 20, and 21, KRAS Exons 2 and 3, and NRAS Exons 2 and 3 because it can simultaneously perform thermal cycling at 6 different Tc’s on a single 96-well plate. This was followed by hemi-nested PCR using a single thermal cycling program, if required, to provide sufficient sample for Sanger sequencing and NGS using an Ion Torrent. Results: Limits of detection experiments using the Horizon Cell Line DNA and serial dilution of this DNA indicated that samples containing 0.01% mutation in the starting material were easily confirmed using both Sanger and NGS sequencing platforms. This was true for the point mutations as well as the EGFR Exon 19 E746_A750delGGAATTAAGAGAAGC. Concordance of cfDNA and matched FFPE tumor DNA is also presented. Conclusion: ICE COLD-PCR can be used in a multiplex fashion for the sensitive detection of all mutations in a region. This is important when investigating regions with multiple mutations such as EGFR Exon 19 deletions. The sensitivities achieved indicate that ICE COLD-PCR is an ideal tool for detection of low level mutations found in cfDNA and potentially circulating tumor cells. The ability to confirm these mutations by either Sanger or NGS platforms allows flexibility in rapid confirmation when few or many gene regions need to be interrogated. 228 POSTER (Board P008) Her2−3 heterodimer is a new and better than HER2 IHC score for clinical outcome prognosis G. Weitsman1 , P.R. Barber2 , K. Lawler3 , C. Gillett4 , N. Woodman4 , B. Kholodenko5 , L.K. Nguyen5 , T. Santra5 , B. Vojnovic2 , T. Ng1 . 1 Richard Dimbleby Department of Cancer Research Randall Division & Division of Cancer Studies Kings College London, Guy’s Medical School Campus, London, United Kingdom; 2 CRUK & MRC Oxford Institute for Radiation Oncology Gray Laboratories, Department of Oncology, Oxford, United Kingdom; 3 Institute for Mathematical and Molecular Biomedicine Kings College London, Guy’s Medical School Campus, London, United Kingdom; 4 Research Oncology Division of Cancer Studies King’s College London, Guy’s Hospital, London, United Kingdom; 5 Systems Biology Ireland at UCD Conway Institute, University College Dublin, Dublin, Ireland The efficacy of anti-Her2 therapies in Her2-positive breast cancer patients is proven and well documented. However, some patients with Her2-negative tumours also benefit from the same therapies (NSABP B-31) and there is no solid hypothesis to explain those observations. Furthermore, the definition of positive vs. negative Her2 status actually reflects overexpression of Her2 above levels detected in normal and non-malignant tissues. Her2 can heterodimerize with other members of the EGFR family, regardless of expression levels, but the dimerization is dependent upon availability of ligand(s). Her2-Her3 dimer has been shown to drive proliferation of breast cancer cells. We have developed a new FRET/FLIM (Forster ¨ resonance energy transfer/fluorescence-lifetime imaging microscopy) based assay reporting protein–protein interaction at distance below 10 nm for detection of Her2-Her3 dimer in formalinfixed paraffin embedded (FFPE) patient samples. Fluorophore labelled antibodies against Her2 and Her3 allow us to measure a FRET signal, which is dependent upon the number of interacting molecules. Using FFPE samples from the METABRIC cohort, we found that the interaction between two proteins does not correlate with expression levels of interacting partners as judged using standard IHC scoring system. The FRET signal measurements were evenly distributed across all samples with 0, 1, 2 and 3-plus scores for Her2 expression. Mathematical modelling suggests that the absence of correlation between Her2 and Her3 protein and their dimer levels, although can be quite unintuitive, is in principle possible, particularly under scenarios when both Her2 and Her3 compete for binding with other receptor tyrosine kinases including the other ErbB family receptors. Interestingly, we also did not find any correlation with known genetic signatures associated with cancer progression. However, a low FRET signal significantly correlated with longer metastasis free survival, when patients with metastatic events up to 10 years are considered. Our findings may pave the way for better understanding of the biology of EGFR family receptor’s adaptation to drug treatment, helping to predict individual patient response to select the right patient for appropriate treatment. 229 POSTER (Board P009) DNA methyltransferase 1 expression in human solid tumors and lymphomas by immunohistochemistry S.X. Yang1 , D. Nguyen1 , L. Rubinstein1 , S. Kummar1 , J.E. Tomaszewski1 , J.H. Doroshow1 . 1 National Cancer Institute NIH, Division of Cancer Treatment and Diagnosis, Bethesda Maryland, USA Background: CpG island methylation in some tumor suppressor genes has been linked to the development or progression of certain human

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cancers. DNA methyltransferase 1 (DNMT1) is the most abundant enzyme among the three members of the DNMT family. Anti-tumor activity of DNMT inhibitors has been demonstrated largely in myelodysplastic syndrome and acute myeloid leukemia in unselected patient populations, and less frequently in solid tumors. Currently, there are no biomarkers capable of predicting efficacy of DNMT inhibitors and available for prospective patient selection. This study was undertaken to investigate the expression of DNMT1, the putative target of some DNMT inhibitors, in a spectrum of human malignancies in an effort to identify a biomarker for efficacy. Materials and Methods: NCI-H23 non-small cell lung cancer cells were treated with and without a novel DNMT inhibitor, 4 -thio-5-aza-2 deoxycytidine (5-aza-T-dCyd), and changes in DNMT1 expression were assessed by western blot and immunocytochemistry (ICC). Expression of DNMT1 was also examined by immunohistochemistry in formalin-fixed and paraffin-embedded primary lung and ovarian cancers as well as lymphomas. Results: Expression of DNMT1 in NCI-H23 cells was nearly depleted by treatment with 1 mM aza-T-dCyd for 96 h revealed by both western blot and ICC (mean staining index, 56.7 versus 4.3; P<0.0001; 2-sided t test). These suggest that 5-aza-T-dCyd is a potent novel DNMT inhibitor and the antibody used detects the difference, indicative of its specificity. DNMT1 was primarily localized in the nucleus, and constitutively expressed in tumor cells at intermediate and high levels in 12.5% (7/56) of lung cancers, 7.7% (3/39) of ovarian tumors, and 15% (12/78) of lymphomas across histologies including diffuse B-cell, Burkitt-like, T-cell, lymphocyte predominant Hodgkin’s, or mixed cellularity Hodgkin’s lymphoma. Conclusions: We have established a DNMT1 immunohistochemical assay that covers a dynamic range from undetectable to weak, intermediate or strong staining in paraffin-embedded tumor samples. The data demonstrate that intermediate to high levels of DNMT1 expression are found in 15% or fewer patients with either lung cancers, ovarian tumors or lymphomas. The application of this assay holds promise for evaluating DNMT1 expression levels as a potential pharmacodynamic and efficacy biomarker for DNMT inhibitors.

230 POSTER (Board P010) High correlation between clinical responses to first line AML patients treated with cytarabine and idarubicin and their pharmacological profiles in patient samples measured by ExviTech J. Ballesteros1 , P. Hernandez1 , D. Primo2 , A. Robles1 , A.B. Espinosa2 , E. Arroyo2 , V. Garcia-Navas1 , J. Sanchez-Fenoy1 , M. Jimenez1 , M. Gaspar1 , J.L. Rojas1 , J. Martinez-Lopez3 , J. Gorrochategui1 . 1 Vivia Biotech, Tres Cantos-Madrid, Spain; 2 Vivia Biotech, Salamanca, Spain; 3 Hospital 12 de Octubre, Hematology, Madrid, Spain Background: Complete remission (CR) after induction therapy is the first treatment goal in acute myeloid leukemia (AML) patients. The aim of this study is to determine the ability of the Vivia’s novel ex vivo drug sensitivity platform Exvitech to predict the CR rates after induction chemotherapy with cytarabine (Ara-C) and idarubicin (Ida) in 1st line AML. Material and Methods: Bone marrow samples from adult patients diagnosed with de novo AML in Spanish centers from the PETHEMA group were included. Whole marrow samples were incubated for 48 h in well plates containing Ara-C, Ida, or their combination. Pharmacological responses are calculated using pharmacokinetic population models. Induction response was assessed according to the Cheson criteria (2003). Patients attaining a CR/CRi were classified as responders and the remaining as resistant. Results: 180 patient samples were used to calculate the dose–response (DR) curves for Ara-C alone, Ida alone, and their synergism. For clinical correlation we used 77 patients with a median age of 55 years. Many samples had a significant number (>20%) of resistant cells to Ara-C. This is a strong clinical predictor of resistance because in the patient the drug will never be present at these high doses for 48 h. The second variable that is a good predictor of response is the synergism between these 2 drugs. The generalized additive model identified an algebraic combination of these 2 variables that yielded the best marker to separate both groups of patients. The probability density functions had minimal overlap. The area under the corresponding ROC curve was 0.935 (0.872, 0.997), and the classification probabilities for the optimal cut point, were 87% (68%to 95%) and 91% (80% to 96%) for sensitivity and specificity, respectively. 54 patients (70.1%) achieved CR after Ida+Ara-C, and the remaining 23 (29.9%) were resistant. 20 of the 23 (86.9%) patients who fail to achieve CR were predicted as resistance in the ex vivo test. 49 of the 54 patients (90.74%) who achieved CR showed good ex vivo sensitivity to Ida+Ara-C predicting for CR. When the ex vivo test predicted a patient as sensitive it was correct in 49/52 cases (94.23%), and when it predicted resistant it was correct 20/25 cases (80%). Overall, 69/77 patients (89.61%) had an accurate prediction of their response to treatment.

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Conclusions: This novel ex vivo pharmacological profile test is able to predict the clinical response to Ida+Ara-C induction. We are increasing the number of patients in this ongoing study, and we are planning a personalized medicine test-adapted Clinical Trial. 231 POSTER (Board P011) Clinical pharmacodynamic assay development for the first in class investigational ubiquitin activating enzyme (UAE) inhibitor MLN7243 B. Bahamon ´ 1 , F. Gao2 , B. Stringer3 , Y. Yang3 , J. Shi4 , K. Burke5 , J. Huck4 , T. Traore4 , D. Bowman5 , H. Danaee1 , M. Millhollen5 , M. Hyer4 , N. Bence5 , Y. Ishii1 . 1 Takeda Pharmaceuticals International Co., Translational Medicine, Cambridge, USA; 2 Takeda Pharmaceuticals International Co., Biostatistics, Cambridge, USA; 3 Takeda Pharmaceuticals International Co., Molecular Pathology, Cambridge, USA; 4 Takeda Pharmaceuticals International Co., Cancer Pharmacology, Cambridge, USA; 5 Takeda Pharmaceuticals International Co., Molecular & Cellular Oncology, Cambridge, USA MLN7243 is a first in class investigational small molecular inhibitor of the ubiquitin-activating enzyme (UAE) that is currently in Phase I. UAE controls cellular ubiquitin conjugation and inhibition of UAE by MLN7243 induces cell cycle arrest, endoplasmic reticulum stress, defects in DNA repair pathways and ultimately apoptosis. In nonclinical studies, MLN7243 behaves as a strong apoptosis inducer in vitro and also exhibited antitumor activity against a broad range of human tumor xenograft models representing solid and hematologic tumor types. The first in human trial of MLN7243, a Phase I dose escalation study in patients with advanced solid tumors is underway. Here we describe development of pharamacodynamic (PD) biomarker assays for clinical studies of MLN7243. PD immunohistochemistry (IHC) biomarkers were selected to examine MLN7243 target engagement (MLN7243ubiquitin adduct) as well as pathway inhibition (cellular polyubiquitin and monoubiquitin histone 2B (Ub-H2B)). Each IHC assay was optimized and tested for dynamic range using a panel of xenograft samples treated with MLN7243 for varying time points. Reproducibility and specificity were calculated using xenograft tumor tissues of known varying expression (5 low, 5 medium and 5 high expressers for polyubiquitin or Ub-H2B). Baseline expression of the pathway inhibition biomarkers using resected human tumor samples from four cancer types (non-small cell lung cancer, ovarian, colon and breast; each n = 30) was evaluated. The stained slides were scanned using the automated Aperio System, relevant tumor area was selected by a pathologist and analyzed by Definiens Software for positive index (% positive) and histological score. All three PD biomarker assays displayed a large dynamic range, small day-to-day and operator-to-operator variation (largest CV = approx. 13%) in both positive index (% positive) or histological scores in a xenograft model. Power to detect 20% change in both positive index (% positive) and histological score was calculated for each marker to determine the number of slides needed in the clinical study (MLN7243-ubiquitin adduct = 2, polyubiquitin = 5, Ub-H2B = 4) based on assay variability. Polyubiquitin and Ub-H2B image analysis revealed a wide variation within each cancer type while between cancer types variation was relatively small (similar mean/median) with the exception of ovarian cancer. It is possible that the baseline variation stems from differences in sample fixation, storage and age or a biological variation in basal ubiquitin pathway flux between patient samples. Supported by these studies, these PD biomarker IHC assays have been implemented in the MLN7243 first in human study. 232 POSTER (Board P012) Development of a targeted NGS assay system for patient enrollment to the NCI-MATCH study M. Williams1 , D. Sims1 , J. Lih1 , A. Datta1 , S. Hamilton2 , A.J. Iafrate3 , J. Sklar4 , S. Sadis5 , N. Takabe6 , J. Tricoli7 , J. Doroshow8 , B. Conley7 . 1 National Cancer Institute-Frederick, Molecular Charterization and Clinical Assay Development Laboratory, Frederick, USA; 2 MD Anderson Cancer Center, Molecular Pathology, Houston, USA; 3 Massachusetts General Hospital, Molecular Pathology, Boston, USA; 4 Yale University Medical Center, Molecular Pathology, New Haven, USA; 5 Life Technologies, Compendia, Foster City, USA; 6 NCI, CTEP, Bethesda, USA; 7 NCI, CDP, Shady Grove, USA; 8 NCI, DCTD, Bethesda, USA Background: NGS provides a tool for multi-analyte testing and is well suited for identifying predictive biomarkers. The NCI and ECOG-ACRIN are leading an effort to launch a U.S. national cancer trial for the treatment of cancer patients who have progressed after standard treatments. NCIMATCH will encompass multiple (20+) treatment arms and rely on an NGS screening assay to identify biomarkers with sufficient levels of evidence associated with treatment selection. This study will be open to all of the

Poster Session – Clinical Methodology U.S. Oncology Cooperative groups. Patient treatment will be open at all NCTN Cancer Centers (>2,400 sites) including community cancer centers. Four clinical laboratories have been selected and have established a clinical laboratory network. Material and Methods: The screening NGS assay uses the Oncomine Cancer Panel NGS Assay which is a targeted NGS assay. Patient biopsies will serve as the source of RNA and DNA for the assay. A single tissue preanalytics center will process all specimens prior to shipment to the clinical laboratories. NGS data will be uploaded to a central data analysis pipeline, where data will be mapped and variants called. Treatment actionable variants will be identified. Upon laboratory verification of the variant calls, the data will uploaded to a rules engine, ‘MATCHBOX’, where treatment will be selected and the results sent to the treating physician. Details of this process will be discussed. Results: The NGS assay will assess 137 genes (including gain of function and loss of function/tumor suppressors). The assay will report out approximately 10,822 different cancer variants (SNV, small indel, large indel, CNV and gene fusions). Details of the levels of evidence required for selection of treatment actionable variants will be provided. Feasibility studies will be completed and data will be presented demonstrating the performance of the NGS assay from the four clinical laboratories. The analytical validation plan will be discussed. 233 POSTER (Board P013) Analytical validation and application of the MPACT assay, a next generation sequencing based targeted mutation detection assay for treatment selection C. Lih1 , D.J. Sims1 , R.D. Harrington1 , E.C. Polley2 , Y. Zhao2 , R.M. Simon2 , M.G. Mehaffey1 , T.D. Forbes1 , W.D. Walsh1 , V. Datta1 , B.A. Conley3 , A.P. Chen4 , S. Kummar4 , J.H. Doroshow4 , P.M. Williams1 . 1 Leidos Biomedical Inc. Frederick National Laboratory for Cancer Research, Molecular Characterization Lab, Maryland, USA; 2 National Cancer Institute, Biometric Research Branch, Maryland, USA; 3 National Cancer Institute, Cancer Diagnosis Program, Maryland, USA; 4 National Cancer Institute, Division of Cancer Treatment and Diagnosis, Maryland, USA Background: Robust and analytically validated assays are essential for development of molecular targeted cancer therapies. Here we described the development, analytical performance and application of a clinical diagnostic assay for MPACT (Molecular Profiling based Assignment of Cancer Therapeutics) trial. MPACT is a pilot and randomized trial that applies a next generation sequencing assay to select therapy. Materials and Methods: Using Life Technologies’ Ampliseq technology and PGM sequencer, we developed a custom NGS assay, called the MPACT assay and assessed the performance metrics (sensitivity, specificity, accuracy, reproducibility) for each type of actionable mutations of interest. The assay utilizes core needle biopsies shipped and processed as formalin fixed paraffin embedded specimens. Results: The MPACT assay interrogates a total of 59,150 bp that represents 391 treatment actionable variants. The rules for treatment selection will be discussed. The analytical sensitivity study showed the MPACT assay achieved 100% sensitivity for SNVs, SNVs at homopolymeric region (HP), and large Insertions and deletions (large indels, >3 bp) and 83.3% for indels and 93.3% in Indels at HP. Confirmation of 39 known variants by Sanger sequencing demonstrated 95% accuracy. The MPACT assay achieved 100% specificity for all 5 variant types. Inter and intraoperator concordance was 100% and 97.15% respectively with greater than 0.99 R square values in allele frequency correlation for detected variants. Analysis of 10 clinical specimens that contained 11 known mutations identified previously by another validated sequencing assay demonstrated the MAPCT assay identified all known mutations. By testing 10 core needle biopsies biopsied 4 independent times by two operators, we demonstrated the MPACT assay showed 100% reproducibility in the detected mutations and treatment selection between all replicates. These data were submitted to the FDA as part of the study IND. Recent upgrades of the data analysis pipeline to TS4.0 have increased sensitivity and specificity of the assay to 100%. The MPACT Study is ongoing and data will be presented discussing the frequency of detection of treatment actionable variants and overall assay success rates. Conclusions: This validation study demonstrated the MPACT assay was well-suited for the intended investigational trial use. The MPACT Study is open and aMOI incidence will be presented.

Poster Session – Clinical Methodology 234 POSTER (Board P014) Kinetic analysis of dynamic 11 C-verapamil PET study: Compartmental v adaptive mixture models comparison F. Hernandez1 , D. Hawe1 , S. Murphy1 , J. O’Sullivan1 , E. Wolsztynski1 , J. Huang1 , M. Muzi2 , J. Eary3 , K. Krohn2 , F. O’Sullivan1 . 1 University College Cork, Statistics, Cork, Ireland; 2 University of Washington, Radiology, Seattle, USA; 3 University of Alabama, Radiology, Birmingham, USA Background: Temporal aspects of dynamic positron emission tomography (PET) generally rely on compartmental models (CM), with questionable assumptions to summarise functional kinetics of injected radio-tracers in living tissues. Material and Methods: The tracer signal time-course is a combination of vascular delivery and tissue retention effects which do not always satisfy the assumptions of tissue homogeneity and instant mixing within compartments required by the CM. This tissue activity time-course can typically be expressed as a convolution between the signal of the tracer in the arterial supply and the tissue residue function. The residue represents the amount of tracer remaining in the tissue and provides a description of the tracer kinetics measurable by the PET scan. Thus, in statistical terms, the residue can be thought of as a survival function for the residence of the tracer in the tissue which does not require the physiological constraints intrinsic in the exponential-like residue needed for CM. Accordingly more flexible novel approaches − nonparametric and later adaptive mixture models − to estimate the residue based on a piecewise linear form have been developed. The new approaches have been successfully probed on two of the most well-established PET radiotracers, 15 O-H2 O and 18 F-fluorodeoxyglucose, used for perfusion and glucose metabolism respectively. This study shows the extension of the new approaches to the multiple-drug resistance transporter P-glycoprotein (P-gp) highly present at the blood–brain barrier (BBB) using 11 C-Verapamil (Vp) in healthy humans before and after inhibition of the P-gp by Cyclosporine (CsA) infusion. Previous PET studies on measuring BBB activity by CsA infusion using Vp have revealed difficulties for the CM to recover metabolic information as they require a full understanding of the tracers metabolisation in the tissue, which is still not certain for Vp. This problem is especially latent when the inhibition of the P-gp allows the tracer through the BBB where it is retained once at the brain tissue level. Results: This work examines and evaluate adaptive mixture model as an alternative to conventional compartment techniques in recovering metabolic information from dynamic PET studies with less biological restrictions. Key bioparameters such as flux, flow, blood volume and volume of distribution from the two different approaches are compared. Cross-validation is used to make regional comparisons and evaluate the fit of the two different estimated residue functions to the tissue activity curves. Conclusions: The accuracy of the standard models for Vp PET studies can be questionable, likely because behaviour of Vp metabolites in tissue is still unclear. Significant statistical evidence in favour of the new adaptivemixture models has been found. Supported by Science Foundation Ireland under SFI-PI 11/27 and by the National Institute of Health (NCI) under PO1-CA-42045. 235 POSTER (Board P015) Kinetic analysis of dynamic 11 C thymidine PET imaging studies: Compartmental and nonparametric approaches S. Murphy1 , D. Hawe1 , F. Hernandez1 , E. Wolsztynski1 , J. Huang1 , J. O’Sullivan1 , M. Muzi2 , J. Eary3 , K. Krohn2 , F. O’Sullivan1 . 1 University College Cork, Statistics, Cork, Ireland; 2 University of Washington, Radiology, Seattle, USA; 3 University of Alabama, Radiology, Birmingham, USA Background: Positron emission tomography (PET) with 11 C thymidine (TdR) as a radiotracer has been used to quantify cell proliferation and DNA synthesis in a variety of cancer types such as malignant brain tumours. Materials and Methods: Given time-dependent records of the tracer activity in blood and tissue, compartment models are used to describe the mechanisms for transfer and metabolism of the radiotracer. These models are based on assumptions such as homogeneity of the tissue and instant mixing within compartments, which can be difficult to justify. This may lead to the model being a poor fit to the data and hence give misleading results. A nonparametric approach was developed to alleviate these restricting assumptions. This technique has been validated in the case of the 15 OH2 O and the 18 F-fluorodeoxyglucose (FDG) PET radiotracers. For TdR, this estimation problem is more complex as the tracer is metabolised in the body, which leads to the presence of both TdR and metabolites in blood and tissue, with the activities of each being jointly observed by the scanner. Therefore a multiple-input function compartment model is required to capture and summarise metabolic activity. Calibration of the

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multiple-input compartment model is achieved using an arterial time-course measured by arterial sampling, which allows the activity in the arterial blood to be separated into TdR and metabolites. An adapted version of the nonparametric approach that is used for 15 O-H2 O and FDG is applied to the TdR data. Results: Simulations were undertaken to examine the efficacy of the multiple input compartment model and also to gain an understanding of the rates of convergence of the parameters of interest. Similar simulations are carried out on the nonparametric model. Comparisons between the models are generated by cross-validation. In TdR-PET studies the key parameter of interest is the thymidine flux constant. Further numerical analysis was carried out to examine the bias and variance of the models for this parameter as well as others estimated in this modelling. Conclusions: Residual analysis shows non constant variance for the compartment model, which leads one to conclude the validity of the model is questionable. Performance analysis of the nonparametric method suggests it is a relevant candidate alternative for the estimation of flux as imaged by thymidine. Supported by Science Foundation Ireland under SFI-PI 11/27 and by the National Institute of Health (NCI) under PO1-CA-42045. 236 POSTER (Board P016) Whole genomic assay on endoscopic ultrasound-guided fine needle aspiration samples of unresectable pancreatic cancer J.M. Ha1 , K.H. Lee1 , J.K. Lee1 , K.T. Lee1 , W.Y. Park2 , J.S. Bae2 , J.K. Jung2 , D.H. Park2 , Y.K. Seong3 , E. Kim1 . 1 Samsung Medical Center, Gastroenterology, Seoul, South Korea; 2 Samsung Medical Center, Genomic Medicine, Seoul, South Korea; 3 Sung Kyun Kwan University Medical School, Internal Medicine, Seoul, South Korea Background: Targeted therapy according to an individual’s genetic profile can improve the oncological outcomes. Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) has been widely used to obtain tumor tissue to diagnose unresectable pancreatic cancer. We tested the feasibility of whole genomic assay on EUS-FNA samples for personalized therapy of unresectable pancreatic cancer. Material and Methods: Whole exome and whole transcriptome sequencing were performed with ten EUS-FNA samples from retrospectively collected pancreatic cancer (9 ductal adenocarcinomas, 1 undifferentiated carcinoma). We compared these results with the reported genomic and transcriptomic profiles of 57 pancreatic cancer tissues in The Cancer Genome Atlas (TCGA) to find novel mutations, differentially expressed genes and gene fusions. Results: Genomic profiles were successfully generated from 7 of 10 (70%) pancreatic EUS-FNA samples by whole genomic sequencing. The frequency of observed genomic mutations was concordantly distributed with that of TCGA; KRAS, TP53, RNF43, SMAD4, MEN1, MLL3, APC and ARID1A, sequentially, and the cluster dendrogram of combining 7 EUSFNA samples’ profiles with TCGA data showed intergroup similarity after TMM (trimmed mean of M-values) normalization. We also identified several new fusion genes from analysis of our whole transcriptome dataset. Conclusions: We were able to perform whole genomic and transcriptomic assay on most EUS-FNA samples from pancreatic cancers and identify that genomic alterations were well correlated with those listed in TCGA of pancreatic cancer. 237 POSTER (Board P017) Impact of phase 1 expansion cohorts on probability of success in phase 2 and time-to-drug-approval: analysis of 385 new drugs in oncology D. Bugano1 , K. Hess2 , L.L. Siu3 , F. Meric-Bernstam4 , A.R.A. Razak3 , D.S. Hong4 . 1 MD Anderson Cancer Center, Hematology/Oncology Fellowship, Houston, USA; 2 MD Anderson Cancer Center, Department of Biostatistics Division of Quantitative Sciences, Houston, USA; 3 Princess Margaret Cancer Centre, Drug Development Program, Toronto, Canada; 4 MD Anderson Cancer Center, Department of Investigational Cancer Therapeutics, Houston, USA Background: Expansion cohorts (EC) are becoming more common in the design of oncology phase 1(P1) trials. Our objective was to determine their impact on phase 2 (P2) trials and on time-to-drug-approval (TDA). Material and Methods: Systematic review of MEDLINE and EMBASE for P1 trials and their corresponding P2 trials. We included single-agent dosefinding adult oncology P1 trials published in 2006–2011. ECs were defined as the enrollment of additional patients (pts) after identification of the P2 dose. Drug approval status and date were based on the FDA website by 04.31.2014. TDA was measured since the first publication of a P1 trial. Positive P2 trials were those that met their primary endpoints. ‘Probability

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Poster Session – DNA Repair Modulation (including PARP, CHK, ATR, ATM)

Table (abstract 237): Characteristics of phase 1 trials, ‘probability of positive phase 2’ and ‘time-to-drug-approval’

Targeted agent Industry-sponsored Multicenter Pub >2008 Pts in dose escalation part Tumor type

Pts in EC

Y vs N Y vs N Y vs N Y vs N 21−37 vs <21 >37 vs <21 Hematologic vs solid Hematologic + solid vs solid Specific histology vs any solid 2−20 vs 0 21–271 vs 0

Prob Positive P2 OR (95% CI)

p

Time-to-approval HR (95% CI)

p

0.9 1.7 0.9 1.0 0.9 1.4 4.1 0.6 1.2 2.4 3.3

0.78 0.12 0.73 0.84 0.62 0.31 0.03 0.36 0.49 0.0054 0.0008

1.0 2.1 2.4 1.0 0.9 1.4 2.4 0.6 2.1 2.1 6.6

0.95 0.33 0.17 0.94 0.88 0.46 0.17 0.62 0.066 0.14 <0.0001

(0.5; 1.6) (0.9; 3.2) (0.5; 1.6) (0.7; 1.7) (0.5; 1.5) (1.3; 4.4) (1.1; 14) (0.2; 1.7) (0.7; 2.0) (1.3; 4.4) (1.6; 6.6)

(0.4; 2.4) (0.5; 9.5) (0.7; 8.5) (0.5; 2.2) (0.3; 2.6) (0.6; 3.4) (0.7; 8.8) (0.1; 4.6) (1.0; 4.4) (0.8; 5.4) (2.9; 15)

All values are after multivariate analysis. of positive P2’ was evaluated with logistic regression and TDA with Cox proportional hazards regression. Results: We identified 515 P1 and 608 P2 trials. There were 385 drugs and 115 (30%) had at least one P1 with EC. A higher proportion of drugs with EC moved into P2 (62% v 37%, p < 0.0001) and had at least one randomized P2 trial (34% v 15%, p = 0.0001). We classified drugs according to the combined number of pts enrolled in all EC for different trials of that drug: No EC (267 drugs), EC2 − 20pts (60), EC21 − 271pts (44), missing (14). Enrolling more than 20 pts in EC was associated with higher probability of a positive P2 and shorter TDA (table). The probabilities of approval at 5 years were: No EC 5%(2−8%), EC2−20 16%(4−26%), EC21– 271 31%(15−44%). Conclusions: Expansion cohorts were associated with a higher probability of positive phase 2 trials and shorter time-to-drug-approval. Factors involved in the decision to open an expansion cohort might influence this association.

DNA Repair Modulation (including PARP, CHK, ATR, ATM) 238 POSTER (Board P018) Reversal of primary and acquired PARP-inhibitor resistance in BRCA-mutated triple-negative breast cancers by inhibition of transcriptional cyclin-dependent kinases (CDKs) S. Johnson1 , N. Johnson2 , D. Chi3 , B. Primack4 , C. Cruz5 , D. Stover1 , ˜ 5, A.K. Greifenberg6 , S. Cao7 , K. O’Connor1 , J. Baselga8 , J. Balmana V. Serra5 , M. Geyer6 , A. D’Andrea9 , E. Lim10 , G.I. Shapiro1 . 1 Dana-Farber Cancer Institute, Department of Medical Oncology, Boston, USA; 2 Fox Chase Cancer Center, Clinical Therapeutics, Philadelphia Pennsylvania, USA; 3 Dana-Farber Cancer Institute, Department of Medical Oncology, Boston MA, USA; 4 Dana-Farber Cancer Institute, Department of Radiation Oncology, Boston MA, USA; 5 Vall d’Hebron Institute of Oncology, Experimental Therapeutics Group, Barcelona, Spain; 6 Max Planck Institute of Molecular Physiology, Department of Physical Biochemistry, Bonn, Germany; 7 Dana-Farber Cancer Institute, Medical Oncology, Boston, USA; 8 Memorial Sloan-Kettering Cancer Center, Human Oncology & Pathogenesis Program, New York City, USA; 9 Dana-Farber Cancer Institute, Radiation Oncology, Boston, USA; 10 Ludwig Institute of Cancer Research, Olivia Newton John Cancer & Wellness Center, Melbourne, Australia Background: PARP-1 is synthetically lethal with homologous recombination (HR) defects. Tumors harboring mutations in BRCA1 and BRCA2 show sensitivity to PARP inhibition to varying degrees. Additionally, acquired resistance to PARP inhibition occurs via emergence of reversion mutations, re-expression of mutated BRCA proteins or alterations in expression of negative HR regulators. CDKs regulate cell cycle progression and transcription, and recent evidence has shown that cyclin K-CDK12 controls transcription of HR genes. We show that CDK12 is a target of the CDK inhibitor dinaciclib, which causes transcriptional repression of multiple DNA damage response and repair pathways. CDK inhibition is known to confer HR deficiency to HR-competent tumors, rendering them PARP inhibitor sensitive. Here, we tested whether dinaciclib-mediated transcriptional inhibition could (1) augment the response of BRCA-mutated triple-negative breast cancer (TNBC) cell lines and patient-derived xenograft (PDX) models that demonstrate modest sensitivity to PARP inhibitor monotherapy; or

(2) restore PARP-inhibitor sensitivity in BRCA-mutated TNBC models that have acquired PARP inhibitor resistance. Results: In MDA-MB-231 cells, dinaciclib reduced phosphorylation of the C-terminal domain of RNA polymerase II in a concentration-dependent manner. RNA was collected from vehicle and dinaciclib-treated cells and levels of global transcription expression change were analyzed using Affymetrix U133A 2.0 arrays. Ingenuity Pathway Analysis (IPA) was used to assess networks of gene transcripts significantly repressed after dinaciclib. The top canonical pathways downregulated were comprised of DNA damage response networks. CDK12 kinase assays using pSer7 CTD as substrate were performed in the presence of vehicle or dinaciclib, demonstrating potent inhibition with IC50 61.8 nM. Dinaciclib was used in combination with veliparib or olaparib in BRCAmutated cell lines and PDX models. These combinations were synergistic in HCC1937 and SUM149 TNBC cells (modestly sensitive to PARP inhibitor monotherapy), as well as in a PARP inhibitor-resistant derivative of highly sensitive MDA-MB-436 cells. Additionally, these combinations demonstrated substantial efficacy in two PDX models with somatic BRCA1 and BRCA2 mutations, and limited de novo PARP inhibitor sensitivity, as well as in a model derived from a BRCA1 germline carrier whose tumor was slowly progressive on PARP inhibitor treatment. Conclusions: PARP inhibitor monotherapy in BRCA-mutated TNBCs is complicated by both de novo and acquired resistance. Transcriptional CDK inhibition represents a promising strategy for augmenting responses and reversing resistance. These results have justified clinical exploration of the combination of dinaciclib and veliparib in both BRCA-proficient and BRCAmutated tumors (NCT NCT01434316). 239 POSTER (Board P019) Niraparib, a selective PARP 1/2 inhibitor, is efficacious in pre-clinical models of small-cell lung cancer Y. Wang1 , J. Ricono2 , K. Admunson2 , S. Agarwal1 , R.J. Fram1 , T. Broudy2 , K.M. Wilcoxen1 . 1 Tesaro Inc., Waltham, USA; 2 Molecular Response, San Diego, USA Purpose: Small cell lung cancer (SCLC) is an aggressive form of cancer that accounts for approximately 15% of all lung cancers. Treatment options have not benefited from the development of currently approved targeted agents, and platinum based chemotherapeutic regimens still dominate treatment in both limited and extensive-stage disease. The PARP inhibitor niraparib, currently in phase 3 studies in ovarian and breast cancer, was investigated for its ability to inhibit the proliferation of SCLC in vitro and in vivo. Methods: Niraparib was evaluated for its ability to inhibit proliferation and colony formation of SCLC cell lines with known deficiencies in p53, Rb1, and PTEN as well as amplifications in FGFR and MYC genes. Low passage SCLC patient-derived xenograft (PDX) models in mice were utilized to evaluate the in vivo efficacy of niraparib as a monotherapy and in combination with common first and second line therapies cisplatin/ etoposide and irinotecan. Molecular characteristics of individual tumors were determined through RNAseq analysis and correlated with niraparib response. Results: Niraparib exhibited in vitro anti-proliferative effects on selected SCLC lines with mutations in p53, Rb1, PTEN, PIK3CA and STK11. However, niraparib activity in these cells was not definitively correlated with a known mutationsor platinum sensitivity. In a SCLC PDX model derived from a metastatic tumor, daily 50 mg/kg niraparib demonstrated antitumor activity in combination with both irinotecan and cisplatin/ etoposide. A cohort treated with a single cycle of etoposide (8 mg/kg, days 1−3) and cisplatin (4 mg/kg, day 1) and 4 weeks of niraparib (50 mg/kg, QD) showed significant tumor growth inhibition. Upon rechallenge with niraparib (75 mg/kg) after a dosing holiday and observable

Poster Session – DNA Repair Modulation (including PARP, CHK, ATR, ATM) tumor regrowth, tumor growth inhibition was again observed despite the presence of large tumors (>500 mm3 ). Additional SCLC PDX models were selected for evaluation of niraparib efficacy based on in vitro sensitivity to platinum agents. Tumor growth inhibition and/or regression during treatment of PDX mice with niraparib (100 mg/kg, QD) was observed in 75% of evaluable models. The determination of niraparib efficacy in SCLC platinum responsive maintenance treatment and molecular characterization of niraparib sensitive models is ongoing. Results will be discussed in the context of developing translational tumor marker classifier candidates for niraparib sensitivity in clinical studies of SCLC. Conclusion: Niraparib exhibited anti-cancer activity against SCLC as an in vitro and in vivo as a monotherapy and in combination with standard of care agents. These results support the clinical investigation of niraparib in SCLC. 240 POSTER (Board P020) The combination of Chk-1 and ATR inhibitor synergistically kills cancer cells K. Sanjiv1 , A. Hagenkort1 , P.M. Reaper2 , T. Koolmeister1 , O. Mortusewicz1 , N. Schultz1 , M. Scobie1 , U.W. Berglund1 , P.A. Charlton2 , J.R. Pollard2 , T. Helleday1 . 1 Science for Life Laboratory Karolinska Institutet, Division of Translational medicine and Chemical Biology MBB, Stockholm, Sweden; 2 Vertex Pharmaceuticals, (Europe), Abingdon Oxfordshire, United Kingdom Inhibition of Chk1 has been reported to lead to phosphorylation of a series of ATR substrates. We therefore hypothesized that a combination of ATR and Chk1 inhibition may provide benefit. Herein we show that treatment of cancer cells with the Chk-1 inhibitor, AZD7762, led to replication stress, which was converted to replication catastrophe and apoptosis when cells were co-treated with the ATR inhibitor VE-821. Synergism was observed in a variety of cancer cell lines but not in normal fibroblast cells. In contrast to previous studies that have shown ATR or Chk-1 inhibitors are most effective in cells defective for p53 function, we observed that the combination of ATR and Chk1 inhibition was equally effective in cancer cells with or without a functional p53 response. Co-treatment with AZD7762 and VE-821 in U2OS cancer cells induced massive loading of RPA onto chromatin and pan nuclear gH2AX, indicative of high levels of ssDNA formation and severe DNA damage. This was associated with stalled replication fork progression, S-phase arrest and cell death. In contrast, non-cancer fibroblast VH-10 cells tolerated the combination well with no DNA damage or cell death observed. Under conditions of induced replication stress, by treatment with hydroxy urea, AZD7762 plus VE-821 caused marked nuclear fragmentation and early onset of apoptosis in the U2OS cancer cells but not in normal fibroblast cells. In mouse xenograft models of lung and breast cancer, treatment with VE-822 (an analog of VE-821) in combination with AZD7762 significantly delayed tumor growth and increased overall survival when compared with mice treated with vehicle or either agent alone. These data support the clinical development of ATR and Chk-1 inhibitor combinations to complement existing DNA damage based chemotherapy. 241 POSTER (Board P021) Epigenetic loss-of-function BRCA1 mediates tumor cure by single dose radiotherapy C. Campagne1 , T.H. Thin1 , J.D. Fuller2 , K. Manova-Todorova3 , A. Haimovitz-Friedman1 , S.N. Powell1 , R.N. Kolesnick2 , Z. Fuks1 . 1 Memorial Sloan-Kettering Cancer Center, Radiation Oncology, New York, USA; 2 Memorial Sloan-Kettering Cancer Center, Molecular Pharmacology and Chemistry, New York, USA; 3 Memorial Sloan-Kettering Cancer Center, Molecular Cytology, New York, USA Background: The mechanism of tumor cure by ionizing radiation is regarded tumor cell autonomous, effected by misrepair of radiation-induced DNA double strand breaks (DSBs) via the function of error prone nonhomologous end joining (NHEJ). This model prevails at the low dose range (8 Gy), with cure depending on tumor propensity for NHEJ misrepair, requiring repeated exposures for tumor ablation. Here we report high (>10 Gy) single dose radiotherapy (SDRT) engages an alternative dual target model. Material and Methods: DSB repair was analyzed in situ by quantitative assessment of the time-dependent buildup and resolution of ionizing radiation-induced foci (IRIF) of specific NHEJ or homology-driven repair (HDR) mediators. Effect of SDRT on the tumor microvasculature was assessed by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). Engagement of microvascular dysfunction in DSB repair was assessed using acid sphingomyelinase (ASMase)-deficient mice, refractory to vascular endothelial injury. Western blot analysis of Small Ubiquitin-like Modifiers (SUMO) in tumor extracts and studies of SUMO conjugating enzymes IRIF in situ were used to evaluate effects of SDRT on SUMOylation.

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Results: SDRT concomitantly induces DSBs in tumor cells and an early wave of ASMase-mediated microcirculatory ischemia/reperfusion (I/R). Reactive oxygen species (ROS) generated therein in parenchymal tumor cells oxidize and dysfunction SUMO conjugating enzymes, leading to catastrophic reprograming of DSB repair. Ku- and Tumor Suppressor p53Binding Protein 1 (53BP1)-mediated NHEJ are not affected, although 53BP1 resolution is delayed. In contrast, HDR is aborted, as SUMO dysfunction impairs recruitment of Receptor-Associated Protein 80 (RAP80), Breast Cancer 1 protein (BRCA1), Replication Protein A (RPA) and RAD51 recombinase into DSB repair foci, reversible by genetic I/R inhibition or pharmacological post-I/R ROS scavenging. The epigenetic loss-offunction BRCA1, coupled with abnormally persistent 53BP1 function, diverts DSB repair to an aberrant 53BP1-dependent lethal pathway, mediating tumor cure. Conclusions: We present a new mechanism of tumor cure by ionizing radiation, in which high SDRT engages a co-dependent dual target model, involving both DNA DSBs and a transient tumor microvasculature injury. It represents an alternative to the classical single target mechanism operating at the low dose range, and provides new targets for modulation of the radiation response, with a potential for yielding new cures in cancer. 242 POSTER (Board P022) BMN 673 as single agent and in combination with temozolomide or PI3K pathway inhibitors in small cell lung cancer and gastric cancer models Y. Feng1 , L.E. Post1 , R. Cardnell2 , L.A. Byers2 , B. Wang1 , Y. Shen1 . 1 BioMarin Pharmaceutical Inc., Research and Drug Discovery, Novato CA, USA; 2 MD Anderson Cancer Center, Thoracic/Head & Neck Medical Oncology, Houston TX, USA BMN 673 is a potent, specific PARP1/2 inhibitor with an antitumor cytotoxic mechanism that includes efficient trapping of PARP-DNA complexes. BMN 673 shows significant clinical activity in patients with germline BRCA mutation ovarian and breast cancer; partial responses were also reported in non-BRCA small cell lung cancer (SCLC) patients treated with BMN 673 (ASCO 2014). To explore BMN 673 anti-tumor activity beyond BRCA, we further investigated the combination potential of BMN 673 with temozolomide (TMZ) or PI3K pathway inhibitors in SCLC and gastric cancer (GC) models. TMZ potentiates the activity of PARP inhibitors including BMN 673 in various tumor models and has shown single-agent activity in SCLC patients. Here we evaluated the combination of BMN 673 with TMZ using human SCLC cell-derived xenograft models. In NCI-H209 xenografts, BMN 673 (0.25 mg/kg, qd×4) plus TMZ at much reduced dosage (3 mg/kg, qd×4) resulted in tumor shrinkage, while each drug alone had no inhibitory effect on tumor growth under the same dosage and schedule. Recent studies show that PARP inhibitor treatment activates the PI3K/mTOR pathway in SCLC cell lines and animal models (CSHL, 2014). We therefore assessed anti-tumor activity of BMN 673 in combination with several PI3K pathway inhibitors including Pan-PI3K, isoform-specific PI3K, and Akt inhibitors in SCLC cells. Additive or weak synergistic effect was observed in different lines based on combination index (CI) value at ED50. In vivo studies are currently being conducted to confirm the in vitro observation. Beyond BRCA1/2 mutations, defects in other components of homologous recombination DNA repair pathway could induce sensitivity to PARP inhibition. Report has suggested that low ATM expression in some GC cell lines may contribute to Olaparib sensitivity in vitro. We examined 14 GC cell lines with various mutational backgrounds and found many of them are highly sensitive to BMN 673 with IC50 values similar to those of BRCA mutant cell lines. Western analysis of protein markers in these cell lines suggests a correlation of BMN 673 sensitivity with reduced ATM protein level and activity (p-ATM). Consistent with the SCLC findings, in vitro assays also demonstrated a synergistic activity of BMN 673 and PI3K pathway inhibitors in some of the GC lines. Together, these findings suggest that combination of BMN 673 with TMZ or PI3K inhibitor(s) have better anti-tumor activity than either single agent alone in SCLC and GC models. 243 POSTER (Board P023) Modulation of PI3K/mTOR pathway following PARP inhibition in small cell lung cancer R.J. Cardnell1 , Y. Feng2 , L. Diao3 , Y. Fan1 , F. Masrorpour1 , S. Mukherjee1 , J. Shen2 , J. Wang3 , L.A. Byers1 . 1 MD Anderson Cancer Center, Thoracic/Head & Neck Medical Oncology, Houston TX, USA; 2 BioMarin Pharmaceutical Inc., Novato CA, USA; 3 MD Anderson Cancer Center, Bioinformatics & Computational Biology, Houston TX, USA Background: Small cell lung cancer (SCLC) is the most aggressive form of lung cancer, accounting for 13% of new lung cancers in the US. We have

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Poster Session – DNA Repair Modulation (including PARP, CHK, ATR, ATM)

previously shown that PARP1 is expressed at high levels in SCLC and that preclinical models of SCLC are sensitive to PARP inhibition. Based on this work, clinical trials of single agent BMN 673 are ongoing and have shown partial responses in a subset of SCLC patients (Wainberg, Z., et al., ASCO 2014). Recently, we also identified an association between higher baseline activation of the PI3K pathway and PARP inhibitor resistance. Here, we investigate changes in PI3K/mTOR pathway signaling in SCLC following PARP inhibition and the impact of combined PARP−PI3K targeting. Material and Methods: >140 total and phospho-proteins were measured by reverse phase protein array (RPPA) pre- and post-treatment with three PARP inhibitors (olaparib, rucaparib, BMN 673) in cell lines and xenograft tumors. Results: In cell lines, treatment with PARP inhibitors caused a significant increase in several phosphorylated proteins in the PI3K/mTOR pathway, including p-mTOR, pAKT and pS6 (p  0.02). This observation was recapitulated in H1048 xenografts treated with BMN 673, with increases in p-mTOR and p-AKT (p  0.02). In contrast, the LKB1 pathway was down-regulated after treatment with decreased levels of LKB1, p-AMPK, and p-TSC (p  0.04). Because LKB1 negatively regulates PI3K/mTOR, reduced levels of LKB1 may play a role in PI3K/mTOR activation. On the basis of these results, we then tested the combination of BMN 673 with the PI3K inhibitor BKM120. Preliminary in vitro analyses show an additive effect of BKM120, with a greater decrease in SCLC proliferation from combination treatment vs either drug alone. Conclusions: In conclusion, we have demonstrated activation of the PI3K/mTOR pathway in response to treatment with multiple PARP inhibitors in SCLC. These results, along with our published work showing greater PARP inhibitor resistance in SCLC with baseline PI3K/mTOR activity, suggest a potential role of this pathway in both primary and acquired PARP inhibitor resistance. We propose that PI3K inhibition may increase clinical activity of PARP inhibitors in SCLC. Based on these results, in vivo testing of PI3K−PARP targeting is ongoing. 244 POSTER (Board P024) Development of xenoimplants from germline BRCA1/2 mutant breast cancer (BC) for the identification of predictive biomarkers, mechanisms of resistance against poly(ADP-ribose) polymerase (PARP) inhibitors and evaluation of novel therapies C. Cruz1 , Y. Ibrahim2 , B. Morancho3 , P. Anton2 , J. Grueso2 , P. Cozar2 , ´ 2 , P.M. Aviles ´ 4 , M.J. Guillen ´ 4 , C. Galmarini4 , J. Arribas3 , M. GuzmAn ˜ 1 , V. Serra2 . 1 Vall d’Hebron Institute of Oncology, J. Baselga5 , J. Balmana High Risk Cancer Prevention Group, Barcelona, Spain; 2 Vall d’Hebron Institute of Oncology, Experimental Therapeutics Group, Barcelona, Spain; 3 Vall d’Hebron Institute of Oncology, Growth Factors Group, Barcelona, Spain; 4 PharmaMar S.A., Non Clinical Pharmacology and Toxicology Department, Colmenar Viejo Madrid, Spain; 5 Memorial Sloan Kettering Cancer Center, NY, New York, USA BRCA1/2-mutant BC is characterized by deficient homologous recombination (HR) DNA repair, resulting in synthetic lethality upon treatment with inhibitors of the single-strand DNA repair enzyme PARP and remarkable clinical responses. Nevertheless, PARP inhibitors (PARPi) have failed to show primary efficacy and/or durable responses in a subset of patients. Several mechanisms of resistance to PARPi have been described both in vitro and in vivo, but their significance in the clinic and their impact on subsequent treatments is unknown. In order to improve current treatment strategies for individual BRCA1/2-mutation carriers, clinically relevant preclinical models are required, which allow the study of PARPiresistance mechanisms, the identification of predictive biomarkers and the assessment of novel therapies. PM01183 is a novel transcription inhibitor with promising activity in BRCA1/2-mutated BC that is currently in phase II clinical trials, and a potential therapeutic strategy for PARPiresistant tumors. Material and Methods: BRCA1/2 mutation carriers with BC or ovarian cancer (OvCa) treated at our institution for primary surgery or metastatic disease were selected for the study and signed the corresponding IRBapproved informed consent. Primary or metastatic tumor samples from these patients were implanted in immunosupressed mice to establish patient-derived xenograft models (PDXs). To characterize PDX sensitivity to the PARPi olaparib, tumor-bearing mice were treated with olaparib 50 mg/kg po 6 days per week or vehicle; bi-weekly tumor growth measurements were performed. Olaparib treatment was maintained in olaparib-sensitive models to generate PDX models with acquired resistance to the PARPi. PM01183 antitumor activity was tested in an olaparibresistant model (PDX196) derived from a BRCA1-mutated OvCa (PM01183 0.18 mg/kg iv per week). Results: Ten PDX models were obtained from BRCA1/2-related cancer patients. Six PDX models were tested for PARPi response with good correlation with the corresponding patient response. PM01183 showed antitumor activity in an olaparib-resistant PDX.

Conclusions: PM01183 might show efficacy in patients with BRCA1/2mutant metastatic disease resistant to PARPi. 245 POSTER (Board P025) Chk1 is a potential novel therapeutic target that regulates cell survival and potentiates chemotherapy in osteosarcoma (OS) models S.J. Strauss1 , P. Mistry1 , A. Mendoza1 , M. Robson1 , H. Holme1 , P. Nandabhiwat1 , B. Kwok2 , M. Qadir2 , R.B. Pedley1 , J.S. Whelan3 , P.H.B. Sorensen2 . 1 UCL Cancer Institute, Dept of Oncology, London, United Kingdom; 2 BC Cancer Research Centre, Dept of Molecular Oncology, Vancouver, Canada; 3 University College Hospital, Dept of Medical Oncology, London, United Kingdom Background: Outcome for patients with osteosarcoma (OS) is improved when chemotherapy is given to treat micrometastatic disease. However, micrometastatic cells differ from those of the primary tumour and appear more resistant to chemotherapy. The aim of this study was to identify potential novel therapeutic targets through the use of genetic loss of function screens in primary and ‘micrometastatic’ in vitro OS tumour models. Methods: An siRNA kinome screen was performed in an OS cell line, MNNG grown as monolayers (M) and ‘micrometastatic’ anchorageindependent cultures (S). Genes that significantly reduced cell survival in both conditions were validated using a panel of OS cell lines and specific inhibitors. Results: Silencing of the cell cycle checkpoint kinase 1 (Chk1) had the most significant effects in M and S cultures inhibiting growth by 60% in 5 of 6 OS cell lines tested. Chk1 is a serine/threonine-protein kinase that regulates S and G2/M phases of the cell cycle. It plays a role in regulation of the DNA damage response and is highly expressed in OS tumours with a poor response to neo-adjuvant chemotherapy (Man et al., Cancer Res, 2005). Here, Chk1 protein was expressed in all OS cell lines. OS cell lines were sensitive to the Chk inhibitor LY2606368 mesylate monohydrate (hereafter LY2606368) with IC50 concentrations in the low nanomolar range (3.4−9.3nM). Chk1 siRNA and LY2606368 caused cell cycle arrest in G1/S by 24 hours, and caspase 3 cleavage indicative of apoptosis. Sub-toxic concentrations of LY2606368 (0.31−5nM) potentiated the effect of methotrexate and doxorubicin in M and S cells. Synergistic effects were most marked when the Chk1 inhibitor was administered 24 hours after the cytotoxic agent and with antimetabolite methotrexate (CI 0.0001–0.03). Combining LY2606368 and doxorubicin or methotrexate resulted in significantly greater gH2AX formation than either drug alone demonstrating increased DNA damage (p < 0.0001). Single agent in vivo activity of LY2606368 was demonstrated in a subcutaneous MNNG OS mouse model with administration of 4 mg/kg twice daily for 4 of 7 days for 3 weeks, significantly delaying tumour growth (p < 0.01) with no significant weight loss. Conclusions: Inhibition of Chk1 is a potentially valuable therapeutic strategy in OS and warrants further validation. 246 POSTER (Board P026) PARP inhibitors trap PARP1 onto damaged DNA via catalytic inhibition and not by an allosteric mechanism T. Hopkins1 , L. Solomon1 , Y. Shi1 , L. Rodriguez1 , C. Donawho1 , E. DiGiammarino1 , S. Panchal1 , A. Olson1 , D. Stolarik1 , D. Osterling1 , W. Gao1 , E. Johnson1 , D. Maag1 . 1 AbbVie, Oncology Discovery, North Chicago IL, USA Background: PARP inhibitors potentiate the cytotoxicity of DNA alkylating agents in vitro and in vivo. Trapping of PARP1 onto single-strand breaks in the presence of PARP inhibitors appears to be related to this increased cytotoxicity of PARP inhibitor-alkylator combinations. Two models have been proposed to explain this effect. In the first, catalytic inhibition of PARP1 prevents auto-PARylation, which is known to promote dissociation of PARP1 from DNA. In the second, PARP inhibitors allosterically enhance the affinity of PARP1 for damaged DNA. Direct evidence of allostery has yet to be reported. The aim of this study was to probe the relative contributions of allostery and catalytic inhibition to the trapping phenomenon. Methods: In vivo efficacy and pharmacokinetics were evaluated in HeyA8 xenograft tumor-bearing SCID mice. PARP1 trapping was evaluated by cellular fractionation, TR-FRET and BLI. Results: Veliparib markedly potentiates the activity of TMZ in vivo in a HeyA8 xenograft model. PARP inhibitors (veliparib, olaparib and BMN-673) all potentiate the activity of MMS and TMZ and trap PARP1 in HeyA8 cells in vitro. PARP inhibitors do not directly stabilize the PARP1-DNA complex. Complex stabilization is achieved by inhibition of auto-PARylation in the presence of NAD+ . In HeyA8 cells, NAD+ depletion via NAMPT inhibition traps PARP1 as effectively as PARP inhibitors. PARP inhibitors do not enhance PARP1 trapping after NAD+ depletion.

Poster Session – DNA Repair Modulation (including PARP, CHK, ATR, ATM) Conclusions: These studies reveal no evidence for an allosteric trapping mechanism and indicate that all PARP inhibitors examined trap PARP1 via catalytic inhibition. The potency of PARP inhibitors with respect to trapping and catalytic inhibition is linearly correlated in biochemical systems. In cells, trapping potency is related to concentrations required for potentiation in animal models. Detection of PARP trapping in cells requires supraphysiologic conditions that exhaust cellular NAD+ and PAR, exceed concentrations required to elicit synergism and are not tolerated in vivo. In addition trapping potency appears to be inversely correlated with tolerability. Quantitation of the degree of trapping that is tolerable and is required for therapeutic benefit is under active investigation. Disclosures: All authors are employees of AbbVie. The design, study conduct, and financial support for this research were provided by AbbVie. AbbVie participated in the interpretation of data, review, and approval of the publication. 247 POSTER (Board P027) Selective inhibitors of nuclear export (SINE) block the expression of DNA damage repair proteins and sensitize cancer cells to DNA damage therapeutic agents T. Kashyap1 , M. Crochiere1 , S. Friedlander1 , B. Klebanov1 , W. Senapedis1 , E. Baloglu1 , D. del Alamo1 , S. Tamir1 , T. Rashal1 , D. McCauley1 , R. Carlson1 , M. Kauffman1 , S. Shacham1 , Y. Landesman1 . 1 Karyopharm Therapeutics, Natick MA, USA Background: SINE is a family of small-molecule drugs that inhibit Exportin 1 (XPO1/CRM1) mediated nuclear export, resulting in retention of major tumor suppressor proteins (TSPs) such as p53, FOXO, pRB and IkB and subsequently in specific cancer cell death. Selinexor is the clinical SINE compound currently in human phase I/II clinical trials in patients with solid and hematological malignancies. The goal of this study was to evaluate the effects of selinexor on DNA repair mechanisms and to test the cytotoxic effects of combining selinexor with DNA damaging agent on hematological and solid tumor. Methods: Whole protein cell lysates from solid and hematological cancer cell lines treated with selinexor with or without agents that induce DNA damage were analyzed in Reverse Phase Protein Arrays (RPPA), immunoblots and quantitative PCR. Selinexor treated cells from solid and hematological cancer lines were analyzed by immunofluorescence to evaluate DNA damage. Non-small cell lung cancer A549 Xenografts were treated with the selinexor (5 mg/kg) and radiation (3 Gy) alone or in combination and tumor growth was evaluated for 28 days. Results: Treatment of solid and hematological cancer cell lines with selinexor did not induce DNA damage in cancer cells but reduced the expression of DNA damage repair proteins: MSH2, MSH6, PMS2, MLH1, Rad51, CHK1 and FOXM1. Selienxor regulates the expression of CHK1, RAD51, MSH2, MSH6 and MLH1 on the transcriptional levels and PMS2 expression on the posttranslational level. There was a trend between the degree of DNA-damage-repair-protein reduction to selinexor sensitivity. Knock down of Chk1 alone, induced cytotoxicity whereas silencing of the other DNA repair proteins did not affect cell viability. Selinexor treatment following exposure to DNA damaging agents like doxorubicin and idarubicin inhibited the repair mechanism of DNA damage caused by these agents and resulted in synergistic cell killing as measured by induction of PARP and Caspase 3 cleavage. In vivo, selinexor (5 mg/kg) and radiation (3 Gy) decreased xenograft tumor size of the non-small cell lung cancer A549 by 15% and 43% respectively, relative to vehicle whereas combination of selinexor and radiation resulted in a 96% tumor decrease. Conclusion: Selinexor reduce the DNA repair mechanism in solid and hematological cancer cell lines and combination of selinexor with agents that cause DNA damage induces cancer cell death that is superior to each therapy alone. These data suggest that such a combination treatment is predicted to result with synergistic therapeutic outcome in cancer patients. 248 POSTER (Board P028) Use of ATR inhibitor in combination with topoisomerase I inhibitor kills cancer cells by disabling DNA replication initiation and fork elongation R. Josse´ 1 , S.E. Martin2 , R. Guha2 , P. Ormanoglu2 , T. Pfister3 , J. Morris4 , J. Doroshow4 , Y. Pommier1 . 1 NCI, Developmental Therapeutic Branch, Bethesda MD, USA; 2 NCATS, Division of Preclinical Innovation, Rockville MD, USA; 3 Leidos Biomedical Research Inc, Laboratory of Human Toxicology and Pharmacology, Frederick MD, USA; 4 DTP-DCTD, Drug synthesis and Chemistry Branch, Bethesda MD, USA Camptothecin and its derivatives, topotecan and irinotecan are specific topoisomerase I inhibitors and potent anticancer drugs. These agents produce well-characterized double-strand breaks upon collision of replication forks with topoisomerase I cleavage complexes. In an attempt to

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develop novel drugs combinations, we conducted a synthetic lethal siRNA screening using a library that targets nearly 7000 human genes. Depletion of ATR, the main transducer of replication stress-induced DNA damage response came at the top candidate gene with synthetic lethality with camptothecin. Validation studies showed that ATR siRNA exacerbated cytotoxic response to both camptothecin and the indenoisoquinoline LMP400 (indotecan), a novel topoisomerase inhibitor in clinical trial. Inhibition of ATR by the recently developed specific inhibitor VE-821 induced synergistic antiproliferative activity when combined with either topoisomerase inhibitor. Cytotoxicity induced by the combination with LMP-400 was greater than with camptothecin. Using single cell analysis and DNA fiber spread, we show that VE-821 abrogates the S-phase checkpoint, and restores origin firing and replication fork progression in cells treated with camptothecin or LMP-400. Moreover, the combination of topoisomerase inhibitors with VE-821 inhibited the phosphorylation of ATR and ATR-mediated Chk1 phosphorylation, while strongly inducing gH2AX. Single cell analysis revealed that the gH2AX pattern changed over time from well-defined focus to a pan-nuclear staining. The change in gH2AX pattern can be useful as a predictive biomarker to evaluate the efficacy of therapy. The key implication of our work is the mechanistic rationale it provides to evaluate the combination of topoisomerase I inhibitors with ATR inhibitors. 249 POSTER (Board P029) Preclinical efficacy of the PARP inhibitor rucaparib (CO338/AG014699/PF-01367338) in pancreatic cancer models with homologous recombination deficiencies (HRD) L. Robillard1 , K. Lin1 , P.P. Lopez-Casas2 , M. Hidalgo2 , T.C. Harding1 . 1 Clovis Oncology, San Francisco CA, USA; 2 CNIO, Madrid, Spain Rucaparib is an oral, potent, small molecule inhibitor of poly (ADP-ribose) polymerase (PARP) being developed for the maintenance treatment of platinum-sensitive ovarian cancer in patients with homologous recombination deficient (HRD) tumors, including those with BRCA1 and BRCA2 mutations. Mutations in BRCA and other homologous recombination HR pathway genes are frequently observed in other tumor types including breast and pancreatic (Alexandrov et al., 2013; Nature). Here, we investigated the efficacy of rucaparib as single agent in pancreatic cancer (PC) cell lines and xenograft models with HRD. Sensitivity to rucaparib was determined in a panel of PC lines (SU86.86, Panc-1, MIA PaCa-2, Panc 10.05) following siRNA knock-down of HR genes (ATM, ATR, BRCA1, BRCA2, PALB2 and RAD51C) to model gene mutations frequently observed in PC. BRCA1, BRCA2, PALB2 and RAD51C siRNA knockdownshowed synthetic lethality (30% of control GI50 ) in the majority of PC cell lines examined and correlated with the induction of HRD as assessed by gH2AX and RAD51 foci formation. As a complement to the cell line data we examined the efficacy of rucaparib in 3 BRCA2 mutant patient-derived xenograft (PDX) PC models (Oncotest, GmbH): PAXF_1876 (BRAF, PTEN, HRAS), PAXF_2005 (KRAS, TP53) and PAXF_2094 (KRAS). Models were selected on BRCA2 frameshift or nonsense mutations that should be functionally deleterious. Rucaparib administration to animals bearing preestablished tumors at 150 mg/kg/day BID (modeled Phase 2/3 dose in mice) resulted in significant monotherapy activity in all models examined with a 44, 96 and 67% reduction in tumor growth at 28 days post-dosing for PAXF_1876, 2005 and 2094, respectively. Expansion of rucaparib preclinical efficacy data beyond BRCA2 mutated PDX models is currently on-going in a PALB2 mutated PDX PC model (JH033; Villarroel et al., 2010; Mol. Ca. Ther.). In addition, the activity of rucaparib was examined in a BRCA2 mutant pancreatic ductal adenocarcinoma patient following FOLFIRINOX progression in a Phase I study (NCT01482715) was shown to be consistent with preclinical observations; a 56% reduction in the patient’s target lesions and a PFS of 6.4 months was observed with no significant toxicity. A Phase 2 study of rucaparib in patients with pancreatic cancer and a known deleterious BRCA mutation is currently on-going (NCT02042378). These findings support the hypothesis that pancreatic cancers exhibiting HRD are sensitive to rucaparib inhibition.

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Poster Session – DNA Repair Modulation (including PARP, CHK, ATR, ATM)

250 POSTER (Board P030) Comprehensive genomic profiling of pancreatic acinar cell carcinomas identifies recurrent RAF fusions and frequent inactivation of DNA repair genes J. Chmielecki1 , K.E. Hutchinson2 , G.M. Frampton3 , Z.R. Chalmers3 , A. Johnson3 , C. Shi4 , J. Elvin5 , S.M. Ali5 , J.S. Ross5 , O. Basturk6 , S. Balsubramanian7 , D. Lipson3 , R. Yelensky3 , W. Pao2 , V.A. Miller8 , D.S. Klimstra6 , P.J. Stephens9 . 1 Foundation Medicine, Cambridge, USA; 2 Vanderbilt University, Cancer Biology, Nashville, USA; 3 Foundation Medicine, Computational Biology, Cambridge, USA; 4 Vanderbilt University, Pathology, Nashville, USA; 5 Foundation Medicine, Pathology, Cambridge, USA; 6 Memorial Sloan Kettering Cancer Center, Pathology, New York, USA; 7 Foundation Medicine, Strategic Alliances, Cambridge, USA; 8 Foundation Medicine, Medical Affairs, Cambridge, USA; 9 Foundation Medicine, Cancer Genomics, Cambridge, USA Background: Pancreatic acinar cell carcinomas (PACCs) account for ~1% (~500 cases) of pancreatic cancer diagnoses annually in the United States. Oncogenic therapuetic targets have proven elusive in this disease, and chemotherapy and radiation have demonstrated limited efficacy against these tumors. Materials and Methods: We performed comprehensive genomic profiling of a large series of PACCs (n = 44), including closely related mixed acinar carcinomas (16 pure PACC, 14 mixed acinar/neuroendocrine, 6 mixed acinar/ductal, 2 mixed acinar/neuroendocrine/ductal, and 6 samples with incomplete histological analysis), using FoundationOne® , a nextgeneration sequencing (NGS)-based platform. DNA was analyzed for base substitutions, insertions/deletions, copy number alterations, and select rearrangements; eleven samples had sufficient material for broad fusion detection using targeted RNA-sequencing. Results: Recurrent rearrangements involving BRAF and RAF1 (CRAF) were observed in 10 samples (23%) of mixed and pure histology, and were mutually exclusive with other known driver events. Biochemical characterization of the most prevalent fusion, SND1-BRAF (n = 5), resulted in activation of the mitogen activated protein kinase (MAPK) pathway which could be abrogated with MEK inhibition. SND1-BRAF was transforming, and cells dependent on this fusion were sensitive to treatment with the MEK inhibitor, trametinib. Broad analysis of recurrent cancer-related genomic alterations in PACC revealed a unique genomic landscape compared to other subtypes of pancreatic cancer. Notably, we observed lower frequencies of KRAS and NF1 alterations compared to pancreatic ductal adenocarcinoma and neuroendocrine tumors, respectively. Inactivating alterations in DNA repair genes were observed in 45% of PACCs, including mixed and pure histologies, and were mutually exclusive with RAF genomic alterations. Conclusions: These findings have immediate clinical impact for PACC patients. RAF fusions in other diseases have demonstrated clinical sensitivity to targeted inhibitors; these agents may represent potential treatment options for the 23% of PACCs driven by these fusions. To our knowledge, this is the first report of RAF fusions in any form of pancreatic cancer. DNA repair deficiencies (45% of PACCs) are associated with sensitivity to platinum-based therapies and may also predict susceptibility to PARP inhibitors currently in late-stage clinical development. Although these alterations have been implicated in other forms of pancreatic cancer, they have been described only rarely in PACC. Collectively, these data suggest multiple potential therapeutic options for over two-thirds of PACC patients, and provide a rationale for using personalized therapies in this disease. 251 POSTER (Board P031) A combined in vitro and mathematical modelling approach for understanding the impact of an inhibitor of ATR on DNA damage and repair after ionising radiation J. Yates1 , S. Checkley2 , L. MacCallum2 , R. Odedra1 , J. Barnes3 , A. Lau1 . 1 AstraZeneca, iMED Oncology, Macclesfield, United Kingdom; 2 AstraZeneca, Discovery Sciences, Macclesfield, United Kingdom; 3 AstraZeneca, Drug Safety and Metabolism, Macclesfield, United Kingdom Background: AZD6738 is a potent specific inhibitor of ATR. As part of clinical development it is planned to investigate the combination of AZD6738 with ionising radiation (IR) in head and neck cancer patients. We have developed a novel cell cycle model to predict cellular responses to combination AZD6738/IR treatment. Materials and Methods: A simple mathematical model of the cell cycle, incorporating DNA damage and repair, was proposed. The model was formulated so that AZD6738 was assumed to inhibit the repair of replication stress induced damage during S-phase of the cell cycle. The model was calibrated using in vitro dose–response data generated using a colon

carcinoma cell line. gH2AX biomarker data was used to measure DNA damage, with cell count as the indicator of tumour proliferation. The in vitro calibrated model was incorporated into a solid tumour growth model and AZD6738 time-varying concentration informed by observed plasma pharmacokinetics in the mouse. Validation of model predictions was against gH2AX changes over time in the same tumour cell line xenografted in mice in vivo and the resulting efficacy after repeated doses of AZD6738 and IR Results: The model was successfully parameterised using in vitro data generated at a range of concentrations of AZD6738 as well as after replenishing with AZD6738 free media to simulate drug washout. The resulting in vivo tumour growth model was capable of accurately predicting in vivo mouse xenograft data, without requiring any additional modification of model parameters. Conclusions: Our prediction of AZD6738/IR combination efficacy has informed on the minimum dosing levels required in the clinic to be pharmacologically active. Minimum efficacious dose will minimize the risk of overdosing and so toxicological effects such as mucositis. The model also predicts drug efficacy and tumour proliferation rates in response to intermittent dose schedules, thus optimizing drug exposure for tumour regression. The model provides a framework that can be extended across other targetted therapy classes, supplementing mathematical models of low throughput in vivo data with high throughput in vitro assays. 252 POSTER (Board P032) The DNA damage response gene Schlafen 11 (SLFN11) is a transcriptional target of ETS transcription factors in Ewing’s sarcoma and other cancers Y. Pommier1 , S.W. Bilke2 , F. Sousa3 , M. Yamade3 , J. Murai3 , V. Rajapakse3 , L. Helman4 , P. Meltzer2 . 1 National Cancer Institute, Laboratory of Molecular Pharmacology, Bethesda, USA; 2 National Cancer Institute, Genetics Branch, Bethesda, USA; 3 National Cancer Institute, Developmental Therapeutics Branch, Bethesda, USA; 4 National Cancer Institute, Pediatric Oncology Branch, Bethesda, USA SLFN11 is a critical determinant of response to DNA targeted therapies including topoisomerase I and II inhibitors (camptothecins, etoposide, doxorubicin) and cisplatin. Ewing’s sarcoma (EWS), which is characterized by expression of the chimeric transcription factor EWS-FLI1, has notably high SLFN11 expression. This led us to investigate whether EWSFLI1 is causative for elevated SLFN11 expression. ChIP-Seq analysis of EWS-FLI1 in A673 EWS cells showed that EWS-FLI1 binds near the transcription start site of SLFN11. We further demonstrate that EWSFLI1 is a positive transcriptional regulator for SLFN11 and that EWS-FLI1mediated SLFN11 overexpression is responsible for high sensitivity of EWS to the topoisomerase I inhibitor camptothecin. The correlated expression between SLFN11 and FLI1 extends to leukemia, pediatric, breast and prostate cancers. These analyses suggest that, in addition to FLI1, several ETS members, including ETS1 regulate SLFN11 expression. Together, our results suggest the emerging relevance of SLFN11 for therapeutic response to DNA damaging agents in ETS-activated cancers. 253 POSTER (Board P033) Phase 1 correlative study of ARQ761, a b-lapachone analogue that promotes NQ01-mediated programmed cancer cell necrosis D. Gerber1 , Y. Arriaga1 , M.S. Beg1 , J.E. Dowell1 , J.H. Schiller1 , A.E. Frankel1 , R. Leff2 , C. Meek2 , J. Bolluyt3 , O. Fatunde3 , R.T. Martinez3 , P. Vo4 , F. Fattah4 , V. Sarode5 , Y. Zhou6 , Y. Xie6 , M. McLeod7 , B. Schwartz7 , D.A. Boothman4 . 1 University of Texas Southwestern Medical Center, Hematology-Oncology, Dallas Texas, USA; 2 Texas Tech University, School of Pharmacy, Dallas Texas, USA; 3 University of Texas Southwestern Medical Center, Hematology-Oncology, Dallas Texas, USA; 4 University of Texas Southwestern Medical Center, Harold C. Simmons Cancer Center, Dallas Texas, USA; 5 University of Texas Southwestern Medical Center, Pathology, Dallas Texas, USA; 6 University of Texas Southwestern Medical Center, Clinical Sciences, Dallas Texas, USA; 7 ArQule Inc., Woburn Massachusetts, USA Background: NAD(P)H:quinone oxidoreductase 1 (NQO1) is a twoelectron oxidoreductase expressed in multiple tumor types at levels 5- to 200-fold above normal tissue. ARQ761 (ArQule, Woburn, MA, USA) is a highly soluble intermediate b-lapachone hydroquinone analogue, complexed in hydroxypropyl-b-cyclodextrin, that exploits the unique elevation of NQO1 found in solid tumors to cause tumor-specific cell death by eliciting a futile redox cycle generating high levels of reactive oxygen species and ultimately PARP1 hyperactivation-dependent cell death. Materials and Methods: We initiated a 3+3 dose escalation study of 3 schedules (weekly, every other week, 2/3 weeks) of ARQ761 monotherapy as a 1-hr or 2-hr infusion. Eligible patients had refractory advanced solid

Poster Session – Drug Delivery tumors, ECOG 0−1, adequate organ function, and central venous access. Patient blood samples were analyzed for ARQ761 levels and NQO1 polymorphisms. Archival tumor tissue was analyzed for NQO1 staining intensity and prevalence. Results: As of May 2014, a total of 31 patients were enrolled and had received at least one regimen of ARQ761. For weekly administration, the maximum tolerated dose was 195 mg/m2 . For every other week and 2/3 week administration, dosing at 540 mg/m2 is ongoing. Mean ARQ761 halflife was approximately 50 minutes. Among 23 patients with response data available, best response was stable disease in 5 patients. Two patients had minor responses, including one heavily pretreated patient with bladder cancer with >50% shrinkage of lung metastases at the 195 mg/m2 dose level. Tissue biomarker analysis was performed for 20 cases. Clinical benefit was correlated with tissue NQO1 expression: disease control rate was 65% in NQO1-positive tumors and 18% in NQO1-negative tumors (P = 0.06). No patients were homozygous for *3 and/or *2 NQO1 polymorphisms. Hemolytic anemia and apparent methemoglobinemia (transient hypoxemia observed by peripheral saturation monitor but not confirmed by arterial blood gas) were the principal toxicities Conclusions: ARQ761 has clinical activity in NQO1-positive tumors. Principal toxicities include hemolytic anemia and methemoglobinemia. Monotherapy and combination studies in NQO1-positive tumors are underway. 254 POSTER (Board P034) Molecular analysis in breast cancer: correlation with immunohistochemical classification and pathologic complete response (pCR) to neoadjuvant chemotherapy (NAC) S. Baulies1 , M. Gonzalez-Cao2 , N. Karachaliou2 , A. Rodriguez Capitan2 , M.A. Molina-Vila2 , M.T. Cusido3 , C. Teixido2 , S. Viteri2 , R. Fabregas3 , X. Gonzalez2 , R. Rosell2 . 1 Institut Universitari Dexeus Quiron, Barcelona, Spain; 2 Institut Universitari Dexeus Quiron, Oncology, Barcelona, Spain; 3 Institut Universitari Dexeus Quiron, Gynecology, Barcelona, Spain Background: Breast cancer is a heterogeneous disease. Dysregulated genes, via expression alterations, affect cellular signalling pathways, leading to chemoresistance. In the present study, we analysed expression levels of a panel of genes implicated in molecular pathways that could have a role in chemoresistance. High expression levels of RTKs as AXL (and its ligand GAS6), and FGFR1 have been described as a poor prognostic factors, although they are mainly expressed in HR+ breast cancer. PTPN12 tumor supresor gene inhibits expression of erb2 or EGFR, and it has been described as frequently downregulated in Triple Negative (TN) tumors. BRCA1 and RAP80 genes are implicated in DNA repair pathway and they are downregulated in TN breast cancer conferring enhanced chemosensitivity. Lower expression levels of BIM (a proapoptotic protein) can be found after response to chemotherapy. As described in TN, EZH2 marks poor prognosis. BTRCP leds to EZH2 degradation through ubiquitination. ZNF217 is a transcription factor that confers poor prognosis in all breast cancer subtypes. YAP/TAZ is implicated in Hippo pathway and it has a role in breast cancer proliferation. Patients and Methods: Gene expression analysis was performed in pretreatment samples of 92 breast cancer patients treated with NAC. Expression levels of AXL/GAS6, FGFR1, PTPN12, BRCA1, RAP80, Bim, EZH2, BTRCP, YAP and ZNF217 genes were analysed by RT-PCR. We explored the correlation of molecular markers with immunohistochemical classification and with pCR. Results: 53 patients were classified as hormone receptor positive (HR+) (57%), 28 HER2+ (31%) and 11 patients as TN (12%). HR+ tumors had higher levels of AXL (p = 0.006), GAS6 (p = 0.05), FGFR1 (p = 0.006), RAP80 (p = 0.007), BTRCP (p = 0.003), PTPN12 (p = 0.023) and ZNF217 (p = 0.005). TN tumors had lower levels of BRCA1 (p = 0.18) and Bim (p = 0.05). Predictor of response: High levels of Bim (p = 0.014), or low levels of AXL (p = 0.003), low FGFR1 (p = 0.014), low YAP (p = 0.010), low BRCA1 (p = 0.008) and PI3K mutations (p = 0.031) were predictive of pCR. Interestingly, patients with high levels of FGFR1 had an OS at 10 years of 0% vs 78% (p = 0.051). Conclusions: Gene expression profile varies by breast cancer subtype. Chemosensitivity is higher in tumors with high levels of Bim or low levels of FGFR1, BRCA1, AXL and YAP.

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Drug Delivery 255 POSTER (Board P035) Pre-clinical in vivo characterization of MLN7243, an investigational ubiquitin activating enzyme inhibitor, in solid tumor models T. Traore1 , J.H. Huck1 , J.S. Shi1 , D.S. Sappal1 , J.D. Duffey1 , Y.Y. Yang1 , E.K. Kadakia1 , A.C. Chakravarty1 , B.S. Stringer1 , Y.I. Ishii1 , R.G. Griffin1 , C.X. Xia1 , M.M. Milhollen1 , J.C. Ciavarri1 , P.F. Fleming1 , N.B. Bence1 , M.H. Hyer1 . 1 Takeda Pharmaceuticals International Company, Oncology, Cambridge, USA Clinical results of VELCADE (bortezomib) For Injection have prompted evaluation of other enzymes within the ubiquitin proteasome system (UPS) as druggable targets for human cancer. We have identified a first in class investigational drug (MLN7243) which targets the ubiquitin activating enzyme, UAE (aka UBA1), an essential cellular enzyme responsible for activating >99% of all cellular ubiquitin. Ubiquitin is involved in multiple cellular processes including, but not limited to: ubiquitin-dependent protein turnover, cell cycle progression, regulation of apoptosis, protein localization and response to DNA damage. In vitro, MLN7243 has single digit nanomolar potency against the UAE enzyme and double digit nanomolar potency in cell-based viability assays. In vivo, MLN7243 administration leads to anti-tumor activity in a wide variety of cell-line-derived and primary human xenograft tumor models grown in mice. Pharmacokinetic analysis in mice indicates MLN7243 is quickly cleared from plasma but maintains a long half-life in xenograft tumor tissues. MLN7243, once in cells, binds to UAE and forms a MLN7243-ubiquitin adduct; this adduct species inhibits the UAE enzyme and remains detectable in tumors for days after the compound has cleared from plasma. A series of tumor biomarkers have been utilized to directly track MLN7243 target engagement (MLN7243ubiquitin adduct), measure global cellular polyubiquitin levels, measure mono-ubiquitinated Histone 2B and measure tumor cell apoptosis (cleaved caspase-3). Pre-clinical PK/PD/EF relationships have been established to help guide clinical decisions. MLN7243 is currently being evaluated in a phase I clinical trial that will evaluate safety, tolerability, pharmacokinetics, pharmacodynamics, and anti-tumor activity in patients with advanced solid tumors. ClinicalTrials.gov identifier: NCT02045095. 256 POSTER (Board P036) Minicell packaged targeted delivery of shRNA to cancer cells M. Jivrajani1 , N. Shrivastava2 , M. Nivsarkar1 . 1 B.V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Pharmacology and Toxicology, Ahmedabad, India; 2 B.V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Pharmacognosy and Phytochemistry, Ahmedabad, India Background: The genetic nature of cancer provides solid support for the rationale of si/shRNA-mediated gene therapy. However, a major hurdle to develop RNAi as cancer therapeutics is successful in vivo delivery. The aim of this study was to explore the efficacy of folate-conjugated bacterial minicells as a novel targeted delivery system for shRNA. Materials and Methods: In this work, plasmid based shRNA against VEGF A (psNIPERDU6A2) was packaged in minicells. These minicells were linked with folic acid for active targeting of tumor cells via folate receptor in vitro and in vivo. Cancer cell lines, LNCaP, HeLa and KB have been selected as positive control whereas A549 was selected as negative control in terms of folate receptor overexpression. In vitro delivery was studied by delivering 109 minicells from each the group, (1) FA minicellspSUPERneo , (2) minicellspsNIPERDU6A2 , (3) FA minicellspsNIPERDU6A2 in selected cell lines and expression of VEGF A was analysed by RT-PCR. Tumor xenograft of A549, LNCaP and KB cells were developed in immunosuppressed C57BL/6 mice, where the animals of each xenograft group were treated with (1) saline, and 109 of respective minicells, i.e. (2) FA minicellspSUPERneo , (3) minicellspsNIPERDU6A2 , (4) FA minicellsPSNIPERDU6A2 intravenously to study in vivo delivery. Tumor volume (mm3 ) was measured in each group every week during the study. At the end of study, tumor was excised to observed relative angiogenesis. Subsequently, expression of VEGF A was analyzed from tumor by RT-PCR. Eventually, In vivo biodistribution of FA minicellspsNIPERDU6A2 was studied. Results: Expression of VEGF A did not change in any of the groups in A549 cell line. In contrast, expression of VEGF A reduced significantly in FA minicellspsNIPERDU6A2 treated group when compared with other two groups in positive control cell lines. There was a gradual increase in the tumor volume till the end of treatment in all four treatment groups of A549 xenograft. Whereas in case of LNCaP and KB xenograft, there was

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a significant decrease in tumor volume inFA minicellspsNIPERDU6A2 treated group as compared to other groups. Relative angiogenesis was decreased significantly in FA minicellspsNIPERDU6A2 treated LNCaP and KB xenograft as compared to A549 xenograft. Similarly, expression of VEGF A was found to be same in all the groups when compared with the saline treated group in A549 xenograft. On the contrary, significant downregulation of VEGF A was found in FA minicellsPSNIPERDU6A2 treated LNCaP and KB xenograft. In vivo biodistribution study revealed that majority of FA minicellspsNIPERDU6A2 localized in the tumor followed by liver and heart. Conclusions: FA minicellspsNIPERDU6A2 have delivered psNIPERDU6A2 effectively in vitro and in vivo in folate receptor overexpressing cancer cells through receptor mediated endocytosis which was confirmed by reduced gene expression of VEGF A and tumor regression. 257 POSTER (Board P037) Development of peptide-mediated drug delivery systems for colon cancer targeted imaging and therapy H.C. Wu1 , C.H. Wu1 , Y.H. Kuo1 . 1 Academia Sinica, Institute of Cellular and Organismic Biology, Taipei, Taiwan Colorectal cancer is one of the most common diagnosed cancers and a leading cause of cancer mortality worldwide. Lack of tumor specificity remains a major problem for chemotherapies in which side effects prevent the delivery of the drug dosages needed to eliminate the majority of cancer cells. Thus, targeted drug delivery system is necessary to effectively deliver the anticancer drugs to the tumor. In this study, we have successfully identified specific peptides binding to the human colorectal carcinoma (hCRC) cells through in vitro biopanning using phage-displayed peptide library. Three high affinity phage clones to colorectal carcinoma were identified, and their binding activities were confirmed by cellular ELISA and flow cytometry. The hCRC-targeted phages recognized five colorectal carcinoma cell lines and surgical specimens from colorectal cancer patients. The tumor homing ability of hCRC-targeted phages was confirmed by xenograft model in vivo. To investigate whether hCRCtargeted peptides could be used to enhance the therapeutic efficacy of anticancer drugs, we synthesized the peptide-mediated liposome encapsulated anti-cancer drugs. Notably, hCRC-targeted peptides conjugated liposomal drugs markedly inhibited hCRC tumor growth in mouse xenograft models. Combination treatment of peptide-mediated targeting liposomes was able to completely eradicate tumors in three-sixth of the total number of tumor-bearing mice without any signs of recurrence. Targeting liposomes improved the therapeutic index by enhancing therapeutic efficacy, reducing side effects, and increasing the survival rate of tumor-bearing mice in orthotopic animal model. Biodistribution studies in tumor-bearing mice indicated that chemotherapeutic drugs were localized in tumor tissue following administration of hCRC targeting peptides conjugated liposomal doxorubicin. Our findings indicate that hCRC-targeted peptides have great potential to be developed into targeted drug delivery systems and imaging for colorectal carcinoma. 258 POSTER (Board P038) Pancreatic cancer cells expressing the OATP1B3 transporter show promising sensitivity to the highly cytotoxic microcystin-LR molecule V. Kounnis1 , G. Chondrogiannis2 , M.D. Mantzaris3 , D. Fokas4 , N.A. Papanikolaou3 , I. Sainis1 , E. Briasoulis1 . 1 University of Ioannina, Cancer Biobank Center, Ioannina, Greece; 2 University of Ioannina, Department of Anatomy-Histology-Embryology Medical School, Ioannina, Greece; 3 University of Ioannina, Laboratory of Biological Chemistry Medical School, Ioannina, Greece; 4 University of Ioannina, Department of Materials Science and Engineering, Ioannina, Greece Background: Microcystins are natural cyclopeptides synthesized mainly by cyanobacteria and are considered health hazardous due to their toxic effects after acute consumption or chronic exposure. From a pharmacologic point of view, microcystins are extremely stable, hydrophilic cyclic heptapeptides with the ability to cause extensive cell damage once uptaken by the specific Organic Anion Transporting Polypeptides (OATP). Their main toxicity mechanisms are the inhibition of the protein phosphatases PP1 and 2A, the reduction of glutathione levels and the increase of Reactive Oxygen Species (ROS), ultimately leading to global hyperphosphorylation and increased cellular oxidative stress. The present study focused on the investigation of microcystin-LR as a potential anticancer compound to target pancreatic cancer cell lines proved to express the OATP1B3. Materials and Methods: For the purposes of this study, we used the characterized cell lines Bx-PC3, MIA PaCa-2 and CHO. The expression of OATP1B3 on the protein level was assessed by Western Blotting and

Poster Session – Drug Delivery the monoclonal antibody mMDQ, whereas its endogenous expression was assessed by real time PCR and specifically designed primers. xCELLigence and flow cytometry (Annexin/PI) methods were used to study the cell toxicity of microcystin-LR, Gemcitabine and their combination. Results: Data resulted from the experimental procedures confirmed the expression of OATP1B3 in both cancer cell lines Bx-PC3 and MIA PaCa-2 with the first showing more prone expression by 38% on the protein and 2.4 fold on the mRNA level. In addition the two pancreatic cancer cell lines, showed a relative proportional − to the transporter expression − sensitivity to microcystin-LR with IC50 of 83.5nM and 2.1mM respectively. Of interest, microcystin LR surpassed the toxic effect of the approved chemotherapeutic agent gemcitabine for the Bx-PC3 cell line, which IC50 was 226nM. Furthermore the combination of microcystin-LR at low micromolar concentrations and gemcitabine at upper nanomolar concentrations showed an increased cytotoxic effect for both cell lines when compared to that of each agent alone with relevant IC50 of 2.9nM and 4.4nM for Bx-PC3 and MIA PaCa-2. Annexin/PI flow cytometry confirmed the proportional − to the transporter expression − dose response for microcystin-LR. Conclusions: The expression of OATP1B3 in cancer cells, could be considered as a candidate therapeutic target. We propose that structural modification of the highly cytotoxic MC-LR molecule − especially its conjugation with N-acetylcysteine, could result in the development of novel compounds with activity against pancreatic cancers that express OATP1B3. 259 POSTER (Board P039) Factors that limit delivery of Cdk4/6 inhibitor palbociclib to GBM K. Parrish1 , J. Pokorny2 , R. Mittapalli1 , K. Bakken2 , J. Sarkaria2 , W. Elmquist1 . 1 University of Minnesota Medical School, Minneapolis, USA; 2 University of Minnesota, Pharmaceutics, Minneapolis, USA Background: Developing effective therapies for glioblastoma multiforme (GBM) is significantly hampered by the blood–brain barrier (BBB), which limits delivery of many potentially effective anti-cancer agents to infiltrative tumor cells. The cyclin-dependent kinase 4 (Cdk4) pathway is hyperactivated in approximately 75% of GBM tumors in association with homozygous deletion of p16 (52%) and amplification of Cdk4 (18%) and Cdk6 (1%). Palbociclib (PD0332991) is a potent Cdk4/6 inhibitor that has shown efficacy in treating some non-brain tumors. The purpose of this study is to determine the mechanisms limiting delivery and efficacy of palbociclib therapy in an orthotopic xenograft model of patient-derived GBM. Methods: Palbociclib distribution to the brain was examined in FVB wildtype (WT), and triple-knockout (TKO; Mdr1a/b(−/−) Bcrp1(−/−) ) mice following escalating oral doses (10, 50, 100 or 150 mg/kg). Concentrations of palbociclib in plasma and brain were determined by LC-MS/MS. Survival studies were conducted in patient-derived primary GBM22 xenograft model in athymic nu/nu mice, and additional drug distribution studies were conducted in these tumor-bearing mice to correlate delivery with efficacy. Results: The brain exposure of palbociclib in TKO mice following a 10 mg/kg oral dose (AUCbrain-to-AUCplasma ratio) was 150-fold higher than in WT mice [WT: 0.044; TKO: 6.24]. Two hours post-dose, the brainto-plasma ratio was constant in WT and TKO mice over a dose range of 10 to 150 mg/kg [brain-to-plasma ratio (10 mg/kg: WT: 0.15±0.06; TKO: 10.3±1.7), (50 mg/kg: WT: 0.19±0.06; TKO: 8.5±3.6), (100 mg/kg: WT: 0.17±0.02; TKO: 4.5±1.7), (150 mg/kg: WT: 0.17±0.03; TKO: 7.8±0.52)]. For efficacy studies, palbociclib was dosed at 150 mg/kg/day continuously. Consistent with sub-therapeutic delivery across the BBB, palbociclib did not prolong the median survival of an orthotopic GBM22 xenograft model. Conversely, treatment of GBM22 xenografts grown as flank tumors resulted in a significant (45 day) survival benefit. Additionally, the brain concentrations following a 150 mg/kg dose were intentionally comparable to the flank tumor concentrations following a 10 mg/kg dose [770±230 ng/mL and 730±510 ng/mL, respectively], and neither provided a therapeutic response. Conclusion: These data suggest that efflux transport at the BBB limit the brain delivery of palbociclib and hence, efficacy, of this Cdk4/6 inhibitor in the treatment of GBM. This has important translational implications in the use of palbociclib in either mono or combination therapies for either primary or secondary brain tumors.

Poster Session – Drug Delivery 260 POSTER (Board P040) Hsp90 Inhibitor Drug Conjugates (HDC): Payloads and possibilities D. Chimmanamada1 , W. Ying2 , J. Zhang2 , D. Proia3 , T. Przewloka1 , J. Jiang1 , D. Vutukuri1 , G. Lu1 , S. Osman1 , S. Chen1 , J. Chu3 , P. Rao3 , D. Zhou3 , T. Inoue4 , L. Shin Ogawa4 , R. Singh4 , N. Tatsuta4 , A. Sonderfan4 , C. Cortis5 . 1 Synta Pharmaceuticals, Chemistry, Lexington MA, USA; 2 Synta Pharmaceuticals, Chemistry, Lexington MA, USA; 3 Synta Pharmaceuticals, Biology, Lexington MA, USA; 4 Synta Pharmaceuticals, DMPK, Lexington MA, USA; 5 Synta Pharmaceuticals, Business Development, Lexington MA, USA Background: Despite the emergence of various therapies, chemotherapy still plays a significant role in the treatment of cancer. Though toxic, the potency and broad applicability of chemotherapeutic drugs make compelling rationale for using them as payloads in drug delivery systems. Several recent technologies, especially antibody drug conjugates (ADC) have been very successful, however for narrow sets of indications. We have recently disclosed the HSP90 inhibitor Drug Conjugate (HDC) platform, which utilizes the unique pharmacokinetic property of HSP90 inhibitors, where the drug is selectively retained in tumor in high concentrations while clearing from plasma and normal tissues relatively quickly. By combining the overexpression of HSP90 and longer residency of its inhibitors in tumor, we conceived an idea to conjugate small molecule anticancer drugs (payload) to HSP90 inhibitor in such a way that the payload is released selectively and gradually in tumor having a prolonged exposure. Results: In this presentation, the rationale for payload selection, their applicability and preliminary assessments will be discussed. Though virtually any small molecule anticancer agent qualifies as payload for HDC technology, the expensive drug development paradigm calls for a strong rationale every time. It has been shown by multiple groups including us that a combination therapy of an HSP90 inhibitor with a suitable drug, such as docetaxel is superior compared to the single agent. Hence, the HDC of taxanes make sense. Other categories include but not limited to, are the drugs that have poor solid tumor penetration such as proteasome inhibitors, drugs that have poor pharmacokinetics (highly metabolized) such as gemcitabine and other antimetabolites, drugs that develop resistance due to efflux (e.g., pgp) from cancer cells such as camptothecins all make good payloads. Several cell cycle inhibitors, such as pan-CDK inhibitors and pan-PI3K inhibitors (such as staurorporine) are also considered. A lead SN-38 conjugate for which detailed evaluations has been done showed that the payload is released in tumor for a prolonged period of time (0.22uM and 0.38uM at 24 h and72 h respectively in tumor against 30nM at 24 h and no drug quantified at 72 h for Irinotecan, at their maximum tolerated doses (MTDs). In vivo efficacy in multiple tumor models also exhibited remarkable activity for the HDC compared to Irinotecan. Toxicological evaluations also revealed that the HDC is safer than Irinotecan at their efficacious doses. Conclusion: The HDC platform has the potential to utilize a number of commonly used small molecule anticancer agents as payloads; expanding applicability through improved efficacy and toxicity profiles. It also has the potential to resuscitate the development of drugs that were abandoned due to toxicity. 261 POSTER (Board P041) Lesion characterization with ferumoxytol MRI in patients with advanced solid tumors and correlation with treatment response to MM-398, nanoliposomal irinotecan (nal-IRI) R.K. Ramanathan1 , R.L. Korn2 , J.C. Sachdev1 , G.J. Fetterly3 , G. Jameson1 , K. Marceau1 , V. Marsh1 , N. Raghunand4 , J. Prey3 , S.G. Klinz5 , J. Kim5 , E. Bayever5 , J.B. Fitzgerald5 . 1 Scottsdale Healthcare/TGen, Virginia Piper Cancer Center, Scottsdale AZ, USA; 2 Imaging Endpoints, Scottsdale AZ, USA; 3 Roswell Park Cancer Institute, Buffalo NY, USA; 4 Arizona Cancer Center, Translational Cancer Imaging, Tucson AZ, USA; 5 Merrimack Pharmaceuticals, Inc., Cambridge MA, USA Introduction: MM-398, a nanoliposomal irinotecan (nal-IRI), is designed to exploit leaky vasculature for enhanced drug delivery to tumors. Deposition of nal-IRI and subsequent conversion to SN-38 in both neoplastic cells and tumor associated macrophages (TAM) may positively correlate with activity. Predictive biomarkers to measure tumor deposition could identify patients likely to benefit from nal-IRI. Ferumoxytol (FMX), a 30 nm ironoxide nanoparticle with MRI contrast properties, is taken up by TAMs and has similar biodistribution patterns to nal-IRI in preclinical models. We have shown the feasibility of quantitative FMX MRI of tumor lesions and report here the correlation of FMX levels with nal-IRI activity in the study population. Patients and Methods: Eligible patients (n = 15; 4M, 11F; median age 58 [28−80] years) with refractory solid tumors had FMX MRI scans prior

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to and following (1, 24, 72 h) FMX infusion. T2* signal was used to calculate FMX levels in plasma and lesions by comparison to a standard curve. A mechanistic PK model built on these values indicated that tissue permeability to FMX contributed to early FMX MRI signals at 1 h and 24 h, while FMX binding contributed at 72 h. Patients continued on nal-IRI at 80 mg/m2 q2w until progression. Core biopsies were obtained 72 h after both FMX and nal-IRI infusions. RECIST v1.1 evaluation was done by CT every 8 weeks. Results: 13/15 (87%) patients received nal-IRI, and 11/13 (85%) were evaluated for response with 1 (7%) partial response (PR), 5 (38%) stable disease and 5 (38%) progressive disease. FMX levels were measured in 31 lesions from 9 patients and compared to lesion size changes after nal-IRI treatment. Median FMX levels in tumor lesions at 1 h and 24 h were 34.1 and 33.6 mcg/mL, respectively. FMX levels above the median were significantly associated with better lesion responses as measured by change in lesion size (p < 0.001 at 1 h; p < 0.003 at 24 h); no relationship was observed at 72 h. Receiver operating characteristics for lesion classification according to PR or lesion size reduction had an AUC 0.8 for early FMX measurements. Levels of irinotecan and SN-38 averaged 3.73 mcg/g [0.13–12.75 mcg/g] and 14.67 ng/g [1.2–64.0 ng/g], respectively, at 72 h. SN-38 levels in biopsies were 5-fold higher than in plasma at 72 h (p = 0.013). Prussian Blue staining of ferumoxytol in biopsies was predominately observed at the stroma-tumor interface where it colocalized with TAMs. Conclusions: Clinical activity of nal-IRI was observed in refractory solid tumors. Our findings are consistent with local activation of MM-398 in the tumor. The relationship between FMX levels in tumor lesions and nal-IRI activity suggests that lesion permeability to FMX may be a useful biomarker for tumor response to nal-IRI in patients with solid tumors. 262 POSTER (Board P042) Improved cytotoxic activity of Nor-b-lapachone-loaded PLGA microcapsules in PC3M prostate cancer cell line C. Pessoa1 , A.C.S. Feitosa1 , M.P. Costa1 , F.C. Evangelista1 , 2 F.A.M. Sales1 , I.S. Bomfim1 , E.N. Silva Junior ´ , G.G. Dias2 , V.N. Freire3 , W.S. Caetano4 . 1 Ceara Federal University, Pharmacology and Physiology, Fortaleza, Brazil; 2 Minas Gerais Federal University, Chemistry, Fortaleza, Brazil; 3 Ceara Federal University, Physics, Fortaleza, Brazil; 4 Instituto ´ Physics, Fortaleza, Brazil Federal do Ceara, Background: b-lapachone is one of the most widely studied naphthoquinones. However, several efforts have been made to find new lapachone analogues, more potent and less toxic. Nor-b-lapachone (NbL), a semisynthetic naphthoquinone derivative from b-Lapachone, is a cytotoxic agent against several cancer cell lines. Material and Methods: To overcome its liposolubility and non-specific toxicity, this drug was formulated in Poly(d,l)-lactide-co-glycolide (PLGA) microparticles by the emulsion-solvent evaporation technique. Surface morphology, particle size distribution, zeta potential, Raman spectroscopy, Fourier transform infrared (FT-IR) spectroscopy, drug encapsulation efficiency, drug release kinetics and in vitro cytotoxicity of the drug-loaded microcapsules were determined. Results: Spherical microcapsules with a size range of 1.03±0.46 mm were obtained. The drug encapsulation efficiency was approximately 19%. The NbL-loaded PLGA microcapsules exhibited a pronounced initial burst release. After the in vitro treatment with the NbL-loaded PLGA microcapsules, a clearly phagocytosis of the spheres was observed in a few minutes. The cytotoxic activity of NbL against PC3M cells was greater when delivered by PLGA microcapsules compared to the free drug. After incubation for 72 h, the IC50 values of the free and encapsulated forms of NbL were 2.045 (1.97–2.12) and 1.046 (0.82–1.34) mg·mL−1 , respectively. We suggest that PLGA microcapsules containing NbL could be a promising drug delivery system to be studied using in vivo models of prostate cancer. Conclusions: The results suggest that NbL-loaded PLGA microcapsules could be used as a promising delivery system for NbL administration in in vivo tests of prostate cancer. 263 POSTER (Board P043) Controlled release of cisplatin using hyaluronic oligosaccharidescoated gold nanoparticles as an efficient delivery system applied to the treatment of pancreatic tumors 2 ˜ , H. Parkkola1 , L. Sobrevals Amieva1 , L. Vivero2 , R. Minana J. Sendra3 . 1 Endor Nanotechnologies, Preclinical, Barcelona, Spain; 2 Endor Nanotechnologies, Nanomaterials, Barcelona, Spain; 3 Endor Nanotechnologies, R&d Director, Barcelona, Spain

Many chemotherapeutic agents currently used in oncology can produce toxicity inducing many side effects, even at the optimal therapeutic doses.

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This problem could be addressed by targeting cellular differences between cancer and normal cells and controlling the delivery of cancer drugs. One such difference is the selective overexpression of cell surface receptors in tumor cells that can act as specific targets for the delivery of a drug. It is well known that CD44, the main receptor for hyaluronic acid (HA), is overexpressed in a wide range of malignancies and in cancer tumor initiating cells, playing an important role in promoting cancer cell proliferation, migration, invasion, and angiogenesis. The aim of our therapeutic approach is based on the need to develop new therapies that can efficiently target tumors, reducing the associated side effects and increasing the antitumor efficacy. In this line, the use of engineered HAcoated gold nanosystem for the delivery of chemotherapeutics is eligible to achieve those objectives. Advanced gold nanosystem uptake by tumoral cells was shown to be CD44-dependent, this was proven by studies with cell lines that exhibit differing CD44 expression levels as well as downregulating the expression of the HA receptor in a cell line that expresses high levels of CD44. Moreover, in vitro studies showed no signs of genotoxicity or oxidative damage and even though very high concentrations showed a slow decrease in cell viability. Advanced gold nanosystem carrying cisplatin was stable in solution and indicated a gradual drug release. Conjugation of cisplatin did not prevent the drug from performing its action in vitro, the drug showed slightly higher toxicity when encapsulated compared to the free drug. In vivo studies conducted in pancreatic cancer xenografts showed that the antitumoral effect was increased compared to free cisplatin, however we detected also increased toxicity when higher doses were administered, suggesting a fine tight therapeutic dosage schedule. Highest gold accumulation was in liver, followed by skin and tumor in pancreatic cancer xenografts. Therefore our data provide evidences of the relevance of using advanced drug delivery nanosystem for the treatment of pancreatic cancer. 264 POSTER (Board P044) Vasculogenic mimicry in small cell lung cancer F. Trapani1 , R.L. Metcalf1 , R. Polanski1 , A. Fusi2 , C. Hodgkinson1 , D. Nonaka3 , M.J. Hendrix4 , C. Morrrow1 , F. Blackhall3 , K.L. Simpson1 , C. Dive1 . 1 Paterson Institute for Cancer Research, Clinical and Experimental Pharmacology, Manchester, United Kingdom; 2 Christie NHS Foundation Trust, Manchester, United Kingdom; 3 Institute of Cancer Studies University of Manchester, Manchester, United Kingdom; 4 Cancer Biology and Epigenomics Program Ann and Robert H. Lurie Children’s Hospital of Chicago Research Center Robert H. Lurie Comprehensive Cancer Center Northwestern University Feinberg School of Medicine, Chicago, USA Introduction: Small cell lung cancer (SCLC) causes 25% of lung cancerrelated deaths worldwide. Despite initial responses to chemotherapy, most patients relapse with drug resistant disease. A minority of SCLC patients are inherently chemorefractory. Improved treatments are urgently required. Targeting tumour vasculature in SCLC with anti-angiogenic drugs produced disappointing results. Angiogenesis-independent tumour vascularisation pathways, including vasculogenic mimicry (VM), warrant further investigation. Aggressive cancer cells displaying VM mimic characteristics of host endothelial cells forming fluid conducting vascular channel-like structures. We sought to determine the prevalence of VM in SCLC and explore any associations of VM with chemotherapy sensitivity and patient survival. Methods: VM was evaluated using CD31/periodic acid-Schiff (PAS) staining in a tissue micro-array (TMA) from 41 limited stage SCLC chemonaive patients and in tumours from 2 chemosensitive and 2 chemorefractory Circulating Tumour Cell (CTC) Derived Explant (CDX) models that mimic donor patient responses to platinum/etoposide chemotherapy (Hodgkinson et al Nature Medicine, 2014). The relative abundance of VM channels (CD31-ve/PAS+ve) compared to host derived blood vessels (CD31+ve/PAS+ve), (VM/total vessels) in the TMA was compared to patient overall survival (OS) and in CDX models, to chemotherapy sensitivity. Results: In the TMA, a VM/Total Vessels score >16% was a poor prognostic factor for OS by univariate (p = 0.011) and multivariate (p = 0.014) analyses. In CDX models, chemo-sensitive tumours had a more VM structures than chemo-refractory tumours. Conclusion: We present the first evidence of VM in SCLC patient biopsies and of the VM phenotype in our unique CDX models. The correlation between high VM in patient biopsies and poorer OS corresponds with findings in other cancers notably melanoma, and may reflect a ‘cancer stem cell’ phenotype linked to VM. We are pursuing the testable hypothesis that high versus low VM levels in chemo-sensitive versus chemo-refractory CDX respectively could affect tumour drug delivery.

Poster Session – Drug Delivery 265 POSTER (Board P045) Pretargeted nanoparticles to deliver both chemotherapeutics and radiation for the treatment of lymphoma C. Fang1 , J.C. Jones1 , S.M. Frayo1 , M.H. Hylarides1 , M. Zhang2 , O.W. Press1 . 1 Fred Hutchinson Cancer Research Center, Clinical Research Division, Seattle WA, USA; 2 University of Washington, Materials Science & Engineering, Seattle WA, USA Background: We developed a new generation of therapeutic nanoparticles (NPs) that can deliver both chemotherapeutics and radionuclides specifically to non-Hodgkin lymphoma (NHL) cells. Only one third of NHL patients are presently cured with chemotherapy and antibody (Abs) treatments. Radioimmunotherapy (RIT) delivers radiation specifically to tumor cells using Abs conjugated to a radioisotope that can be therapeutically effective against resistance to chemotherapy. However, the therapeutic window of RIT is limited by slow clearance of unbound radiolabeled Abs from the circulation, resulting in substantial radioactivity in normal tissues. Codelivery of both chemotherapeutics and radiation will result in therapeutic synergism that will maximize tumor cell killing while reducing resistance to single-agent therapy. Targeting NPs to the CD20 or CD19 antigen that is widely expressed on the NHL cell surface will result in effective delivery of the therapeutic payloads and thus maximize cell killing. Material and Methods: We use lymphoma xenograft athymic mouse model to demonstrate the efficacy of NPs. The NPs are based on multifunctional biodegradable chitosan polymer with well-characterized molecular weight and composition. Chemotherapeutic drug monomethyl auristatin (MMAE) is incorporated to polymer via disulfide linkages while therapeutic radionuclide yttrium-90 (90 Y) is attached via chelating agents. The size and zeta potential of NPs were characterized by dynamic light scattering. We administrate the NPs utilizing ‘pre-targeted’ delivery strategy by inject antibody-streptavidin conjugates (Ab-SA) first, then clearing agents to remove Ab-SA from circulation, and then inject NPs 24 h after. The pharmacokinetics (PK), biodistribution (BD), toxicity and therapeutic effects of NPs are examined. Results: The NPs were monodisperse and with neutral surface charge with sufficient drug loading. BD and PK studies showed rapid clearance of NPs in circulation and preferential accumulation of NPs in tumor (5% I.D./g) of xenograft mice. Pretargeted approach improves the tumor-to-normal organ ratio to over 1 for all clearance organs. The NPs show no sign of toxicity in terms of circulating blood cell analysis and blood chemistry panel. To date, the NPs with both chemo and radiation eliminated tumor at lower dose than the effective dose of single agents. Conclusions: The pretargeted NPs co-delivering MMAE and Y-90 to the antigens that are widely expressed on the NHL cell surface results in effective internalization of the therapeutic payloads and maximize tumor cell killing with minimum side effects. Data table Name

Unit

Data

Hydrodynamic size Polydispersity Index Zeta Potential MMAE Loading

nm

27.9 0.150 1.56 8.2

Figure: Structure.

mV (w/w)%

Poster Session – Drug Design 266 POSTER (Board P046) Auxiliar treatment by targeting the extracellular matrix to improve drug delivery and therapeutic response E. Henke1 , F. Roehrig1 , H. Hoffmann1 , F. Escorcia2 , M. Wartenberg3 , S. Volova4 , S. Gaetzner5 , A. Rosenwald3 , S. Erguen6 , D.A. Scheinberg2 , ¨ Z.V.I. Granot7 . 1 Institute for Anatomy and Cell Biology, Universitat Wurzburg, ¨ Wurzburg, ¨ Germany; 2 Molecular Pharmacology, Memorial Sloan-Kettering Cancer Center, New York, USA; 3 Institute for Pathology, ¨ Wurzburg, Universitat ¨ Wurzburg, ¨ Germany; 4 Institut for Clinical ¨ Biochemistry, Universitatsklinikum Wurzburg, ¨ Wurzburg, ¨ Germany; 5 ¨ Wurzburg, Institute for Tissue Engineering, Universitat ¨ Wurzburg, ¨ ¨ Germany; 6 Institute for Anatomy and Cell Biology II, Universitat Wurzburg, ¨ Wurzburg, ¨ Germany; 7 Developmental Biology and Cancer Research, Hebrew University, Jerusalem, Israel During the formation of a new tumor, tumor cells do not only proliferate, but also form their own microenvironment. The newly established microenvironment not only supports the further growth and proliferation of the tumor cells, but it also protects the tumor from the effects of therapeutic agents. An important part of the tumor microenvironment is the extracellular matrix (ECM). Besides providing structural support, the ECM is also directly involved in regulating cell behavior like motility and invasiveness. Lysyl oxidases are a family of enzymes that catalyze the cross linking of proximal fibrous protein strands. As a result, the ECM becomes more rigid. Lysyl oxidases are up-regulated in tumors and it has been shown that the resulting increased rigidity of the tumor tissuedirectly contributes to invasiveness and metastatic behavior. We found recently that crosslinking catalyzed by lysyl oxidases also increases the physical barrier function of ECM proteins. Lysyl oxidase activity reduces interstitial diffusion, thereby protecting tumor cells from exposure to drugs. Inhibition of lysyl oxidases can improve drug transport within the tumor and dramatically enhance treatment efficacy. We treated early lung metastasis in mouse models with the pan lysyl oxidase inhibitor 3-aminopropionitrile (BAPN). Lysyl oxidase inhibition reduced build-up of fibrous matrix and left the lesions in a more immature state. Drug diffusion and oxygenation was significantly improved. Adding lysyl oxidase inhibition to chemotherapeutic treatment strongly improved efficacy of the cytotoxic drugs both in primary tumors and in established metastatic disease. Impotantly, BAPN-treatment reversed the negative impact of antiangiogenic therapy on drug delivery in an anti-VEGF refractory tumor model, synergistically improving drug transport and treatment efficacy in a study that combined lysyl oxidase inhibition with VEGF-ablation and cytotoxic treatment. Our results indicate that interfering with the formation of the supportive tumor microenvironment might be an effective way to slow improve drug delivery and response. Especially, in combination with anti-angiogenic treatment it has the potential to significantly improve the effficacy of the therapeutics already at our disposal to combat cancer. 267 POSTER (Board P047) Novel adjuvant therapy with leptin peptide receptor antagonist-2 conjugated to nanoparticles (IONP-LPrA2) to minimize chemoresistance in triple negative breast cancer T. Harmon1 , A. Harbuzariu2 , L. Yang3 , R.R. Gonzalez-Perez1 . 1 Morehouse School of Medicine, Atlanta GA, USA; 2 Morehouse School of Medicine, MBI, Atlanta GA, USA; 3 Emory University, Atlanta GA, USA Background: Triple negative breast cancer (TNBC) is a life-threatening disease with no targeted therapy, which is linked to obesity incidence. Leptin, a cytokine produced by adipose tissue and overexpressed in breast cancer, induces TNBC growth and survival. Leptin binds to its receptor, OB-R, inducing expression of Notch and breast cancer stem cell (BCSC) genes. Notch is a pro-angiogenic factor, which interacts with leptin and IL-1 signaling crosstalk (NILCO). NILCO induces the expression of several pathways involved in cell proliferation, metastasis, and angiogenesis; notably vascular endothelial growth factor (VEGF/VEGFR2). We have created leptin peptide receptor antagonists linked to iron oxide nanoparticles which will inhibit leptin signaling, thus decreasing the expression of Notch and BCSC genes and proteins. Methods: Several human TNBC cell lines were treated with the chemotherapeutics (IC50 doses: taxanes, doxorubicin, cyclophosphamide and anti-angiogenic/chemotherapeutic, sunitinib) combined with IONPLPrA2 for 24−72 h. Cell viability, apoptosis rate and the expression levels of NILCO molecules were analyzed using the Cellometer Vision Image Cytometer® . Results: IONP-LPrA2 improved the effects of chemotherapeutics on cell viability and apoptosis, which could allow the reduction of effective dosage.

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The expression of several NILCO components was significantly reduced by IONP-LPrA2 alone and in combination with chemotherapeutics. Conclusion: These findings suggest that IONP-LPrA2 may be useful as an adjuvant therapy in the treatment of TNBC. Results also indicate that IONP-LPrA2 may work in concert with chemotherapeutics and antiangiogenic drugs in vivo. Present findings suggest that IONP-LPrA2 adjuvant therapy could be a novel way to specifically target TNBC, and to reduce chemoresistance as well as the undesired side effects of chemotherapeutics. This may be particularly important for obese patients that show a higher incidence and poorest prognosis of TNBC. Acknowledgements: This work was partially supported by the National Institutes of Health and National Cancer Institute Grant 1SC1CA138658−05 and U54 CA118638, and DOD Idea Award BC 123427 to RRGP; and facilities, and support services at Morehouse School of Medicine (NIH RR03034 and 1C06 RR18386) and NIH/NCRR grant 1G12RR026250−03.

Drug Design 268 POSTER (Board P048) A potent and highly efficacious bivalent Smac Mimetic APG-1387 in Phase I clinical development J. Lu1 , S. Rong1 , H. Sun1 , L. Liu1 , D. McEachern1 , G. Wang2 , J. Wen2 , Y. Zhai2 , M. Guo2 , D. Yang2 , S. Wang1 . 1 University of Michigan, Comprehensive Cancer Center, Ann Arbor MI, USA; 2 Ascentage Pharma Group, Preclinical development, Taizhou Jiangsu, China Background and Aim: Inhibitors of apoptosis proteins (cIAP1/2 and XIAP) are key regulators of apoptosis and are attractive cancer therapeutic targets. Targeting IAPs with small molecules represents a promising new strategy for cancer treatment. APG-1387 was designed a potent smallmolecule inhibitors that mimic the natural dimeric Smac protein. We report the design and preclinical characterization of APG-1387 for its anticancer activity and mechanism of action. Methods and Results: Using a structure-based computational drug design method, we designed and synthesized SM-1387 (renamed as APG-1387 by Ascentage Pharma) as a bivalent Smac mimetic and a potent inhibitor of cIAP1, cIAP2 and XIAP. Biochemical assays showed that APG-1387 binds to XIAP, cIAP1, cIAP2 proteins with Ki values of 1, 30 and 20 nM, respectively. APG-1387 potently antagonizes XIAP proteins containing both the BIR2 and BIR3 domains and induces cIAP-1 degradation at 1−10 nM in human cancer cell lines. Cell growth assays showed that APG-1387 inhibits cell growth in a subset of human cancer cell lines and achieves IC50 values of 10 and 23 nM, respectively, in MDA-MB-231 human breast cancer and SK-OV-3 ovarian cancer cell lines. Flow cytometry and western blotting assays showed that APG-1387 at 10–100 nM induces profound apoptosis in the MDA-MB-231 and SK-OV-3 cell lines. In vivo study showed that APG1387 is capable of inducing tumor regression even at weekly dosing in the MDA-MB-231 xenograft model. APG-1387 shows a strong combination synergy with docetaxel and achieves tumor regression in the HCC1954 triple negative breast cancer model with no signs of toxicity. Conclusion: Our preclinical data suggest that APG-1387 is a promising anticancer agent. A phase I clinical trial of APG-1387 is ongoing to evaluate its safety, pharmacokinetics, PD biomarkers and preliminary efficacy in patients with advanced cancers. 269 POSTER (Board P049) The NCI-60 as an effective tool for scaffold hopping: A phenotypic systems-based approach to the design of novel chemotherapeutics D.G. Wishka1 , V. Kumar2 , B. Teicher3 , G. Kaur4 , B. Fang5 , P. Risbood6 , M. Hollingshead7 , J. Zais7 , J. Morris6 . 1 National Cancer Institute, Drug Synthesis and Chemistry Branch, Frederick MD, USA; 2 Leidos Biomedical Research, Drug Synthesis and Chemistry Branch, Frederick MD, USA; 3 National Cancer Institute, Molecular Pharmacology Branch, Rockville MD, USA; 4 National Cancer Institute, Molecular Pharmacology Branch, Frederick MD, USA; 5 M.D. Anderson Cancer Center, Houston TX, USA; 6 National Cancer Institute, Drug Synthesis and Chemistry Branch, Rockville MD, USA; 7 National Cancer Institute, Biological Testing Branch, Frederick MD, USA The indole-3-carbinol NSC-743380 [(1-(3-chlorobenzyl)-1H-indol-3-yl)methanol] causes regression in the mouse A498 renal xenograft model. Although its mechanism of action is not well understood, NSC-743380 produces a distinct growth inhibition pattern in the NCI-60 cell line screen. NSC-743380 acts very rapidly in cell culture, with changes observed in multiple signaling pathways within 5 minutes of exposure.

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Using cell lines derived from the NCI-60 panel, a search for a second xenograft model sensitive to NSC-743380 was unsuccessful. In addition, a significant medicinal chemistry effort examining modifications to the indole-3-carbinol structure of NSC-743380 failed to produce a suitable alternative. The COMPARE algorithm is a powerful tool for identifying compounds that share common mechanisms of action by matching their respective growth inhibition patterns in the NCI-60 cell panel. Running a COMPARE analysis on the existing NCI-60 data for the NCI compound collection with NSC-743380 as the exemplar identified a number of alternative chemotypes, including one represented by the mustard, NSC101490 [(E)-N -(4-(bis(2-chloroethyl)amino)-2-chlorobenzylidene)nicotinohydrazide]. Using the NCI-60 growth inhibition patterns as a guide to overlay of the SAR features from indole-3-carbinol series of NSC-743380 with that of the mustard series of NSC-101490 provided the basis for the scaffold hop to the new Benzyl-Amino-Benzyl-Alcohol (BABA) class of chemotherapeutics. Herein, we report the identification of a potent subtype of the BABA chemotype that shares with NSC-743380 the ability to cause regression in the A498 xenograft model as well as its distinctive NCI60 growth inhibition pattern. In addition, evidence will be presented to show that sulfotransferase activation may be a component involved in the antiproliferative activity of the BABA series against sensitive cell lines. 270 POSTER (Board P050) Discovery, development and optimization of low molecular weight EPH−ephrin protein–protein inhibitors M. Tognolini1 , C. Giorgio1 , I. Hassan-Mohamed1 , E. Barocelli1 , M. Mor1 , D. Pala1 , S. Russo1 , M. Incerti1 , A. Lodola1 . 1 University of Parma, Pharmacy, Parma, Italy Background: The EPH−ephrin system plays a key role in tumorigenesis and its de-regulation correlates with a poor clinical prognosis in many solid tumors. Agents targeting the EPH−ephrin system could be potentially useful for the inhibition of different facets of cancer progression. However, even though some classes of small molecules targeting EPH−ephrin interactions have been reported, their use is hampered by poor chemical stability and low potency. Stable and potent ligands are essential to obtain strong pharmacological data. Materials and Methods: We recently identified lithocholic acid (LCA) as a competitive and reversible compound able to disrupt EPH−ephrin interaction showing a Ki value of 49 mM. Using LCA scaffold as a reference structure and starting from a docking model of the EPHA2−LCA complex we designed and synthetized a series of amino acid conjugates of lithocholic acid and the new compounds were characterized for their ability to disrupt the EPHA2–ephrin-A1 interaction by means of an ELISA binding assay. The most promising compounds were further characterized for their cellular functional properties including: aspecific cytotoxicity, antiproliferative effect, cell cycle, inhibition of Eph kinase phosphorylation, induction of morphological changes, angiogenesis and migration. In order to check for the overall selectivity of the new compounds selected molecules were tested for their ability to interact with physiological-targets of bile acids (FXR, TGR5) as well as some other kinases (EGFR, VEGFR). Results: The L-homo-Trp-conjugated of LCA (UniPR129) emerged as a potent and selective antagonist of EPH receptors, inactive on the FXR, TGR5, EGF and VEGF receptors. UniPR129 reversibly and competitively disrupted EPHA2–ephrin-A1 interaction with Ki = 370 nM in an ELISA binding assay and it showed low micromolar potency in cellular functional assays, including inhibition of EPHA2 activation, cell migration and cell rounding and disruption of in vitro angiogenesis without cytotoxic effects.

Structure of UniPR129 was modified, with the aim to improve its pharmacokinetic properties, leading to the identification of a new orally bioavailable compound. Conclusions: The discovery of UniPR129 represents not only a major advance in potency compared to the existing EPH−ephrin antagonists but also an improvement in terms of cytotoxicity, making this molecule a useful pharmacological tool and a promising lead compound. Moreover, steroid scaffold can be modified in order to obtain potent, selective, and soluble bioavailable compounds.

Poster Session – Drug Design 271 POSTER (Board P051) Data integration and graph analysis for cancer genomics and drug discovery B. Bernard1 , M. Miller1 , H. Rovira1 , I. Shmulevich1 . 1 Institute for Systems Biology, Seattle, USA Overview: Each tumor type being studied by TCGA consists of tens of thousands of molecular data points derived from thousands of primary human tumor samples, from which we identify millions of statistically significant molecular associations. From these data, we know that each tumor harbors numerous somatic mutations and copy number aberrations that contribute to loss and gain of function in the cancer cells. While certain genes are more frequently aberrated than others, each tumor sample has a unique mutation and copy number profile that should inform patient stratification, treatment planning, novel target identification, and drug repurposing for personalized therapeutic strategies and drug discovery. The search space across millions of associations for tens of thousands of genes and thousands of potential drugs is tremendously underexplored. Consequently, promising targets and therapies are not identified or evaluated. Approaches to integrate and query molecular associations from heterogeneous sources of data pose a significant research and computational challenge in drug discovery. To this end, we have integrated statistical analysis of molecular data from thousands of primary tumor samples in TCGA with a large and heterogeneous cancer knowledge base, including cell line screening, protein–inhibitor interactions, semantic-based information in the literature, and protein interaction databases. We model these heterogeneous relationships in a graph database, and query this large graph to generate novel and testable predictions for target identification, drug repurposing, and tumor type prioritization for therapeutic intervention. Example 1. Identification of novel targets in primary human tumors: We have combined the Cancer Cell Line Encyclopedia (CCLE) and Achilles shRNA screening projects to infer synthetic lethal (SL) relationships in cell lines with characterized mutation profiles. Integration of these SL relationships with molecular associations in TCGA enables the identification of graph patterns and subsequent prioritization of potential cross-cancer therapeutic targets with direct evidence for SL relationships in both cell line and primary human tumor samples. Example 2. Patient stratification and prioritization of tumor types for clinical trials: Analysis of targeted drug screening in cell lines reveals that the sensitizing genes (i.e., those genes harboring mutations and copy number aberrations in drug-sensitive cell lines) are invariably not limited to the drug target itself. For example, AKT1 inhibition is most effective in cell lines with PTEN mutations. By analyzing the integration of cell line screening hits with accompanying relationships derived from primary tumor sample data in TCGA, we can stratify patients based on their aberration profiles and prioritize clinical trials for tumor types with evidence for relationships between the drug target and sensitizing aberrations. 272 POSTER (Board P052) 8-(1-Anilino)ethyl)-2-morpholino-4-oxo-4H-chromene-6-carboxamides as PI3Kbeta/delta inhibitors: structure–activity relationships and identification of AZD8186, a clinical candidate for the treatment of PTEN deficient tumours B. Barlaam1 , S. Cosulich1 , S. Degorce1 , M. Fitzek1 , S. Green1 , U. Hancox1 , C. Lambert-van der Brempt1 , J.J. Lohmann1 , M. Maudet1 , R. Morgentin1 , M.J. Pasquest1 , A. Peru1 , P. Ple1 , T. Saleh1 , M. Vautier1 , M. Walker1 , L. Ward1 , N. Warin1 . 1 AstraZeneca, Oncology, Macclesfield, United Kingdom Several studies have highlighted the dependency of PTEN deficient tumours to PI3Kb activity and specific inhibition of PI3Kd has been shown clinical efficacy against human B-cell cancers. We previously reported the identification of a new series of pyrido[1,2-a]pyrimidin-4-ones and our efforts1−2 to optimize this series as orally active PI3Kb/d inhibitors, leading to compounds 1 and 2. Combining high potency, high solubility and high metabolic stability was a significant challenge in the pyrido[1,2-a]pyrimidin4-one series, possibly because of the intrinsic lipophilicity of the pyrido[1,2-a]pyrimidin-4-one core. In this poster, we describe the discovery and optimization of a new series of 8-(1-anilino)ethyl)-2-morpholino-4oxo-4H-chromene-6-carboxamides as PI3Kb/d inhibitors, leading to the identification of the clinical candidate AZD8186, a potent inhibitor of p-Akt in cells sensitive to PI3Kb inhibition (IC50 0.003 uM (+/− SEM: 0.001) in PTEN null breast adenocarcinoma MDA-MB-468 cells) and in cells sensitive to PI3Kd inhibition (IC50 0.017 uM (+/− SEM: 0.004) in Jeko B cell line), but not to cells sensitive to PI3Ka inhibition (IC50 0.752 uM (+/− SEM: 0.293) in PIK3CA mutant human breast ductal carcinoma BT474). On the basis of the lower lipophilicity of the chromen-4-one core compared to the previously utilised pyrido[1,2-a]pyrimid-4-one core, this series of compounds (and AZD8186 in particular) displayed high metabolic stability and suitable

Poster Session – Drug Design physical properties for oral administration. AZD8186 showed profound pharmacodynamic modulation of p-Akt in PTEN-null PC3 prostate tumour bearing mice after oral administration and showed complete inhibition of tumour growth in the mouse PTEN-deficient PC3 prostate tumour xenograft model. AZD8186 was selected as a clinical candidate for treatment of PTEN-dependent cancers and has recently entered phase I clinical trials.

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cell growth of HCT116 cells and also significantly suppressed tumor growth in the HCT116 xenograft model. Those results suggest that the STLC derivatives with two linked phenyl rings could be a novel lead compound to design further clinical candidates of the next generation of KSP inhibitors for antitumor chemotherapies.

273 POSTER (Board P053) Significance of serine-167 and cysteine-129 residues in the active site of the immune-suppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1) for the binding of novel inhibitors P. Tomek1 , B.D. Palmer1 , J.U. Flanagan1 , L. Ching1 . 1 Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand Background: IDO1 is an enzyme expressed by a broad range of cancers to suppress the host’s immune system. It is a validated target for cancer therapy, with two IDO1 inhibitors already in human clinical trials. The IDO1 active site amino acids Ser-167 and Cys-129 are predicted to form hydrogen bond interactions with inhibitors. We have screened for inhibitors to probe the importance of these residues in the IDO1 active site for ligandbinding and functional activity of the enzyme. Material and Methods: The National Cancer Institute Diversity Set III library (1597 compounds) was screened for inhibition of IDO1 enzymatic activity using our recently developed fluorescence assay automated with a JANUS robotic workstation. Mutant IDO1 proteins were prepared using Stratagene QuikChange® Site-Directed Mutagenesis Kit. Results: The NCI library was screened against wild-type IDO1 and alanine replacement mutants of serine-167 (S167A) and cysteine-129 (C129A). This identified three new classes of IDO1 inhibitors (pyrimidinones, phenanthroimidazoles and benzoxadiazoles). Compounds from the phenanthroimidazoles and benzoxadiazoles classes exhibited a tightbinding, reversible mode of inhibition that was strikingly different to that of a well-characterised IDO1 inhibitor, 4-phenyl-1H-imidazole. When screened against the S167A mutant, 18 of the 69 compounds that inhibited wildtype IDO1 (>25% at 20 mM) exhibited decreased activity against the S167A mutant. Structure–activity studies around two of these compounds, 2-(1H-phenanthro[9,10-d]imidazol-2-yl)phenol and 6-carboxy-5-((4-iodo-2methylphenyl)amino)benzo[c][1,2,5]oxadiazole 1-oxide, were performed, and showed that the hydroxy and N-oxide substituents on these two molecules respectively, were related to the decreased activity. When screened against C129A mutant, no compounds in the library showed decreased activity compared to wild-type IDO1, however three compounds showed increased inhibition. Conclusions: Three IDO1 inhibitors identified in this study displayed tight-binding reversible mode of inhibition, one of the more desirable mechanisms of action for therapeutic agents. The hydroxyl group of serine167, but not the thiol of cysteine-129 in the IDO1 active site appears to be important for interactions with a range of different inhibitors. 274 POSTER (Board P054) Novel cysteine derivatives for the next generation anticancer agents acting on KSP N. Ogo1 , J. Sawada1 , Y. Ishikawa1 , K. Matsuno1 , A. Hashimoto2 , A. Asai1 . 1 University of Shizuoka, Graduate School of Pharmaceutical Sciences, Shizuoka, Japan; 2 Taiho Pharmaceutical Co. Ltd., Tsukuba Research Center, Ibaraki, Japan Kinesin spindle protein (KSP), known as human Eg5, plays an important role in the early stages of mitosis. It is responsible for the formation and maintenance of the bipolar spindle. Inhibition of KSP leads to cell cycle arrest during mitosis and causes cells with a monopolar spindle followed by cell death. A large number of KSP inhibitors have been reported and some of them entered into clinical trials. Although clinical efficacy of these inhibitors has been limited to date, recently better results have been obtained in the treatment of hematological malignancies with thiadiazole based KSP inhibitors such as ARRY-520. We previously identified S-tritylL -cysteine (STLC) as a selective KSP inhibitor from our HTS campaign and reported that several STLC derivatives induced M-phase accumulation and subsequent apoptosis in leukemic cells (BMCL 2007, 17, 3921–3924, BMCL 2010, 20, 1578–1580, Cancer Lett. 2010, 298, 99–106). The objective of this study is to improve the potency of these leads and to confirm the antitumor efficacy in vivo. As a result of further structure–activity relationship studies, we identified novel derivatives with two fused phenyl rings which showed remarkably potent KSP inhibitory activity with IC50 values in nanomolar ranges. Docking simulation of these novel derivatives using AMBER12 indicated that cross-linking of the phenyl rings in the trityl group allowed for better binding by occupying a hydrophobic pocket in the L5 allosteric binding site. The representative derivatives potently inhibited

Figure: Novel cysteine derivatives. 275 POSTER (Board P055) Modulation of PIP2 levels through small molecule inhibition of PIP5K D. Andrews1 , S. Cosulich2 , N. Divecha3 , D. Fitzgerald3 , V. Flemington2 , C. Jones1 , D. Jones3 , O. Kern4 , E. MacDonald4 , S. Maman4 , J. McKelvie5 , K. Pike1 , M. Riddick6 , G. Robb1 , K. Roberts2 , J. Smith4 , M. Swarbrick4 , I. Treinies7 , M. Waring1 , R. Wood6 . 1 AstraZeneca, Chemistry, Macclesfield, United Kingdom; 2 AstraZeneca, Bioscience, Macclesfield, United Kingdom; 3 Cancer Research UK, Inositide Laboratory, Manchester, United Kingdom; 4 Cancer Research Technology, Chemistry, Cambridge, United Kingdom; 5 Cancer Research Technology, Chemistry, London, United Kingdom; 6 Cancer Research Technology, Bioscience, Cambridge, United Kingdom; 7 Cancer Research Technology, Bioscience, London, United Kingdom Background: Phosphatidyl inositol (4,5)-bisphosphate (PIP2 ) is a key phospholipid signalling molecule, involved in cellular processes such as cell proliferation and survival. Cellular PI(4,5)P2 is synthesised by phosphorylation of PI(4)P on the D-5 position of the inositol head group by phosphatidylinositol-4-phosphate 5-kinases (PIP5Ks). The family of PIP5K is comprised of 3 isoforms a, b and g regulated by membrane receptors, phosphorylation and small GTPases of the Rho and ARF family. Activation of this pathway is known to promote growth and invasion of cancer cells, rendering PIP5K an attractive therapeutic target for antitumour therapies Material and Methods: Two separate focussed screening campaigns identified a number of hit series. Structure Activity Relationships (SAR) was built around three chemotypes, (A, B & C) with biochemical assays routinely run against all three isoforms of PIP5K, two isoforms of PI4K and PI3Ka. Compounds were also routinely assayed cellular assays, measuring surrogate markers of cellular PIP2 levels. Results: Potent, selective inhibitors (pIC50 >8), of PIP5K kinases have been developed in three chemical series. The SAR of series B will be described in detail. Live cell imaging results will be presented. Conclusions: Cell data demonstrate that PIP5K inhibition slows the rate of PIP2 synthesis.

PIP5Ka (pIC50 ) PIP5Kb (pIC50 ) PIP5Kg (pIC50 ) PI3Ka (pIC50 ) PI4Ka (pIC50 ) PI4Kb (pIC50 ) PIP2 inhib @ 3mM

Series A

Series B

Series C

5.8 7.2 7.2 5.3 5.1 5.1 3.7%

7.8 8.6 8.9 4.5 4.8 5.4 21%

9.3 8.8 8.9 <4.0 4.0 4.0 17%

276 POSTER (Board P056) An X-ray crystal structure-based understanding of the inhibition of the MDM2−p53 protein–protein interaction by isoindolinones B. Anil1 , E. Blackburn1 , T. Blackburn2 , S. Cully2 , J. Liu3 , C.J. Drummond2 , J.A. Endicott1 , B.T. Golding4 , R.J. Griffin2 , K. Haggerty2 , J. Lunec2 , D.R. Newell2 , C.H. Revill2 , C. Riedinger1 , A.F. Watson2 , Q. Xu2 , Y. Zhao2 , I.R. Hardcastle2 , M.E.M. Noble1 . 1 University of Oxford, Department of Biochemistry, Oxford, United Kingdom; 2 Newcastle University, Northern Institute for Cancer Research, Newcastle upon Tyne, United Kingdom; 3 Newcastle University, Northern Istitute for Cancer Research, Newcastle upon Tyne, United Kingdom; 4 Newcastle University, School of Chemistry, Newcastle upon Tyne, United Kingdom The p53 tumor suppressor plays a pivotal role in responding to cellular stress. Activation of p53 protein results in the transcription of a number of

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genes that govern progression through the cell cycle, the initiation of DNA repair, and apoptosis. The activity of p53 is tightly regulated by the MDM2 protein, in an auto-regulatory feedback loop. Inhibition of the MDM2−p53 protein–protein complex by small molecule inhibitors has been shown to reactivate normal p53 pathways in cells overexpressing MDM2, and so exert an anti-cancer effect. A number of series of potent small-molecule inhibitors have been developed as far as clinical trials. Understanding of the structural basis for inhibition from ligand-bond protein X-ray structures has been important in the optimisation of most of these series. We have developed a class of inhibitors of the MDM2−p53 interaction, based on an isoindolinone scaffold [J. Med. Chem. 2006, 49, 6209–6221, Bioorg. Med. Chem. Letters 2011, 21, 5916– 5919]. NU8406A, (R)-4-chloro-3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (IC50 = 43.8±6.2 nM) shows comparable in vitro activity to Nutlin-3a. Recently, we reported the MDM2 co-crystal structure of Nutlin-3a using a surface-entropy reduction mutant [Acta Cryst 2013, D69, 1358–1366]. We have applied these MDM2 mutants to solve the co-crystal structure of NU8406A. The structure shows that the binding mode of the isoindolinone is comparable to that for Nutlin-3a, and confirms the (R)-stereochemistry of the ligand. Preliminary structure–activity studies have revealed significant differences between the two series in the way that the Phe19 pocket is occupied. 277 POSTER (Board P057) The discovery and pre-clinical development of the first clinical stage EZH2-inhibitor, EPZ-6438 (E7438) K. Kuntz1 , H. Keilhack2 , R. Pollock2 , S. Knutson2 , N. Warholic2 , V. Richon2 , R. Chesworth1 , R. Copeland1 , M. Porter-Scott1 , C. Sneeringer1 , T. Wigle1 . 1 Epizyme Inc, Molecular Discovery, Cambridge Massachusetts, USA; 2 Epizyme Inc, Biology, Cambridge Massachusetts, USA Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). Cancers harboring these genetic alterations are dependent on EZH2 enzymatic activity for cellular survival. Here, we disclose the discovery of EPZ-6438 (E7438), as a potent, selective and oral bioavailable small molecule inhibitor of EZH2 in preclinical models of NHL. Previously we have disclosed the properties of EPZ005687, a tool compound useful for exploring the in vitro biology of EZH2 inhibition. Multiparametric optimization of the potency, pharmokinetics, oral biovailability and safety properties of this series led to the discovery of the clinical compound, EPZ-6438. Modulation of the log P was required to reach the optimal balance between clearance and bioavailability while maintaining the requisite potency. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. EPZ-6438 recently entered clinical testing as E7438 in a dose escalation phase 1 trial in relapsed or refractory malignancies.

Poster Session – Drug Design 278 POSTER (Board P058) Sentinel lymph nodes mapping of macrophage targeted mannosyl human serum albumin-indocyanine detected by combined color and near infrared fluorescence imaging system in esophagus Y. Quan1 , Y. Oh2 , J.I.H.O. Park3 , J. Park4 , J. Jeong4 , B. Kim5 , H. Kim1 . 1 Korea University Guro Hospital, Department of Thoracic Cardiovascular Surgery, Seoul, Korea; 2 Korea University, Department of Bio-Convergence Engineering, Seoul, Korea; 3 Korea Advanced Institute of Science and Technology, Department of Bio and Brain Engineering, Seoul, Korea; 4 Seoul National University, Department of Transdisciplinary Studies Program in Biomedical Radiation Science, Seoul, Korea; 5 Korea University, Department of Biomedical Engineering, Seoul, Korea Objective: Esophageal cancer is one of the most poor prognosis diseases. Therefore, 3-field lymph node dissection is performed for surgically curable esophageal cancer. However these extended lymphadenectomy is leads to high morbidity. Thus, sentinel lymph node (SLN) concept is essential point for esophageal cancer surgery to avoid unnecessary lymph node dissection. There are some pre-clinical trials use fluorescence indocyanine green (ICG) or radioisotope to selective removing SLN. In this study, we used mannosyl human serum albumin (MSA)-ICG as a new SLN targeting tracer to investigate esophageal SLN mapping using intraoperative color and fluorescence merged imaging system (ICFIS). Methods: We activated macrophage U937 cells and treated ICG or MSAICG respectively. The distributions of ICG and MSA-ICG in cells were investigated by Infrared Imaging System. In-vivo study, the ICG or MSAICG was injected into footpad of nude mice and then the lymphatic flow was investigated by Infrared Imaging System. Additionally, 5 Yorkshire pigs underwent thoracotomy and received submucosal injection of MSA-ICG with endoscopic needle through an esophagoscope. The distribution of MSA-ICG in SLN of pig esophagus was investigated by ICFIS. Results: Our study found that the MSA-ICG were specific bound to macrophage than free ICG, and also the detected lymph node imaging more distinguishable than free ICG in mice. The pig esophageal SLN which received MSA-ICG submucosal injection were detectable from 15 min. In 3 of 5, two SLN were identified, however in 2 of 5, one SLN was detected. Discussion: In fluorescence guided esophageal surgery, the MSA-ICG provided precise real-time imaging of SLN. Therefore the MSA-ICG may be a reliable tracer for SLN identification in esophagus cancer. 279 POSTER (Board P059) Discovery of multiple kinases inhibitors, DBPR114, as the novel anti-cancer agent H.P. Hsieh1 , C.C. Kuo2 , J.J. Chiu2 , T.A. Hsu1 , T.K. Yeh1 , C.T. Chen1 . 1 National Health Research Institutes, Institute of Biotechnology and Pharmaceutical Research, Zhunan Miaoli County, Taiwan; 2 National Health Research Institutes, Institute of Cellular and Systems Medicine, Zhunan Miaoli County, Taiwan Purpose: The goal of targeted therapy is to identify the suitable target molecules to be inhibited, in order to achieve the best antitumor effect. The classical viewpoint is that inhibitor acting on single target allows selectivity and less adverse effect. However, clinical experience revealed that almost relapsed cancer patients developed drug resistance, often due to the activation or development of alternative signaling pathways or mutations that single target drugs are unable to effectively inhibit them. A current trend in the development of kinase inhibitors is the assumption that multi targeted therapy, which targets at several signaling pathways simultaneously, is more effective than single targeted therapy. Thus, paradigm in designing new anticancer drug is shifted: The drugs that act on multiple targets might have a better chance of inhibiting cancer cell proliferation than drugs that act on a single target. The objective of this research is aimed at identifying a multi-targeted kinase inhibitor as a development candidate for cancer therapy. Material and Method: We established Structure–Activity Relationship (S.A.R.) study based on 800 compounds that were synthesized by High Throughput Parallel Synthesis. Scaffold hopping approach by changing pharmacophore moiety had led us to discover DBPR114 as a multipletargeted kinase inhibitor. Results: We discovered a novel multiple-targeted kinase inhibitor, DBPR114, that effectively inhibited 15 kinases in a panel of 57 oncogenic related kinases profiling, particularly against to Aurora kinase A and B, FLT-1, FLT-3 and c-Met in nanomolar range. In pharmacological study, DBPR114 significantly shrank tumor in 8 different xenograft models including Mia-Paca2, AsPC-1 (pancreatic carcinoma), Hep3B (hepatocellular carcinoma), MKN-45 (gastric carcinoma), MOLM-13 and MV4;11 (acute myeloid leukemia), NTUB-1 (bladder cancer), and Colo205 (colorectal carcinoma) at a dose of 3 to 20 mg/kg by intravenous

Poster Session – Drug Design administration without significant adverse effect in most group of in vivo evaluations. Conclusion: This multiple targeting inhibitory properties plays a major role to shrinks tumor growth of various cancer cells in vivo and in vivo such as MOLM-13, MV4;11, MKN45, Colo-205, Mia-Paca2 and NTUB-1. The broad spectrum of anti-tumor activity of BPR1K0871 provides a great potential to become a promising multi-targeted kinase inhibitors as next generation of anti-cancer drug. 280 POSTER (Board P060) A hybrid drug design approach to overcome imatinib resistance for treating leukemia Y.M. Wei1 , K.K.W. To2 , S.C.F. Au-Yeung1 . 1 The Chinese University of Hong Kong, Department of Chemistry, New Territories, Hong Kong; 2 The Chinese University of Hong Kong, School of Pharmacy, New Territories, Hong Kong Background: Imatinib, a multi-targeted tyrosine kinase inhibitor, exhibits potent anticancer activity against leukemia harboring the bcr-abl oncogene and some solid tumors overexpressing c-kit and PDGFR. However, its clinical efficacy is severely hindered by the emergence of resistance primarily due to acquired mutations in the bcr-abl kinase domain. Material and Methods: By applying our established ‘Platinum (Pt) drug − Bioactive ligand’ drug design platform, novel Pt-imatinib hybrid compounds were synthesized and fully characterized by 1 H-NMR and ESI+ MS. In silico docking analysis was performed to investigate the interaction of the new compounds with wild-type bcr-abl and its various resistance-causing mutants. Anticancer activity was evaluated by the colorimetric MTT assay. Kinase inhibition profiling was performed to assess the specificity of the new compounds on various receptor tyrosine kinases. Cell cycle regulation and apoptosis were studied by propidium iodide and Annexin V-7AAD staining, respectively. Reaction between the new compounds and DNA was studied by NMR using 5 -GMP as a model nucleotide. Results: The new Pt-imatinib hybrid compounds were found to be highly effective against the leukemia cell line K562 harboring bcr-abl, but not in another non-bcr-abl expressing leukemia RPMI8226 and some other solid tumors. Importantly, the new compounds maintained their anticancer activity in an imatinib-selected resistant K562 subline. Moreover, the new compounds did not appreciably affect cell growth of normal HEK293 and LLC-PK1 cell lines. Kinase profiling revealed that the new compounds maintain selectivity towards bcr-abl, c-kit and PDGFR, similar to imatinib. By docking simulation, the new compounds were predicted to interact with both wild-type bcr-abl and some of its resistance-mediating mutants. This was verified by biochemical kinase inhibition assay on selected bcr-abl mutants. In K562 cells, the new compounds produced G1 phase arrest, in contrast to G2/M arrest mediated by classical Pt drugs. Unlike classical Pt compounds, the Pt-imatinib hybrids only form monofunctional Pt-DNA adducts, presumably contributing to the minimal toxicity in normal cells. Conclusions: New lead Pt-imatinib hybrid compounds were found to be specific towards bcr-abl-expressing leukemia cells and capable of circumventing resistance mediated by clinically identified mutations. Their good safety profile and unique mechanisms advocate further clinical development.

Figure: General formula of the designed compounds.

281 POSTER (Board P061) Novel hybrid drug design strategy to circumvent erlotinib resistance and to optimize its pharmacokinetic properties for treating lung cancer D.C.S. Poon1 , S.C.F. Au-Yeung2 , K.K.W. To1 . 1 The Chinese University of Hong Kong, School of Pharmacy, New Territories, Hong Kong; 2 The Chinese University of Hong Kong, Department of Chemistry, New Territories, Hong Kong Background: Erlotinib is an EGFR tyrosine kinase inhibitor (TKI) highly potent towards lung cancer carrying the sensitizing EGFR mutations. However, its usefulness is severely compromized by resistance mediated primarily by the secondary EGFR T790M mutation. Most EGFR TKIs are substrates of the efflux transporters Pgp and ABCG2. Induction of these

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transporters can remarkably decrease cellular accumulation of erlotinib, thereby conferring drug resistance. Moreover, EGFR TKIs are not effective for brain metastasis from primary lung cancer because of poor drug penetration restricted by Pgp and ABCG2 at the blood–brain barrier. Material and Methods: Novel platinum (Pt)–erlotinib hybrid compounds were synthesized by our established ‘Pt-bioactive ligand’ drug design approach. They were fully characterized by 1 H-NMR and ESI+ MS. Anticancer activity in a panel of non-small cell lung cancer cell lines was evaluated by sulforhodamine B assay. Docking simulation was performed to investigate the interaction of the new compounds with EGFR harboring different mutations. Inhibition of different EGFR mutant kinases was evaluated by biochemical kinase assay. Drug uptake and transport across cell monolayer specifically overexpressing P-gp/ABCG2 were studied to examine whether the new compounds are substrate of the transporters. Results: Similar to erlotinib, new Pt-erlotinib hybrid compounds were found to be highly effective against HCC827 harboring the sensitizing EGFR L858R mutation (IC50 : 0.006–0.16 mM) but become inactive in H520 with minimal EGFR expression (IC50 ~15 mM). Importantly, they were much less affected than erlotinib by acquired resistance mediated by the secondary EGFR T790M mutation in H1975 cells (fold resistance: ~200 versus ~12,000 for erlotinib). By docking simulation, the new compounds were predicted to maintain their binding with the EGFR T790M mutant. Results from biochemical kinase assay reveal that the new compounds inhibited EGFR in an ATP-competitive manner and that the inhibition was only minimally reduced by the EGFR T790M mutation. Unlike erlotinib, cellular uptake and transport of the hybrid compounds were not affected by Pgp and ABCG2 overexpression. Conclusions: Novel Pt-erlotinib hybrids were found to circumvent resistance mediated by the acquired EGFR T790M mutation. By escaping P-gp/ABCG2-mediated efflux, the new compounds may be effective for treating metastatic brain cancer. Further development of the hybrid compounds is warranted.

282 POSTER (Board P062) Membrane anchorage of Stat3 via artificial protein lipidation M. Avadisian1 , S. Fletcher1 , B. Liu1 , W. Zhao2 , J. Turkson2 , C. Gradinaru1 , P. Gunning1 . 1 University of Toronto, Chemistry and Physical Sciences, Mississauga, Canada; 2 University of Central Florida, College of Medicine, Orlando, USA Lipidation localizes cytosolic proteins within cellular membranes through a covalent attachment of sterol or lipid groups. This post-translational modification restricts the motility of otherwise soluble proteins to the cell membrane. Using artificial lipidation, we propose to mimic Nature by artificially inducing protein–membrane anchorage through the use of a rationally designed Protein–Membrane Anchor (PMA) to inhibit a protein’s motility and function within the cell. We hypothesized that induced membrane anchorage of proteins can hold significant therapeutic value when applied to cancer-promoting cell-signaling proteins. To demonstrate the protein–membrane anchorage strategy, we choose to target Stat3 because of its constitutive activity in a number of cancers. Thus, our goal was to develop an inhibitor that could sequester Stat3, a 93 kDa protein, at the plasma membrane and suppress its motility through PMA-induced protein–membrane association. Our proto-type PMA 1 was composed of two binding modules: a recognition motif to bind the protein and an anchor to sequester the protein complex to the membrane. PMA 1 was composed of a potent Stat3 inhibitor covalently attached to a cholesterol membrane anchor. PMA 1 was able to anchor Stat3 to the cell membrane in MDA-MB-231 breast cancer cells that are known to have constitutively-active Stat3. Most excitingly, in the presence of 25mM concentration PMA 1, we observed complete sequestration of Stat3 to the cell membrane through PMA−Stat3 association. To improve metabolic stability, we synthesized a new library of PMAs with varying Stat3 binding and membrane anchoring moieties. Currently, we are conducting biophysical and immunofluorescent studies on the new PMAs.

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283 POSTER (Board P063) The development of the first selective inhibitors of the UBA5 enzyme to probe for E1 activity in diseased cells S.R. da Silva1 , S.L. Paiva1 , M. Bancerz2 , M. Geletu2 , A.M. Lewis2 , J. Chen3 , Y. Cai3 , H. Li3 , P.T. Gunning1 . 1 University of Toronto, Chemistry, Toronto, Canada; 2 University of Toronto Mississauga, Chemical and Physical Sciences, Mississauga, Canada; 3 Georgia Regents University, Biochemistry and Molecular Biology, Augusta, USA Cancer cells produce and degrade proteins more rapidly than most normal cells, and are in turn more sensitive to changes in protein regulation. Similarly to secretory cells, cancer cells have the propensity to undergo endoplasmic reticulum (ER) stress due to higher protein turnover that, if left uncorrected, can result in apoptosis. To avoid these fates, cells have developed support systems such as the conjugation of the ubiquitin-fold modifier 1 (UFM1) ubiquitin-like protein to other protein targets, which has been implicated in counteracting apoptotic ER stress in pancreatic secretory cells. Our research has recently focused on the development of the first inhibitor of the UFM1 pathway by targeting its E1 enzyme, UBA5, in order to counteract the anti-apoptotic effects of this system and to further study the role of UFM1 conjugation in cancer cells. We have successfully identified a lead inhibitor, 5C-Z, which incorporates an adenosine moiety coupled to a zinc(II) polyazamacrocylic coordination complex. Through enzymatic assays that evaluate the transthiolation ability of UBA5 in the first step of UFM1 activation, our inhibitor exhibits low micromolar activity against UBA5 while demonstrating potent selectivity over other E1 enzymes (>20fold). Kinetic assays reveal that 5C-Z acts non-competitively on UBA5, indicating that the compound could possibly be binding to and inhibiting the “inactive” subunit of the UBA5 homodimer. Furthermore, treatment of lung and leukemia cells that exhibit high levels of UBA5 protein expression leads to a decrease in cell proliferation yet does not induce cell death in diseased or healthy cells, up to 200 mM. This novel strategy of inhibiting UBA5 and UFMylation would make cancer cells that are highly dependent on this system more susceptible to treatment regimens of principle drugs, which could lead to the use of milder drug dosing strategies. Our novel inhibitors produced from this research can be used as a probe to further investigate the role of E1 activating enzymes in cancer progression.

Figure: General formula of the compounds.

284 POSTER (Board P064) Poly(ADP-ribose) glycohydrolase (PARG) inhibitors increase nuclear poly(ADP-ribose) after methylating DNA damage A. Jordan1 , B. Acton1 , E. Fairweather1 , N. Hamilton1 , S. Holt1 , J. Hitchin1 , C. Hutton1 , D. James1 , S. Jones1 , A. McGonagle1 , H. Small1 , K. Smith1 , A. Stowell1 , I. Waddell1 , B. Waszkowycz1 , D. Ogilvie1 . 1 Cancer Research UK Manchester Institute, Drug Discovery, Manchester, United Kingdom Background: DNA single strand breaks (SSBs) are the most common type of damage occurring in cells. Poly(ADP ribose) polymerase (PARP) binds to SSBs and auto-ribosylates itself using NAD+ as a substrate. This creates chains of poly ADP ribose (PAR) which provide a signal for other proteins to repair the lesion. Sequential removal of the PAR chains is accomplished by Poly(ADP-ribose) glycohydrolase (PARG). Failure to complete the DNA repair process, either by inhibition of PARP (e.g. with olaparib) or inhibition of PARG (with shRNA), can lead to cell death. PARG is the only enzyme known to efficiently catalyse the hydrolysis of O-glycosidic linkages of ADP-ribose polymers and exists (unlike PARP) as a single gene. We therefore sought to exploit this vulnerability and as part of a fostering agreement with AstraZeneca have developed novel small molecule inhibitors of PARG activity. Methods: A screening cascade for small molecule inhibition of PARG was developed. Biochemical inhibition of PARG was measured using a bespoke HTRF assay. In addition, selected PARG inhibitors were tested against other enzyme substrates (ARH3, PARP1) to assay for selectivity. Active compounds were then taken forward and tested in cells for PAR chain persistence and for cytotoxicity using a 3-day HeLa assay. Suitable compounds were then evaluated for their physico-chemical properties and their in vivo PK profiles determined.

Poster Session – Drug Design Results: Early PARG inhibitors increased nuclear PAR chain levels in cells after 1 h treatment with the methylating agent methyl methanesulfonate (MMS) but also displayed off-target cytotoxicity in the absence of MMS. Applying computational chemistry enabled us to discover novel series of compounds with sub-micromolar potency with a wide differential to acute cytotoxicity. Compounds were also highly selective against PARP1 and ARH3 in vitro. PAR chain persistence in cells was also maintained after using the more clinically-relevant methylating agent temozolomide (TMZ). PARG inhibitors displayed satisfactory in vitro and in vivo PK profiles. Conclusions: We have developed PARG inhibitors that block the breakdown of PAR chains in cells after exogenous DNA damage by methylating agents. These tool compounds are potent and selective and enable us to both explore in more detail the cellular mode of action and investigate pre-clinical in vivo models for PARG inhibition. 285 POSTER (Board P065) A nanomolar-potency small molecule inhibitor of the STAT5 protein A.A. Cumaraswamy1 , A. Lewis2 , M. Geletu2 , A. Todic2 , D.B. Diaz2 , X.R. Cheng3 , C.E. Brown2 , R. Laister4 , D. Muench5 , K. Kerman3 , H.L. Grimes5 , M.D. Minden4 , P.T. Gunning1 . 1 University of Toronto, Department of Chemistry, Mississauga ON, Canada; 2 University of Toronto, Chemistry, Mississauga ON, Canada; 3 University of Toronto, Department of Physical & Environmental Sciences, Scarborough ON, Canada; 4 Princess Margaret Cancer Institute, Ontario Cancer Institute, Toronto ON, Canada; 5 Cincinnati Children’s Hospital Medical Centre, Division of Experimental Hematology, Cincinnati OH, USA Signal Transducer and Activator of Transcription 5 (STAT5) protein has gained notoriety for its aberrant role in many human cancers. In contrast to normal cells where STAT5 activity is rapid and transient, in cancer cells, including leukemias, STAT5 activity is routinely hyper-activated, conferring resistance to cell death and driving excessive expression of proto-oncogenes. Despite significant evidence showing STAT5’s role in human cancers, there has been little progress in developing direct inhibitors of STAT5 function. Potent and selective small molecule inhibitors of STAT5 will be effective therapeutics for treatment of hematological cancers and overcoming the side effects of current treatments. STAT5 is activated extracellularly by ligand-receptor binding, which results in STAT5 recruitment to intracellular receptor sites via their Src Homology 2 (SH2) domain. STAT5 is then phosphorylated, facilitating STAT5-STAT5 dimerization via reciprocal phosphotyrosine-SH2 interactions. The dimer shuttles to the nucleus and induces target gene transcription. In cancer cells, STAT5 is persistently activated, leading to the aberrant expression of STAT5 target genes that promote cancer cell survival and prevent cell death. To achieve this goal, we have employed a structure-based drug design strategy using computational analysis, medicinal chemistry and synthetic methods amenable to generating a diverse library. Herein, we report the first nanomolar, STAT5-selective inhibitor, AC-3-019, possessing a phosphotyrosyl-mimicking salicylic acid group, which potently and selectively binds to STAT5 over STAT3, inhibits STAT5-SH2 domain complexation events in vitro, silences activated STAT5 in leukemic cells, as well as STAT5’s downstream transcriptional targets, including MYC and MCL1, and as a result, leads to apoptosis. We believe AC-3-019 represents a useful probe for interrogating STAT5 function in cells as well as being a potential candidate for advanced preclinical trials.

Poster Session – Drug Design 286 POSTER (Board P066) Potent and selective non-sulfamate-containing small molecule inhibitors of the ubiquitin activating enzyme S. Paiva1 , S.R. da Silva1 , M. Bancerz1 , H. Quereshi1 , G.W. Xu2 , A.D. Schimmer2 , P.T. Gunning1 . 1 University of Toronto, Chemical and Physical Sciences, Mississauga, Canada; 2 University Health Network, Princess Margaret Cancer Centre, Toronto, Canada Within the cell, many essential regulatory functions including protein recycling, translocation and cell signaling are mediated by the conjugation of ubiquitin (Ub) and ubiquitin-like (Ubl) proteins to target substrates. Ub/Ubl protein conjugation occurs through a cascade of events involving three enzymes: the ubiquitin activating enzyme (E1), the ubiquitin conjugating enzyme (E2) and the ubiquitin ligase (E3). In cancer cells, many of these systems are hyperactive making them attractive targets for the design of novel cancer therapeutics. More specifically, in recent years there has been a plethora of interest in the development of strategies that focus on designing inhibitors specific for different E1 isoforms. To this end, there has been some success, namely the mechanistic inhibitor MLN4924 and PYZD4409 that target the NEDD8 and Ub activating enzymes, respectively (NAE and UAE). MLN4924 is a sub-micromolar NAE inhibitor currently in Phase I clinical trials for the treatment of leukemia, lymphoma, multiple myeloma and solid tumours, while PYZD-4409 is a micromolar UAE inhibitor that promotes disease regression in leukemia and myeloma models. Given the great success of MLN4924 and the promising results of PYZD-4409, targeting E1s is an excellent strategy for controlling cancer cell growth and disease progression. However, the design of an inhibitor with potent and selective UAE activity still remains an elusive goal in this field of research. Our research has primarily focused on the rational design of selective and potent non-mechanistic UAE inhibitors by targeting a novel druggable site on the UAE protein using a two-prong approach. We have successfully developed inhibitors that display low nanomolar inhibition of UAE in purified enzymatic based assays (IC50 ~120–500 nM), as well as exhibiting >10fold selectivity for UAE over other E1 enzymes. To date, these are the most potent small molecule non-sulfamate-based inhibitors of UAE reported.

287 POSTER (Board P067) The discovery and optimization of small molecule antagonists of the WDR5−MLL interaction R. Al-Awar1 , R.S. Al-Awar1 , M. Getlik1 , D. Smil2 , Y. Bolshan2 , G. Poda1 , G. Senisterra2 , H. Wu2 , A. Allali-Hassani2 , G.A. Wasney2 , D. Barsyte-Lovejoy2 , L. Dombrovski2 , A. Dong2 , H. He2 , A. Seitova2 , I. Chau2 , F. Li2 , J.F. Couture2 , E. Kuznetsova2 , R. Marcellus1 . 1 Ontario Institute for Cancer Research, Drug Discovery, Toronto, Canada; 2 Structural Genomics Consortium, University of Toronto, Toronto, Canada ABSTRACT. Among other cellular processes, gene expression is regulated by epigenetic histone modifications. Histone methyltransferases catalyze the transfer of the methyl group from S-adenosylmethionine to specific lysine residues on histones. Mixed lineage leukemia 1 (MLL1) is a methyltransferase that methylates lysine 4 on histone H3 (H3K4me3) and is an important regulator of the haemopoietic system. Deregulation of MLL1 is often associated with acute myeloid and lymphoid leukemias and was proposed as a novel therapeutic target. WD40 repeat protein 5 (WDR5) is a component of the multiprotein MLL1 complex that is essential for its methyltransferase activity. Thus, therapeutic intervention of the WDR5/MLL1 interaction may lead to possible novel therapeutic agents for MLL-dependent leukemias. Using a structure-based design approach our structure activity relationship studies identified selective and cell-permeable compounds that bind to WDR5 with low nanomolar affinities. In future chemical biology studies these molecules will serve as valuable molecular probes to dissect the biological role of WDR5. 288 POSTER (Board P068) Synthetic isomalyngamide A analogs that inhibit breast cancer migration W. Li1 , T. Chang1 , C. Hung1 , C. Chen1 , S. Jao2 . 1 Academia Sinica, Institute of Chemistry, Taipei City, Taiwan; 2 Academia Sinica, Institute of Biological Chemistry, Taipei City, Taiwan Background: Because of frequent relapses in patients due to drug resistance and characteristically aggressive in very young women, breast

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cancers trigger enormous burden in health system, especially in the family of patient. Considerable progress has been made in the development of synthetic glycosylated isomalyngamide A analogs-related antitumor agents for medicinal interest. Based on the previous observations, we hypothesized that the isomalyngamide A analog might be a perfect substance with which to examine the role played by glycosylation and various linkers on antimigratory properties. Material and Methods: A series of synthetic glycosylated isomalyngamide A analogs with different linkers were prepared using the method of solution-phase synthesis and evaluated for their biological activities in human breast cancer cells. The effects of synthetic isomalyngamide A analogs on cell viabilities were determined by using the MTT assay. The inhibition of in vitro migration and invasion activities of synthetic isomalyngamide A analogs were examined by using a transwell assay. The potency in inhibition of cancer cell adhesion were studied by using the adhesion assay. Furthermore, the complete molecular mechanism for synthetic isomalyngamide A analog-mediated antimigratory property was explored using western blot analysis. Results: The findings show that the flexible rather than the rigid linkers to isomalyngamide A backbone more critically affect cell migration and invasive ability. Two synthetic isomalyngamide A analogs were observed to suppress migration, invasion and adhesion events in human breast adenocarcinoma MDA-MB-231 cells. Evidence has been achieved for a mechanism for inhibition of metastatic activities in MDA-MB-231 cells by synthetic isomalyngamide A analogs through the integrin-mediated antimetastatic pathway, suppression of the expression of p-FAK and paxillin. Conclusions: The observations made in this study demonstrate how the impact of the flexible and the rigid linker upon backbone of molecule affects the ability of synthetic isomalyngamide A analogs to modulate motility, migration, invasive capacity and adhesive property. In addition, the results of a complete western blot analysis of the in vitro antimigratory activities of synthetic isomalyngamide A analogs, show that these substances are potential antimetastatic agents for the treatment of breast cancer. 289 POSTER (Board P069) Progress in drugging CYP1A1, 1B1 and CYP2W1 overexpressed in cancer K. Pors1 , V. Le Morvan2 , S. Travica3 , S.D. Shnyder1 , M. Sutherland1 , H.M. Sheldrake1 , M. Searcey4 , I. Johansson3 , S. Mkrtchian3 , P.M. Loadman1 , J. Robert5 , M. Ingelman-Sundberg3 , L.H. Patterson1 . 1 University of Bradford, Institute of Cancer Therapeutics, Bradford, United ´ Bradford, France; Kingdom; 2 University of Bordeaux, Institut Bergonie, 3 Karolinska Institute, Stockholm, Sweden; 4 University of East Anglia, School of Chemical Sciences and Pharmacy, Norwich, United Kingdom; 5 ´ Bordeaux, France University of Bordeaux, Institut Bergonie, Background: The cytochrome P450 (CYP) enzymes are responsible for the oxidation of a diverse range of xenobiotic and endogenous compounds. Although CYPs operate mainly to detoxify xenobiotics and endogenous molecules, members of the CYP1 family are also known to catalyse one of the first steps in the metabolism of carcinogens originating from exposure to xenobiotics with the risk of developing cancer. Another member of the CYP family, CYP2W1, has been found to be expressed in adrenal gland, gastric, lung, rhabdomyosarcoma and colon cancer while the expression in adult non-transformed tissues remains absent. The high expression of CYP1A1, 1B1 and 2W1 in tumour tissue and surrounding stroma compared to nearby normal tissue provides an opportunity for development of selective cancer therapeutics. Materials and Methods: Briefly, these include synthetic chemistry necessary for synthesis of duocarmycin bioprecursors, use of recombinant CYP bactosomes for metabolite identification using LC/MS, transfected CYP1A1, 1B1 and 2W1 cancer cell lines and analysis of primary tissues. Results: At this meeting we will present both published and unpublished work, which is focussed on re-engineering the duocarmycin family of natural products for tumour-selective therapy. Novel data include progress on using ICT2726 as a biomarker to detect CYP2W1 functional activity and a range of agents that are activated in CYP1B1-transfed cell lines (Cal27 and Cal33). We are also disclosing information on our ongoing research of R and S-enantiomers of several duocarmycin bioprecursor compounds in both in vitro and in vivo models. Conclusions: Our findings reveal opportunities in targeting CYP1A1, 1B1 and 2W1 with personalized molecular cancer therapeutics based on the duocarmycin pharmacophore.

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Poster Session – Molecular Targeted Agents I

290 POSTER (Board P070) Exosome analysis in cancer patients: From the preclinical towards the clinical application: Trial design I. Mertens1 , M. Castiglia2 , A.P. Carreca2 , G. Baggertman1 , M. Peeters3 , P. Pauwels2 , C. Rolfo4 . 1 University Antwerp − VITO, Center for Proteomics − CFP-CeProma, Antwerp, Belgium; 2 Antwerp University Hospital, Molecular Pathology Unit Department of Pathology, Antwerp, Belgium; 3 Antwerp University Hospital, Oncology Department, Antwerp, Belgium; 4 Antwerp University Hospital, Phase I − Early Clinical Trials Unit Oncology Department, Antwerp, Belgium Background: Cancer cells produce a heterogeneous mixture of vesicular, organelle-like structures (extracellular vesicles, EVs) into their surroundings including blood and other body fluids. Exosomes are small (40 to 100 nm) membrane derived vesicles that develop from exophytic budding of the cellular membrane after the fusion of multivesicular bodies or mature endosomes with the cellular membrane. It has been shown that tumour cells exposed to hypoxia secrete exosomes with enhanced angiogenic and metastatic potential. Thus exosomes might be involved in tumor progression and they can potentially be used for prognosis and therapy selection, as they contain a variety of molecules such as signal proteins and/or peptides, microRNAs, mRNAs and lipids, which could be potential biomarkers. Materials and Methods: To evaluate the biomarker potential of the exosome derived RNA and protein content, we first optimized an extraction protocol for exosomes in plasma based on the Optiprep density gradient protocol. After purification, the exosomes are characterized and quantified using Western Blot and Nanosight analysis. Later, the RNA and protein fraction of the extracted exosomes from 60 NSCLC, 60 pancreatic cancer and 60 colorectal cancer patients is compared to 60 healthy controls. The proteome content is evaluated using mass spectrometry based quantitative shotgun proteomics. The RNA profiles are generated using next generation sequencing. After profound bioinformatics analysis, the potential of the RNA and protein profiles will be evaluated for diagnostic, prognostic and therapy purposes. Results: We are able to purify and characterize exosome material from plasma samples derived from patients. The data from nanosight analysis and Western blot indicate that we are able to work with very pure exosome samples for RNA and protein axtraction. Currently, RNA and protein profiles from different cancer types are being compared to healthy controls. Conclusion: The first step in bringing exosome analysis to the clinic is optimized: exosome purification and characterization. In a next step, RNA and protein profiles are being evaluated as potential biomarkers.

Molecular Targeted Agents I 291 POSTER (Board P071) Aflibercept has anti-tumor activity in bevacizumab-escaping tumors of colorectal cancer: Molecular profiles and mechanisms 1

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C. Dib , R.G. Bagley , P. Mankoo , J. Pollard , J. Watters , M. Chiron . 1 Sanofi R&D, Oncology, Vitry-Sur-Seine, France; 2 Sanofi R&D, Oncology, Cambridge, Usa; 3 Sanofi R&D, Oncology, Vitry-Sur-Seine, France; 4 Oncotest Gmbh, Oncology, Freiburg, Germany Background: Aflibercept (Afl, ziv-aflibercept in the US) is a potent multiple angiogenic factor trap that prevents VEGF-A, VEGF-B, and placental growth factor (PlGF) from activating the native receptors. Based on an OS benefit in the phase 3 VELOUR trial, Afl + FOLFIRI was approved to treat metastatic colorectal cancer (mCRC) following a prior oxaliplatincontaining regimen. In prior preclinical studies, Afl demonstrated superior anti-tumor activity relative to bevacizumab (Bev) in 39/48 CRC patientderived xenografts (PDX). The PDX were profiled for KRAS status and genomic analysis to elucidate mechanisms and potential biomarkers behind the differential activity. Methods: Primary or metastatic CRC tumors were engrafted and passaged subcutaneously into NMRI nude mice (Chiron et al., Mol Cancer Ther, 2014). In 3 PDX models showing some response to Bev, mice were subsequently randomized to either continue treatment with Bev or switched to Afl treatment. Tumors collected 1 day after last treatment were profiled by RNA sequencing. An informatics workflow was devised to associate RNA expression changes to either the human tumors or the mouse stroma, and differentially expressed genes were identified in each of the two treatments (fold change magnitude of 2 by Wilcoxon test and BH corrected P-value <0.05). Profiling for KRAS status and genomic analysis was done by whole exome sequencing and/or Sanger technique. Results: In 48 CRC PDX models tested, the differential activity of Afl and Bev showed no association with KRAS mutation status (Fisher-exact test

p-value >0.05): 24 were KRAS mutant and 24 were wild type. In 3 PDX models where treatment was either switched to Afl or maintained with Bev, Afl demonstrated greater anti-tumor activity than Bev (t-test: P < 0.0001, 2 being KRAS mutant and 1 KRAS WT). RNA sequence analysis revealed differential effects between Afl and Bev in both the murine host (643 genes, 374 up-regulated Afl vs. Bev) and human tumors (82 genes, 19 upregulated Afl vs. Bev). A number of angiogenesis-promoting genes were down-regulated by Afl vs. Bev including: VEGF receptor genes (FLT1, FLT4, KDR), Notch pathway genes involved in vessel formation control (Dll4, Notch 3 & 4), the Ephrin family that regulate cell migration (Epha2, Ephb1), and the matrix metalloproteinases (MMP10, 13, 15, 17, 28). Conclusions: The superior anti-tumor activity of Afl vs. Bev in CRC PDX was independent of KRAS status. Gene expression profiling of stroma and tumor genes of CRC PDX tumors showed clearly different modulation patterns of both stroma and tumor between Afl and Bev. The results suggest that increased control of expression of angiogenesis-promoting genes by Afl may be associated with its superior anti-tumor activity relative to Bev in CRC PDX. 292 POSTER (Board P072) Salmonella typhimurium A1-R decoys quiescent cancer cells to cycle rendering them chemosensitive S. Yano1 , Y. Zhang1 , M. Zhao1 , Y. Hiroshima1 , S. Miwa1 , F. Uehara1 , H. Kishimoto2 , H. Tazawa3 , T. Fujiwara2 , R.M. Hoffman4 . 1 AntiCancer Inc., San Diego California, USA; 2 Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Department of Gastroenterological Surgery, Okayama, Japan; 3 Okayama University Hospital, Center for Innovative Clinical Medicine, Okayama, Japan; 4 University of California San Diego, Department of Surgery, San Diego California, USA Background: A major problem with solid tumors is that most cancer cells within them are not cycling and therefore resistant to currently-used chemotherapy. Material and Methods: Tumor sphere models as well as tumors in vivo were infected with tumor-targeting Salmonella typhimurium A1-R (A1-R) and the cell cycle phase and drug sensitivity of the cancer cells within the spheres or tumors were determined. Results: Fluorescent ubiquitination-based cell cycle indicator (FUCCI)expressing cancer cells in tumor spheres in serum-free medium were quiescent. After A1-R infection, the cancer cells in the spheres entered S-phase and became sensitive to cisplatinum (CDDP) to which they were resistant before A1-R infection. A1-R infection of FUCCI-expressing subcutaneous tumors also induced them to cycle. The combination of A1-R followed by CDDP reduced tumor size compared with A1-R monotherapy or CDDP alone. Conclusions: This study demonstrates that A1-R decoys quiescent cancer cells to cycle and thereby become sensitive to conventional chemotherapy. 293 POSTER (Board P073) Inhibition of the cell cycle regulated Cdc7 kinase pathway is an efficacious therapeutic approach for hematologic malignancies and solid tumors R. Santos1 , D. Shum2 , D. Carrillo1 , R. Zhang1 , M. Churchill1 , S. Mukherjee1 , R. Brentjens3 , C. Radu2 , T.J. Kelly4 , H. Djaballah2 , M.G. Frattini1 . 1 Columbia University Medical Center, Medicine, New York NY, USA; 2 Memorial Sloan Kettering Cancer Center, Molecular Pharmacology and Chemistry, New York NY, USA; 3 Memorial Sloan Kettering Cancer Center, Medicine, New York NY, USA; 4 Memorial Sloan Kettering Cancer Center, Molecular Biology, New York NY, USA Background: Despite extensive drug discovery efforts, drug-candidate failure and patients relapsing in the clinic remain as persistent problems likely secondary to escape mechanisms focused on the control of both DNA replication and DNA repair pathways. Cdc7 is a cell cycle regulated serine/threonine kinase whose activity is required for the initiation of DNA replication and has also been implicated in the control of the DNA damage response, and therefore linked to the maintenance of genomic integrity. Both Cdc7 and its known substrate, the mini-chromosome maintenance (MCM) complex that functions as the replicative DNA helicase, are overexpressed in the majority of solid tumors, leukemias, and lymphomas making Cdc7 kinase activity a potential therapeutic target in both hematologic malignancies and solid tumors. Materials and Methods: In order to identify a small molecule inhibitor of the Cdc7 kinase, we performed a high throughput screen for inhibitors of Cdc7 kinase activity. Chemo-informatic analysis of the hits revealed enrichment in one chemical cluster made up of several naturally occurring allosteric inhibitory compounds, that we termed MSK-747 and MSK-777.

Poster Session – Molecular Targeted Agents I Results: MSK-747 and 777 cytotoxic activities were assessed against panels of cancer cell lines and primary patient samples representing the majority of solid and liquid tumors and demonstrated efficacy, with potencies (EC50) in the low nanomolar range. We have shown that both 747 and 777 are effective against cell lines harboring over-expression of the multidrug resistance (MDR) efflux pump, demonstrating that Cdc7 inhibition can overcome a major mechanism of chemotherapy resistance in human tumor cells. In addition, cell lines and patient samples harboring high-risk structural and molecular mutations and samples from relapsed and/or chemotherapy refractory patients were also found to be susceptible to Cdc7 kinase inhibition. Cell cycle analysis in both cancer cell lines and primary patient samples exposed to the compounds revealed an S phase arrest, cell cycle dependent caspase-3 activation, and apoptotic cell death. In vivo dose-dependent anti-tumor activity of 747 and 777 was shown in mouse models of leukemias (ALL and AML), NSCLC, melanoma, and ovarian carcinoma. In addition, biomarker identification and the mechansim of cancer cell selectivity over normal cells with these compounds will be discussed. In all studies, MSK-777 was found to be more efficacious and therefore was chosen as the lead candidate to continue preclinical and clinical development. Conclusions: Cdc7 kinase inhibition is a novel and efficacious mode of therapy for both hematologic malignancies and solid tumors. MSK-777, a naturally occurring allosteric inhibitor of this kinase, is scheduled to enter Phase I clinical trials in early 2015. 294 POSTER (Board P074) Synthetic lethal screen identifies Aurora A as a selective target in HPV driven cervical cancer B. Gabrielli1 , F. Bokhari2 , M. Ranall2 , A. Stevenson2 , M. Murell3 , M. Kelly3 , S. McKee2 , G. Leggatt2 , T. Gonda4 , N. McMillan3 . 1 University of Queensland, Diamantina Institute for Cancer Immunology and Metabolic Medicine, Brisbane Qld, Australia; 2 University of Queensland, Diamantina Institute, Brisbane Qld, Australia; 3 Griffith University, Griffith Health Institute, Gold Coast Qld, Australia; 4 University of Queensland, School of Pharmacy, Brisbane Qld, Australia HPV has been identified as the definitive agent in cancers of the cervix, penis, vulva, vagina, anus, skin, eye, and head and neck, and is responsible for 5% of the total cancer burden worldwide. HPV oncogenes disable a number of tumour suppressor pathways, including p53 and Rb, contributing to the transformed phenotype. We have performed an siRNA screen using the kinome (779 genes) library to identify genes that when depleted are synthetically lethal with HPV transformation. The primary and validations screens have confirmed Aurora A kinase (AURKA) as a potential synthetic lethal target selective for HPV transformed cells. In vitro research using the investigational selective small molecule AURKA inhibitor alisertib found alisertib to be significantly more potent towards the HPV transformed cells, and selectively promoted apoptosis in the HPV cancers. The drug was shown to target the HPVE7 oncogene, the level of expression of this oncogene possibly influencing sensitivity. Apoptosis was sensitive to Mcl-1 but not Bcl-2 over expression, indicating that the mechanism is associated with the proteolytic destruction of Mcl-1 in the extended mitosis in the alisertib treated HPV cancer lines. Xenograft experiments with cervical cancer cell lines showed alisertib inhibited growth of HPV and non-HPV xenografts during treatment. The non-HPV cancer growth was delayed, but in two separate HPV cancers models, regression and no resumption of growth was detected at even 50 days post-treatment. A second transgenic model of premalignant disease driven solely by HPVE7 similarly demonstrated sensitivity to drug treatment. These findings provide preclinical evidnce that alisertib warrants evaluation as a potential targeted compound with activity in HPV-transformed cervical cancer and premalignant disease that may have application to other HPV driven cancers. 295 POSTER (Board P075) Combining forces: Study of the cytotoxic effect of the MDM2 inhibitor Nutlin-3 in combination with CDDP in non-small cell lung cancer cell lines C. Deben1 , C. Rolfo2 , V. Deschoolmeester1 , A. Wouters1 , M. Peeters3 , I. Gil-Bazo4 , F. Lardon1 , P. Pauwels5 . 1 University Antwerp, Center for Oncological Research, Antwerp, Belgium; 2 Antwerp University Hospital, Phase I − Early Clinical Trials Unit Oncology Department, Antwerp, Belgium; 3 Antwerp University Hospital, Oncology Department, Antwerp, Belgium; 4 Cl´ınica Universidad de Navarra, Oncology Department, Pamplona, Spain; 5 Antwerp University Hospital, Molecular Pathology Unit Department of Pathology, Antwerp, Belgium Background: The p53/MDM2 interaction has been a well-studied target for new drug design leading to the development of, among others, the small

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molecule inhibitor Nutlin-3. Clinical phase 1 and 2 studies show promising results for drugs of the Nutlin-3 family administrated as monotherapy in several tumor types. Furthermore, combining targeted therapies with conventional treatments has received much attention in the past few years. Materials and Methods: Therefore, we combined Nutlin-3 with the chemotherapeutic drug CDDP, a known activator of the p53 pathway, in a series of non-small cell lung cancer cell lines with different p53 background. We focused on the importance of the dosing regime and the role of wild type p53. Thus we used the commonly used NSCLC cell line A549 as wild type p53 cell line and used a p53 shRNA lentiviral vector to obtain a p53 deficient sub cell line. In addition we used CRL-5908, harboring a p53 mutant genotype (R273H). Cells were treated with both CDDP and Nutlin-3 or CDDP followed by Nutlin-3 for 24 hours. The SRB-assay was used to determine the cytotoxic effect of both mono- and combination therapies after which possible synergism was calculated using the Chou– Talalay method, both under normoxic and hypoxic conditions. In addition we determined the p53 protein levels as well as the mRNA and protein levels of its main transcription targets MDM2, p21, PUMA and BAX. The induction of apoptosis and cell cycle arrest were determined by flowcytometric analysis. Results: The strongest synergistic effect was observed in the p53 wild type cell lines when treatment with CDDP (IC20 : 2mM) was followed by Nutlin-3 treatment under normoxic (CI: 0.486±0.138, 5mM Nutlin-3), and hypoxic conditions (CI: 0.625±0.082, 5mM Nutlin-3). However, when administrated simultaneously, this effect was only present at very low concentration of CDDP and induced an average additive effect under normoxic (CI: 0.990±0.082, 5mM Nutlin-3) and hypoxic conditions (CI: 1.068±0.361, 5mM Nutlin-3). After sequential treatment, the mRNA and protein levels of p53’s transcription targets MDM2, p21, PUMA, and BAX were markedly increased compared to CDDP monotherapy, as for the number of apoptotic cells (27.37±7.97% Ann V positive cells vs. 9.36±2.93%) and cells in G2/M phase arrest (66.67±0.64% vs. 18.15±7.89%). These findings were not observed in the p53 mutant and deficient cell lines, confirming the role of p53 in this synergistic effect. Conclusions: Our results point towards a promising combination therapy, being the induction of DNA damage by CDDP, followed by an increase in p53 levels by Nutlin-3, leading to a synergistic cytotoxic effect in a wild type p53 NSCLC cell line. Translationally, a lower dose of CDDP could be used in combination with Nutlin-3, potentially reducing side effects for NSCLC patients. 296 POSTER (Board P076) A first-in-Asian phase I dose escalation study to evaluate the safety and pharmacokinetics of VS-6063 (defactinib), a focal adhesion kinase inhibitor in subjects with non-hematologic malignancies T. Shimizu1 , H. Aida2 , J. Horobin3 , M. Keegan3 , M. Padval3 , A. Poli3 , C. Hashii2 , K. Nakagawa1 . 1 Kinki University Faculty of Medicine, Phase I Unit at Department of Medical Oncology, Osaka, Japan; 2 Japan Clinical Research Operations (JCRO), Tokyo, Japan; 3 Verastem Inc., Cambridge MA, USA Background: Defactinib (VS-6063) has been shown to be a potent, reversible inhibitor of focal adhesion kinase (FAK) and proline-rich tyrosinekinase-2. Blockade of FAK reduces tumor growth and metastasis through inhibition of tumor cell survival, proliferation and invasion as well as tumor angiogenesis. Treatment with FAK inhibitors has been demonstrated to reduce the proportion of cancer stem cells (CSCs) in a dose dependent manner while chemotherapy standard of care agents (SOC) enrich for CSCs. The ability of CSCs to survive exposure to chemotherapy but remain susceptible to novel drugs suggests a unique therapeutic approach whereby SOC may be combined or sequenced with targeted drugs to kill surviving CSCs, prevent tumor recurrence and metastasis. This is a single-center, phase 1, open-label, dose-escalation study to investigate the safety and pharmacokinetics (PK) of defactinib in first-in-Asian (Japanese) subjects. Methods: Subjects with no further standard of care options and a life expectancy of 3 months with advanced non-hematologic malignancies were enrolled. Defactinib was administered continuously at a starting dose of 200 mg BID, escalated to 400 mg BID and then to 600 mg BID. Patients continued treatment with defactinib until disease progression. Pharmacokinetics were collected on Day 1 and 15 during cycle 1. Results: Nine subjects were enrolled (n = 3 each; 200, 400 and 600 mg cohorts): median age was 60 years (38−75); ECOG PS was 0 or 1. Defactinib was well tolerated and no dose limiting toxicities were observed at any of the 200 mg, 400 mg or 600 mg dose levels. Common toxicities included: Fatigue, headache, increased bilirubin and diarrhea. PK analyses confirmed the exposure at the recommended phase 2 dose (RP2D) of 400 mg BID was comparable to that previously reported in non-Japanese subjects. Durable stable disease (SD) 24 weeks was confirmed in two patients (malignant mesothelioma and colon cancer) with improvement of clinical symptoms.

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Conclusions: Overall PK and AE profiles of Japanese subjects are consistent with those previously reported in non-Japanese subjects. Data from this study supports the entry of Japanese subjects at the RP2D to the ongoing multinational trial (COMMAND) of defactinib in malignant mesothelioma patients. Clinical Trial Information: NCT01943292 297 POSTER (Board P077) Genetic and pharmacologic inhibition of EPHA2 promotes apoptosis in NSCLC J. Chen1 , K. Amato2 , S. Wang1 , V. Youngblood2 , D. Brantley-Sieders1 , R. Cook2 , L. Tan3 , N. Gray3 . 1 Vanderbilt University Medical Center, Medicine, Nashville TN, USA; 2 Vanderbilt University Medical Center, Cancer Biology, Nashville TN, USA; 3 Dana Farber Cancer Institute, Biochemistry & Molecular Pharmacology, Boston MA, USA Background: Genome-wide analyses determined previously that the receptor tyrosine kinase (RTK) EPHA2 is commonly overexpressed in nonsmall cell lung cancers (NSCLCs). EPHA2 overexpression is associated with poor clinical outcomes; therefore, EPHA2 may represent a promising therapeutic target for patients with NSCLC. Material and Methods: The EphA2 knockout mouse model was used to determine lung cancer progression in Kras-mutant knockin mice. RNAimediated depletion or pharmacologic inhibition of EPHA2 was employed to determine cell viability and signaling in human lung cancer cell lines in vitro and tumor growth in vivo. Tumor cell proliferation was measured by BrdU incorporation ELISA or PCNA immunohistochemistry and apoptosis by TUNEL assay or Cell Death ELISA. We assessed tumor growth in vivo using H358 human lung cancer cells xenografts in mice. Results: Targeted disruption of EphA2 in a murine model of aggressive Kras-mutant NSCLC impairs tumor growth. Knockdown of EPHA2 in human NSCLC cell lines reduced cell growth and viability, confirming the epithelial cell autonomous requirements for EPHA2 in NSCLCs. Targeting EPHA2 in NSCLCs decreased S6K1-mediated phosphorylation of cell death agonist BAD and induced apoptosis. Furthermore, an ATP-competitive EPHA2 RTK inhibitor, ALW-II-41−27, reduced the number of viable NSCLC cells in a time-dependent and dose-dependent manner in vitro and induced tumor regression in human NSCLC xenografts in vivo. Conclusion: Collectively, these data demonstrate a role for EPHA2 in the maintenance and progression of NSCLCs and provide evidence that ALWII-41−27 effectively inhibits EPHA2-mediated tumor growth in preclinical models of NSCLC. 298 POSTER (Board P078) Met degradation by SAIT301, a Met monoclonal antibody, reduces the invasion and migration of nasopharyngeal cancer cells via inhibition of EGR-1 expression C. Kim1 , B.S. Lee1 , K.A. Kim2 , Y.J. Song2 , K.H. Cheong2 . 1 Ajou Univ. Hospital, Otolaryngology, Suwon, South Korea; 2 Samsung Advanced Institute of Technology (SAIT)/Samsung Electronics Co., Bio Therapeutics Lab, Suwon, South Korea Nasopharyngeal carcinoma (NPC) is a common malignant tumor with high invasive and metastatic potential. The hepatocyte growth factor (HGF)-Met signaling pathway has a critical role in mediating the invasive growth of many different types of cancer, including head and neck squamous cell carcinoma. HGF also stimulates NPC cell growth and invasion in the cell line model. In this study, we determined the inhibitory effect of Met, using a Mettargeting monoclonal antibody (SAIT301), on the invasive and growth potential of NPC cell lines. Met inhibition by SAIT301 resulted in highly significant inhibition of cell migration and invasion in both the HONE1 and HNE1 cell lines. In addition, we also found that co-treatment of SAIT301 and HGF decreased the anchorage-independent growth induced by HGF in HNE1 cell lines. After SAIT301 treatment, Met, together with its downstream signaling proteins, showed downregulation of p-Met and p-ERK, but not p-AKT, in both HONE1 and HNE1 cell lines. Interestingly, we found that HGF treatment of NPC cell lines induced early growth response protein (EGR-1) expression, which is involved in cell migration and invasion. In addition, co-treatment with SAIT301 and HGF inhibited the HGF-induced expression of EGR-1. Next, knockdown of EGR-1 using small-interfering RNA inhibited HGF-induced cell invasion in NPC cell lines, suggesting that the expression level of EGR-1 is important in HGF-induced cell invasion of NPC cells. Therefore, the results support that SAIT301 inhibited Met activation as well as the downstream EGR-1 expression and could have therapeutic potential in NPC. Taken together, we suggest that Met is an anticancer therapeutic target for NPC that warrants further investigation and clinical trials and SAIT301 may be a promising tool for NPC therapy.

Poster Session – Molecular Targeted Agents I 299 Tyk2-src dependence of kidney cancer

POSTER (Board P079)

B. Krishnan1 , S.C. Hanna2 , H.L. Wilson1 , S.T. Bailey1 , J.S. Damrauer1 , T. Simamura3 , R.L. Levine4 , K.K. Wong5 , G.L. Johnson2 , W.Y. Kim1 . 1 University of North Carolina Chapel Hill NC USA, Lineberger Comprehensive Cancer Center, Chapel Hill NC, USA; 2 University of North Carolina Chapel Hill NC USA, Department of Pharmacology, Chapel Hill NC, USA; 3 Loyola University Chicago, Oncology Institute Department of Molecular Pharmacology and Therapeutics, Maywood IL, USA; 4 Memorial Sloan-Kettering Cancer Center, Human Oncology and Pathogenesis Program Leukemia Service Department of Medicine, New York NY, USA; 5 Dana-Farber/Harvard Cancer Center, Department of Medicine Harvard Medical School Department of Medical Oncology and Lowe Center for Thoracic Oncology, Boston MA, USA Renal cell carcinoma (RCC) accounts for 209,000 new cases and 102,000 deaths worldwide each year. Cytotoxic chemotherapy and radiotherapy are largely ineffective, especially for metastatic disease, where the 5-year survival is only 10%. This represents an important need to develop better therapies against advanced RCC. Inhibitors of the vascular endothelial growth factor receptor (VEGFR) tyrosine kinase and the mammalian target of rapamycin (mTOR) delay the progression of advanced renal tumors. Given these therapeutic successes and that targeting kinase signaling cascades in cancer remains a promising avenue for drug development, we performed a high-throughput siRNA screen of the kinome to identify novel kinase targets for therapy. RNAi of the Janus kinase (JAK) family member tyrosine kinase 2 (TYK2) was highly lethal to RCC cells. Both shRNA as well as pharmacologic TYK2 inhibition were effective in decreasing in vitro and in vivo RCC tumor growth. Additionally RCC tumors were significantly enriched for the GNF2_TYK2 genes compared to adjacent normal kidney in 2 independent datasets (TCGA & GSE33093), also TYK2 is activated in a subset of RCC tumors; this increased activation correlates with poor overall survival of patients (TCGA). Moreover, we noted that mTORC1 inhibition resulted in IL6 mediated induction of pTYK2 and that dual inhibition of mTORC1 and TYK2 significantly reduced RCC cell growth over either alone, suggesting that TYK2 can mediate mTOR inhibitor resistance. We looked to further elucidate the effects of TYK2 knockdown on the reprogramming of RCC kinome. To this end, the phosphorylation status of 43 phospho-kinases was simultaneously assessed by phosphokinase arrays in RCC cells stably expressing shRNA’s to NS or TYK2. Of the probed phospho kinases, 4 of the 8 SRC family kinases (SFK) were significantly decreased relative to shNS. Furthermore treatment with rapamycin induced SFK activation as did knock-down of raptor. Given the lack of a clinically available selective TYK2 inhibitor, we explored the effect of combined mTOR and SRC inhibition in vivo; SRC + mTOR inhibition significantly decreased tumor growth over single agent treatment. In summary, our studies provide evidence that TYK2 is activated in RCC, demonstrate that TYK2 signals to regulate pSFK, and show that the TYK2−SRC axis is upregulated in the face of mTOR inhibition. In aggregate, we have uncovered a TYK2 and SRC dependence of RCC with important implications for therapy. 300 POSTER (Board P080) Preclinical pharmacologic characterization of GSK2849330, a ® monoclonal AccretaMab antibody with optimized ADCC and CDC activity directed against HER3 N. Clarke1 , C. Hopson2 , A. Hahn2 , K. Sully3 , F. Germaschewski3 , J. Yates1 , C. Akinseye1 , B. Mangatt2 , Z. Jonak4 , C. Matheny4 . 1 GlaxoSmithKline, Biopharm Discovery, Stevenage Herts, United Kingdom; 2 GlaxoSmithKline, Oncology R&D, Collegeville, USA; 3 GlaxoSmithKline, Biomarker Discovery, Stevenage Herts, United Kingdom; 4 GlaxoSmithKline, Biopharm R&D, Upper Merion PA, USA Background: HER3 is expressed in a broad range of solid tumors where its signaling is important in tumorigenesis and drug resistance. GSK2849330 is an IgG1/Ig3 chimeric, glycoengineered humanized monoclonal antibody directed against HER3 with enhanced potency to mediate antibodydependent cell cytotoxicity (ADCC) and complement dependent cytoxicity (CDC), resulting in three potential modes of anti-tumor activity. Methods: Various in vitro and in vivo studies were conducted in tumor cell lines and tumor xenograft models to characterize GSK2849330 pharmacology. Results: GSK2849330 selectively bound HER3 at extracellular Domain III with ~2 nM binding affinity, blocked ligand (heregulin) binding, receptor dimerization and activation. In tumor cell lines, GSK2849330 antagonized heregulin-induced phosphorylation of the HER3 receptor and its downstream effector, AKT, and inhibited heregulin-induced proliferation.

Poster Session – Molecular Targeted Agents I GSK2849330 dose-dependently inhibited tumor growth in a variety of mouse xenograft models. Compared with the non-ADCC/CDC enhanced antibody, GSK2849330 had a ~17-fold greater binding affinity to human FcgRIIIA (initiation of ADCC) and to human complement protein C1q (initiation of CDC). In ADCC assays using human leukocytes as effector cells and various tumor target cells, GSK2849330 was up to two orders of magnitude more potent than the WT antibody. GSK2849330 caused complement-mediated lysis of HEK293 cells expressing HER3, which was proportional to HER3 protein expression on the cell surface. Conclusions: GSK2849330 inhibits HER3 signal transduction in vitro and in vivo and is up to two orders of magnitude more potent in mediating ADCC and CDC than WT antibody. This is the first AccretaMab® antiHER3 antibody to initiate phase 1 clinical studies. The enhanced ADCC and CDC potency is intended to increase anti-tumor activity compared to a non-enhanced antibody as the immune mechanisms of action are directed toward killing dividing and non-dividing cells in addition to inhibition of HER3 signaling. All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals. 301 POSTER (Board P081) Using PK/PD/efficacy modeling to predict potential of AZD9291 to target brain metastases from advanced NSCLC with EGFR sensitizing mutations (EGFRm+) J. Yates1 , P. Ballard1 , S. Ashton1 , D. Cross1 , R. Dattani1 , M. Mellor1 , J. Wilson1 , P. Yang2 , L. Xie2 . 1 AstraZeneca, iMED Oncology, Macclesfield, United Kingdom; 2 AstraZeneca, Innovation Center China, Shanghai, China Background: AZD9291 is an oral, potent, irreversible EGFR-TKI selective for sensitising (EGFRm+) and T790M resistance mutations. It is metabolised in vivo to AZ5104 that has a similar profile to AZD9291. The aim of this work was to understand whether AZD9291 has the potential to be efficacious against EGFRm+ tumours that have metastasized to the brain. Materials and Methods: Quantitative whole body autoradiography (QWBA) and LC-MS was used to assess brain pharmacokinetics. The subcutaneous PC9 (ex19del) xenograft tumour model in immunocompromised mice was used to assess the phospho-EGFR reduction after a single dose of AZD9291 and its anti-tumour effect on repeat dosing. A mathematical Pharmacokinetic–Pharmacodynamic (PKPD) and efficacy model was developed using these data. Efficacy data from a PC9 based brain metastases (BM) model was used to confirm the predictions of the model. An evaluation of an active clinical dose was performed by simulating the model with human PK substituted for mouse. Results: QWBA and LC-MS based studies demonstrated that AZD9291 had significant exposure in the brain with the active metabolite AZ5104 showing more modest brain exposure. AZD9291 dosed at 5 mg/kg/day in a mouse EGFRm+ BM xenograft model (PC9) resulted in tumour regressions, further suggesting that AZD9291 may have potential to penetrate the brain and target EGFRm+ BM patients. The mouse subcutaneous xenograft PKPD-efficacy model was adapted to account for the differential exposure and binding of AZD9291 and AZ5104 in brain compared to plasma and was found to predict the regressions observed in the mouse BM model. With the model validated, inclusion of clinical PK predicted that a clinical dose of 80 mg would be sufficient to target EGFRm+ brain metastases. Conclusions: Experimental data demonstrates that AZD9291 and its metabolite AZ5104 distribute into the rodent brain and are efficacious in a mouse model of EGFRm+ BM. A mathematical model based integration of these data with other pre-clinical and clinical data suggests that further investigation of the potential for AZD9291 to target brain metastases in the clinic is warranted. 302 POSTER (Board P082) FAK inhibitor VS-6063 (defactinib) targets mesothelioma cancer stem cells which are enriched by standard of care chemotherapy J. Pachter1 , V.N. Kolev1 , P. Baas2 , D.T. Weaver1 , Q. Xu1 . 1 Verastem Inc, Research, Cambridge, USA; 2 Netherlands Cancer Institute, Thoracic Oncology, Amsterdam, Netherlands Malignant pleural mesothelioma (MPM) is an aggressive tumor in the lining of the lung often resulting from prior exposure to asbestos. Median overall survival with standard of care chemotherapy is only 12 months from diagnosis. This poor prognosis may be attributable at least in part to cancer stem cells (CSCs) which are resistant to chemotherapy and can mediate cancer recurrence and progression. Focal adhesion kinase

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(FAK) has been shown to play an essential role in the survival, selfrenewal and tumor-initiating capability of CSCs. Accordingly, the FAK inhibitor VS-6063 is currently being tested in patients with mesothelioma following disease control on standard pemetrexed/platinum chemotherapy (COMMAND, ClinicalTrials.gov NCT01870609). Aldehyde dehydrogenase (ALDH) activity was validated as a CSC marker in mesothelioma by assessment of tumor-initiating capability in mice. As compared to ALDH-negative cells, sorted ALDH-positive MM87 mesothelioma cells showed 8-fold greater self-renewal capacity in an in vitro tumorsphere assay, and 35-fold greater tumor initiating capacity when implanted in limiting dilutions in immunodeficient mice. Indeed, 50 ALDH-positive cells were sufficient to generate sizeable tumors in 3 weeks illustrating the aggressive nature of mesothelioma CSCs. Treatment of a human mesothelioma cell line with pemetrexed enriched ALDH-positive CSCs 6-fold, with similar CSC enrichment by cisplatin and other chemotherapeutic agents. In direct contrast, the FAK inhibitor VS-6063 markedly reduced the proportion of CSCs. The enrichment of mesothelioma CSCs was similarly observed in samples from 11 patients following first line chemotherapy. Patient specimens post treatment with pemetrexed and cisplatin showed an elevated ALDH1 immunohistochemistry H-score (p = 0.036) as compared to matched mesothelioma biopsies taken from the same patients prior to chemotherapy, suggesting the clinical relevance of CSCs in mesothelioma. To assess drug effects on tumor-initiating capacity, MM87 merlin-low mesothelioma and H28 merlin-high mesothelioma cell lines were treated in vitro with VS6063, pemetrexed or the combination and subsequently implanted into mice. While control or pemetrexed-treated mesothelioma cells showed robust tumor initiation, cells treated with VS-6063 alone or VS-6063 plus pemetrexed showed little or no tumor initiating capacity. These data provide strong rationale for the current clinical testing of VS6063 following treatment with pemetrexed plus platinum to potentially delay time to relapse and improve outcome for patients with mesothelioma. 303 POSTER (Board P083) Genomic and epigenomic analysis identify potential therapeutic targets in luminal B breast cancer molecular subtype L. Addou Klouche1 , A. Belhadj1 , A. Bendaoud1 , M. Benyelles1 , I. Ziane1 , M. Chaffanet2 , D. Birnbaum2 . 1 UDL-Sba, Biotoxicology, Sidi-Bel-Abbes, ´ Algeria; 2 IPC, Oncologie moleculaire, Marseille, France Background: Breast cancer (BC) is a complex and heterogeneous disease whose therapeutic approach must be refined. Luminal B breast cancer molecular subtype are estrogen receptor-positive (ER+), highly proliferative, resistant to standard therapies and have a poor prognosis. Although they express hormone receptors, their metastatic risk and resistance to hormone therapy and to conventional chemotherapy demand to develop appropriate therapies. To better understand this subtype we compared DNA copy number aberrations (CNAs), DNA promoter methylation and gene expression profiles in luminal B tumors with those observed in BCs of the other molecular subtypes. Material and Methods: To further define molecular alterations associated with the luminal B subtype we studied, with high-Troughput molecular analysis (CGHarray, promoter arrays and next-generation sequencing), copy number aberrations, DNA promoter methylation alterations and gene expression deregulation in 188 primary BC samples. An integrated analysis of genomic, epigenomic and expression profiling from DNA microarrays has contributed to the identification of candidate genes. Results: Frequent CNAs included 8p11-p12 and 11q13.1-q13.2 amplifications, 8q21.12-q24.23, 17q11.1-q25.1 gains and 6q14.1-q24.2, 18p11.31p11.32 losses were found. A total of 237 and 101 luminal B-specific candidate oncogenes and tumor suppressor genes (TSGs) presented a deregulated expression in relation with their CNAs, including 11 genes previouslyreported associated with endocrine resistance. Interestingly, 88% of the potential TSGs are located within chromosome arm 6q, and seven candidate oncogenes are potential therapeutic targets. Twenty-four genes presented a deregulated expression in relation with a high DNA methylation level. Conclusion: This refined molecular dissection of luminal B BCs has pointed to both new and well-known specific candidates. we have reported luminal B candidate genes that may play a role in the development and/or hormone resistance of this aggressive subtype. Further characterization of the 6q TSGs is an important goal. This should help better understand pathways and mechanisms affected, and find new therapeutic targets.

100 Thursday 20 November 2014 304 POSTER (Board P084) PI3 -Kinase inhibition forestalls the development of drug resistance in BRAFV600E/PTENNull melanoma M. Deuker1 , V. Marsh Durban2 , W. Phillips3 , M. McMahon1 . 1 University of California San Francisco, Helen Diller Family Comprehensive Cancer Center and Department of Cell and Molecular Pharmacology, San Francisco, USA; 2 Cardiff University, European Cancer Stem Cell Research Institute, Cardiff, United Kingdom; 3 University of Melbourne, Peter MacCallum Cancer Centre, Victoria, Australia The phosphatidylinositol 3 -kinase (PI3K) signaling pathway is frequently dysregulated in BRAF mutated melanomas and serves to promote the growth of tumors initiated by expression of BRAFV600E . The most prevalent mechanism of PI3K pathway activation occurs via silencing expression of the phosphatase and tensin homolog (PTEN) tumor suppressor, but mutational activation of PIK3CA, encoding the 110kDa catalytic subunit of PI3 -kinase-a, has also been observed at low frequency. However, the PI3K catalytic isoform dependence of BRAF mutated melanoma remains poorly understood, as does the differential isoform dependence imbued by discrete nodes of PI3K pathway dysregulation. To characterize the PI3K isoform dependence of BRAFV600E /PTENNull as well as BRAFV600E /PIK3CAH1047R melanoma, we administered a panel of isoform-selective PI3K inhibitors to cultured melanoma cells and to genetically engineered mouse (GEM) models of melanoma of both genotypes. These studies revealed that, while BRAFV600E /PIK3CAH1047R melanoma was sensitive to p110a-selective inhibition, inhibition of BRAFV600E /PTENNull melanoma cell proliferation required combined blockade of p110a (PIK3CA), p110d (PIK3CD) and p110g (PIK3CG). Moreover, in GEM models of BRAFV600E /PIK3CAH1047R or BRAFV600E /PTENNull melanoma, isoform-selective PI3K inhibition elicited only a cytostatic effect as monotherapy, but enhanced melanoma regression in response to BRAFV600E →MEK→ERK pathway-targeted inhibition. Most interestingly, treatment with GDC0032, a p110b-sparing PI3K inhibitor, forestalled the onset of MEK inhibitor (GDC-973, cobimetinib) resistant disease in the BRAFV600E /PTENNull GEM model of melanoma. Together, these data suggest that PIK3CA mutational status may be used to select for melanoma patients that will respond to a p110a-selective inhibitor. By contrast, PTENNull melanoma patients will require a p110b-sparing pan-class I PI3K inhibitor. Moreover, although monotherapy PI3K inhibition did not elicit dramatic tumor regression, blockade of this pathway may be a useful strategy to extend the duration of response to BRAFV600E pathway-targeted therapy. 305 POSTER (Board P085) Selinexor, a novel selective inhibitor of nuclear export, potentiates the antitumor activity of gemcitabine against pancreatic cancer by nuclear retention of p27 M. Amit1 , S. Kazim2 , H. Kazim2 , S. Shacham3 , M. Kauffman3 , M.P. Malafa2 . 1 Moffitt Cancer Center and Research Institute, Tampa FL, USA; 2 Moffitt Cancer Center and Research Institute, GI Oncology, Tampa FL, USA; 3 Karyopharm Therapeutics, Natick MA, USA Background: Despite recent advances, pancreatic cancer is a lethal malignancy associated with chemoresistance and poor prognosis. Gemcitabine has remained the primary therapeutic agent for treatment of patients with pancreatic cancer. Export of TSPs by XPO1 is one of the important resistant mechanisms employed by tumor cells. Selinexor (KPT330) specifically inhibits exportin-1 (XPO1) resulting in nuclear retention and activation of tumor suppressor proteins and growth regulatory proteins including p53, p27, p21, and FOXO. XPO1 is frequently elevated in multiple tumors types including pancreatic cancer and correlates with poor prognosis. Material and Methods: The antitumor effect of the combination of selinexor and gemcitabine was evaluated in in vitro and in vivo orthtopic mouse models of human pancreatic cancer. MiaPaca-2 and L3.6pl pancreatic cancer cell lines were treated with different concentrations of selinexor and gemcitabine and MTT assays were used to assess anchorage dependent growth. Athymic nude mice were injected orthotopically into the pancreas with human pancreatic cancer cell lines L3.6pl with luciferase. Mice were randomly assigned to one of the four treatment arms: (1) Vehicle (PBS 1 ml/kg, ip, 2×/week and PVP/PF68 1ml/kg, po, 3×/week), (2) KPT330 (20 mg/kg, oral, 3×/week), (3) gemcitabine (100 mg/kg, ip, 2×/week) and (4) KPT330 (10 mg/kg, oral, 3×/week + gemcitabine (50 mg/kg, ip, 2×/week) for 4 weeks. The tumor volumes and tumor weights were recorded at the end of treatment. Expression of p21 and other apoptotic proteins were determined by immunohistochemistry and western blot. Results: Gemcitabine and selinexor inhibited cell proliferation in concentration dependent manner and acted synergistically to inhibit anchorage

Poster Session – Molecular Targeted Agents I dependent and independent cell growth. Selinexor depleted XPO1, induced the proapoptotic protein (Bax) and depleted the anti-apoptotic protein survivin. Both selinexor and gemcitabine alone significantly decreased the tumor volume and tumor weights as compared to the placebo (p < 0.05). The combination of selinexor and gemcitabine potentiated the antitumor activity of each other to further reduce tumor weight and volume (p < 0.0001). Higher percentage of cells expressed p27 in the nucleus in selinexor treated mice (median: 50%) and combination group (80%) as compared to vehicle or gemcitabine group (35%). No cytoplasmic staining of p27 was observed in selinexor treated groups but was present in gemcitabine and vehicle groups. Conclusions: Selinexor enhances the antitumor activity of gemcitabine in pancreatic cell lines and orthotopic xenograft pancreatic cancer models, possibly by nuclear retention of proapoptotic proteins including p27. Clinical trial evaluating the combination of gemcitabine and selinexor in patients with pancreatic cancer is being planned. 306 POSTER (Board P086) CC-115 inhibits DNA damage and repair pathways in vitro T. Tsuji1 , L. Sapinoso1 , S. Sankar1 , D.S. Mortensen2 , S. Xu1 . 1 Celgene, Discovery Oncology, San Diego, USA; 2 Celgene, Medicinal Chemistry, San Diego, USA Nonhomologous end-joining (NHEJ) is one of the most important DNA double strand break (DSB) repair pathways in mammalian cells and is controlled by the DNA-dependent protein kinase (DNA-PK). We have developed a dual inhibitor of DNA-PK and mTOR kinase (CC-115) that is currently undergoing Phase 1 dose escalation. Here we describe our efforts to characterize the DNA-PK inhibitory activity of CC-115. Using biochemical and cellular assays to measure NHEJ activity, CC-115 shows NHEJ disruption by inhibiting DNA-PK. Inhibition of DNA-PK by CC-115 results in inhibition of known substrates including Ku80, XRCC4 and autophosphorylation of DNA-PK. All three proteins reportedly play critical roles in NHEJ. In addition, CC-115 inhibits the dissociation of DNA-PK, XRCC4, and DNA ligase IV from DNA ends, potentially critical to the inhibition of NHEJ activity. Interestingly, treatment of cells with CC-115 results in inhibition of phosphorylation of ATM at S1981, which is required for ATM kinase activity, and inhibition of phosphorylation of ATM substrates such as Chk2 (T68), Nbs1 (S343), 53BP1 (S25/29), and SMC1 (S957) in HOP92, HT-29, and MBA-MB-436 cancer cell lines. Another specific inhibitor of DNA-PK, NU7441, also inhibits ATM kinase cellular activity. Inhibition of ATM by CC-115 and NU7441 appears to be an indirect consequence of DNA-PK inhibition since neither NU7441 nor CC-115 directly inhibits ATM kinase. We demonstrate that CC-115 inhibits DNAdamage-induced translocation of Rad51, a homologous recombination (HR) marker, suggesting that CC-115 inhibits HR pathway presumably by indirect inhibition of ATM. In conclusion, CC-115 inhibits two major DSB repair pathways, NHEJ and HR, by direct inhibition of DNA-PK and indirect inhibition of ATM, respectively. 307 POSTER (Board P087) Stromal delivery of long Pentraxin-3 impairs FGF/FGFR-dependent tumor growth and metastasis A. Giacomini1 , E. Di Salle1 , D. Coltrini1 , S. Rezzola1 , M. Belleri1 , M. Presta1 , R. Ronca1 . 1 University of Brescia, Molecular and Translational Medicine, Brescia, Italy The FGF/FGFR system contributes to cancer progression by inducing tumor growth and neovascularization, thus representing an emerging therapeutic target. Long Pentraxin-3 (PTX3) is a soluble pattern recognition receptor expressed by endothelial and immune cells in inflammatory contexts. Among various ligands, PTX3 binds different members of the FGF family, acting as a natural FGF ligand trap. Here, we generated transgenic mice expressing human (h)PTX3 under the control of endothelial specific Tie2/Tektranscription regulatory sequences (Tie2-hPTX3 mice). These animals accumulate significant levels of hPTX3 in perivascular stroma and in the blood stream. On this basis, Tie2-hPTX3 mice were used to investigate the impact of stroma delivery of hPTX3 on tumor growth, vascularization and metastasis. The anti-angiogenic activity of endothelium-derived hPTX3 was confirmed by ex vivo aorta ring and in vivo matrigel plug assays. Next, different syngeneic FGF-dependent tumor cell lines, including TRAMP-C2 prostate carcinoma, B16-F10 melanoma and Lewis Lung carcinoma cells, were subcutaneously injected in Tie2-hPTX3 mice. Notably, the growth of all tumor grafts was significantly reduced in Tie2-hPTX3 mice when compared to wild type animals and was accompanied by a significant reduction of FGFR1 phosphorylation, decrease of tumor vascularity and tumor cell proliferation. Also, B16-F10 melanoma and M5076 ovarian sarcoma cells showed a dramatic decrease of their capacity to form experimental

Poster Session – Molecular Targeted Agents I metastases in the lung and liver, respectively, after intravenous injection in Tie2-hPTX3 mice. Also, the orthotopic growth of syngeneic pancreatic and mammary tumor cells was significantly reduced after injection in Tie2hPTX3 mice and led to increased survival compared to control mice. Finally, double transgenic TRAMP/Tie2-hPTX3 mice showed a significant delay of multistage prostate tumor onset and progression in respect to TRAMP mice. Our findings demonstrate for the first time that in vivo delivery of PTX3 exerts a dramatic impact on tumor growth, vascularization and metastasis. These results have set the basis for the identification of a low molecular weight PTX3-derived molecule that recapitulates the FGF-trap activities of PTX3 and exhibits promising therapeutic potential for FGF-dependent tumors. 308 POSTER (Board P088) Androgen deprivation therapy sensitizes prostate cancer cells to T-cell killing through androgen receptor dependent modulation of the apoptotic pathway S. Gameiro1 , A. Ardiani1 , A.R. Kwilas1 , R.N. Donahue1 , J.W. Hodge1 . 1 National Cancer Institute, CCR/LTIB, Bethesda MD, USA Despite recent advances in diagnosis and management, prostrate cancer remains the second most common cause of death from cancer in American men, after lung cancer. Failure of chemotherapies and hormonedeprivation therapies is the major cause of death in patients with castrationresistant prostate cancer (CRPC). Currently, the androgen inhibitors enzalutamide and abiraterone are approved for treatment of metastatic CRPC. Enzalutamide is a second-generation androgen antagonist that has no agonist activity. Abiraterone is a cytochrome P450 inhibitor that blocks adrenal and intratumoral androgen production. Here we show for the first time that both enzalutamide and abiraterone renders prostate tumor cells more sensitive to T cell-mediated lysis through immunogenic modulation, and that these immunomodulatory activities are androgen receptor (AR)-dependent. In studies reported here, the NAIP gene was significantly down-regulated in human prostate tumor cells treated in vitro and in vivo with enzalutamide. Functional analysis revealed that NAIP played a critical role in inducing CTL sensitivity. Amplification of AR is a major mechanism of resistance to androgen-deprivation therapy (ADT). Here, we show that enzalutamide enhances sensitivity to immune-mediated killing of prostate tumor cells that overexpress AR. The immunomodulatory properties of enzalutamide and abiraterone provide a rationale for their use in combination with immunotherapeutic agents in CRPC, especially for patients with minimal response to enzalutamide or abiraterone alone, or for patients who have developed resistance to ADT. 309 POSTER (Board P089) RNA sequencing and in silico analysis identifies an unannotated antisense long non-coding RNA involved in cancer progression S. Inoue1 , K. Horie-Inoue2 , K. Ikeda2 . 1 Univ of Tokyo, Dept of Anti-aging Med, Tokyo, Japan; 2 RCGM Saitama Med Univ, Div of Gene Regulation & Signal Transduction, Saitama, Japan Background: Long non-coding RNAs (lncRNAs) are RNAs that have no obvious coding capacities with their length >200 bp and widely transcribed across the entire genome in spatial and temporal specific manners. The biological roles of lncRNAs as tumor suppressors or activators are emerging in various types of cancer. Identification and functional analyses will be required for lncRNAs involved in cancer progression. Material and Methods: Here we attempt to identify a functional hormonedependent lncRNA in hormone-dependent cancer. We performed RNA sequencing in estrogen-sensitive or hydroxytamoxifen-resistant MCF7 breast cancer cells and conducted in silicoscreening of lncRNAs based on our sequencing data together with integrative annotation open databases of lncRNAs. The contribution of hormonal regulation to the expressions of lncRNAs is also investigated by mapping estrogen receptor binding sites across the genome. lncRNA expression was examined in several cancer cells by qRT-PCR. Effects of lncRNA knockdown on cell proliferation, cell cycle, and cell migration were evaluated by the transduction of specific siRNAs into cancer cells. Whether lncRNA knockdown influences in vivo tumor formation was evaluated by the injection of siRNAs into xenograft tumors in nude mice. Animal studies were approved by the Institutional Animal Care and Use Committee. Results: An unannotated estrogen-dependent lncRNA, denoted as BClnc-X, was identified, which is located in the vicinity of transcriptional start site and in the antisense direction of a coding gene. The genomic position of BClnc-X also includes a functional estrogen binding site. BClnc-X is expressed in prostate cancer cells as well as breast cancer cells. We also found that BClnc-X is expressed in clinical breast cancer samples. Specific siRNAs against BClnc-X substantially repressed the proliferation

Thursday 20 November 2014 101 and migration of MCF7 cells, as well as decreased the percentages of cells in S phase of cell cycle. BClnc-X knockdown could also reduce tumor formation in mice models. Conclusions: We consider that BClnc-X will contribute to the progression of cancer by putatively associating with hormone signaling. BClnc-X will be applied to clinical management of breast cancer as a potential molecular target for diagnostic and therapeutic options for the advanced disease. 310 POSTER (Board P090) Inhibition of Trk-driven tumors by the pan-Trk inhibitor RXDX-101 D. Anderson1 , M. Ciomei2 , P. Banfi2 , S. Cribioli2 , E. Ardini2 , A. Galvani2 , G. Li1 . 1 Ignyta Inc, San Diego CA, USA; 2 Nerviano Medical Sciences, Nerviano, Italy The Trk family of kinases, which include TrkA, TrkB and TrkC, are high affinity receptors for the neurotrophin family of nerve growth factors. Deregulated kinase activities of Trk family members due to chromosome rearrangements, gene mutations, splicing variants and overexpression have been shown to be associated with tumorigenesis and poor prognosis in a number of cancer types. Particularly, several chromosomal rearrangements involving TrkA have been reported in lung, colorectal, papillary thyroid, glioblastoma, melanoma and other cancers, and are believed to be the key oncogenic driver in these tumors. Therefore oncogenic Trk may represent a promising therapeutic target in Trk-driven tumors. RXDX-101 is an orally available, potent and selective ATP-competitive panTrk, ROS1 and ALK inhibitor, with comparable activities against TrkA, TrkB and TrkC in biochemical and cell based assays (IC50s <10 nM). In KM12, a human colorectal cancer cell line driven by constitutively active TrkA fusion TPM3-NTRK1, RXDX-101 exhibited in vitro anti-proliferative activity with an IC50 of 17 nM, accompanied by inhibition of TrkA phosphorylation and concomitant inactivation of downstream effectors, PLCgamma1, AKT and ERK, as well as cell cycle arrest and apoptosis. In mice bearing KM12 xenografts, treatment with RXDX-101 resulted in tumor regression and durable stasis under either intermittent or continuous dosing regimens, accompanied by sustained intratumoral inhibition of phospho-TrkA and PLCgamma1. In these studies, RXDX-101 was well tolerated during the course of treatment. In conclusion, our data indicates that RXDX-101, a potent and selective pan-Trk inhibitor currently in clinical development, is an attractive targeted agent for Trk-driven tumors. 311 POSTER (Board P091) IGF-1R inhibition induced activation of Yes/SFK acts as a by-pass resistance pathway in rhabdomyosarcoma X. Wan1 , C. Yeung1 , C. Heske1 , A. Mendoza1 , L.J. Hlman1 . 1 National Cancer Institute, Pediatric Oncology Branch, Bethesda, USA Objective: Development of clinical resistance has been common in IGF-1R block and represents a significant hindrance and limits treatment efficacy in the clinic. Thus, identifying the mechanisms of acquired resistance to IGF-1R blockade is a major goal. The aim of this study was to identify the molecular mechanisms responsible for acquired resistance to IGF-1R targeted therapy in rhabdomyosarcoma. Method: Expression profiles of IGF components and SFKs were analyzed by cDNA microarray. Antiproliferative effects of anti-IGF-1R agents and Src family kinase inhibitors alone or in combination were tested in vitro in multiple rhabdomyosarcoma (RMS) cell lines and in vivo using RD and Rh30 xenografts. Western blot and immunoprecipitation were performed to identify potential resistance mechanisms to IGF-1R inhibition. Results: cDNA microarray revealed that both IGF components and Yes are highly expressed in RMS. IGF-1R blockade either with R1507, an antibody against IGF-1R, or with BMS-754807, a small molecular inhibitor against of IGF-1R/IR, results in an increase in Yes activation, which is associated with resistance to IGF-1R blockade in RMS. Combining anti-IGF-1R agents with SFK inhibitors resulted in blockade of IGF-1R inhibition induced activation of Yes/SFK and displayed enhanced antitumor activity in vitro and in vivo. Conclusions: Our data demonstrate that IGF-1R inhibition induced activation of Yes/SFK may act as a by-pass resistance pathway. Cotargeting both IGF-1R and SFK shows advantageous antitumor activity in vitro and in vivo. Our findings may be of particular relevance clinically since both Yes and IGF components are overexpressed in RMS. Dual inhibition of IGF-1R and SFK may have a broader and enhanced clinical benefit for RMS patients.

102 Thursday 20 November 2014 312 POSTER (Board P092) The prognostic role of mesothelin expression and its association with KRAS mutation in advanced lung adenocarcinoma A. Thomas1 , Y. Chen2 , S. Steinberg3 , J. Luo4 , G. Giaccone5 , I. Pastan6 , M. Miettinen7 , R. Hassan8 . 1 National Cancer Institute, Bethesda MD, USA; 2 National Cancer Institute, Thoracic and GI Oncology Branch, Bethesda MD, USA; 3 National Cancer Institute, Center for Cancer Research, Bethesda MD, USA; 4 National Cancer Institute, Laboratory of Cancer Biology and Genetics, Bethesda MD, USA; 5 Georgetown University Washington DC USA, Oncology, Washington DC, USA; 6 National Cancer Institute, Laboratory of Molecular Biology, Bethesda MD, USA; 7 National Cancer Institute, Laboratory of Pathology, Bethesda MD, USA; 8 National Cancer Institute, Thoracic and GI Oncology Branch, Bethesda MD, USA Background: Mesothelin is a cell surface glycoprotein which is highly expressed in several epithelial cancers and may have a role in cell adhesion and metastases. Mesothelin-targeted therapies have yielded promising preliminary results in pancreatic adenocarcinoma and mesothelioma. In this study, we used prospectively obtained clinical and pathological data to characterize mesothelin expression in advanced lung adenocarcinoma. Material and Methods: Tissue was obtained from patients with stage III or IV lung adenocarcinoma who underwent molecular profiling of potentially actionable genes on a pilot trial of molecular profiling and targeted therapies in advanced thoracic malignancies (NCT01306045). Mesothelin expression was assessed by immunohistochemistry (IHC) using monoclonal antibody 5B2 (Novocastra/Leica, Bannockburn, IL). The intensity of expression and the percentage of cells expressing mesothelin were assessed. Mutations of EGFR, KRAS, BRAF, AKT1, PIK3CA and HER2 were assessed by pyrosequencing; HER2 amplification and ALK translocation by fluorescence in situ hybridization. Results: 93 patients who enrolled in the trial had adenocarcinoma histology, stage III or IV at diagnosis and had adequate formalin fixed paraffin embedded tissue available for further studies. Median age of patients was 61 years; 40 (43%) patients were male and 53 (57%) female. The patient population was predominantly Caucasian (74%) and 33% were never-smokers. EGFR, KRAS mutations and ALK translocations were found in 20%, 27% and 10% cases respectively. 49 of 93 (53%) tumors had mesothelin expression in at least 1% cells. High mesothelin expression, defined as mesothelin expression in >25% of cells was seen in 22 of 93 (24%) cases. 15 of 27 (56%) KRAS mutant-tumors had high mesothelin expression compared with only 7 of 65 (11%) KRAS wild-type tumors (P < 0.0001). None of the 20 EGFR mutant tumors had high mesothelin expression compared with 22 of 70 (46%) of EGFR wild-type tumors (P = 0.002). High mesothelin expression was independently associated with inferior survival (median 18.2 months vs. 32.9 months; P = 0.014). Conclusions: High mesothelin expression, found in 24% of advanced lung adenocarcinoma is a poor prognostic factor and is independently and strongly associated with KRAS mutation and EGFR wild-type status. Mesothelin targeted therapy needs further evaluation in advanced lung adenocarcinomas, especially the KRAS mutant subtype. 313 POSTER (Board P093) Development of a one-step isolation platform for exosomal RNA and circulating cell-free DNA from cancer plasma samples D. Enderle1 , K. Brinkmann1 , T. Koestler1 , S. Bentink1 , C. Berking2 , J. Skog3 , M. Noerholm1 . 1 Exosome Diagnostics GmbH, Martinsried/Munich, Germany; 2 Ludwig-Maximilian University, Dept. of Dermatology and Allergology, Munich, Germany; 3 Exosome Diagnostics Inc., Cambridge, USA Background: Circulating nucleic acids in the bloodstream of cancer patients are of great interest to medical research because of their potential to yield information on the patient’s disease status and treatment options without requiring a tissue biopsy. Any diagnostic test that seeks to utilize body fluids for mutation analysis needs a platform that can maximize the capture of tumor derived mutations in circulation. Blood plasma contains at least two cell-free sources of nucleic acids: circulating cell-free DNA (cfDNA), generated from apoptotic or necrotic cells, and RNA enclosed in extracellular vesicles including exosomes (exoRNA), which are actively secreted by cells in the body. Since the total amount of nucleic acids in biofluids is very limited and tumor mutations are reflected on both RNA and DNA, we devised a method to co-isolate all exoRNA and cfDNA out of blood plasma samples into a volume small enough for effective downstream processing by RT-qPCR and targeted re-sequencing by NGS. Materials and Methods: We developed a novel spin column-based method, EXO52, to co-isolate the RNA and DNA from 1−4 mL of blood plasma and serum. The purification is based on membrane affinity-binding of extracellular vesicles, which protect the exoRNA from degradation, and

Poster Session – Molecular Targeted Agents I simultaneous capturing of the circulating cfDNA. Purification of high quality nucleic acids and subsequent reverse transcription allows the analysis of mutations present in both fractions of nucleic acids by allele specific RTqPCR and ultra deep sequencing (UDS) of disease-specific gene panels. The spectrum of mutations covering 15 mutation hotspots from 8 genes was analyzed using a custom library preparation method and bioinformatics pipeline. Results: EXO52 spin columns efficiently capture high quality nucleic acids from biofluids and a single column can be used to deplete cfDNA and exoRNA from plasma volumes up to 4 mL. The eluate contains cfDNA as well as a non-degraded small and large RNAs including mRNA, miRNA, and rRNA. As a proof of principle, we demonstrate the detection of high and ultra-low frequency mutations in blood samples of three different cancer types including malignant melanoma and colorectal cancer. Somatic mutations can readily be detected in both exoRNA and cfDNA, with the combination of both as the superior choice for mutation signal and yield. Conclusions: In this study we demonstrate the utility of a blood-based liquid biopsy using EXO52 spin columns for detection of somatic mutations in the co-purified exoRNA and cfDNA. Since the total amount of genetic material in plasma is a limiting factor for current development of genetic tests, we show the combination of high quality RNA from vesicles and cfDNA to be of advantage for detection of somatic mutations in various cancers in addition to enabling expression-based analysis and detection of fusion transcripts. 314 POSTER (Board P094) Optimal sequencing schedules for combining BRAF inhibition with BCL-2 inhibition A. Schalck1 , D.T. Frederick1 , M.R. Hammond1 , I. Ferreiro-Neira2 , Z.A. Cooper3 , J.C. Cusack2 , D.P. Lawrence1 , K.T. Flaherty1 , J.A. Wargo3 , R.J. Sullivan1 . 1 Massachusetts General Hospital Cancer Center, Center for Melanoma, Boston MA, USA; 2 Massachusetts General Hospital, Surgical Oncology, Boston MA, USA; 3 MDACC, Surgical Oncology, Houston TX, USA Background: BRAF inhibitors (BRAFi), MEK inhibitors (MEKi), and their combination are effective and approved by the FDA for the treatment of BRAF mutant melanoma. However, resistance to these therapies typically develops within a year. We previously showed that pre-treatment BCL-2 expression correlates inversely with outcome to BRAFi, and that MCL-1 expression decreases upon BRAF inhibition. We hypothesized that targeting BCL-2 using navitoclax (ABT263) would prime high BCL-2 tumors to BRAFi. Likewise, since ABT263 does not inhibit the anti-apoptotic protein MCL-1 and is known to be ineffective against tumors expressing high levels of MCL-1, we hypothesized that lowering MCL1 with BRAFi would prime high MCL-1 tumors to ABT263. We were able to demonstrate synergy between the two drugs when used in combination in a panel of BRAF mutant cell lines, in vitro and in vivo. The work presented here builds on these findings focusing pretreatment BCL-2 and MCL-1 expression levels as potential predictive biomarkers of benefit to various sequencing and dosing schedules of the two drugs. Material and Methods: mRNA expression of patient tumors (obtained pre- and 10−14 days after BRAFi therapy) and cell lines was measured by qPCR. Protein expression of patient samples was quantified by RPPA analysis. Melanoma cell lines were treated with vehicle control (DMSO), ABT263 (1mM), PLX4720(1mM), and the combination. Annexin V staining was performed and measured by flow cytometry to assess apoptotic effect. MTT assays were used to assess cell proliferation for different drug schedules. Results: For both qPCR and RPPA analysis, samples expressing the highest pre-BRAFi mRNA and protein levels of MCL-1 showed the greatest fold decreases of MCL-1 with BRAF inhibitor treatment (r=-0.56, p = 0.023, n = 16 and r=-0.81, p = 0.003, n = 11, respectively). A similar correlation was also demonstrated across a panel of cell lines (n = 9). Furthermore, for both patient samples and cell lines, BRAFi therapy neutralized MCL-1 over-expression to levels comparable to the baseline of low pre-treatment MCL-1 expressing patient samples and cell lines. Additionally in cell lines, high pre-treatment MCL-1 levels were associated with minimal apoptosis in the context of ABT263. This was overcome with either concurrent or prior PLX4720, but not when these cell lines were subjected to a drug schedule of ABT263 followed by PLX4720. Conclusions: Melanoma cells highly expressing MCL-1 may become sensitized to ABT263 treatment with BRAFi. Conversely, BCL-2 inhibitor treatment may reduce the threshold for apoptosis increasing sensitivity to MAPK pathway inhibition. BCL-2 and MCL-1 expression may prove useful biomarkers to guide drug scheduling for patients who might otherwise succumb quickly to de novo resistance.

Poster Session – Molecular Targeted Agents I 315 POSTER (Board P095) Biological characterization of TAS3681, a new type of androgen receptor antagonist with androgen receptor downregulating activity K. Minamiguchi1 , M. Seki1 , H. Aoyagi1 , T. Mori1 , D. Kajiwara1 , N. Masuko1 , R. Fujita1 , S. Okajima1 , Y. Hayashi1 , E. Sasaki1 , T. Utsugi1 . 1 Taiho Pharmaceutical Co. Ltd, Tsukuba Research Center, Ibaraki, Japan Background: Recent evidence from both preclinical and clinical studies is consistent with the importance of reactivation of androgen receptor (AR) signaling in a majority of castration-resistant prostate cancer (CRPC). This knowledge has driven the clinical development of new inhibitors of androgen production (e.g., abiraterone) and AR signaling (e.g., enzalutamide). However, resistance to these new therapies has already been reported. Reactivation of AR signaling occurs by several mechanisms in CRPC. In the present study, we describe the biological characterization of TAS3681, which is a new AR antagonist with AR downregulation activity, and propose this concept as a potential new approach for the treatment of CRPC. Methods: For cell growth assay, prostate cancer cells were treated with androgen and TAS3681 in steroid depleted media for 3 days, and the living cells were determined by using wst-8 reagent. For assay of AR transactivation, COS-7 cells were transiently transfected with androgenresponsive reporter gene construct and expression vectors encoding wt or mu ARs. The transfected cells were treated with TAS3681 and androgen in steroid depleted media for 24 h, and luciferase activity was measured. For an in vivo pharmacodynamic (PD) assay to confirm AR downregulation in tumor, TAS3681 was orally dosed in CRPC tumor xenograft model. Tumor was isolated and AR level in tumor was determined by Western blot. Results: In cell growth assay, TAS3681 suppressed the growth of AR positive prostate cancer cells but did not affect that of AR-negative DU145 prostate cancer cells, indicating a dependency on AR for efficacy. TAS3681 did not stimulate AR nuclear translocation and suppressed wt and mu ARs (including F876L) transactivation in cells, indicative of its pure AR antagonist profile. In contrast to enzalutamide and bicalutamide, TAS3681 effectively suppressed androgen-independent AR transactivation by growth factors and cytokines via AR downregulation activity. Interestingly, in prostate cancer cells which express full-length and splice variant AR, TAS3681 reduced the expression of both ARs. Moreover, TAS3681 treatment effectively decreased AR level in CRPC tumors in vivo. Conclusion: Our findings suggest that TAS3681, a new type of AR antagonist with AR downregulation activity, has the potential to circumvent resistance to current and 2nd-generation therapies targeting AR signaling and could provide a new therapeutic strategy in the treatment of CRPC. 316 POSTER (Board P096) Potent, partial agonists at ERalpha as selective estrogen mimics for treatment of tamoxifen-resistant breast cancer G. Thatcher1 , R. Xiong1 , H.K. Patel1 , J. Zhao1 , X. Liang1 , Y. Wang1 , M.E. Molloy2 , D. Tonetti2 . 1 University of Illinois College of Pharmacy, Medicinal Chemistry, Chicago IL, USA; 2 University of Illinois College of Pharmacy, Biopharmaceutical Sciences, Chicago IL, USA The estrogen receptor (ER) status of a mammary tumor is highly correlated with the aggressiveness of breast cancer and with prognosis and mortality. Up to 80% of breast cancers are ER+, in which estrogen fuels tumor growth. Tamoxifen (TAM) has been the standard of care for therapy for over 30 years; however, 30−50% of patients are unresponsive to TAM or develop resistance, leaving chemotherapy as the final treatment option. Paradoxically, prior to the introduction of TAM, both estradiol (E2 ) and the ER agonist diethylstilbestrol (DES) achieved similar success in the treatment of breast cancer, but with unacceptable side effects. One such side effect, uterine growth, is also shared by TAM itself. Development of a Selective Estrogen Mimic (SEM) that is effective in breast cancer without uterotrophic and other side effects is a novel treatment option that we hypothesized to be effective in causing regression of TAMresistant breast cancer. A rational approach would be an ER ligand with pharmacological partial agonist actions, able to mimic E2 in causing regression of tamoxifen-resistant breast cancer, but with antagonist actions in the face of excessive ER activation. We used the benzothiophene scaffold common to the Selective Estrogen Receptor Modulator (SERM), raloxifene used in clinically in postmenopausal osteoporosis, to develop a prototype SEM: TTC-352. Both cell-based and biochemical assays using coregulator recruitment validated TTC-352 as a partial agonist at ERa. In two mouse xenograft models of TAM-resistant breast cancer, this SEM caused complete and irreversible regression of established breast tumors. Impressively TTC-352 did not fuel growth of estrogendependent T47D xenografts and did not cause uterine growth. TTC352, therefore, selectively mimicked E2 in causing tumor regression, but without the adverse effects of fueling growth of uterine tissues and of endocrine-sensitive mammary tumors. To better understand the

Thursday 20 November 2014 103 mechanistic implications of these results and to develop a predictive SAR, in vitro studies were conducted in TAM-resistant and parent cell lines using ERE-reporters, and measurements of gene regulation, and cell viability. In MCF-7:5C TAM-resistant cell cultures, a strong correlation was observed between cell viability and agonist potency at ERa and a computational model was developed to account for partial agonist activity. The results collected to date on SEMs indicates that partial agonists at ERa represent a new approach to breast cancer therapy.

Figure: Structure of TTC-352.

317 POSTER (Board P097) IKKb inhibition suppresses sphere formation and self-renewal of lung cancer initiating cells T. Carneiro-Lobo1 , A.C.P. Salviatto1 , A.S. Baldwin2 , D.S. Basseres1 . 1 ˜ Paulo, Biochemistry, Sao ˜ Paulo, Brazil; 2 University University of Sao of North Carolina at Chapel Hill USA, Lineberger Comprehensive Cancer Center, Chapel Hill, USA Background: Activating mutations in KRAS are prevalent in cancer, but therapies targeted to oncogenic RAS have been ineffective to date. An alternative route for blocking RAS-driven oncogenic pathways is to target downstream effectors of RAS. Recent studies have shown that oncogenic RAS activates the NF-úB transcription factor pathway and that KRASinduced lung tumorigenesis is suppressed by expression of a degradationresistant form of the IúBa inhibitor or by genetic deletion of IKKb or the RELA/p65 subunit of NF-úB. Since RAS signaling has been shown to be enhanced in cancer initiang cells (CICs), we analyzed the potential of IKKb inhibition therapy to target lung CICs. Material and Methods: We used serum-free growth-factor supplemented media in ultra low attachment plates to analyze tumorsphere formation, clonogenic growth and self-renewal of KRAS positive lung cancer H358 and A549 cells in the absence or presence of a highly specific IKKb inhibitor (compound A, Bayer), that we have recently shown is able to reduce KRASinduced lung tumor growth. To evaluate the tumor initiating potential of tumorsphere forming cells, identical numbers of parental and tumorsphere forming cells were injected subcutaneously in nude mice. Stem cell markers were evaluated both by quantitative real time PCR and by flow cytometry. Results: Both A549 and H358 cells were able to form tumorspheres with increased tumor initiating ability in a xenograft model in Balb/c-nude mice. In addition, sphere forming cells were able to self-renew, and when compared to the parental cell lines, had increased clonogenic potential and displayed increased expression of stem cell surface markers CD44 and CD24, as well as increased expression of stem cell transcription factors Sox2, Oct4 and Nanog. Interestingly, IKKb signaling inhibition by Compound A resulted in specific downregulation of CD24 and suppressed in a concentration-dependent manner tumorsphere formation and growth, as well as self-renewal growth. Furthermore, Compound A reduced the clonogenic ability of tumorsphere forming cells. Conclusion: Our results suggest that IKKb inhibition therapy can reduce KRAS positive lung CICs, and, therefore might contribute to a more lasting therapeutic effect in KRAS-induced lung cancer. 318 POSTER (Board P098) Novel therapeutic targets for ARID1A mutant ovarian clear cell carcinoma (OCCC) R.E. Miller1 , I. Bajrami1 , R. Brough1 , A. Konde1 , J. Campbell1 , R. Rafiq1 , A. Ashworth1 , C.J. Lord1 . 1 Institute of Cancer Research, Gene Function, London, United Kingdom Background: Ovarian clear cell carcinoma (OCCC) is characterised by low response rates to standard chemotherapy and poorer prognosis than the more common high-grade serous ovarian cancer subtype. Next generation sequencing has identified ARID1A loss of function mutations in up to 57% of OCCC. ARID1A is a component of the SWI/SNF chromatin-remodeling complex, which regulates many cellular processes including differentiation, proliferation and DNA repair. ARID1A functions as a tumour suppressor and the high incidence of this mutation makes it an attractive therapeutic target for this disease.

104 Thursday 20 November 2014 Based on our current understanding of ARID1A function, it is difficult to predict a therapeutic strategy for these tumours. Therefore we adopted an unbiased approach using integrated functional and molecular profiling of ARID1A mutant and wild-type tumour cell models. Methods: We profiled two parallel systems; a panel of ARID1A mutant and wild-type OCCC cell lines models and an isogenic pair of ARID1A proficient and deficient HCT116 cell lines, using high throughput screening with chemical and siRNA libraries. This has been integrated with molecular profiling data generated from exome and RNA sequencing to aid the discovery of novel targets. Results: Using functional screens we have identified critical genetic dependencies and potential therapeutics in a series of clear cell ovarian cancer models. Specifically, we have established that dasatinib is selective for OCCC models with loss of function ARID1A mutations both in vitro and in vivo. Dasatinib is not only selective for ARID1A mutant OCCC models, but experimental induction of ARID1A deficiency drives dasatinib sensitivity in ARID1A wild-type OCCC models as well as in isogenic cell systems (both mouse and human) where ARID1A has been rendered dysfunctional by gene targeting. Conclusions: These results provide a framework for the discovery of therapeutic targets for clear cell ovarian cancer characterised by ARID1A mutations. Using an unbiased drug and siRNA sensitivity screening approach, we have identified dasatinib as a candidate therapeutic approach for OCCC. This Dasatinib/ARID1A synthetic lethal approach can be observed both in in vitro and in vivo and suggests that dasatinib should be assessed in clinical trials in OCCC patients. 319 POSTER (Board P099) Genomic profiling of uterine leiomyosarcomas reveal frequent alterations in Akt/mammalian target of rapamycin (mTOR) pathway genes and other actionable genomic abnormalities linked to targeted therapies J.A. Elvin1 , Z.R. Chalmers2 , J. Chiemlicki3 , K.A.I. Wang4 , N. Palma5 , S.M. Ali5 , A. Huho6 , C.E. Sheehan6 , V.A. Miller7 , P.J. Stephens8 , J.S. Ross1 . 1 Foundation Medicine, Pathology, Cambridge MA, USA; 2 Foundation Medicine, Biomarker and Companion Diagnostics Development, Cambridge MA, USA; 3 Foundation Medicine, Cancer Genomics, Cambridge MA, USA; 4 Foundation Medicine, Computational biology, Cambridge MA, USA; 5 Foundation Medicine, Medical Affairs, Cambridge MA, USA; 6 Albany Medical College, Dept of Pathology and Laboratory Medicine, Albany NY, USA; 7 Foundation Medicine, Clinical Development, Cambridge MA, USA; 8 Foundation Medicine, Research and Product Development, Cambridge MA, USA Background: Uterine leiomyosarcoma (LMS) is a rare, highly aggressive tumor arising from uterine wall smooth muscle, which frequently recurs, spreads hematogenously, and responds poorly to standard adjuvant chemotherapy. We performed comprehensive genomic profiling of 62 clinical uterine LMS samples to identify genomic-derived drug targets of therapy for patients with this lethal cancer and insight into the biology of this tumor. Methods: Hybridization capture from 236 cancer-related genes and 19 genes commonly rearranged in cancer (FoundationOne® , n = 30) or from 405 cancer-related genes and 31 genes commonly rearranged in cancer (FoundationOne® Heme DNA only, n = 32) was applied to  50 ng of DNA extracted from 62 FFPE uterine LMS tumor specimens and sequenced to high, uniform coverage. Genomic alterations (base substitutions, small indels, rearrangements, copy number alterations) were determined and then reported for these patient samples. Actionable alterations are defined as those identifying anti-cancer drugs on the market or in registered clinical trials. Results: 62 female LMS patients with a median age 55 years (range 37−74 years) with predominantly high tumor grades and advanced stage had sequencing of their primary tumor (n = 26) or metastasis (n = 36). Analysis revealed 61 (98%) of LMS harbor at least one alteration (mean 4.0; range 0−10). Inactivating mutations of at least one critical tumor suppressor gene TP53 (71%) or RB1 (50%) or chromatin remodeling regulator ATRX (31%) was observed in 85% of cases. 39 uterine LMS (63%) had at least one actionable alteration (mean 1.4; range 0−5). Alterations predicted to result in activation of the Akt/mammalian target of rapamycin (mTOR) pathway were identified in 35% of cases, predominantly as a result of loss of function of negative regulators (PTEN 11.3%, NF1 4.8%, TSC1 4.8%, STK11 3.2%, PIK3R1 1.6%) and less frequently through gain of function of positive regulators (amplification of RICTOR 3.2%; AKT2 3.2%; RPTOR 1.6%; AKT1 1.6%, IGF1R 1.6%, PIK3CA 1.6%) Potentially druggable fusions were identified in three patients (TPM3-NTRK1, STK32B-ALK and ACTG2-ALK ). Conclusions: Approximately one third of uterine LMS patients harbored mTOR pathway alterations corroborating prior preclinical and immunohistochemical studies supporting the importance of mTOR signaling

Poster Session – Molecular Targeted Agents I in smooth muscle proliferation. The almost universal co-occurrence of tumor suppressor loss suggests a combinatorial effect is necessary for tumorigenesis. Given the relative chemoinsensitivity, radioresistance and poor prognosis uterine LMS, comprehensive genomic profiling has the potential to identify therapeutic targets in a significant subset of patients. 320 POSTER (Board P100) First-in-human study with ARQ 092, a novel pan AKT-inhibitor, in subjects with advanced solid tumors or recurrent malignant lymphoma M. Saleh1 , K. Papadpoulos2 , A. Arabnia1 , A. Patnaik2 , R.M. Stein1 , F. Chai3 , M. Lamar3 , R. Savage3 , G. Abbadessa3 , A. Tolcher2 . 1 Georgia Cancer Specialist/Northside Hospital Cancer Institute, Sandy Springs, USA; 2 START − South Texas Accelerated Research Therapeutics LLC., San Antonio, USA; 3 ArQule Inc., Woburn, USA Background: ARQ 092 is an oral allosteric, ATP-independent, potent and selective AKT inhibitor. Data from cell lines and xenografts support the exploration of its anti-tumor activity on a broad range of solid and hematological tumors. Material and Methods: This is an ongoing phase 1, open label, dose escalation study. A traditional 3+3 dose escalation schema was employed. Multiple dose levels and schedules were explored. MTD is defined as the dose level at which 1 of 6 subjects has a DLT(s) during the first 28 days. Endpoints include safety, pharmacokinetics, biomarkers, efficacy. Treatment is continued until disease progression or unacceptable toxicity. Results: As of 22 Apr 2014, 63 pts (39.7% male; median age 62.6 yrs, 11 endometrial, 7 colon, 4 breast, 4 lymphoma, 4 neuroendocrine, 4 NSCLC, 3 ovarian and 26 others) were treated in 13 dose cohorts with 3 dosing schedules (table). Dosing schedule

Dose range (mg)

Frequency

No. of cohorts

No. of patients

Continuous Intermittent Weekly

10−80 80–270 250–350

QOD/QD QD, 1 week on, 1 week off BID, 1 day on, 6 days off

6 5 2

28 27 8

The most frequent drug related adverse events (>10%) included hyperglycemia 28.6%, macula-papular (MP) rash 27.0%, nausea 22.2%, pruritus 17.5%, diarrhea 15.9% and stomatitis 12.7%.10 pts experienced 13 DLTs (all grade 3) including 4 MP rash, 3 hyperglycemia needing insulin, 3 elevated ALT/AST, 2 stomatitis and 1 congestive heart failure. The MTDs were determined to be 60 mg once daily (QD) and 220 mg QD for the continuous and intermittent dosing schedule respectively. The MTD for the weekly dosing schedule has not been defined, but the 350 mg BID dose was not well tolerated. One pt with lymphoma on the intermittent schedule had a partial response, 21 had stable disease including 3 pts with 18−20%tumor reduction. For pts dosed from 10 mg QOD (every other day) to 270 mg QD, 1 week on/off the steady state Cmax and AUC(0−24 h) ranged from 29 nM to 1,658 nM and 489 h·nM to 29,649 h·nM, respectively. Increase in glucose correlated with drug PK levels over time. Biomarker data will be presented, including the use of glycemia to modulate the dose. Conclusions: MTDs for 2 of the 3 dosing schedules has been achieved. Safety profile was in line with other drugs in the class. The correlation between the glucose change, rise in plasma insulin levels and drug exposure that portends clinical activity, and preliminary antitumor activity support proof of concept for AKT inhibition by ARQ 092. 321 POSTER (Board P101) Potent and selective inhibition of EZH2 by AU-2121 leads to significant tumor growth inhibition in mutant EZH2 dependent non-Hodgkin lymphoma S. Ahmed1 , K. Narayanan2 , A. Gadakh1 , S. Dodheri3 , S. Surendranath4 , S. Nathan4 , S. Mukherjee5 , S. Marappan6 , R. Sushmita3 , S. Dhadunia3 , K.M. Obilisetti1 , J. Anil1 , J. Reddy1 , K. Reddy1 , A. Basavaraju7 , C. Naik7 , C. Pandit1 , M. Ramachandra2 . 1 Aurigene Discovery Technologies Ltd., Medicinal Chemistry, Bangalore, India; 2 Aurigene Discovery Technologies Ltd., Pre-clinical Biology, Bangalore, India; 3 Aurigene Discovery Technologies Ltd., DMPK, Bangalore, India; 4 Aurigene Discovery Technologies Ltd., Cell and Molecular Biology, Bangalore, India; 5 Aurigene Discovery Technologies Ltd., Molecular Modeling, Bangalore, India; 6 Aurigene Discovery Technologies Ltd., Biochemistry, Bangalore, India; 7 Aurigene Discovery Technologies Ltd., In Vivo Pharmacology, Bangalore, India Background: Hyperactivity of mutant EHZ2 results in trimethylation of histone H3 lysine 27 (H3K27), which is well correlated with lymphoma-

Poster Session – Molecular Targeted Agents I

Thursday 20 November 2014 105

genesis in patients. Effective pharmacological inhibition of the activity with a small molecule has become a challenging task, because of the poor cellular potency and/or sub optimal pharmacokinetic properties of existing inhibitors. Materials and Methods: We have successfully identified a series of compounds that inhibit EZH2 using iterative medicinal chemistry efforts and SAR based approaches. Optimization of the series for target inhibition and anti proliferative activity in mutant EZH2 B cell lymphoma cell lines, coupled with a desirable pharmacokinetic profile resulted in identification of AU-2121 as the lead compound Results: AU-2121 is an S-adenosyl methionine (SAM) competitive inhibitor that potently inhibits mutant forms of EZH2 identified in nonHodgkin lymphoma patients. Lymphoma cell lines karpas 422 and Pfeiffer, dependent on mutant EZH2 exhibited dose and time dependent inhibition of trimethylation of H3K27 that translated into potent inhibition of proliferation with an EC50 of 78 and 3 nM respectively. The translation of enzyme potency to cellular potency can be attributed to high permeability (11.2×10−6 ) and low efflux (6.4×10− ) exhibited by the compound. AU-2121 exhibits 40 fold selectivity over closely related enzyme EZH1 and is more than 1000 fold selective over other related histone methyl transferases. AU-2121 exhibits good pharmacokinetic properties and moderate oral bioavailability. Initial tolerability studies in Balb/c mice showed that AU2121 was well tolerated at a high dose of 600 mg per kg per day for 7 days without significant loss in bodyweight and major pathological findings. Oral administration of AU-2121 to mice bearing pfeiffer tumors resulted in dose dependent inhibition of tumor growth with maximum inhibition of 98% observed at a dose of 300 mg per kg per day as compared to vehicle controls on day 28. Conclusions: AU-2121 and its close analogues are being optimized further to develop a new treatment option for non-Hodgkin lymphoma expressing mutant EZH2.

323 POSTER (Board P103) The development of short form of mimic microRNA for lung cancer therapy

322 POSTER (Board P102) Novel alternative splice variants as potential biomarkers and therapeutic targets in aggressive prostate cancer in men of African descent

X. Zhou1 , A.J. Olszanski2 , M. Middleton3 , R. Gonzalez4 , M.J. Bargfrede1 , M. Kneissl5 , V. Bozon ´ 6 , E. Gangolli7 , K. Venkatakrishnan1 , M. Patel8 , C. Zopf8 , D.W. Rasco9 . 1 Takeda Pharmaceuticals International Co., Clinical Pharmacology, Cambridge, USA; 2 Fox Chase Cancer Center, Phase 1 Early Clinical Development Program Medical Oncology, Philadelphia, USA; 3 Oxford University Hospitals NHS Trust, Department of Oncology, Oxford, United Kingdom; 4 University of Colorado Cancer Center, Medicine/Medical Oncology, Aurora, USA; 5 Takeda Pharmaceuticals International Co., Clinical Research, Cambridge, USA; 6 Takeda Pharmaceuticals International Co., Oncology Clinical Research, Cambridge, USA; 7 Takeda Pharmaceuticals International Co., Translational Medicine, Cambridge, USA; 8 Takeda Pharmaceuticals International Co., Drug Metabolism and Pharmacokinetics, Cambridge, USA; 9 South Texas Accelerated Research Therapeutics (START), Department of Oncology, San Antonio, USA

S. Patierno1 , J. Freedman2 , B. Wang3 , N. Lee3 , D. George2 . 1 Duke Comprehensive Cancer Center, Durham NC, USA; 2 Duke Comprehensive Cancer Center, Medicine, Durham NC, USA; 3 George Washington University School of Medicine and Health Sciences, Pharmacology and Physiology, Washington DC, USA Background: African American (AA) men exhibit 2-fold higher incidence and 3-fold higher mortality rates from prostate cancer compared to Caucasian American (CA) men and disparities in tumor aggressiveness remain after controlling for social determinants of health. We utilized a transcriptome approach to this population-based difference to identify molecular mechanisms of tumor aggressiveness and associated biomarkers and molecular targets. Material and Methods: RNA was isolated from prostate biopsy cores positive for cancer from a cohort of AA and CA men. Exon arrays were used to identify alternative splicing events in AA versus CA prostate cancer and RT-PCR was used to validate predicted alternative splicing patterns defined by the exon arrays. The biological significance of alternative splicing events was established by assessing the effect of specific siRNA knockdown of the alternative isoforms, and splice-switching oligonucleotides, on proliferation and invasion of prostate cancer cell lines derived from AA and CA patients. Results: We identified 934 differentially expressed exons in 861 corresponding genes between AA and CA prostate cancer. Using pathway analysis of PCa-associated genes, a subset of eight genes exhibiting alternative splicing patterns was validated and the biological significance of alternative splicing of one of the genes, PIK3CD, has been established. A long splice variant of this gene is the sole isoform in CA prostate cancer, whereas AA prostate cancer exhibits a short splice variant in addition to the long splice variant. Knockdown of the long isoform in CA prostate cancer results in a decrease in proliferation and invasion. In AA prostate cancer, if the long splice variant is knocked down the short splice variant drives proliferation and invasion. Knockdown of the short isoform in AA prostate cancer results in a decrease in proliferation and invasion. Studies are underway to examine the therapeutic potential of manipulating alternative splicing using splice-switching oligonucleotides. Conclusions: These studies suggest that population-specific alternative splicing is associated with increased growth and more aggressive cancer invasion characteristics of prostate cancer in AA men. Establishing the underlying genetic differences between AA and CA prostate cancer and the biologic relevance of these differences to tumor biology will identify biomarkers and/or therapeutic targets that may have profound implications for the prevention, screening, diagnosis and management of prostate cancer for men of all races.

S. Ohno1 , K. Sudo2 , M. Kuroda1 . 1 Tokyo Medical University, Pathology, Tokyo, Japan; 2 Tokyo Medical University, Animal Research Center, Tokyo, Japan MiR-34a is a potent tumor suppressor microRNA (miRNA) and inhibited in many cancers. Over-expression of miR-34a inhibits proliferation and induces apoptosis in lung cancer cell lines, therefore the replacement therapy of miR-34a as a promising approach for the treatment of lung cancer. However, the systemic administration of the nucleic acid drugs is typically limited by unwanted immune responses to nucleic acid via TLR and RIG-I/MDA5. Because the intensity of immune response to nucleic acid correlates with their length, the shortening of nucleic acid enable the inhibition of the production of inflammatory cytokines. Therefore, the aim of the study was to develop the shortened miR-34a. The shortening of mimic miRNA with stable predicted RNA secondary structure that enabled shortening the length of mimic miRNA around 30-nucleotides while keeping the inhibitory activity and the broad range of targets. In addition, the immune responses against nucleic acids were decreased in the shortened mimic miRNA compared with normal mature form of miRNA. The results suggested that the shortening of miRNA is a promising approach for the development of safer nucleic acid drugs. 324 POSTER (Board P104) Clinical pharmacokinetics (PK), translational PK/pharmacodynamics (PD), and exposure–adverse events (AEs) relationship of MLN2480, an oral investigational pan-RAF kinase inhibitor

Background: MLN2480 is an investigational pan-RAF kinase inhibitor being evaluated in an ongoing Phase 1 study (NCT01425008). The maximum tolerated dose (MTD) has been identified as 200 mg administered every other day (Q2D) in 28-day cycles in patients with advanced solid tumors. Emerging clinical data have revealed several AEs consistent with MAPK pathway inhibition. Two of these AEs (i.e., acneiform rash and CPK elevation) have been specifically associated with MEK inhibition (MEKi). This analysis explores the relationship between these AEs and MLN2480 systemic exposure. Materials and Methods: Nonclinical PK/PD relationships were evaluated in mice with A375 BRAF- or SKMEL NRAS-mutant melanoma xenografts. Tumor PD was characterized by decrease in phosphorylated ERK (pERK) levels from baseline. Clinical PK was previously characterized and a base population PK (PPK) model was developed. Translational PK/PD simulations using clinical PPK and nonclinical PD parameters were conducted to predict clinical tumor PD effects at 200 mg Q2D (MTD). Logistic regression assessed the relationship between MLN2480 exposure and MEKi-like AEs (e.g. rash, CPK elevation), and defined exposures associated with these clinical phenotypes. Simulations explored alternative intermittent dosing schedules to find exposures associated with MAPK pathway inhibition for a reasonable window of time without compromising overall dose density. Results: MLN2480 exhibited dose-linear PK over 20–280 mg in cancer patients. Translational PK/PD simulations predicted an average 48−60% decrease in clinical tumor pERK levels with MLN2480 at the MTD, which is within the reported range for vemurafenib and trametinib. Exposure−AE analysis indicated a higher incidence of clinical effects associated with MAPK pathway inhibition at higher MLN2480 exposure (N = 43, P < 0.05). Per PK/PD simulations, MLN2480 700 mg once weekly is anticipated to offer the same overall exposure as 200 mg Q2D, to reduce tumor pERK levels (average 46−57%, maximum 69−76%), and to provide exposures associated with AEs reflecting robust MAPK pathway inhibition for 20−40% of the dosing interval.

106 Thursday 20 November 2014 Conclusions: Translational PK/PD simulations indicated that MLN2480 exposures achieved at 200 mg Q2D (MTD) are within the pharmacodynamically active range. Exposure−AE analysis suggests the potential for more robust MAPK pathway inhibition at higher exposures of MLN2480, which is consistent with pan-RAF kinase inhibition by MLN2480. 325 POSTER (Board P105) Identification and characterization of an irreversible inhibitor of CDK2 M. Martin1 , E. Anscombe2 , E. Meschini3 , D. Staunton2 , M. Geitmann4 , U.H. Danielson5 , L.Z. Wang6 , R. Mora Vidal6 , T. Reuillon3 , B.T. Golding3 , D.R. Newell6 , S. Wedge6 , M.E.M. Noble6 , J.A. Endicott6 , R.J. Griffin3 . 1 Newcastle University, Northern Institute for Cancer Research, Newcastle-upon-Tyne, United Kingdom; 2 University of Oxford, Department of Biochemistry, Oxford, United Kingdom; 3 Newcastle University, NICR and Department of Chemistry, Newcastle upon Tyne, United Kingdom; 4 Beactica AB, Uppsala, Sweden; 5 Uppsala University, Department of Chemistry, Uppsala, Sweden; 6 Newcastle University, Newcastle Cancer Centre NICR, Newcastle upon Tyne, United Kingdom Background: Irreversible protein kinase inhibitors that modify cysteine or lysine residues within the ATP binding site offer, through their different mode of action, an alternative approach to ATP-competitive compounds. Deregulation of the cell cycle is a characteristic of most human tumors, occurring frequently through disruption of the pRb signaling circuit, and aberrant control of cyclin-dependent protein kinases (CDKs) has been directly linked to cancer development. NU6102 is a potent and selective CDK2 ATPcompetitive inhibitor in which the sulfonamide moiety is positioned close to a pair of lysine residues, suggesting that there is an opportunity to develop irreversible CDK2 inhibitors that target these amino acids. Materials and Methods: Guided by the CDK2/NU6102 structure a library of 2-(aminoethyl)sulfone derivatives were synthesized from which 6-(cyclohexylmethoxy)-N-(4(vinylsulfonyl)phenyl)-9H-purin-2-amine (NCL00006300) was generated as an intermediate. Surface plasmon resonance, mass spectrometry, X-ray crystallography and biochemical assays carried out using authentic and mutant CDK2/cyclin A complexes have been used to characterize the interaction of NCL-00006300 with CDK2/cyclin A. The inhibitor mode of action has been characterized against a panel of cell lines by clonogenic assays and western blotting. Results: Determination of the CDK2/cyclin A/NCL-00006300 co-crystal structure reveals the inhibitor binding mode through a covalent interaction to Lys89, a residue that is not well conserved across the protein kinase family. Cellular studies support an irreversible inhibitor mode of action and provide evidence that selective irreversible CDK2 inhibition can be achieved in cells. Importantly, NCL-00006300 was cytotoxic following short-term exposure, consistent with irreversible CDK2 inhibition. Conclusions: We describe the first example of an irreversible inhibitor that selectivity targets CDK2 by grafting a reactive vinylsulfone moiety onto the structure of a potent and selective reversible CDK2 inhibitor. The structure suggests routes by which the reactive vinylsulfone moiety could be grafted onto other CDK2-selective pharmacophores to develop inhibitors that specifically target CDK2. 326 POSTER (Board P106) A novel non-ligand competing anti-EGFR antibody for cancer treatment X. Wang1 , S. Rust1 , F. Comer1 , V. Muniz-Medina1 , Q. Du1 , A. Yuan1 , K. Senthil1 , R. Fleming1 , R. Minter1 , S. Coats1 . 1 Medimmune LLC, Gaithersburg MD, USA Background: Clinical application of the current anti-EGFR antibodies (mAb) are mainly for head and neck cancer and treatment of colorectal cancers that express wild type KRAS. The two currently approved antibodies, cetuximab and panitumumab, compete with ligands binding to EGFR and block EGFR signaling. Acne form skin rash is the main side effect associated with anti-EGFR mAb treatment which is due, in part, to inhibition of the EGF/EGFR pathway in normal skin tissues. Severe acne form skin rash often leads to sub-optimal dosing or termination of treatment. Here we describe a novel anti-EGFR mAb, D-BLOCK, that does not compete with EGF binding but has similar anti-proliferative activity in tumor cells. The discovery and development of D-BLOCK may bring new therapeutic options for patients with tumors expressing EGFR. Methods: A panel of anti-EGFR scFv molecules were isolated from a phage library and converted to fully human IgGs. Binding affinity to EGFR, epitope binning and ligand competition were determined by standard ELISA, SPR and FACS analyses. The affinity to EGFR was further optimized for selected antibodies through site-directed mutagenesis. D-BLOCK was conjugated with a novel anti-microtubule tubulysin warhead

Poster Session – Molecular Targeted Agents I to generate an antibody drug conjugate (ADC). Anti-tumor activity of armed and un-armed D-BLOCK was tested in in vitro cell killing assays, and in vivo in both standard tumor cell line xenograft and patient derived primary tumor models. Results: D-BLOCK mAb demonstrated similar anti-proliferative activity in both in vitro and in vivo tumor assays to the current anti-EGFR mAbs. However D-BLOCK does not compete with EGF binding to EGFR. We clearly demonstrated that D-BLOCK and EGF ligand can co-bind a monomeric form EGFR. D-BLOCK also binds EGFR simultaneously with Panitumumab − suggesting they recognize non-overlapping epitopes. Further analyses indicated that D-BLOCK disrupts EGFR:EGFR dimer formation. Conjugation of D-BLOCK with cytotoxic tubulysin enhanced its cell killing activity in cultured tumor cells including those with KRAS mutations. Conclusion: D-BLOCK is a non-ligand competing anti-EGFR antibody. It inhibits EGFR signaling primarily through disrupting EGFR:EGFR dimer formation. This novel antibody may be explored in combination with current therapies or developed as an ADC to increase its tumor cell killing activity. 327 POSTER (Board P107) Combination of MDM2 antagonists with RAS pathway inhibitors in colorectal cancer B. Higgins1 , C. Tovar2 , K. Kolinsky2 , Y. Zhang3 , S.A. Middleton1 , G. Nichols1 , K. Packman2 , F. Su2 , L. Vassilev2 . 1 Roche Innovation Center New York, Translational Medicine Oncology, New York, USA; 2 Roche Pharma Research and Early Development, Discovery Oncology, Nutley, USA; 3 Roche Pharma Research and Early Development, Galenics, Nutley, USA Activation of the RAS/RAF/MEK/ERK pathway confers resistance to the apoptotic activity of p53 when activated by MDM2 antagonists. Combinations of MEK inhibitors (RO5068760, GDC0973 [cobimetinib]) or a BRAF mutant inhibitor (vemurafenib) with nutlin family MDM2 antagonists (RG7112, RG7388) were tested in colorectal (CRC) models for enhancement of apoptotic activity leading to increased anti-tumor activity. Enhanced response correlated with the presence of activating KRAS or BRAF mutations, in conjunction with functional p53 signaling typical to the p53 wildtype cell lines tested. MDM2 antagonist combined with MEK inhibitor significantly enhanced the apoptotic activity over either agent alone in CRC cells with activated RAS pathway signaling (KRAS G13D and BRAF V600E). This combination overcame the resistance to p53 apoptotic activity that is mediated via RAS pathway activation by increasing pro-apoptotic (Puma) and decreasing anti-apoptotic (Bcl2, Mcl1) proteins. In vitro, synergy was seen with MDM2 antagonist + MEK inhibitor in KRAS mutant CRC cells, and with MDM2 antagonist + vemurafenib in a BRAF mutant, vemurafenib de novo resistant CRC cell line (RKO) (CI = 0.034 at ED90 and CI = 0.471 at ED90 , respectively). For both scenarios, there was also a significant enhancement of antitumor activity in vivo. A search of the TCGA database for p53 wt and RAS pathway mutations in CRC cancer (n = 423) showed 7% p53 wildtype + KRAS mutant and 7% p53 wildtype + BRAF mutant. With the spectrum of KRAS mutations recently expanding beyond G12 and G13, the percentage of CRC with activating mutations in a Ras-family member may be as high as 60%, hinting that the population of p53 wildtype + KRAS mutant patients could be larger. Patients with functional p53 mutations may also respond, expanding it even further. These novel combinations provide an opportunity to address unmet need in KRAS mutant and BRAF mutant CRC and support clinical exploration. 328 POSTER (Board P108) Jagged1 expression and its relevance in metastatic progression of breast cancers N. Bednarz-Knoll1 , A. Efstathiou1 , F. Gotzhein1 , H. Wilkman1 , V. Mueller2 , Y. Kang3,4 , K. Pantel1 . 1 University Medical Center Hamburg-Eppendorf, Institute for Tumor Biology, Hamburg, Germany; 2 University Medical Center Hamburg-Eppendorf, Department of Gynecology, Hamburg, Germany; 3 Princeton University, Department of Molecular Biology, Princeton, USA; 4 Cancer Institute of New Jersey, Breast Cancer Program, New Brunswick, USA Background: Jagged1 (Jag1) is reported to be involved in the formation of bone metastasis and therefore, hypothetically associated with worse prognosis of tumor patients. In this study, Jag1 expression was assessed in primary tumors and circulating tumor cells (CTCs) of breast cancer patients and compared to clinico-pathological parameters and patients’ outcome. Material and Methods: Jag1 expression was evaluated by immunohistochemistry on tissue microarrays comprising tumor samples of 411 breast cancer patients and compared to clinico-pathological parameters and patients’ outcome. Additionally, CTCs were isolated from peripheral blood of 107 metastatic breast cancer patients and analysed for Jag1 expression.

Poster Session – Molecular Targeted Agents I Results: No, weak, moderate and strong Jag1 expression was detected in 97 (42%), 76 (33%), 38 (17%) and 17 (8%) of 228 informative tumors, respectively. Jag1 strong expression correlated to lymph node positivity (p = 0.027) and cancer-related death (p = 0.030). It tended also to be expressed more frequently in patients with disseminated tumor cells detected in their bone marrow (p = 0.082). Seventeen (16.9%) of 107 patients with metastatic breast cancer were positive for CTCs; 12 (70.6%) of these 17 patients had Jag1(+) CTCs and 2 (11.8%) showed Jag1(+) and Jag1(−) CTCs. Ten (76.9%) of 13 patients with progressive disease and two (100%) patients with recurrence had Jag1(+) CTCs, respectively. Conclusions: Jag1 strong expression seems to be a signature of more advanced disease and tumor cell dissemination in breast cancer patients. Mechanism and clinical relevance of this finding has to be further investigated, especially in context of targeted therapies (Wan et al., Nature Med., 2013). 329 POSTER (Board P109) A first-in-human study evaluating the safety and pharmacology of MM-151, a novel oligoclonal anti-EGFR antibody combination in patients with refractory solid tumors M. Beeram1 , C. Lieu2 , W. Harb3 , L. Power4 , J. Kearns5 , R. Nering4 , V. Moyo4 , B. Wolf4 , A. Adjei6 . 1 The Start Center For Cancer Care, San Antonio Texas, USA; 2 University of Colorado, Medical Oncology, Aurora, USA; 3 Horizon BioAdvance, Medical Oncology, Lafayette, USA; 4 Merrimack Pharmaceuticals Inc., Clinical Operations, Cambridge, USA; 5 Merrimack Pharmaceuticals Inc., Research, Cambridge, USA; 6 Roswell Park Cancer Institute, Medical Oncology, Buffalo, USA Background: MM-151 is an oligoclonal combination of three IgG1, antiEGFR antibodies designed to bind distinct non-overlapping EGFR epitopes and inhibit ligand-mediated signal amplification. MM-151’s molecular composition enables antagonism of clinically relevant EGFR ligand mixtures, EGFR down-regulation and immune effector function (ADCC, CDC). A Phase 1 study was initiated to assess the safety, tolerability, PK, immunogenicity and preliminary clinical activity of MM-151 alone and in combination with irinotecan. Methods: A standard 3+3 design was used to assess safety and tolerability of MM-151 monotherapy (QW, Q2W, Q3W) and MM-151 (QW) in combination with irinotecan 180 mg/m2 (Q2W). Results: Results presented below are based on preliminary data as of June 4, 2014. A total of 82 patients (pts) have been enrolled (median age 62 years; 40 male, 42 female) with 73 pts on monotherapy (36 QW, 23 Q2W, 14 Q3W) and 9 pts on the combination with irinotecan. The most common tumor types were CRC (31 [38%]), NSCLC (12 [13%]) and SCCHN (8 [10%]). A monotherapy MTD has not been defined and dosing continues at 10.5 mg/kg QW and 15 mg/kg Q2W. Most adverse events were CTCAE grades 1 and 2. Infusion related reaction (IRR) was the most common AE (54 [65.9%]); however, this was effectively managed with premedication and an optimized infusion schedule. The most common non-IRR AEs were comprised of EGFR-pathway toxicities, including rash (63 [77%]), hypomagnesemia (21 [26%]), mucositis (11 [13%]) and diarrhea (21 [26%]). Initial biomarker data suggest that IRR relates to engagement of the innate immune system. At doses 9 mg/kg QW, trough total antibody levels at steady state were in the expected therapeutic range. Partial responses (PRs) per RECIST criteria were observed in 2 CRC pts, and a total of 8 (26% of mCRC) pts had SD for >4 months (2 of the 8 had SD for 20.1 and 19.8 months). Combination therapy with irinotecan has begun with MM-151 at 6 mg/kg QW, and a PR was observed at this dose level in a SCCHN pt. Initial safety data are consistent with known toxicities of MM-151 and irinotecan. Conclusions: Results to date demonstrate that MM-151 monotherapy has an acceptable tolerability profile and preliminary indications of objective clinical activity. Updated monotherapy results and additional data of the combination with irinotecan will be presented. 330 POSTER (Board P110) Preclinical development of BIS-1602, first in class growth hormone releasing hormone antagonist Y.Y. Maxuitenko1 , N.L. Block2 , A.V. Schally3 , S.J. Reich1 , P. Goldstein1 . 1 Biscayne Pharmaceuticals, Miami, USA; 2 Biscayne Pharmaceuticals and University of Miami Miller School of Medicine, Miami, USA; 3 Veterans Affairs Medical Center and University of Miami Miller School of Medicine, Miami, USA Local release of growth hormone releasing hormone (GHRH) by tumor cells plays a role in tumor growth and proliferation. GHRH receptors

Thursday 20 November 2014 107 were shown to be expressed in primary tumors including prostate, breast, ovarian, endometrial, colorectal, gastric, pancreatic, renal, lung, bone, glioblastomas, and lymphomas. Thus, antagonists of GHRH provide a new avenuein the development of cancer therapeutics. Here we report results from preclinical research of a novel, highly potent GHRH antagonist, BIS1602. BIS-1602 (formerly known as MIA-602) is a fluorinated lipopeptide GHRH antagonist, whose many amino acid substitutions compared to earlier reported GHRH antagonists increase its half-life and potency. BIS1602 demonstrates anti-tumor activityacross a broad range of human tumor xenograft models: non-small cell lung, androgen-independent prostate, estrogen-independent breast, triple negative breast, ovarian, endometrial, renal, pancreatic, urothelial tumors, melanoma, glioblastoma, and diffuse mixed B cell lymphoma. Additionally, BIS-1602 enhances the efficacy of chemotherapy in multiple human tumor xenograft models without an increase in chemotherapy-related toxicity. The antitumor activity results from blocking the action of autocrine and/or paracrine GHRH via competitive binding to the GHRH receptors. BIS-1602 inhibits cell proliferation by inactivation of ERK and AKT kinases; induces apoptosis of tumor cells by increasing Bax and decreasing Bcl-2; and reduces the invasiveness and metastatic potential by reducing active forms of MMP2 and MMP9. Anti-tumor activity is achieved by daily subcutaneous (s.c.) injections (<5 mg/mouse). Administration of BIS-1602 does not cause overt toxicity, weight loss or myelosuppressive events in rats and dogs as a result of 14 days of s.c. administration. Pharmacokinetics analysis shows that BIS-1602 is absorbed and achieves plasma levels in the range of other clinically used hypothalamic peptide analogs. BIS-1602 is a novel, first in class, GHRH antagonist with an impressive anti-tumor profile and is currently being developed for clinical investigation.

331 POSTER (Board P111) Biomarker discovery and validation for assessing the response to cMet inhibition and functional inactivation 1 2 S. Jung1 , S. Selzer1 , C. Loßner ¨ , K. Kuhn1 , U. Fiedler2 , A. Klotzbucher ¨ , H.D. Zucht1 , S. Koncarevic1 , T. Prinz1 , D. Hertfelder3 , K.A. Boehme3 , 2 1 1 1 3 H. Volkmer , M.H.G. Kubbutat , P. Budde , I. Pike . Proteome Sciences R&D GmbH & Co. KG, Frankfurt, Germany; 2 ProQinase GmbH, Freiburg, Germany; 3 Natural and Medical Sciences Institute, Tubingen, ¨ Germany

Background: The hepatocyte growth factor (HGF) receptor cMet is a receptor tyrosine kinase which regulates cell proliferation, morphogenesis, and motility. Mutations in cMet, over-expression of cMet or aberrant cMet signalling is present in various tumours including prostate cancer and participates in angiogenesis, tumour development, invasion, and metastasis. High cMet expression is observed in late stages and metastases of prostate cancer. We present a biomarker discovery and validation study for measuring cMet activation and inhibition effects. The markers aim at determining if a prostate cancer patient will be susceptible to treatment with a drug targeting the cMet pathway. Material and Methods: We analysed two prostate cancer cell lines: DU145 (moderate metastatic potential) and PC-3 (high metastatic potential). We compared the protein expression patterns of untreated control cells, cells exposed to HGF, and to inhibitors SU11274 and to PHA665752 in the presence of HGF. Supernatant and cell extracts were analysed applying a TMTsixplex quantitative proteomics approach. Complementary analyses of cell extracts were performed applying a 2-DE gel-based approach. The discovered biomarker candidates were further evaluated using the orthogonal approaches ELISA and SRM in cell lines and tumor biopsy samples. Results: Proteomics analysis profiled 1980 (809 in at least two sets) proteins. Bidirectional hierarchical cluster analysis visualized abundance of 45 biomarkers with activated and inhibited or inactivated cMet pathway. Gene ontology, pathway and cellular localisation information constrained a set of potentially secreted markers. In ELISA validation for a subset of markers we showed an increase of protein abundance for 3 markers in DU145 cells after activation of the HGF/cMet signal. This effect was compensated in presence of cMet inhibitors (PHA665752, SU11274, Crizotinib and BMS-77607). In selected reaction monitoring (SRM) validation experiments with 28 proteins a subset showed different abundance in samples +/− inhibitors of the HGF/cMet signalling pathway in cell lines. Markers were tested in biopsy samples (prostate carcinoma, benign prostate hyperplasia, and bladder carcinoma). The SRM analysis of the biomarker panel in biopsy tumor tissues revealed differences between malign and benign types. Conclusions: The results of the described approach can be extended for determining drug sensitivity and treatment efficacy in patients by measuring biomarkers in body fluids and cancer tissue.

108 Thursday 20 November 2014 332 POSTER (Board P112) Dual PI3K delta/gamma inhibition by RP6530 accentuates bortezomib activity in multiple myeloma cell lines S. Viswanadha1 , G. Babu2 , S. Veeraraghavan3 , S. Vakkalanka4 . 1 Incozen Therapeutics Pvt. Ltd., Biology, Hyderabad, India; 2 Incozen Therapeutics Pvt. Ltd., Medicinal Chemistry, Hyderabad, India; 3 Incozen Therapeutics Pvt. Ltd., DMPK, Hyderabad, India; 4 Rhizen Pharmaceuticals S.A., Research, La Chaux-de-Fonds, Switzerland Background: The bone marrow microenvironment contributes to the pathogenesis of Multiple Myeloma (MM) by promoting the oncogenic process including drug resistance. High expression levels of the PI3K delta and gamma isoforms in patient MM cells implicate this target as a novel and attractive intervention strategy aimed at attenuating the progression of the disease. Current treatment options for MM include the use of bortezomib, a proteosome inhibitor that acts by promoting apoptosis in neoplastic cells. Combination of bortezomib with a PI3K d/g inhibitor may therefore have added therapeutic potential. RP6530 is a novel, potent, and selective PI3K d/g inhibitor. RP6530 demonstrates high potency against PI3Kd (IC50 = 25 nM) and g (IC50 = 33 nM) enzymes with selectivity over a (>300-fold) and b (>100-fold) isoforms. In this study, the potential of the combination of bortezomib (BORT) and RP6530 in MM cell lines was evaluated. Methods: Potential of the combination (1 mM RP6530 + BORT between 0.1 nM and 1 mM) was studied in MM cell lines, namely, MM-1S and MM-1R. Proliferation was determined by a MTT assay after incubating with compound for 72 h at 37ºC. Apoptotic potential of the combination was estimated by AnnexinV/PI staining while their role in interfering with cell cycle progression was evaluated using a Guava® cell cycle assay reagent (Millipore). Inhibition of AKT phosphorylation by RP6530 or the combination was determined by Western Blotting. Results: Addition of 1 mM RP6530 caused a significant leftward shift in the EC50 of BORT in MM-1S (3-fold) and MM-1R (1.5-fold) cell lines. While 1 mM RP6530 alone had minimal effect on apoptosis, co-treatment with BORT increased the number of Annexin V+ /PI+ cells in the late apoptotic phase across the cell lines tested (5.6% for 1 nM BORT vs. 12.6% for combination in MM-1S; 5.8% for 1 nM BORT vs. 26.6% for combination in MM-1R). Likewise, an increase in Annexin V+ /PI− cells was also observed upon combining 1 nM BORT with 1 mM RP6530 as opposed to treatment with BORT alone (21% vs. 15% in MM-1S and 60% vs. 11% in MM-1R). Treatment with 1 mM RP6530 resulted in G2/M arrest in MM-1S and MM1R lines with very few cells in the SubG0 phase. Addition of 1 nM BORT to RP6530 increased cytotoxicity manifested by an increase in cells in the SubG0 phase (9% for BORT vs. 20% for combination in MM-1S; 18% for BORT vs. 56% for combination in MM-1R). While RP6530 resulted in a 70−90% inhibition of pAKT in MM-1S and MM-1R cell lines, addition of BORT did not accentuate the effect indicating that the mechanisms of action of both compounds are complementary and non-redundant. Conclusions: RP6530 enhanced the activities of BORT in depleting arresting MM cell proliferation and causing apoptosis and subsequent cell death. Combination therapy of BORT and RP6530 in a mouse MM xenograft model is planned to better define the efficacy in an in vivo setting. 333 POSTER (Board P113) Short antisense oligonucleotides antagonize Lin28 and enable pre-let-7 processing and suppression of cell growth in human hepatocarcinoma cells M. Roos1 , G. Civenni2 , M. Lucic1 , D. Pavlicek1 , U. Pradere1 , H. Towbin1 , C.V. Catapano2 , J. Hall1 . 1 University of Zurich, Department of Chemistry and Applied Biosciences, Zurich, Switzerland; 2 Tumor Biology and Experimental Therapeutics, Institute of Oncology Research (IOR), Bellinzona, Switzerland The let-7 microRNAs (miRNAs) have prominent roles in development and cancer. Let-7 miRNAs are important suppressors of cell growth and their targets include oncogenes like K-Ras, Myc and HMGA-2. Expression of let-7 is frequently lost in tumours and correlates with poor prognosis. Importantly, in many cancers the tumor suppressor function of let-7 is abrogated by oncogenic Lin28, one of several RNA binding proteins, which regulate let-7 biogenesis by interacting with conserved motifs in let-7 precursors close to the Dicer cleavage site. The main objective of this study was to test the possibility of re-establishing the normal let-7 processing by targeting the terminal loop region (TLR) of pre-let-7 with short antisense oligonucleotides (ASOs) and by-pass Lin28 repression. Using new in vitro assays, we have identified a high-affinity binding site for ASOs overlapping the Lin28 binding region in pre-let-7a-2. These ASOs selectively antagonize the docking of Lin28, but, importantly, still allow processing of pre-let-7a-2 by Dicer. Functionally, in hepatocarcinoma Huh7 and HepG2 cancer cells including the stem-like cell component ASOs increased the level of mature

Poster Session – Molecular Targeted Agents I let-7, which, in turn, repressed its targets including the Lin28B and HMGA2 oncoproteins. Strikingly, in Lin28B over-expressing hepatocarcinoma cells ASO treatment produced growth arrest and reduced clonogenic potential. ASOs that bind the terminal loop region of let-7 precursors represent a novel approach to rescue let-7 miRNA biogenesis and tumor suppressor function in Lin28-dependent cancers. This work was partly funded by an Eidgenossische ¨ Technische Hochschule Grant (ETH-01 11−2) and Krebsforschung Schweiz (KFS-2648−08–2010). 334 POSTER (Board P114) Aplidin: first in class compound targeting EEF1A in tumor cells A. Losada1 , J.F. Martinez1 , P. Moral2 , L. Carrasco2 , F. Gago3 , 1 C. Cuevas1 , L.F. Garc´ıa-Fernandez ´ , C.M. Galmarini1 . 1 PharmaMar S.A. Sociedad Unipersonal, Cell Biology, Colmenar Viejo (Madrid), Spain; 2 Centro de Biolog´ıa Molecular Severo Ochoa, Genome Dynamics and Function, Madrid, Spain; 3 University of Alcala´ de Henares, Biomedicine, Alcala´ de Henares, Spain Background: Aplidin (plitidepsin, APL), a cyclic depsipeptide found in the marine tunicate Aplidium albicans, is currently undergoing phase III clinical investigation for multiple myeloma. Oxidative stress, Rac1 activation and MAPKs phosphorylation altogether, trigger the apoptotic death of Aplidintreated tumor cells. Didemnin B, another member of the didemnins family, has been shown to interact with EEF1A and to inhibit protein synthesis in tumor cells. Here we investigated whether EEF1A is a target of Aplidin and whether it has a role in the mechanism of action of the compound. Materials and Methods: A HeLa cell line derivative (HeLa AplR) specifically resistant to Aplidin was previously described. DARTS method was used to analyze the interaction of EEF1A with Aplidin: cell protein extracts were incubated with the compound, digested with proteases and subjected to western blotting with an antibody against EEF1A. Relative amounts of EEF1A1 and EEF1A2 at mRNA and protein levels were analyzed by DNA microarray and iTRAQ respectively. Other components of the eEF1 complex were analyzed by Western blot. Stable cell lines overexpressing EEF1A1-EGFP or EEF1A2-EGFP were generated by transfection with the appropriate plasmids, and their sensitivity to Aplidin analyzed by dose–response experiments. Protein synthesis inhibition was analyzed in vitro with reticulocyte cell lysate, and in vivo in HeLa cells by metabolic labeling with [35 S]-methionine/cysteine in the presence of Aplidin. Results: In the presence of Aplidin, EEF1A was protected from protease digestion, suggesting a direct interaction. EEF1A2 was less expressed both at mRNA and protein levels in HeLa AplR compared to HeLa wt cells. No significant changes were seen in the levels of EEF1A1 or other components of the eEF1 complex. One hour treatment with Aplidin did not inhibit protein synthesis in HeLa cells, in contrast to cycloheximide. HeLa AplR cells with EEF1A2 levels restituted to normal partially recovered their sensitivity to Aplidin. Part of the signaling events induced by Aplidin in tumor cells and absent in HeLa AplR cells, such as p38 and ERK phosphorylation, were also recovered in the EEF1A2-EGFP stably transfected derivative. Others, as JNK phosphorylation and PARP proteolysis, were not recovered. Conclusion: Aplidin interaction with EEF1A2 (and probably EEF1A1) could have a role in its mechanism of action, independently of the canonical protein synthesis activity of the elongation factor. 335 POSTER (Board P115) RICTOR amplification as a novel molecular target for the treatment of lung cancer H. Cheng1 , B. Piperdi1 , Y. Zou1 , A. Verma1 , X. Liu2 , E. Schwartz1 , C. Zhu3 , C. Montagna4 , B. Halmos2 , A. Chachoua5 , R. Perez-Soler1 . 1 Albert Einstein College of Medicine, Oncology, Bronx NY, USA; 2 Columbia University Medical Center, Oncology, New York NY, USA; 3 Albert Einstein College of Medicine, Pathology, Bronx NY, USA; 4 Albert Einstein College of Medicine, Genetics and Pathology, Bronx NY, USA; 5 NYU Langone Medical Center, Oncology, New York NY, USA Background: Oncogenic alterations, such as mutations, translocations and amplification, have been associated with clinical responses to their specific molecularly-targeted therapy for subgroups of cancer patients. We recently identified amplification of RICTOR, a key component of the mTORC2 complex, in 8% of lung cancer patients, either as the only potentially actionable target or concurrently with other genetic changes. We hypothesizethat RICTOR amplification may represent a novel molecular target for the treatment of a unique lung cancer patient subset. Methods: We first performed FISH to identify lung cancer cell lines with RICTOR amplification. Then the effects of transient siRNA-mediated RICTOR knockdown in RICTOR-amplified lung cancer cells were examined. Next, the effects of inducible shRNA-mediated RICTOR knockdown were determined both in vitro and in vivo (xenograft mouse model). Additionally,

Poster Session – Molecular Targeted Agents I we investigated the in vitro cytotoxicity of inhibitors against different components of the PI3K/AKT/mTOR pathways in RICTOR-amplified lung cancer cells. Results: The majority of cells possess normal copy numbers of RICTOR but 3 out of 27 non-small cell lung cancer (NSCLC) cell lines, including H23 (adenocarcinoma) and H1703 (squamous), were found to have ratio of RICTOR/control around 2, suggesting RICTOR amplification in those cell lines. Genetic knockdown of RICTOR by using either siRNA or inducible shRNA was associated with growth inhibition of RICTOR-amplified lung cancer cell lines. The growth of xenograft lung tumor was also inhibited by inducible RICTOR knockdown. Among 6 different kinase inhibitors against the PI3K/AKT/mTOR pathways, agents blocking both mTOR1 and mTOR2 were the most active in RICTOR-amplified lung cancer cells. Conclusions: Targeted genetic blockade of RICTOR led to growth inhibition of RICTOR-amplified lung cancer cells, indicating that selective targeting of RICTOR may represent a novel therapeutic strategy for the treatment of this subgroupof lung cancer patients. Our study also provides the rationale for utilizing dual mTOR1/2 inhibitors as a potential therapy for lung cancer patients harboring RICTOR amplification. 336 POSTER (Board P116) The effect of food on the pharmacokinetics of the investigational Aurora A kinase (AAK) inhibitor, alisertib (MLN8237), in patients (pts) with advanced solid tumors or lymphomas X. Zhou1 , T.M. Bauer2 , S. Goel3 , J. Sarantopoulos4 , B. Zhang5 , V. Kelly6 , J. Mertz1 , K. Venkatakrishnan1 . 1 Takeda Pharmaceuticals International Co., Clinical Pharmacology, Cambridge, USA; 2 Sarah Cannon Research Institute, Tennessee Oncology, Nashville, USA; 3 Albert Einstein Cancer Center, Medical Oncology, Bronx, USA; 4 Institute for Drug Development Cancer Therapy & Research Center, Experimental Developmental Therapeutics, San Antonio, USA; 5 Takeda Pharmaceuticals International Co., Statistics, Cambridge, USA; 6 Takeda Pharmaceuticals International Co., Oncology Clinical Research, Cambridge, USA Background: Alisertib is an investigational, oral, selective AAK inhibitor currently in clinical development for multiple oncology indications. The recommended phase 2 dose (RP2D) for single-agent alisertib is 50 mg twice-daily for 7 days in 21-day cycles. This phase 1 study was conducted to characterize the effects of a high-fat meal on single-dose pharmacokinetics (PK) of the enteric-coated tablet (ECT) formulation of alisertib in pts with advanced cancer (NCT01898078). Materials and Methods: Pts aged 18 years with an ECOG PS 0−1 were eligible. Following overnight fasting for at least 10 hours (h), pts received a single 50 mg dose of alisertib (5×10 mg enteric-coated tablet) under either fasted or fed conditions (standard high-fat meal) using a 2-cycle, 2-way crossover design. Blood samples for PK analysis were collected in cycles 1 and 2, pre-dose and at intervals for up to 72 h post-day 1 dose. Following these assessments, pts were eligible to receive alisertib at the RP2D until disease progression. Primary endpoints included ratios of geometric mean for AUC0−inf and Cmax (fed versus fasted; the associated 90% confidence intervals [CI] were estimated using analysis of variances). Safety and tolerability of alisertib were secondary endpoints. Results: As of April 2014, 26 pts had been enrolled (50% male; 88% White; median age 60 years; mean weight 76 kg). 20 pts were PKevaluable. Following a single oral dose of alisertib, median Tmax was 6 h and 3 h under fed and fasted conditions, respectively. Preliminary PK analysis indicated that, following single-dose alisertib administration, AUC0−inf geometric mean ratio under fed conditions was 113% of that under fasted conditions (90% CI: 93.1%, 137%). Cmax geometric mean ratio under fed conditions was 102% of that under fasted conditions (90% CI: 88.3%, 117%). To date, drug-related adverse events (AEs) were reported in 24 (92%) pts, the most frequent was neutropenia (n = 18; 69%). Most frequent drug-related grade 3 AEs included neutropenia (n = 13; 50%) and leukopenia (n = 6; 23%). 1 pt experienced a drug-related serious AE (abdominal pain). Conclusions: The systemic exposures achieved following administration of a single 50 mg oral dose of alisertibafter a high-fat meal appeared similar to those observed in the fasted state. These preliminary results suggest that alisertib can likely be administered without regard for the timing of meals. Safety and tolerability evaluation of alisertib is ongoing.

Thursday 20 November 2014 109 337 POSTER (Board P117) Sensitisation of HPV+ HNSCC to cytotoxic treatments by targeting the G2/M checkpoint with AZ-1775 to improve survival A. Osman1 , N. Tanaka1 , A. Patel1 , J. Wang1 , A. Fitzgerald1 , T. Xie1 , M. Zhao1 , S. Jasser1 , M. Gadhikar1 , H. Skinner2 , M. Frederick1 , F. Johnson3 , J.N. Myers1 . 1 UT M.D. Anderson Cancer Center, Head and Neck Surgery, Houston Texas, USA; 2 UT M.D. Anderson Cancer Center, Radiation Oncology, Houston Texas, USA; 3 UT M.D. Anderson Cancer Center, Thoracic/Head and Neck Medical Oncology, Houston Texas, USA Background: The incidence of oropharyngeal squamous cell carcinomas (OPSCCs) has dramatically increased in recent years, due to causative association with high risk human papillomavirus (HPV) infection. Although the majority of patients with HPV+ oropharyngeal cancers have a favorable prognosis, some patient’s tumors remain resistant to chemoradiotherapy with locoregional and systemic recurrences. Wee-1 is a kinase that has been linked to DNA damage induced G2-M arrest, owing to its ability to inactivate cyclin dependent kinase 1 (CDK1) through phosphorylation of the Tyr15 residue. AZ-1775 is a small molecule inhibitor of Wee-1 kinase which has been shown to sensitize multiple tumor types to DNA-damaging agents. It is currently being evaluated in a phase II clinical trial for recurrent and metastatic head and neck cancer. We have recently demonstrated that AZ-1775 can overcome cisplatin resistance in HPV-negative HNSCC cells through mitotic arrest followed by senescence rather than apoptosis. Therefore, we assessed the ability of AZ-1775 to sensitize the HPV+ OPSCC cells to cisplatin and/or radiation. Material and Methods: Clonogenic survival assays, and an orthotopic mouse model of oral cancer were used to examine in vitro and in vivo sensitivity of five HPV+ OPSCC cell lines to AZ-1775 in combination with cisplatin and/or radition, while cell cycle analysis, western blotting, were performed to dissect molecular mechanisms. Results: The AZ-1775 significantly reduced the cell survival and enhanced in vitro sensitivity of HPV+ OPSCC cells to chemoradiotheray. Furthermore, addition of AZ-1775 to cisplatin and/or radiation induced prolonged G2/M arrest and shifted the death of HPV+ OPSCC tumor cells from either terminal senescence or mitotic arrest to an apoptotic pathway, by decreasing the expression of apoptosis inhibitory proteins MCl-1 and XIAP. In addition, AZ-1775 dramatically increased the antitumor effect of cisplatin in mice bearing the HPV+ OPSCC xenografts (p < 0.001). Conclusions: Overall, the data demonstrate that AZ-1775 enhances antitumor efficacy of cisplatin in preclinical models of HPV+ head and neck cancer. This study is important because it demonstrates the potential for a therapeutic strategy aimed at targeting the G2/M checkpoint in tumors associated with HPV infection. Through the use of synthetic lethality triggered by Wee-1 inhibition it appears possible to restore apoptotic cell death pathways and improve treatment of HPV+ tumors. 338 POSTER (Board P118) BM-1252 (APG-1252): a potent dual specific Bcl-2/Bcl-xL inhibitor that achieves complete tumor regression with minimal platelet toxicity L. Bai1 , J. Chen1 , L. Liu1 , D. McEachern1 , A. Aguilar1 , H. Zhou1 , C.Y. Yang1 , H. Wang2 , J. Wen2 , G. Wang2 , Y. Zhai2 , M. Guo2 , D. Yang2 , S. Wang3 . 1 University of Michigan, Department of Internal Medicine, Ann Arbor MI, USA; 2 Ascentage Pharma Group, Taizhou Jiangsu, China; 3 University of Michigan, Comprehensive Cancer Center and Departments of Internal Medicine Pharmacology and Medicinal Chemistry, Ann Arbor MI, USA Bcl-xL is overexpressed in many different types of human cancers and represents an attractive cancer therapeutic target. However, on-target thrombocytopenia toxicity caused by Bcl-xL inhibition greatly limits the application of Bcl-xL inhibitors. We report herein a strategy aimed at directly addressing the on-target thrombocytopenia of Bcl-xL inhibitors, while maintaining strong antitumor activity. Starting from a novel class of highly potent dual Bcl-2 and Bcl-xL inhibitors, we developed a ‘masking’ strategy to reduce the cell-permeability of the inhibitors by limiting the cell permeation of the drugs into platelets, while allowing the drugs to convert into more active metabolite in tumor tissue. Extensive selection of a large number of compounds led to the identification of BM-1252 (APG1252), which converts into a more active metabolite BM-1252-M1 (APG1252-M1) in vivo. While both APG-1252 and APG-1252-M1 bind to Bcl-2 and Bcl-xL with sub-nanomolar affinities (Ki < 1 nM), APG-1252 is >10times less active than APG-1252-M1 in cell growth assay in a panel of small cell lung cancer (SCLC) cell lines. Mechanistically, APG-1252-M1 induces Bax/Bak-dependent apoptosis in MEF/MCL1−/− model cell line. In vivo, both APG-1252 and APG-1252-M1 can achieve complete tumor regression in animal models of human cancer even at weekly dosing, but APG-1252

110 Thursday 20 November 2014 is >30-times less effective in platelet killing than BM-1252-M1 in animals. APG-1252-M1 exerts nanomolar single agent activity in a small subset of colorectal cancer (CRC) cell lines and synergizes with MEK inhibitor trametinib in a large subset of CRC cell lines. Taken together, our data show that APG-1252 warrants extensive evaluation as a promising dual Bcl-2/Bcl-xL inhibitor for preclinical and clinical development. 339 POSTER (Board P119) NAMPT inhibition as strategy to impair tumor growth 1 A. Cerezo1 , S. Jimenez ´ , E. Lospitao1 , N. Bravo1 , R. Campos-Olivas2 , 3 ˜ , R. Gilmour4 , S. Geeganage4 , G. Zhao4 , C. Aguilera1 , M. Canamero S. Velasco-Miguel1 . 1 Spanish National Cancer Research Centre, Cell Signalling, Madrid, Spain; 2 Spanish National Cancer Research Centre, Spectroscopy and NMR Unit, Madrid, Spain; 3 Spanish National Cancer Research Centre, Histopathology, Madrid, Spain; 4 Lilly Research Laboratories, Discovery Oncology, Indianapolis, USA

Background: NAD+ participates in electron transfer in redox reactions, mediates energy metabolism, mitochondrial function and different signaling mechanisms in the cell. Some cells synthesize NAD+ de novo, but many tumor cells are highly dependent on the activity of the enzyme NAMPT (nicotinamide phosphoribosyltransferase) to regenerate NAD+ from nicotinamide. Accordingly, NAMPT is over expressed in a large number of tumors from different tissues, and several studies suggest that NAMPT inhibition may be a good strategy to inhibit tumor growth. Material and Methods: pLKO lentiviral vectors were used for constitutive silencing of NAMPT, and pTRIPZ vectors were employed for doxycyclineinduced silencing and the NAMPT pharmacological inhibitor FK866 was used for in vitro experiments. Cell proliferation was analyzed through growth curves, and measuring the ATP content with Celltiter-Glo. Metabolic changes induced by the loss or inhibition of NAMPT were analyzed with a Seahorse XF-analyzer. Quantification of nutrient uptake in cells was performed by high resolution Nuclear Magnetic Resonance (NMR) spectroscopy of cell media. Xenograft experiments were performed in 12week old athymic nude mice implanted with 5×106 cells 786−0 or HCT116 cells Results: NAMPT was silenced by shRNA in a panel of tumor cell lines of different histologies, including the 786−0 renal cell carcinoma cell line, the HT1080 fibrosarcoma, and the HCT116 colon carcinoma cell line among others. Cells with NAMPT knockdown showed lower NAD+ and ATP levels, a slower rate of metabolite exchange, and reduced proliferation in vitro. In addition, the knockdown cells had less capacity to form colonies, and had diminished tumor growth in xenograft experiments. Consistent with the shRNA data, specific inhibition of NAMPT activity by use of the small molecule inhibitor FK866 in vitro resulted in a cell proliferation delay and reduced colony formation. Glycolytic activity and mitochondrial function, however, were not significantly affected. Tumors generated by NAMPT silenced cells showed less Ki67 staining, but similar distribution of hypoxic, glycolytic and oxidative areas within the tumors. Conclusions: NAMPT silencing or specific pharmacological inhibition impairs tumor cell proliferation both in vitro and in xenograft experiments. Immunohistochemistry staining of different metabolic markers in the tumors showed a heterogeneous distribution of metabolic enzymes within the tumor related to the most hypoxic areas, suggesting a microenvironment regulation of tumor cell metabolism that might mediate compensation of inhibitory signals. 340 POSTER (Board P120) Preclinical activity of the VEGFR, FGFR and PDGFR inhibitor lucitanib in FGFR2 aberrant endometrial and gastric cancer models F. Guffanti1 , R. Chila` 1 , E. Bello1 , L. Ceriani1 , M. Zangarini1 , M. Zucchetti1 , C. Saba2 , A. Jacquet-Bescond2 , M. Pierrat2 , G. Damia1 . 1 IRCCS-Mario Negri Institute for Pharmacological Research, Oncology, Milan, Italy; 2 Institut de Recherches Internationales Servier, Oncology Innovation Therapeutic Pole, Suresnes Cedex, France Lucitanib (S 80881, E-3810 and CO-3810) is a tyrosine kinase inhibitor with activity against vascular endothelial growth factor receptors 1−3 (VEGFR1−3) fibroblast growth factor receptor 1−2 (FGFR1−2) and platelet derived growth factor receptor a and b (PDGFRa/b). As predicted from in vitro FGFR1 amplified lung cell lines and in vivo xenografts models, the ongoing phase I/II clinical study of lucitanib (EudraCT#2010–019121−34) has shown activity in tumors reported to benefit from antiangiogenic agents and in breast cancer patients with FGF-aberrant (FGFR1 and/or FGF3/4/19 gene amplified) tumors. FGFR2 is frequently deregulated in tumors by gene mutation (e.g. ~12% endometrial cancers) and amplification (e.g. ~10% gastric cancers) and its inhibition has been advocated as a potential new therapeutic strategy. To explore the activity of lucitanib in FGFR2

Poster Session – Molecular Targeted Agents I aberrant preclinical models, a panel of 4 tumor cell lines (2 endometrial − ANC3A and MFE-296; 2 gastric − KatoIII and SNU16) with FGFR2 mutation or amplification and 2 wild-type (wt) FGFR2 cell lines (endometrial HEC1A and gastric MNK45) were selected. In vitro lucitanib treatment was 10−25 fold more effective in FGFR2 mutated endometrial cells (IC50 values ranging from 0.12−0.3mM) compared to the wt endometrial cell line (3 mM). Similar results were obtained in FGFR2 amplified vs. wt gastric cell lines (0.45mM vs. 3 mM). The in vivo antitumor activity of lucitanib was examined in FGFR2 aberrant (MFE-296 and SNU16) and wt (HEC1A and MNK45) xenografts. When tumors reached a mass of 200 mg, animals were randomized to receive escalating doses of lucitanib (0, 2.5, 5, 10 and 20 mg/kg/day PO) continuously for 30 days. A dose-dependent antitumor activity of lucitanib was observed in all the xenografts tested. Pharmacodynamic and pharmacokinetic assessments were undertaken at the same drug doses in tumor bearing mice. Lucitanib plasma and tumor levels were measured by LC-MS/MS after 4 and 24 hrs of the last dose of a 12 day drug dosing regimen. Drug tumor levels were related to the administered dose. In agreement with previous data generated in lung cancer xenograft models, lucitanib concentrations were higher in the tumor compared to the plasma. In conclusion, this preclinical data indicates that lucitanib demonstrates in vitro efficacy in FGFR2 aberrant endometrial and gastric cancer cells, in vivo efficacy in all the tested models and corroborates the favorable distribution profile of the drug in tumors. 341 POSTER (Board P121) Dinaciclib alters cell cycle dynamics and induces cell death in Soft Tissue Sarcomas N. Mulet Margalef1 , S. Rello-Varona2 , S. Garcia-Monclus ´ 2, M. Fuentes-Guirado2 , O.M. Tirado2 , X. Garcia del Muro1 . 1 ICO-DIR-IDIBELL, Medical Oncology Department, Barcelona, Spain; 2 IDIBELL, Laboratory of Molecular Oncology, Barcelona, Spain Background: Soft Tissue Sarcomas (STS) have generally very bad prognosis. Studying the molecular biology of STS is further required in order to develop better targeted therapies. The aims of this study are: 1. Determine the status of different cell cycle regulators (CDK and cyclins) in STS cell lines. 2. Challenge cells with the CDK inhibitor dinaciclib. 3. Analyze the effects of the treatment on the expression of cell cycle regulators, cell cycle dynamics and cell death induction. Material and Methods: Overall dose–response to dinaciclib in different STS cell lines (synovial sarcoma, liposarcoma, mixoid liposarcoma, leiomyosarcoma and fibrosarcoma) was determined by proliferation assays (WST-1). Treated cells response was further analysed by means of flow cytometry techniques in order to determine changes in cell cycle dynamics and cell death induction. Expression of CDKs, cyclins and other relevant proteins in cell cycle and cell death regulation was determined by Western Blot (WB) before and after treatment with dinaciclib. Results: STS cell lines have different patterns of expression of key regulators of the cell cycle (particularly in the entry to G2 /M). Treatment with dinaciclib induces a variable G2 /M blockade in all STS cell lines tested, although it only triggers cell death in some of them. Resistance to dinaciclib seems to be higher in those cell lines that underexpress G2 /M entry regulators. Cell death induction after dinaciclib treatment is mediated by the intrinsic apoptosis pathway as it is visualized by the loss of the mitochondrial membrane potential. Moreover cell death records are correlated with changes in members of the Bcl-2 family of proteins, one of its anti-apoptotic members was found to be upregulated in Dinaciclibresistant cells lines. Conclusion: As in most of cancers, cell cycle is disregulated in STS. In preclinical terms, cell cycle inhibitors represent a promising therapeutic strategy in STS, but probably not in all subtypes for equal. Dinaciclib is a good candidate for therapy development. Molecular biomarkers related with cell cycle could help to pre-select sensitive patients. The study of resistant phenotypes opens the gates to refined combined therapies able to overcome anti-apoptotic signaling. Further investigation with in vivo models is required.

Poster Session – Molecular Targeted Agents I 342 POSTER (Board P122) A phase 1/2 study evaluating the safety, pharmacokinetics and efficacy of ABT-414 in subjects with advanced solid tumors likely to over-express the epidermal growth factor receptor (EGFR) A. Tolcher1 , G. Goss2 , M. Gordon3 , L. Gandhi4 , K.P. Papadopoulos1 , D. Rasco1 , M. Pedersen5 , J. Fischer5 , W. Ames5 , H. Xiong5 , M. Dudley5 , W. Munasinghe5 , P. Ansell5 , K. Holen5 , E. Vokes6 . 1 South Texas Accelerated Research Therapeutics (START), Clinical Research, San Antonio TX, USA; 2 The Ottawa Hospital Cancer Centre, Ottawa Ontario, Canada; 3 Pinnacle Oncology Hematology, Scottsdale AZ, USA; 4 Dana-Farber Cancer Institute, Boston MA, USA; 5 AbbVie, North Chicago IL, USA; 6 University of Chicago, Chicago IL, USA Background: Multiple tumor types, including lung, head and neck, colon and pancreatic cancer can exhibit aberrant epidermal growth factor receptor (EGFR) signaling. ABT-414 is an antibody drug conjugate designed to treat tumors expressing EGFR. It consists of a unique antibody that targets an epitope within activated wildtype EGFR or mutated EGFRvIII, and is linked to a potent, toxic anti-microtubule agent, monomethylauristatin F (MMAF). Materials and Methods: Objectives of the study were to determine the safety profile, the maximum tolerated dose (MTD), the recommended Phase 2 dose (RP2D), and to evaluate the pharmacokinetic (PK) profile and preliminary anti-tumor activity of ABT-414 in patients with a solid tumor that is likely to overexpress EGFR. Adverse events (AEs) were reported according to NCI-CTCAE. Efficacy parameters were analyzed by RECIST 1.1. Two dosing schedules were evaluated − an every 3 week schedule (Q3W) and an alternate schedule with 2 weeks on, 1 week off. Results: As of May 4, 2014 a total of 41 patients were treated with ABT414. The median age was 59 years (range 39−82) and 42% (17/41) were male. The most common tumor types were non-small cell lung cancer (37%), colon cancer (17%), and head and neck cancer (20%). The most common treatment emergent AEs for Q3W were blurred vision (18/34, 53%), dry eye (15/34, 44%), nausea (14/34, 41%), fatigue (15/34, 44%), and vomiting (11/34. 32%). The most common treatment emergent AEs for the alternate schedule were nausea, vomiting, constipation, blurred vision and fatigue (each in 3/7, 43%). The only grade 3/4 AEs occurring in 2 patients were reversible keratitis, fatigue and dyspnea (each in 2/34, 6%) in the Q3W cohort. A dose limiting toxicity (DLT) of eye pain occurred in 1 patient in the Q3W cohort at 2.0 mg/kg. Although the MTD for the Q3W schedule was not reached, there were frequent and concerning ocular toxicities at 4.0 mg/kg; thus 3.0 mg/kg was determined as the RP2D. There were no reported DLTs in the alternate schedule cohort and the MTD was not reached. The highest cleared dose for the alternate schedule was 1.0 mg/kg. One patient with triple negative breast cancer in the Q3W schedule experienced a partial response. Conclusions: The most common toxicity of blurred vision was related to transient corneal epithelial microcysts. The response in triple negative breast cancer suggests proof-of-concept for an EGFR-antibody drug conjugate. 343 POSTER (Board P123) Salmonella typhimurium A1-R effectively targets experimental breast cancer brain metastasis Y. Zhang1 , S. Miwa1 , N. Zhang1 , R.M. Hoffman2 , M. Zhao1 . 1 AntiCancer Inc., San Diego California, USA; 2 University of California San Diego, Department of Surgery, San Diego California, USA Background: Brain metastasis is often a terminal phase of breast cancer with very few treatment options. The aim of this study was to demonstrate the efficacy of Salmonella typhimurium A1-R (A1-R) on breast cancer brain metastases. Material and Methods: High brain-metastatic variants of murine 4T1 breast cancer cells expressing red fluorescent protein (RFP) were injected into the left ventricle of transgenic nude mice expressing nestin-driven green fluorescent protein (ND-GFP). ND-GFP mice express GFP in nascent blood vessels. At various time points, the tumors and vasculature in the brain were imaged by laser-scanning confocal and stereo fluorescence microscopy. Results: Eighty percent of the cells that reached the brain extravasated and grew perivascularly. Twenty percent of the cells proliferated within the vasculature. Compared to the control group, mice treated with A1-R had significant inhibition of brain metastasis and increased survival (p < 0.05). Conclusions: The results of the present study suggest the clinical potential of bacterial therapy of breast cancer brain metastasis.

Thursday 20 November 2014 111 344 POSTER (Board P124) Search for new blood biomarkers for response to antiangiogenic therapy in non-small cell lung cancer patients ´ no ˜ 2 , S. Ponce1 , A. Rodr´ıguez-Garzotto1 , M.T. Agullo-Ortu 2 2 C.V. D´ıaz-Garc´ıa2 , A. Agudo-Lopez ´ , C. Perez ´ , E. Prieto-Garc´ıa2 , 1 H. Cortes-Funes ´ , L. Iglesias1 . 1 Hospital Universitario 12 de Octubre, ´ ´ Hospital Oncolog´ıa Medica, Madrid, Spain; 2 Instituto de Investigacion 12 de Octubre, Oncolog´ıa, Madrid, Spain Background: Bevacizumab (BV) has shown increased overall response rate and survival in patients (Pt) with non-small cell lung cancer (NSCLC). No biomarkers have been described predictors of response in these Pt. The aim of this study is to investigate the usefulness of gene expression profiles of peripheral blood monocytes from patients with NSCLC before and after chemotherapy plus BV. Material and Methods: A prospective study of blood samples from Stage IV NSCLC Pt treated with BV plus chemotherapy in our institution between 2009 and 2010 was performed. Plasma samples were collected before cycle 1 and after completion of cycle 2 (week 7). Clinical and radiological outcomes were recorded. The TaqMan® Human Angiogenesis Array was used for analysis (microfluidic card). This array contains 96 targets involved in human angiogenesis and lymphangiogenesis. Amplifications were carried out on the AB 7900HT RT-PCR system, and relative gene expression values were calculated by the DDCt method. Results: We present preliminary results of 12 Pt who suffered stability or improvement of disease. Differentially expressed gene between samples before and after chemotherapy is shown in the table.

Gene

P-Value (t-test)

Biological function

F2 PF4 COL4A2 SERPINF1 FGA HSPG2 TNF MDK VEGFB PTN EDIL3 PDGFB TGFA ITGB3 ENPP2

0.0002 0.0015 0.0157 0.0218 0.0235 0.0371 0.0037 0.0071 0.0111 0.0126 0.0194 0.0346 0.0376 0.0121 0.0418

Angiogenesis inhibitor Angiogenesis inhibitor Angiogenesis inhibitor Angiogenesis inhibitor Angiogenesis inhibitor Angiogenesis inhibitor Angiogenic growth factor Angiogenic growth factor Angiogenic growth factor Angiogenic growth factor Angiogenic growth factor Angiogenic growth factor Angiogenic growth factor Angiogenesis target Angiogenesis target

Conclusions: A range of differentially expressed genes associated with BV pluschemotherapy has been identified in our study. These genes could be used to assess the effectiveness of antiangiogenic therapy in addition to standard clinical and radiological evaluations, and even for designing new treatments. 345 POSTER (Board P125) Interaction of plitidepsin with eEF1A in living tumor cells ´ 1, C. Garc´ıa1 , A. Losada2 , J.M. Molina-Guijarro2 , M.A. Sacristan J. Martinez2 , C.M. Galmarini2 , M.P. Lillo1 . 1 Institute of Physical Chemistry Rocasolano, Department of Biological Physical Chemistry, Madrid, Spain; 2 PharmaMar S.A.U., Cell Biology Department, Colmenar Viejo (Madrid), Spain Background: Plitidepsin (Aplidin® ), a cyclic depsipeptide originally from the marine tunicate Aplidium albicans, is currently in a Phase III clinical trial in patients with relapsed or refractory multiple myeloma. It is reported that, in tumor cells, eEF1a is the potential target for Aplidin® . Here, we took advantage of two-photon micro-spectroscopy to localize the compound in treated tumor cells and shed light on its in vivo interaction with eEF1A. Materials and Methods: HeLa wt and HeLa AplR (resistant to Aplidin® ) cell lines have been previously described. HeLa AplR present lower eEF1A2 levels than HeLa wt cells. We stably transfected HeLa AplR cells with plasmids encoding GFP-tagged eEF1A1 or eEF1A2, and selected clones expressing physiological levels of the fusion proteins. A fluorescent Aplidin® derivative, Apl-dmac, was used to track the localization and interactions of the compound. 3D-scanning two-photon microscopy was performed with an Olympus IX71 microscope equipped with an UPLSAPO 60× objective, an excitation laser beam (750–850 nm, MaiTai, SpectraPhysics) and a

112 Thursday 20 November 2014 MicroTime200 (PicoQuant, GmbH). Using a FLIM-phasor approach we identified different plitidepsin species within cells. We could also apply the FLIM-phasor technique to measure FRET between the fluorescent tags dmac and GFP, thus identifying molecular interactions. Results: We detected three Aplidin® molecular species in treated cells. Species A interacts with a component of the plasma membrane both in HeLa wt and HeLa AplR cells. No FRET dmac® GFP was detected for this species, implying a distance between Apl-dmac and eEF1A-GFP ˚ Species B was seen in the inner part of the greater than 60−70 A. plasma membrane in HeLa wt, but not in HeLa AplR cells. This species is present, though, in HeLa AplR cells transfected with eEF1A1 or eEF1A2. Using FRET we could detect complexes Apl-dmac/eEF1A1-EGFP and Apl-dmac/eEF1A2-EGFP in the inner plasma membrane, corresponding with localization of species B. Species C moves free in the cytoplasm both in HeLa wt and HeLa AplR cells, although in the second the concentration is lower. At short times, free Aplidin-eEF1A complexes are seen close to the plasma membrane but then they distribute throughout the cytoplasm and finally accumulate in well defined regions. Conclusion: Aplidin® interacts and forms different complexes with eEF1A in two subcellular regions, first bound to the inner side of the plasma membrane and then free in the cytoplasm of tumor cells.

Poster Session – Molecular Targeted Agents I

346 POSTER (Board P126) Inhibition of Wnt pathway by novel thiazole-based Traf2- and Nck-interacting kinase (TNIK) inhibitor

(TKI). cMET amplification is a known resistance mechanism against EGFRTKI’s and the cMET receptor plays an important role in cancer growth. Therefore, a possible link between the EGFR and cMET pathway was investigated. Materials and Methods: We selected formalin fixed paraffin embedded (FFPE) tissue samples of 164 non-small cell lung cancer patients (NSCLC), never before treated with targeted therapies. The cMET-expression was assessed by immunohistochemistry (IHC) with the anti-total cMET (SP44) monoclonal antibody, with as reference tissue bronchial epithelial cells. cMET -amplification was evaluated by in situ hybridisation with the MET DNP probe and Chromosome 7 DIG probe (Ventana, Basel, Switzerland). In a subset of 70/164 patients, EGFR-expression was assessed by IHC with the anti-EGFR (3C6) antibody (Ventana) with bronchial epithelial cells as reference. EGFR mutational status of 52 EGFR expressing patients was tested using high resolution melting analysis (HRMA) and sequencing. Results: High cMET-expression was found in 48% of the patients and EGFR-expression in 22.5%, while cMET -amplification was only detected in 2.4%. 16 out of 52 (31%) sequenced samples carried an EGFR mutation. A significant correlation (p < 0.0001) was found between cMET- and EGFRexpression. In addition, a significant correlation (p < 0.05) was found between cMET-expression and EGFR-mutation status, with 16/16 EGFRmutant patients showing a high cMET expression. Conclusions: Our results show a significant correlation between cMETexpression, EGFR-expression and EGFR-mutational status in NSCLC patients. This interesting relation possibly opens the door for combination therapy with EGFR and cMET inhibitors.

Y. Uno1 , H. Moriyama1 , S. Kashimoto1 , M. Masuda2 , M. Sawa1 , T. Yamada2 . 1 Carna Biosciences Inc., Drug Discovery Research, Kobe, Japan; 2 National Cancer Center Research Institute, Division of Chemotherapy and Clinical Research, Tokyo, Japan

348 POSTER (Board P128) Two NSCLC-PDXs with different EGFR exon 20-insertions respond differently to different TKIs

Introduction: Aberrant activation of the Wnt pathway has been implicated as the key driver of carcinogenesis, particularly in colorectal cancers, and therefore the Wnt pathway has been thought of as an attractive anticancer drug target. The Wnt pathway is also involved in the regulation of cancer stem cells (CSCs), and therefore, inhibition of the Wnt pathway could be a new therapy for cancer by eradication of CSCs. Recently, we reported Traf2- and Nck-interacting kinase (TNIK) plays important roles in the Wnt signaling pathway by making a complex with beta-catenin and T-cell factor-4 (TCF4) in a nucleus. We also found that TNIK is activated in human colorectal cancer cell lines, and proliferations of those cells are highly dependent upon the expression and catalytic activity of TNIK. Therefore inhibitors of TNIK may represent a promising therapeutic approach for treating Wnt-signal activated cancers by killing CSCs. Here we describe a discovery and characterization of a novel TNIK inhibitor. Methods: To study the effects of the TNIK inhibitor on the Wnt signal pathway, the expressions of Wnt target genes were analyzed by Realtime PCR. Expression levels of Wnt target proteins were also analyzed by Western blotting. Anti-proliferative activity profile of the TNIK inhibitor was studied using a cancer cell panel. Results: Intensive lead optimization efforts resulted in the successful identification of a thiazole-based potent TNIK inhibitor with single digit nanoM IC50 value. The thiazole compound showed potent Wnt signal inhibition in TOP/FOP reporter assays. The Wnt signal inhibition was confirmed by detecting of down-regulation of Wnt target genes/proteins expressions. The thiazole compound showed strong anti-proliferative activity against HCT116, a Wnt-driven colorectal cancer cell line, but no inhibitions were observed under 10 microM concentration against Wntindependent cancer cell line and normal cell line. Detailed results including cancer cell panel studies will be presented in the conference. 347 POSTER (Board P127) Can a link between the EGFR and cMET pathway in non-small cell lung cancer explain resistance against targeted therapies and open new therapeutic opportunities? N. Van Der Steen1 , K. Van Der Steen2 , K. Zwaenepoel3 , E. Giovannetti4 , M. Castiglia3 , A.P. Carreca3 , P. Pauwels3 , C. Rolfo5 . 1 University Antwerp, Center for Oncological Research, Antwerp, Belgium; 2 Onze-Lieve-Vrouw Ziekenhuis, Department of Pathology, Aalst, Belgium; 3 Antwerp University Hospital, Molecular Pathology Unit Department of Pathology, Edegem, Belgium; 4 VU Amsterdam, Pharmacology Lab Cancer Center, Amsterdam, Netherlands; 5 Antwerp University Hospital, Phase I − Early Clinical Trials Unit Oncology Department, Antwerp, Belgium Background: Targeted therapies have achieved promising results in Non Small Cell Lung Cancer. Although they are currently used in the clinic to block the oncogenic mutated epidermal growth factor receptor (EGFR), in time problems arise with resistance against these tyrosine kinase inhibitors

H. Li1 , M. Yang2 , J. Cai2 . 1 Crown Biosciences, Santa Clara CA, USA; 2 Crown Biosciences, Translational Oncology, Santa Clara CA, USA Activating EGFR mutations are oncogenic drivers for many non-small cell lung cancer (NSCLC), including exon 19 deletion mutation, L858R mutation, T790M mutation and exon-20 insertion mutation. A number of EGFR TKIs (gefitinib, erlotinib, afatinib) have been found to effectively target some of the activating mutations and suppress tumor growth. Cetuximab, a monoclonal antibody targeting EGFR, also demonstrated activity against some of activated EGFR. We previously described that we created a large panel of patient derived xenografts (PDXs) from the treatment na¨ıve Asian NSCLC patients, including different histological subtypes of both adenocarcinoma (ADC) and squamous cell carcinoma (SCC), and those with classic activating EGFR mutations of point mutations and deletions. Among these EGFR-mutated PDXs, two harbor two different exon 20insertions, LU0387-ADC with 9-base insertion at 2319, and LU3075-SCC with 6-base insertion at 2316. We were interested in testing their responses to a panel of EGFR inhibitors. In contrast to the great response (complete response) to cetuximab seen for exon-19 deletion mutants, L858R single or L858R/T790M double mutants, both exon 20-insertions are completely resistant to cetuximab. This observation suggested that insertion mutations are fundamentally different from other classic EGFR activating mutations (see Table 1). Both insertion mutants are largely resistant to different TKIs, in similarly contrast to being sensitive for other EGFR mutants (Table 1). This observation is somewhat consistent with the suggested poor inhibitory effect by TKI on the EGFR with insertion mutation as shown in the anecdotal reports in the clinic. In particular, our data showed that the responses to the different TKIs by the two insertion mutants are also different. While LU3075 demonstrated resistance to all the EGFR inhibitors tested, LU0387 showed partial sensitivity to erlotinib and afatinib. One possible interpretation, or our working hypothesis, is that the two different insertions confer different drug–target interaction and thus target inhibitions. If confirmed, a new biomarker strategy could be developed to facilitate individualized patient stratification in TKI treatment. The data from these studies will be presented. Table 1. Responses to different EGFR inhibitors by NSCLC with different EGFR mutations Mutation type

Example

Cetuximab Erlotinib Gefitinib Afatinib

L858R L858R + T790M Deletion mutation Insertion mutation (exon 20−) Insertion mutation exon 20

(LU0858)a LU1868 LU1235 LU0387 LU3075

++ ++ ++ − −

a

++ − ++ +/− −

++ − ++ − −

+ + + + −

A model also with c-met gene amplication, but with when c-MET is inhibited, the model is sensitive to erlotinib.

Poster Session – Molecular Targeted Agents I 349 POSTER (Board P129) Synergistic drug combinations that target beta-catenin-driven and MYC-driven cancers J.C.M. Uitdehaag1 , J.A.P. Spijkers-Hagelstein1 , J.A.D.M. de Roos1 , A.M. van Doornmalen1 , M.B.W. Prinsen1 , J. de Man1 , R.C. Buijsman1 , G.J.R. Zaman1 . 1 Netherlands Translational Research Center B.V., Oss, Netherlands Background: Combination therapy plays an important role in achieving durable responses in anticancer therapy. However, the discovery of synergistic therapy that works in a particular genetic background is still in its infancy. Here we present a rational approach for identifying synergies by combining selective inhibitors that show a common sensitivity for specific genetic backgrounds, especially CTNNB1 (beta-catenin) and MYC mutations. Materials and Methods: We have established a panel of 44 genetically well-characterized cell lines that represents the most frequently occurring oncogenic drivers in cancer, such as CTNNB1, MYC and KRAS mutations (www.ntrc.nl). A collection of more than 80 inhibitors targeting all important oncogenic signaling pathways, including many kinase and epigenetic regulators, was profiled in the cell panel, generating highly reproducible and high-quality cell proliferation data. Response of the inhibitors was linked to the genetic background of the cell lines by Anova analysis. The selectivity of kinase inhibitors was determined by screening on a panel of more than 300 kinases. Follow-up synergy experiments were performed by in vitro proliferation assays on equipotent mixtures, followed by curve shift analysis, isobolograms and combination index scoring. Results: For all (>80) inhibitors, the drug response in the 44-cell line panel (Oncolines™) was analyzed. Notably, the Wnt-pathway inhibitor ICG001 and the MEK inhibitor trametinib were shown to preferentially inhibit CTNNB1-mutated cancer cell lines. Neratinib, a spectrum selective EGFR inhibitor, and GSK1070916, an Aurora kinase inhibitor, were shown to specifically target MYC-mutated cell lines. The pharmacological synthetic lethal effects of these compounds are caused by inhibition of specific pathways. Subsequent combination of these and other inhibitors showed synergy in the inhibition of cell proliferation in a high number of cases. ICG-001 is synergistic with trametinib in the CTNNB1-mutant colon cell line SW48, and not in the CTNNB1-wild type colon cell line SW620, indicating that the combination has an enhanced synthetic lethal effect. Conclusions: Pharmacogenomic analysis of single agent responses identified compounds that show synthetic lethality with CTNNB1-mutated and MYC-mutated cancers. Combination of these compounds in the pertinent genetic background identified compound sets with enhanced targeting of CTNNB1-mutated and MYC-mutated cancers. Panel profiling is therefore an unbiased way of identifying potentially synergistic compound sets. 350 POSTER (Board P130) Oncogenic Ras mutants differentially utilize PLC-dependent calcium flux and PKC activation for MAPK signaling C. Pitt1 , F. McCormick1 . 1 University of California San Francisco, San Francisco CA, USA Ras mutations drive approximately 30% of human cancers by aberrant regulation of the mitogen-activated protein kinase (MAPK) signaling cascade in gene expression, cellular growth and survival. While oncogenic Ras mutations are sufficient for constitutive GTP-loading and plasma membrane localization of the immediate MAPK effector Raf, it is unknown whether Ras variants differentially recruit additional signaling proteins in scaffolding complexes required for MAPK hyperactivation. Phosphoregulation of Raf is a crucial second step in Ras-dependent MAPK signaling. Phospholipase C (PLC) is a key mediator of Raf phosphorylation by generation of diacylglycerol (DAG) and inositol trisphosphate (IP3) activating protein kinase C (PKC) and calcium signaling. K-Raslox (H-Ras−/−; N-Ras−/−; K-Raslox/lox; RERTert/ert) mouse embryonic fibroblasts (MEFs) were kindly provided by the laboratory of Mariano Barbacid (CNIO, Madrid, Spain). Treatment with 4-hydroxytamoxifen (4-OHT) yields ‘Rasless’ cells, devoid of the major endogenous alleles. These cells were lentivirally transduced with various Ras alleles for comparison. Western blot and co-immunoprecipitation were the primary signaling readouts. Intracellular calcium flux in response to extracellular stimuli, ion channel agonists or inhibitors was quantitatively measured using the fluorescent Ca2+ indicator Fluo-4 NW (Invitrogen). Growth of ‘Rasless’ MEFs can be restored by reconstitution with any of the major Ras alleles. ‘Rasless’ cells are disconnected from growth factor or phorbol ester signaling and are deficient in producing a MAPK response. Though oncogenic Ras variants display constitutive GTPloading, unaffected by upstream stimulation, EGF and PMA are both capable of increasing the MAPK output from the basal level. PLC activity

Thursday 20 November 2014 113 is required to sustain basal MAPK signaling and necessary for the maximal response to stimuli. Mutant Ras alleles display differences in the extent of their dependency on PLC for generation of the MAPK response to stimulation. K-Ras mutants differentially bind Kinase Suppressor of Ras (KSR) 1 and 2 scaffolding proteins and degrees of interaction can be modulated by PLC inhibition. Downstream of PLC, calcium signaling is sufficient to generate increases in MAPK signaling in mutant Ras-expressing cells. Further, the calcium-regulated chloride channel TMEM16A modulates the MAPK response to Ca2+ flux in a Ras mutationdependent manner. Though oncogenic Ras does not require growth factor-mediated activation, enhanced MAPK signaling depends on both Ras-GTP status for membrane recruitment of Raf and Raf activation by proximal factors. The latter step requires PLC activity via downstream PKC and calcium signaling. Due to conformational variation of mutant Ras proteins, variable signaling complexes are formed and proximal Raf agonists or inhibiting proteins are differentially regulated by KSR scaffolding interactions in response to stimulation or inhibition of PLC. The discovery that specific Ras mutants may be more susceptible to PLC inhibition or downstream modulation of calcium flux may provide a novel avenue for clinical blockade of growth signaling in certain Ras mutant cancers. 351 POSTER (Board P131) hz515H7, a humanized antibody exerts its antitumor activity via antagonism of the CXCR4/SDF-1 axis, and through effector functions C. Klinguer-Hamour1 , M. Broussas2 , B. Akla2 , S. Berger3 , N. Boute3 , C. Beau-Larvor2 , A. Robert3 , J.F. Haeuw4 , L. Goetsch2 , C. Bailly5 , N. Corvaia5 . 1 Institut de Recherche Pierre Fabre, Physico-Chemistry Department, Saint-Julien-en-Genevois, France; 2 Institut de Recherche Pierre Fabre, Experimental Oncology Department, Saint-Julien-en-Genevois, France; 3 Institut de Recherche Pierre Fabre, UBMC, Saint-Julien-en-Genevois, France; 4 Institut de Recherche Pierre Fabre, Biochemistry Department, Saint-Julien-en-Genevois, France; 5 Institut de Recherche Pierre Fabre, Saint-Julien-en-Genevois, France Background: The chemokine receptor CXCR4 and its ligand stromal cell-derived factor-1 (SDF-1) play a central role in various physiological and pathological processes, including cancer. CXCR4 is over-expressed in a large number of tumors. The CXCR4/SDF-1 axis is directly implicated in migration, invasion leading to metastases, cell proliferation and angiogenesis. Moreover, CXCR4 over-expression is correlated with poor prognosis in many types of cancer. A humanized monoclonal antibody hz515H7 of IgG1 isotype was generated against the human CXCR4 receptor. It displayed potent antagonist properties for all major pathways associated with SDF-1/CXCR4 signaling in vitro. hz515H7 mAb was also evaluated for its ability to induce cancer cell death through ADCC and CDC in vitro. Furthermore, the importance of effector functions in the mechanism of action of hz515H7 mAb was investigated, using in vivo xenograft tumor models. Materials and Methods: The ADCC assay was based on the measurement of lactate dehydrogenase activity released from cytosol of damaged cells and the CDC assay was based on ATP measurements using a luminescent cell viability assay. Xenograft models: 10×106 Ramos cells (Burkitt’s lymphoma) were implanted s.c. into the right flank region of each SCID mouse and allowed to grow to the designated size before administration of antibodies. Tumor volume was calculated using the formula: length × width × height × p/6. For the U937 survival model, the U937 cells (AML) in exponential phase of growth were pelleted and resuspended in sterile PBS. Cells (10×106 in 200 ml) were implanted ip. in female Nod/SCID mice. hz515H7 mAb was injected twice a week and the mice followed for survival. Results: In vitro ADCC assays show that incubation of hz515H7 mAb with tumor cells expressing CXCR4 led to cell killing when purified NK cells were added. In addition, tumor cells incubated with hz515H7 in the presence of human sera, induced cell death via CDC mechanism. Furthermore, both the lymphoma and AML xenograft models showed that effector functions of hz515H7 mAb were essential for its antitumor activity. Conclusions: Our results demonstrate that hz515H7 mAb is unique since it has two major biological properties, (1) interference with CXCR4/SDF-1 axis and (2) triggering effector functions (ADCC and CDC). Its potential of best in class molecule is now investigated in phase I clinical trial.

114 Thursday 20 November 2014 352 POSTER (Board P132) UNC2025, a novel small molecule MerTK and Flt3 tyrosine kinase inhibitor, has therapeutic activity and promotes sensitivity to chemotherapy in animal models of acute leukemia D. Graham1 , D. DeRyckere1 , X. Wang2 , A.A. Hill1 , W. Zhang2 , S.V. Frye3 , H.S. Earp4 . 1 University of Colorado Denver, Department of Pediatrics, Aurora CO, USA; 2 University of North Carolina, Center for Integrative Chemical Biology and Drug Discovery, Chapel Hill NC, USA; 3 University of North Carolina, Center for Integrative Chemical Biology and Drug Discovery and Department of Medicine, Chapel Hill NC, USA; 4 University of North Carolina, Lineberger Comprehensive Cancer Center and Department of Medicine, Chapel Hill NC, USA Background: These studies investigate the potential utility of UNC2025, a dual MerTK and Fms-like tyrosine kinase 3 (Flt3) inhibitor, as a therapy for acute myeloid and acute lymphoid leukemias (AML and ALL). Methods: Inhibition of MerTK and Flt3 phosphorylation was measured using enzymatic and cell-based assays. Induction of apoptosis was determined using flow cytometry and colony-forming potential was assessed in methylcellulose cultures. Orthotopic B-ALL xenografts were generated in NOD/Scid/IL-2 gamma mice using a luciferase-expressing derivative of the 697 cell line. Inhibition of MerTK in bone marrow leukemia cells was determined by immunoblot. Disease burden was assessed using bioluminescence imaging and median survival was determined. Orthotopic patient-derived xenografts (PDX) were generated from primary AML samples and blasts were detected using flow cytometry. Results: UNC2025 inhibited MerTK and Flt3 in enzymatic (Ki = 0.16 and 0.59 nM, respectively) and cell-based (IC50 = 2.7 and 14 nM, respectively) assays and had limited off-target activity against 300 other kinases. Treatment with 100–200 nM UNC2025 induced apoptosis in 40−90% of cells and reduced colony-forming potential by 80–100% in leukemia cell cultures. In animal models, UNC2025 is orally bioavailable and inhibited MerTK phosphorylation in bone marrow leukemic blasts. Treatment with UNC2025 resulted in a dose-dependent reduction in tumor burden and increased median survival from 27 to 70 days after tumor implantation in a B-ALL xenograft model of minimal residual disease (p < 0.0001) and from 27.5 to 45 days when treatment was initiated after establishment of more substantial disease (p < 0.0001). Similarly, in a MerTK-expressing AML PDX model short-term treatment with UNC2025 resulted in a significant decrease in peripheral disease burden (9.3% blasts versus 35.6% for vehicle-treated animals, p = 0.0225) and both the fraction of splenic blasts (28.5 versus 75.7%, p = 0.0005) and spleen mass (170 versus 834 mg, p < 0.0001) were dramatically decreased. UNC2025 was even more effective in a Flt3-ITD mutant, MerTK-expressing AML PDX model, where treatment resulted in disease regression (0.24% peripheral blasts versus 11.4% pre-treatment, p = 0.0094) and prolonged median survival from 16 to >107 days post-treatment (p = 0.0114). In addition, in the B-ALL model treatment with UNC2025 and methotrexate, a chemotherapy currently in clinical use for treatment of ALL, resulted in reduced tumor burden compared to either agent alone and enabled tumor-free survival during an 88 day treatment window. Conclusions: The very high potency, relative selectivity, oral bioavailability, and target inhibition and therapeutic efficacy mediated by UNC2025 in murine models, both alone and in combination with chemotherapy, support continued development of a dual MerTK/Flt3 inhibitor for treatment of patients with acute leukemia. 353 POSTER (Board P133) Protein expression for receptor activator of NFkB (RANK) and its ligand (RANKL) in non-small cell lung cancer (NSCLC) M. D’Arcangelo1 , S. Ekman2 , W. Dougall3 , D. Branstetter4 , M. Bergqvist2 , P. Liv2 , D. Chan5 , J. Botling6 , F. Hirsch7 . 1 University of Colorado, Division of Medical Oncology, Denver, USA; 2 University of Uppsala, Department of Radiology, Uppsala, Sweden; 3 Amgen Inc., Therapeutic Innovation Unit, Thousand Oaks, USA; 4 Amgen Inc., Pathology, Thousand Oaks, USA; 5 University of Uppsala, Department of Medical Oncology, Denver, USA; 6 University of Uppsala, Department of Immunology, Uppsala, Sweden; 7 University of Colorado, Department of Medical Oncology, Colorado, USA Background: The activation of RANK by its ligand is crucial for bone metastasis in several cancers, including lung tumors. The fully human antiRANKL antibody denosumab reduces the risk of skeletal events. Evidence suggests that denosumab therapy may significantly prolong survival of lung cancer patients with bone metastases versus zoledronic acid. A direct anti-tumor effect of denosumab on lung cancer cells cannot be excluded. Therefore, we investigated RANK/RANKL expression and its prognostic significance in a large cohort of NSCLC patients.

Poster Session – Molecular Targeted Agents I Methods: Immunohistochemistry using the mAbs against RANKL (M366, Amgen) or RANK (N1H8, Amgen) was performed on a tissue microarray including 364 treatment na¨ıve resected NSCLC samples collected at University of Uppsala Sweden (median follow up 7.8 years). Evaluation of EGFR and KRAS mutations were performed with Therascreen EGFR RGQ PCR kit (Qiagen) and ALK overexpression by IHC (D5F3, Cell Signaling). Results were obtained for 352 samples. Median age was 67 years and males were 54%. Pathological stages: I 65%, II 15%, III 17%, IV 3%. Histology: 196 adenocarcinomas, 120 squamous cell carcinomas, 41 large cell carcinomas. A certified pathologist scored the protein expression using H-score, which considers staining intensity and percentage of stained tumor cells (range 0–300). Results: RANK (H-score >5) and RANKL (H-score >10) were detected in 106 (30%) and in 181 (52%) tumors, respectively. Concomitant expression of both proteins was observed in 65 (19%) samples. No correlation was observed between RANK/RANKL expression and histology, gender, smoking status, stage and EGFR mutations. RANK and RANKL positivity were associated with presence of KRAS mutation (p = 0.003 and p<.001, respectively). ALK positive tumors more often expressed RANKL (p = 0.005), while RANK expression seemed not to be affected. RANK and RANKL expression had no prognostic significance in this cohort of patients. Conclusions: RANK and RANKL are expressed at the protein level in a considerable number of early stage NSCLC patients, especially in KRAS mutated and ALK positive tumors, but do not affect survival. These data are focused on the tumor and not on RANKL/RANK expression in normal lung and infiltrating immune cells. These findings support and warrant further investigation on RANKL targeting in NSCLC. 354 POSTER (Board P134) PCR-based assay for BRAFV600 mutation analysis in ctDNA: clinical results from plasma and serum samples M. Gonzalez-Cao1 , C. Mayo de las Casas1 , M.A. Molina-Vila1 , L. De Mattos-Arruda2 , J.L. Manzano3 , E. Munoz ˜ 2 , J. Cortes2 , J.P. Berros4 , M. Sanmamed5 , A. Gonzalez5 , C. Alvarez4 , N. Karachaliou1 , N. Jordana-Ariza1 , S. Martin Algarra5 , R. Rosell1 . 1 Institut Universitari Dexeus Quiron, Instituto Oncologico Dr Rosell, Barcelona, Spain; 2 Hospital Vall d’Hebron, Medical Oncology, Barcelona, Spain; 3 Catalan Institute of Oncology Hospital Germans Trias i Pujol, Medical Oncology, Badalona, Spain; 4 Hospital Central Asturias, Medical Oncology, Oviedo, Spain; 5 Clinica Universitaria de Navarra, Medical Oncology, Pamplona, Spain Background: Determination of BRAFV600 mutation in cell free tumor DNA (ctDNA) in serum (s) or plasma (p) has been studied as a surrogate for tumor tissue biopsy. Our aim was to develop a PCR-based assay to analyze BRAFV600 mutation in s and p, compare results in both samples and to clinically validate ctDNA as a minimally invasive tool. Material and Methods: A quantitative 5 -nuclease PCR-based assay for determination of BRAFV600 mutation in ctDNA was developed and validated in 92 patients previously genotyped in tissue. We compare results from serum and plasma in every patient. Samples along BRAF inhibitor treatment were available from 4 melanoma patients. Results: The assay has a limit detection of 2.5 pg mutated DNA/mL and a ratio of V600 versus wild-type allele of 1:20,000. The assay had a clinical sensitivity of 57.7% and a specificity of 100%. Among patients with the BRAFV600 in tumor tissue (n = 52), 30 (57.7%) had BRAFV600E mutation in ctDNA (s or p), 28 (53.8%) in p (p = 0.05) and 23 (44%) in s (p = 0.016). Four melanoma patients were subjected to longitudinal analysis of BRAFV600 in ctDNA over time. Three out of 4 patients had BRAFV600 detectable in s/p at baseline. Patient 1 presented partial response; the absolute concentration of BRAFV600E increased 48 hours after starting BRAF inhibitor treatment, markedly decreased after 7 days and was not detectable after 30 days. The evolution of the BRAFV600E ratio was parallel in this patient, but remained around 40% in plasma and 10% in serum at 48 hours, to drop afterwards. Patient 2 showed complete response, BRAFV600 was detectable in serum/plasma pretreatment but not at the time of analysis performed after 30 days on treatment, at 240 days of follow-up complete response is ongoing and BRAFV600 is not detectable in s/p. Patient 3 showed progressive disease as best response, he was positive for BRAFV600 in pre-treatment plasma but negative in serum. After two months on BRAF inhibitor treatment, an increase in BRAFV600 quantification was observed both in terms of absolute concentration and also according to mutation load both in plasma and serum. Progressive disease was evident in this patient with rapid evolution and death two weeks after diagnosis of disease progression. Conclusion: Our assay has shown a better sensitivity to capture BRAFV600 mutation in ctDNA from p than from serum. Combination of s and p samples improves likehood ratio of positive test. cfDNA may be a potential minimally invasive alternative tool to help with diagnosis and monitoring of BRAFV600 mutant tumors.

Poster Session – Molecular Targeted Agents I 355 POSTER (Board P135) Altiratinib (DCC-2701): a balanced inhibitor of MET, TIE2, and VEGFR2 kinases that exhibits broad anti-tumor and anti-angiogenic activities D. Flynn1 , B.D. Smith1 , C.B. Leary1 , M.D. Kaufman1 , B.A. Turner1 , M.M. Hood1 , W.P. Lu1 , T.J. Rutkoski1 , T. Samarakoon1 , S. Vogeti1 , S.C. Wise1 , O. Rosen1 . 1 Deciphera Pharmaceuticals, Lawrence, USA Background: Altiratinib is a Type II switch pocket inhibitor of MET kinase as well as TIE2 and VEGFR2 kinases. This profile provides an agent that exhibits anti-tumor activity in cancers driven by MET overexpression or genomic mutation, and also blocks angiogenic and metastatic processes mediated by the tumor microenvironment. Material and Methods: Altiratinib was evaluated in MET, TIE2, and VEGFR2 biochemical studies, including evaluation in a number of MET activation mutants. Cellular activity was evaluated in tumor cell lines exhibiting MET amplification (MKN-45 gastric, EBC-1 NSCLC) or overexpression (B16/F10 melanoma; U87 glioblastoma). In vivo pharmacokinetic/pharmacodynamic studies were performed in an MKN45 xenograft model. Efficacy was demonstrated in a battery of xenograft/ allograft models including: gastric (MKN-45), melanoma (B16/F10, A375), ovarian (SKOV-3), colorectal (COLO-205), lung (EBC-1), breast (PyMT), and glioblastoma (U87). Results: Altiratinib afforded balanced inhibition of MET, TIE2, and VEGFR2 kinases in the low nM range (IC50 s 2−9 nM) and blocked HGF-, ANG-, or VEGFA-induced HUVEC activation and capillary tube formation. Altiratinib retained potency versus activation loop mutant forms of MET (D1228X, Y1230X, M1250X), inhibiting all forms with IC50 < 6 nM. Altiratinib inhibited MET activation, as well as proliferation, motility, and survival in a number of cell lines including MKN-45, EBC-1, A439, B16/F10, and U87 (IC50 s < 10 nM). In vivo, altiratinib suppressed MET phosphorylation for >24 hr after a single 10 mg/kg oral dose in an MKN-45 xenograft pharmacodynamic model. Altiratinib exhibited anti-tumor activity in melanoma (B16/F10, A375), gastric (MKN-45), lung (EBC-1), colorectal (COLO-205), breast (PyMT), ovarian (SKOV-3) and GBM (U87) xenograft or allograft models. In these in vivo studies, altiratinib was shown to inhibit tumor growth, angiogenesis, invasion and/or metastasis. Altiratinib also blocked recruitment of TIE2-expressing monocytes in the PyMT breast cancer model. In some models, altiratinib was shown to increase overall survival. In particular, in the i.c.v. orthotopically implanted U87 glioblastoma model, altiratinib extended survival by 1.7-fold vs vehicle (112 days vs 66 days), while the combination of altiratinib + bevacizumab extended survival by 2.5 fold vs vehicle and by 1.9-fold vs bevacizumab single agent (166 days vs. 88 days). Conclusions: Altiratinib is a balanced inhibitor of MET, TIE2, and VEGFR2 kinases. This profile provides robust inhibition of tumors driven by MET amplification or overexpression, and also provides the potential for altiratinib to block tumor microenvironment angiogenic resistance mechanisms and pro-tumoral effects of TIE2-expressing macrophages in the clinical setting. Altiratinib is currently in Phase 1 clinical trials in patients with solid tumors. 356 POSTER (Board P136) Discovery of novel pyrido[2,3-b]pyrazine as fibroblast growth factor receptor (FGFR-1, 2, 3 & 4) kinase inhibitors with nanomolar affinity P. Angibaud1 , O. Querolle1 , V. Berdini2 , G. Saxty2 , A. Cleasby2 , H. Colombel1 , I. Csoka1 , R. Gilissen3 , P. King3 , L. Meerpoel4 , C. Paulussen3 , I. Pilatte1 , V. Poncelet1 , D.C. Rees2 , B. Roux1 , V. Tronel1 , T. Verhulst3 , B. Wroblowski3 , C.W. Murray2 , J. Vialard3 . 1 Janssen R&D, Medicinal Chemistry, Val de Reuil, France; 2 Astex Pharmaceuticals, Discovery, Cambridge, United Kingdom; 3 Janssen R&D, Oncology, Beerse, Belgium; 4 Janssen R&D, Medicinal Chemistry, Beerse, Belgium The receptor tyrosine kinase subfamily of Fibroblast Growth Factor Receptors (FGFR-1, 2, 3 and 4) is involved in a variety of key cellular processes, including proliferation, migration, survival, and differentiation. There is now substantial evidence that aberrant regulation of FGFR signalling is associated with development of several tumor types. This has triggered efforts from academia and the pharmaceutical industry culminating in identification and early development of selective FGFR inhibitors, with diverse kinase inhibition and pharmacological profiles that are currently being evaluated in clinical studies. We recently reported that a quinoxaline moiety can efficiently bind the hinge region of FGFR catalytic sites. In a continuation of our efforts to identify FGFR inhibitors we have discovered that pyrido[2,3-b]pyrazine can replace the quinoxaline moiety. Further optimization of the substitution pattern led us to molecules with favourable drug-like properties and anti-tumoral activity in a FGFR2dependent SNU-16 human gastric carcinoma xenograft model. Here we disclose the structure–activity relationships as well as the chemical

Thursday 20 November 2014 115 synthesis pathway of these novel pyrido[2,3-b]pyrazine inhibitors with nanomolar affinity on FGFR-1, 2, 3 and 4. 357 POSTER (Board P137) Multi-color flow cytometry immunophenotyping for detection of CSC in NSCLC ˜ 2, A. Martinez-Romero1 , J.M. Pardo1 , S. Tejedor1 , S. Calabuig Farinas R. Lucas3 , S. Figueroa4 , E. Jantus-Lewintre2 , C. Camps5 , R. Farras1 . 1 ´ Centro de Investigacion Principe Felipe, Valencia, Spain; 2 Fundacion ´ Hospital General Universitario de Valencia, Valencia, de Investigacion Spain; 3 Universidad de Valencia, Valencia, Spain; 4 Consorcio Hospital ´ General Universitario de Valencia, Servicio Cirug´ıa Toracica, Valencia, ´ de Investigacion ´ Hospital General Universitario de Spain; 5 Fundacion Valencia and Consorcio Hospital General Universitario de Valencia, ´ Servicio Oncolog´ıa Medica, Valencia, Spain Background: Non-squamous cell lung cancer (NSCLC) accounts for almost 80% of all lung tumors and as a whole, the overall survival at 5 years is one of the lowest, being less than 15%. The relatively modest success obtained in the treatment of NSCLC in the past 30 years make urgent the design of new therapeutic strategies. The discovery of a small population of cells with stem cell properties (cancer stem cell, CSC) has led to the beginning of a new emerging area in cancer research. It has been determined that the CSC, are the first components of the tumor which lead to tumor progression and metastasis. In addition to its ability to self-renew and differentiate, these cells are resistant to conventional chemotherapy. Therefore the CSCs hold great promise as a new target with therapeutic potential. In the laboratory, using polychromatic immunophenotyping by flow cytometry with specific CSCs surface markers, we have observed the existence of different cell subpopulations in both cell lines derived from NSCLC and in tumors from patients with NSCLC in resectable stage. Besides, tumor spheres have been obtained from a cell population expressing CD326+/CD90-, confirming the self-renewal capacity of this cell population. Aims and Methods: The purpose of our study is to isolate and characterize at the molecular level the signaling pathways by which NSCLC CSCs are regulated. We have performed immunophenotyping analysis by flow cytometry of 18 NSCLC tumor-derived cells and a panel of NCSLC cell lines to study the expression of surface markers related to stem cell in lung cancer and the distribution of cell subpopulations generated by a combination of different markers. Tumor formation assay, flow cytometry and western blot analysis were used to characterize the different cell subpopulations. Results: The immunophenotyping polychromatic analysis shows the high heterogeneity of the NSCLC tumor samples for specific surface markers. After excluding hematopoietic cells we found expression of CD326, CD90, CD166, E-cadherin CD44 and CD34 surface markers in all samples with high heterogeneity. CD31, CD117, CD133, ABCG2, CD271 and N-cadherin were negative. The cluster analysis data suggest that these cells are classified as CD326+/166+/CD90−, CD326+/166−/CD90− and CD326−/166−/CD90+ subsets. In vitro culture experiments showed that only CD326+/CD90− cells are capable of forming tumor spheres. Conclusions: Our data suggest that CD326 expression is essential for maintenance of NSCLC CSCs. Blocking CD326 signaling pathway appears to be a rationale therapeutic strategy for eliminating NSCLC stem cells. 358 POSTER (Board P138) Knockdown of beta-catenin with dicer-substrate siRNAs down-regulates Wnt/beta-catenin pathway signaling N. Pursell1 , W. Zhou1 , R. Diwanji1 , B. Holmes1 , N. Avitahl-Curtis1 , C. Dutta1 , H. Dudek1 , S. Ganesh1 , M. Abrams1 , W. Wang1 , B. Ying1 , D. Chen1 , S. Shui1 , U. Saxena1 , H. Yang1 , A. Shah1 , R. Arvan1 , M. Koser1 , C. Lai1 , B.D. Brown1 . 1 Dicerna Pharmaceuticals Inc., Watertown MA, USA Our knowledge of cancer-driving molecular pathways has expanded tremendously in recent years, yet this knowledge has been challenging to convert into new therapies due in part to the abundance of so-called undruggable targets. RNA interference (RNAi) offers a promising alternative to traditional cancer therapeutics through its ability to silence expression of traditionally undruggable targets at the messenger RNA (mRNA) level. We have previously identified and characterized high-potency Dicer-substrate short interfering RNAs (DsiRNAs) targeting b-catenin (CTNNB1), which yielded significant reduction of b-catenin expression in liver cancer models when delivered using lipid nanoparticles (LNP). Reduction of b-catenin reduced tumor burden in mouse models as well as reducing expression of b-catenin-regulated genes such as MYC. We have further optimized tumor-centric LNPs used for b-catenin DsiRNA delivery to identify welltolerated delivery methods, resulting in significant knockdown of b-catenin

116 Thursday 20 November 2014 expression specifically in tumor tissue of hepatocellular carcinoma animal models. Analysis of downstream protein markers revealed down-regulation of the cancer-driving Wnt/b-catenin signaling pathway following b-catenin DsiRNA treatment. A decrease in cell proliferation markers and an increase in apoptotic markers was also observed following treatment. b-catenin DsiRNAs were also further evaluated in additional liver and colorectal cancer cell lines both in vitro and in animal models. The results presented in this study demonstrate efficient down-regulation of the Wnt/b-catenin signaling pathway following treatment with DsiRNAs targeting b-catenin. These results support continued development of the b-catenin DsiRNAas a cancer therapeutic and reinforce the need for continued development of DsiRNAs in general as therapeutics for traditionally undruggable cancer targets. 359 POSTER (Board P139) Correlation of tumour-associated macrophage, but not tumourinfiltrating lymphocyte, levels with progression-free survival in patients with metastatic renal cell carcinoma treated with axitinib J.A. Williams1 , J.F. Martini1 , B. Escudier2 , B.I. Rini3 , R.J. Motzer4 , J. Tarazi5 , S. Li6 , P.A. English7 . 1 Pfizer Oncology, Translational Oncology, San Diego, USA; 2 Institut Gustave Roussy, Medical Oncology Department, Villejuif, France; 3 Cleveland Clinic Taussig Cancer Institute, Department of Solid Tumor Oncology, Cleveland, USA; 4 Memorial Sloan-Kettering Cancer Center, Department of Medicine, New York, USA; 5 Pfizer Oncology, Clinical Development, San Diego, USA; 6 Pfizer Oncology, Clinical Development Statistics, Shanghai, China; 7 Pfizer Oncology, Clinical Development Statistics, San Diego, USA Background: Intensive investigations are ongoing to identify and validate predictive molecular markers for anti-angiogenic agents in metastatic renal cell carcinoma (mRCC) and other solid tumour types. However, this proves to be challenging due to multiple resistance mechanisms. Maturing understanding of immuno-oncology functions, including tumour-infiltrating lymphocytes (TIL) and tumour-associated macrophages (TAM), and their influence on mRCC biology provides the opportunity for new insights into sensitivity to anti-angiogenic agents. Materials and Methods: Archival tumour samples were collected on an optional basis and prior to axitinib dosing from 52 patients with mRCC treated with axitinib following 1 prior systemic first-line regimen in the phase III AXIS trial (NCT00678392). Samples were assessed for macrophage (CD68+ ) and lymphocyte (CD3+ ) infiltration using immunohistochemistry. Potential associations between these tumourassociated immune cells and clinical efficacy following axitinib treatment were investigated by Kaplan–Meier analysis using median CD68+ (0.08 cells/mm2 ) and CD3+ (399.5 cells/mm2 ) levels as thresholds and by receiver operating characteristics (ROC) analysis. Results: Higher CD68+ levels were associated with longer median progression-free survival (PFS) of 12.0 months for median CD68+ cells/mm2 vs 3.7 months for
Poster Session – Molecular Targeted Agents I lines and cancer tissues. We have investigated the expressions of these p53 dependent miRs on surgically resected non-small cell lung cancer (NSCLC) tissues and analyzed the correlation with these miRs and clinicopathological characteristics and recurrence-free survival (RFS). Also, we investigated the correlation between these miRs and their possible target genes. Methods: We used reverse transcription-polymerase chain reaction to analyze the relative expression of miR-192, -194, and -215 and possible their target mRNA such as CDK4, CCND1, CDK6 and c-MET in paired tumor and adjacent normal tissue from 42 surgically resected NSCLC patients who received no presurgical treatment. Results: There was no statistical difference between recurrence and non-recurrence group in baseline clinical characteristics. The relative expression of miR192 was correlated with the relative expression of p53 positively (R2 = 0.145; p = 0.005). There was no significant difference in miR192 between tumor tissues and normal tissues (p = 0.624). The relative expression of miR192 was down-regulated in advanced stage (p = 0.025). High miR192 expression group (27.2 months; 95% CI 20.5– 34.4) showed better recurrence free survival than low miR192 expression group (56.3 months, 95% CI 46.0–66.7) (hazard ratio 4.1, 1.0–23.2; p = 0.045). The expression of miR192 were negatively correlated with that of c-MET with statistical significance (R2 = 0.172, p = 0.035). Median RFS was 54.7 months (95% CI 42.6–66.7) in the high miR192 and low c-METexpression group and 20.0 months (14.3–25.5) in low miR192 and high c-MET expression group (4.9, 0.9–25.7; p = 0.059). Conclusion: miR194 could be a reliable prognostic marker on recurrence in surgically resected NSCLC through regulation of c-MET. These results can pave the way to the clinical use of miRNAs as prognostic markers and further therapeutic targets. 361 POSTER (Board P141) Nanoformulations of the PARP inhibitors olaparib and BMN 673 for cancer nanotherapy S. Sridhar1 , S. Tangutoori1 , P. Baldwin1 . 1 Northeastern University, Nanomedicine Center, Boston MA, USA Introduction: PARP (Poly ADP Ribose Polymerase) plays a crucial role in the DNA repair pathways in a cell, rendering it the ‘Achilles Heel’ specifically in cancer cells. Olaparib and BMN 673 are PARP inhibitors that are being tested in several clinical trials. These poorly soluble drugs are currently orally administered, but have poor bioavailability and tumor accumulation. Here we successfully developed novel injectable nanoformulations NanoOlaparib and NanoBMN673, thus enabling a platform which can provide a safe vehicle for intravenous delivery specifically targeted to the tumor, thereby increasing the bioavailability. Methods: NanoOlaparib and NanoBMN673 have been successfully formulated and tested in vitro and in vivo. The NanoOlaparib formulation is comprised of nanoparticles of mean diameter 120 nm, zeta potential ~+15 mV, polydispersity index ~0.05 and loaded with olaparib at clinically relevant concentrations of 400mM to 2.3mM. The efficacy of NanoOlaparib was determined on ovarian cancer cell lines including PA-1, KURAMOCHI etc, and in prostate cancer cell lines PC3, LNCAP and FK01. The functionality of nanoPARPi/combination formulations on all the cancers, reflected by inhibition of PARylation, g-H2AX and RAD-51 was determined by immunoflourescence assays. We have studied their efficacy and toxicity in endometrial OvCa murine model with KRaS-PTEN deletion, prostate Ptenpc−/− ;Trp53pc−/− and breast cancer BrcaCo/Co;MMTV-Cre;p53+/− genetically modified models. Results: NanoOlaparib showed (i) increased early nuclear localization, and (ii) increased radiosensitisation, in PTEN deficient prostate cancer cell lines LNCaP, PC3 and FKO1. Intraperitoneal administration of NanoOlaparib showed ~10 times more tumor accumulation than free Olaparib, and decreased the tumor volumes ~1.5 fold, with minimal toxicity in reticuloendothelial system organs. Conclusions: Robust nanoparticle formulations NanoOlaparib and NanoBMN673 have been successfully demonstrated. We observed a significant enhancement in the efficacy both in vitro and in vivo with the nanoformulations in ovarian, prostate and breast cancers. Strong radiosensitization was observed after several days. These results imply an important role for the nanoOlaparib and nanoBMN formulations as chemo and radio-sensitizers enabling mono- and combination therapeutic approaches for several cancers.

Poster Session – Molecular Targeted Agents I 362 POSTER (Board P142) Phenotypic plasticity in epithelial progenitors and mesenchymal carcinoma is regulated by Axl signaling A. Engelsen1 , K. Wnup-Lipinska1 , C. Tiron1 , F. Pelissier1 , T. Jokela1 , G. Haaland1 , G. Gausdal2 , T. Sandal2 , R. Frink3 , X. Liang2 , S. Hinz3 , L. Ahmed2 , M. Hellesøy2 , D. Mickelm2 , J. Minna3 , M. LaBarge4 , R. Brekken3 , J. Lorens1 . 1 University of Bergen, Biomedicine, Bergen, Norway; 2 BerGenBio, Biomedicine, Bergen, Norway; 3 UT Southwestern, Harmon Cancer Center, Dallas, USA; 4 LBNL, Life Sciences, Berkeley, USA Epithelial plasticity engendered by the epithelial-to-mesenchymal transition (EMT) gene program is important for normal adult tissue homeostasis and is a characteristic of aggressive carcinomas. Phenotypic plasticity facilitates tumor heterogeneity, drug resistance, and metastatic spread; and is utilized by normal and neoplastic epithelial cells to maintain stem cell functions. The Axl receptor tyrosine kinase is prominent in advanced malignancy and often correlated with poor clinical outcome. We demonstrate that Axl signaling is required to maintain EMT-associated features including invasiveness, stem cell-like traits and drug resistance in mammary and lung carcinomas. Remarkably, we show that Axl expression demarcates a distinct population of multipotent progenitor cells with within normal mammary epithelial hierarchies with repopulating capacity. This provides a unique rationale for the role of Axl signaling in the regulation of stemness traits in cancer. Axl inhibitors, including a clinical stage selective Axl kinase inhibitor (BGB324), were evaluated in preclinical models of carcinomas with mesenchymal traits including triple negative breast (TNBC), pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer (NSCLC). Axl inhibition in PDAC and TNBC blocked invasiveness and metastasis; abrogated the tumor initiation capacity of TNBC cells, an activity associated with cancer stem cells; and blocked the emergence of EMT-associated acquired resistance to erlotinib in human NSCLC xenografts. Furthermore, combination treatment of BGB324 with chemotherapy inhibited the growth of human NSCLC xenografts and significantly prolonged survival in orthotopic and genetically engineered mouse models of PDAC. Our results suggest that Axl signaling serves as a novel regulator of epithelial plasticity necessary to maintain EMT-related stemness traits and that Axl inhibition can enhance the efficacy of multiple anti-cancer strategies. Together with the recent results from early clinical safety studies demonstrating BGB324 as being safe and well tolerated, this provides a rationale for further clinical studies. 363 POSTER (Board P143) Cytosolic p21 is a pharmacodynamic marker of CHEK1 and IKKe inhibition in ovarian cancer cells M. Kim1 , D.J. Min1 , G. Wright1 , C. Annunziata1 . 1 NCI, CCR, Bethesda MD, USA Background: The IKK (IkB kinase)-related kinase, IKKe, was previously identified as an oncogene in breast cancer and was associated with poor clinical outcome in ovarian cancer (OC). Recently, we demonstrated that IKKe expression was significantly higher in metastatic ovarian carcinomas compared to primary tumors and promoted tumor invasion and metastasis, while its loss moderately decreased in cellular proliferation. Methods and Results: We screened human kinome shRNA library using IKKe-matched cell line pairs and identified 65 genes that further inhibited the survival of OC cells in combination with IKKe depletion. Of these, CHEK1 loss significantly decreased survival of IKKe-depleted cells in both p53 wild-type and null OC cell lines, A2780 and Ovcar5, respectively. The dramatic overexpression of CHEK1 in The Cancer Genome Atlas containing more than 500 ovarian serous cystadenocarcinoma implies its clinical significance. Consistent with the shRNA screening results, chemical inhibitors BX795 (IKKe) and PF00477736 (CHEK1) recapitulated the effects of shRNAs in 6 OC cell lines. Combined inhibition resulted in a significant increase in p21 protein level and a further increase in apoptosis. The loss of p21 by siRNA rendered cells resistant to the combined inhibition of IKKe and CHEK1, supporting its pro-apoptotic role in OC cells. We further demonstrated that IKKe physically interacted with p21 in cytosolic compartment and lowered the p21 protein level in its kinase dependent manner. The co-inhibition of IKKe and CHEK1 activities, regardless of p53 status, was more effective in killing ovarian cancer cells than single treatment. Interestingly, the inhibition of IKKe activity alone induced dramatic G2/M arrest in all 6 cell lines without DNA damage response, while CHEK1 inhibition resulted in significant DNA damage response with abnormal S phase profile in cell cycle analysis and an increase in g-H2A.X level. Conclusion: We identified CHEK1 as an IKKe dependent lethal gene in ovarian cancer cells. CHEK1 inhibitor as a single agent has not been successful in clinic and this may be due to an increase in a prosurvival pathway such as an IKKe-mediated signaling, which may contribute

Thursday 20 November 2014 117 to resistance and later to relapse. Identification of IKKe dependent signaling will lead to development of combinational therapeutics in the poor prognostic group of patients with a high level of IKKe expression. Importantly, cytosolic p21 increase may be a pharmacodynamic marker for the combined inhibition of IKKe and CHEK1. 364 POSTER (Board P144) First-in-human phase 1 study of MLN2480, an investigational oral pan-RAF kinase inhibitor, in patients (pts) with relapsed or refractory solid tumors, including BRAF/NRAS-mutant melanoma M. Middleton1 , D.W. Rasco2 , A.J. Olszanski3 , P. Corrie4 , P. Lorigan5 , R. Plummer6 , J. Larkin7 , A. Pavlick8 , X. Zhou9 , Z. Yuan10 , E. Gangolli11 , M. Kneissl12 , V. Bozon ´ 13 , R. Gonzalez14 . 1 Oxford University Hospitals NHS Trust, Department of Oncology, Oxford, United Kingdom; 2 South Texas Accelerated Research Therapeutics (START), Department of Oncology, San Antonio, USA; 3 Fox Chase Cancer Center, Phase 1 Early Clinical Development Program Medical Oncology, Philadelphia, USA; 4 Cambridge University Hospitals NHS Foundation Trust, Medical Oncology/Cancer Division, Cambridge, United Kingdom; 5 The Christie NHS Foundation Trust, Medical Oncology, Manchester, United Kingdom; 6 Newcastle Hospitals NHS Foundation Trust, Experimental Cancer Medicine/Medical School, Newcastle upon Tyne, United Kingdom; 7 The Royal Marsden Hospital NHS Foundation Trust, Department of Medicine, London, United Kingdom; 8 New York University Langone Medical Center, Department of Medicine Division of Hematology and Medical Oncology, New York, USA; 9 Takeda Pharmaceuticals International Co., Clinical Pharmacology, Cambridge, USA; 10 Takeda Pharmaceuticals International Co., Global Statistics, Cambridge, USA; 11 Takeda Pharmaceuticals International Co., Translational Medicine, Cambridge, USA; 12 Takeda Pharmaceuticals International Co., Clinical Research, Cambridge, USA; 13 Takeda Pharmaceuticals International Co., Oncology Clinical Research, Cambridge, USA; 14 University of Colorado Denver School of Medicine, Division of Medical Oncology, Denver, USA Background: Activating mutations in the MAPK pathway are common in many solid tumors. RAF kinases play a key role in this pathway and represent a valid target for therapy. This study (NCT01425008) evaluated the safety, MTD, preliminary antitumor activity and pharmacokinetics (PK) of MLN2480. Methods: Pts aged 18 yrs with advanced solid tumors (dose-escalation [DE] and PK cohorts) and inoperable stage 3/4 melanoma (expansion [ME] cohorts) received oral MLN2480 every other day (Q2D) in 22- or 28-d cycles. Dose escalation was based on cycle 1 DLTs. PK and ME cohorts received the MTD; ME cohorts were defined by treatment history (na¨ıve vs treated) and mutation status (NRAS vs BRAF). Results: At data cut-off, 75 pts had enrolled, 30 to DE cohorts, 25 to ME cohorts and 20 to the PK cohort. DE pts received MLN2480 20–280 mg Q2D in 22-d cycles or 200 mg Q2D in 28-d cycles. 2 pts (280 mg Q2D, 22-d cycles) had cycle 1 DLTs of grade 3 periorbital edema and maculopapular rash. Drug-related AEs of interest included arthralgia in 23% of pts and nevi in 10% (only at low doses 20 mg and 40 mg). PK data from the DE cohorts indicated dose-proportional (d21) exposure with MLN2480 20– 280 mg Q2D. MLN2480 MTD was 200 mg Q2D. In 45 pts of the ME/PK cohorts, median cycles received were 2 (1−12) and 7 pts received 6 28-d cycles. Common drug-related AEs in ME/PK pts included maculopapular rash (40%), fatigue (31%) and anemia (22%); grade 3 (29% of pts) included maculopapular rash (7%) and anemia (4%). At 200 mg, frequency of drug-related arthralgia and nevi decreased to 7% and 0%, respectively; CPK elevations were seen in 11%. No squamous cell carcinomas were reported. In ME/PK pts, 11% discontinued due to AEs; 2 pts died on study (1 drug-related: respiratory failure at 280 mg). In na¨ıve BRAF-mut RECISTevaluable ME pts (n = 10), there were 4 PRs, 1 SD and 5 PDs. In na¨ıve NRAS-mut ME pts (n = 7), there were 1 PR, 3 SDs and 3 PDs. Response duration ranged from 1.5−5.4+ mos. A pt with NRAS-mut thyroid cancer has been on study for 24 cycles with SD. 6 pts overall had SD for 4.7–18.9+ mos. Conclusions: The safety profile of MLN2480 at up to 200 mg Q2D (MTD) was acceptable. Safety observations at low and high doses support the hypothesis that MLN2480 may overcome the paradoxical activation of the MAPK pathway in the clinic and may block RAF heterodimerization, unlike BRAF-specific inhibitors. Preliminary response data suggest evidence of antitumor activity in BRAF- and NRAS-mut melanoma.

118 Thursday 20 November 2014 365 POSTER (Board P145) Pim-1 kinase: Validated as a therapeutic cancer target for MYC-driven tumours O. Renner1 , Y. Cecilia1 , M.C. Rodriguez de Miguel1 , S. Peregrina1 , B. Garcia-Serelde1 , M.I. Albarran1 , A. Cebria1 , D. Cebrian1 , F. Ramos-Lima1 , A. Carnero1 , J. Pastor1 , C. Blanco-Aparicio1 . 1 Spanish National Cancer Research Centre (CNIO), Experimental Therapeutics Program, Madrid, Spain Provirus integration site for Moloney murine leukemia virus 1 (Pim-1) kinase belongs to a family of constitutively active serine/threonine kinases. The overexpression of Pim-1 can contribute to tumorigenesis, thereby establishing Pim-1 as a proto-oncogene. In B-cell lymphoma, colorectal cancer, pancreatic cancer and other tumours, the over expression of Pim-1 is linked to poor prognosis. Proviral integration experiments suggested the cooperation between Pim-1 and the protooncogene c-myc that was subsequently confirmed by double transgenic mice. Pim-1 acts synergistically with the transcription factor c-myc by phosphorylation and by interaction with c-myc on the chromatin level, leading to elevated trancription of c-myc target genes and cell proliferation. Pim-1 also promotes cell cycle progression. Pim-1-mediated phosphorylation of p27 leads to its binding to 14−3−3 protein, its nuclear export and proteasome-dependent degradation. The aim of our study was to analyze the effect of kinase-inhibition of Pim-1 on tumour growth in a mouse model of haematological malignancy. To this end, we constructed a conditional knock-in mouse model in which a single amino acid exchange can be induced by expression of the Cre-recombinase, leading to expression of an inactive form of Pim-1 kinase. These Pim-1 kinase-dead (KD) mice do not display phenotypic abnormalities or reduced live span even after homozygous exchange by CMV-Cre expression of the wild type form of Pim-1 against the mutated form. As expected, elevated levels of the cell cycle inhibitor p27 can be observed in the organs of Pim-1 kinase-dead mice by immunohistochemistry (IHC). In order to study the effect of the Pim-1 kinase inhibition on tumour development driven by c-myc, we crossed the Pim-1 KD mice with the Em-cmyc transgenic mouse line, which expresses high levels of the oncoprotein c-myc leading to the development of a rapidly growing B-cell lymphoma that resembles the human Burkitt-lymphoma. Even the heterozygous switch to the kinase-dead form of Pim-1 doubles the survival of the Em-c-myc mice; the homozygous switch brings survival back close to normal. On the molecular level, we observed elevated p27-levels in tumours of hetero- and homozygous Pim-1 kinase-dead mice carrying the Em-c-myc transgene. The tumours of these mice also displayed areas with reduced levels of c-myc (Western blot and IHC). Based upon these results, we set-up an allogenic Em-c-myc mouse model that allowed us to study the effect of kinase inhibitors in an efficient and standardized way. We can show that novel, selective and potent Pim-1 kinase inhibitors that have been developed in our department are able to efficiently inhibit c-myc-driven tumour growth, as proposed by the genetic ablation of Pim-1 kinase activity. In summary, the presented study proofs inhibition of Pim-1 kinase activity to be a promising strategy to inhibit cancer growth driven by c-myc, one of the most frequently deregulated oncogenes in human cancer. 366 POSTER (Board P146) Targeting colorectal and pancreatic cancer stem cells with the LGR5 monoclonal antibody BNC101 P. Chu1 , F. Shojaei1 , K. Smith1 , J. Norton1 , C. Walsh1 , J. Iglesias1 , C. Reyes1 . 1 Bionomics Inc, San Diego CA, USA BNC101 is a monoclonal antibody (mAb) targeting LGR5 currently undergoing IND-enabling studies in preparation for Phase I clinical studies in 2014. LGR5 is a validated cancer stem cell (CSC) receptor overexpressed in colorectal cancer (CRC), pancreatic cancer and most other solid tumors. Loss and gain-of-function studies indicate that LGR5 is a functional cancer receptor involved in the growth and survival of colon cancer cells in vitro and in vivo. Sorted LGR5+ primary CRC are highly tumorigenic compared to LGR5− cells in limiting dilution in vivo xenograft studies. Using Bionomics’ CSC Rx Discovery™ platform, a panel of high affinity LGR5 mAbs were generated and screened for in vitro and in vivo activity against CSCs from patient-derived CRC tumors. LGR5 mAb therapeutic drug candidates were first screened and identified based on their ability to inhibit the formation of CRC CSC colonies in vitro. Top LGR5 mAb candidates were then tested as single agents and in combination with standard of care (SOC) chemotherapy in primary xenograft efficacy studies. Tumors from these studies were also harvested and re-implanted in limiting dilution assays (LDA) to assess in vivo anti-CSC activity of antiLGR5 mAbs. A number of functional LGR5 mAbs with significant antitumor and anti-CSC activity were identified through this process. A lead

Poster Session – Molecular Targeted Agents I BNC101 mAb clone was selected for clinical development based on its ability to inhibit CSC growth and key signaling pathways from multiple patient tumors both in vitro and in vivo. In LDA studies using two different CRC patient tumors, 5/8 (63%) and 6/8 (75%) mice implanted with serially diluted cells from BNC101 treated tumors remained tumor free, compared to 1/8 (13%) and 2/8 (25%) in the control groups. Combination with SOC was shown to further improve BNC101 activity in one CRC model, where 8/8 mice re-implanted for LDA remained tumor-free in a 6 month followup. BNC101 was also found to be highly active as a single agent and in combination with SOC in multiple pancreatic models and the MDA-MB231 triple-negative breast cancer (TNBC) xenograft model. In one primary pancreatic model, BNC101 combined with SOC completely eradicated 3/7 established tumors, compared to 0/7 tumors eradicated with control SOC alone. Additional in vivo translational data in CRC, pancreatic and TNBC supporting our hypothesis that BNC101 targeting of CSCs will significantly improve survival and increase duration of response in cancer patients will be presented. 367 POSTER (Board P147) Induction of apoptosis and inhibition of angiogenesis by novel fusion protein − AD-O54.9 as a new preclinical strategy in cancer treatment P. Rozga1 , B. Zerek1 , A. Pieczykolan1 , M. Galazka1 , K. Bukato1 , S. Pawlak1 , M. Szymanik1 , A. Jaworski1 , M. Teska-Kaminska1 , K. Poleszak1 , A. Grochot-Przeczek2 , W. Strozek1 , J. Pieczykolan1 . 1 Adamed, Drug Discovery, Warsaw, Poland; 2 Jagiellonian University, Department of Medical Biotechnology, Krakow, Poland Background: For almost two decades tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) has been under extensive development as a potential therapeutic agent. TRAIL/Apo2L is a member of the TNF superfamily with unique ability to induce apoptosis in cancer cells while remaining neutral to normal cells. Tumor growth is tightly related to new blood vessel formation and tissue remodeling. Platelet derived growth factor (PDGF) is important in vascular physiological and pathological development. Inhibition of PDGF pathway blocks angiogenesis what results in reduction of tumor growth. We proposed a novel fusion protein (AD-O54.9) with dualistic proapoptotic and antiangiogenic activity. Our molecule consists of the soluble recombinant TRAIL/Apo2L variant linked with an effector peptide sequence and an activation motif recognized by tumor-specific proteases (MMPs, uPa) between. The effector peptide is composed of 19-amino acid fragment derived from human PDGF, which binds the PDGF receptors competitively to the natural ligand while being itself devoid of activity. As a consequence, angiogenic activity of PDGF is blocked, stimulation of new blood vessels formation does not occur and finally tumor growth is inhibited. Materials and Methods: AD-O54.9 protein was expressed in E. coli, using pET expression system, and purified by IEX chromatography. The biophysical properties were verified by circular dichroism (CD) and interactions with receptors were analyzed using surface plasmon resonance (SPR). Cytotoxic activity of AD-O54.9 was examined using a propidium iodide assay and its antiangiogenic activity was evaluated by HUVEC tube formation and ring aortic assays. Safety was tested on human primary hepatocytes. The proapoptotic and antiproliferative activity was tested using molecular biology and flow cytometry methods. In vivo potential was examined on mice xenograft models of human cancers. Results: The molecule showed in vitro specific cytotoxic effect on various primary cancer cell lines at IC50 below 0.01 ng/ml with no activity against human hepatocytes. This protein efficiently interacted with TRAIL and PDGF receptors. We demonstrated that AD-O54.9 is a very potent apoptosis inducer and inhibitor of angiogenesis. This fusion protein showed superior efficacy displaying significant tumor volume regression compared with TRAIL/Apo2L and standard chemotherapeutic agents. Conclusions: The obtained results confirm that we developed a very promising fusion molecule with a high potential of anticancer activity that could be considered as a novel therapeutic agent. 368 POSTER (Board P148) NP137, the first humanized monoclonal antibody directed against netrin-1, exhibits antitumor activity by inducing dependence receptors-mediated cell death J.G. Delcros1 , B. Ducarouge1 , R. Abes2 , D. Goldschneider1 , B. Gibert1 , J.G. Blachier2 , D. Neves2 , P. Mehlen1 , A. Bernet2 , S. Depil2 . 1 Cancer Research Center of Lyon, Dependence receptors cancer and development, Lyon Cedex 08, France; 2 Netris Pharma, Lyon Cedex 08, France Background: Some receptors are active in the absence of ligand and actively trigger cell death through apoptosis. These receptors are called

Poster Session – Molecular Targeted Agents I ‘dependence receptors’ (DRs) as their expression at the cell surface renders cells critically dependent for their survival on ligand availability. Deleted in Colorectal Carcinoma (DCC) and UNC5H are prototypic DRs which induce apoptosis unless their ligand netrin-1 is present. It has been shown that netrin-1 is up-regulated in a large fraction of tumors and that interference with netrin-1/receptors interaction is associated with inhibition of tumor growth and metastasis in various preclinical models. We have generated the first humanized netrin-1 antibody, NP137, and characterized its antitumor activity. Preclinical development and Pharmacological results: Mice with boosted immune system were used for immunization against netrin-1 recombinant protein. A monoclonal IgG1 antibody 4C11, with favorable preclinical characteristics, was obtained. 4C11 specifically targets netrin-1 with high affinity (Kd: 13 nM) and efficiently inhibits binding of netrin-1 to its receptor UNC5H2 (IC50: 0.5 nM). 4C11 binds an epitope present in the second laminin-type EGF-like repeat of netrin-1. In vitro, 4C11 induced apoptosis in netrin-1-dependent cell lines as assessed by caspase 3 activation. In vivo, 4C11 had a significant anti-tumor effect associated with good pharmacokinetic properties. Tumor growth inhibition was observed in several solid and liquid tumor models using 4C11 as a single agent. A strong synergy with doxorubicin was shown in a mouse syngeneic model of osteosarcoma, reversing its chemoresistant phenotype. NP137 was generated by 4C11 CDR grafting with a selection based on potency, immunogenicity and manufacturability. Antitumor activity of 4C11 is maintained after humanization. Several experiments are ongoing to further characterize the mechanism of action of NP137 and identify potential biomarkers predictive of response. In this context, we have shown that combination of chemotherapy with netrin-1 inhibition is associated with a control of the residual disease in breast and ovarian models. Conclusion: We have generated the first humanized antibody targeting netrin-1 and inducing dependence receptors-mediated tumor cell death. Toxicological studies are ongoing with first results in rodents suggesting a high therapeutic margin. A first-in-human trial is planned at the end of 2015, in collaboration with EORTC. 369 POSTER (Board P149) Diagnosis and molecular targeting for individualized treatment of patients with pre-neoplastic lesions and locally advanced cervical cancer P.M.A. Moreno-Acosta1 , A.R.R. Alfredo Romero-Rojas2 , A.H.S. Antonio Huertas Salgado1 , D.M. Diana Mayorga1 , N.M. Nicolas Morales1 , J.A. Jinneth Acosta3 , O.G. Oscar Gamboa4 , N.M. Nicolas Magne5 , M.M. Monica Molano6 . 1 Instituto Nacional de Cancerologia, Research on Cancer Biology, Bogota´ D.C., Colombia; 2 Instituto Nacional de Cancerologia, Group of Pathology, Bogota´ D.C., Colombia; 3 Instituto Nacional de Cancerologia, Pathology National University of Colombia, Bogota´ D.C., Colombia; 4 Instituto Nacional de Cancerologia, Research ´ Branch, Bogota´ D.C., Colombia; 5 Institut de la Loire Cancerologie, Department of Radiotherapy, Saint Priest en Jarez, France; 6 The Royal Women’s Hospital, The Royal Women’s Hospital, Melbourne VIC, Australia Background: The natural history of HPV infection and development of cervical intraepithelial neoplasia indicate that most lesions disappear without treatment in contrast to a significant proportion of high-grade lesions that progress to invasive cancer if not treated. Persistent infection with high-risk HPV may be necessary for the development of cervical cancer. The purpose of the study was make a retrospective molecular diagnosis that include detection of HPV16 variants, polymorphism Arg72Pro of P53, expression of anti-apoptotic markers such as IGF-1R and Survivin, and hypoxic markers and glycolytic as GLUT1 and CAIX and markers of progression as hTERT in samples invasive cancer and preneoplastic lesions taken in 2002 and 1986; the results may be useful to propose an appropriate therapeutic management. Methods: From a prospective study of invasive squamous cell carcinoma of the cervix and predictive markers of response to radiotherapy published in 2012 of which the frequency of HPV 16 variants was reported, we study retrospectively some cases that present the Asian-American variant (AA). One of these cases correspond to a 43-year-old woman, whom in 1986 he took two biopsies of the transformation zone of the cervix, each at an interval of three months; the first biopsy was diagnosed as a high-grade lesion supports changes consistent with HPV infection, and the second biopsy was diagnosed as low-grade lesion. Gynecological medical board decided that the patient should be subjected to extended abdominal hysterectomy, being referred to another hospital. After 16 years of progression, in 2002, the patient returns to INC. In this year the presence of the Asian-American variant of subclass C of HPV 16 in a frozen biopsy tissue transformation zone was determined. Results: HPV16 E6 E-r and AAc mixed variants were detected in samples took in 1986. A HPV16 E-r and AAc variant was detected in 2002, which indicate persistence of the infection. Polymorphism analysis of Arg72Pro

Thursday 20 November 2014 119 p53 in 1986 and 2002 showed an Arg/Pro genotype. An increment of expression of IGF1R, Survivin, GLUT1, and CAIX was observed in biopsies taken in 2002 compared with the analysis done in biopsies of 1986; hTERT expression in pre-neoplastic lesions was detected at 100%. Conclusions: In the adequate therapeutic management of preneoplastic lesions and cervical cancer, the presence of the AAc-HPV16 could be used as a prognostic marker of persistent infection, progression and treatment, as well hTERT expression as tumor progression marker. The analysis of molecular profiles that include biomarkers prognostic, predictive as IGF1R expression and molecular targets, and employed in this work could ensure early diagnosis and better therapeutic management of cervical lesions and cervical cancer. 370 POSTER (Board P150) Discovery of the genes that underpin the transition to malignant phenotype of breast tissues in a highly consanguineous region I. Gupta1 , S. Shanmuganathan1 , A. Ouhtit1 . 1 Sultan Qaboos University, Department of Genetics, Muscat, Oman In Oman, the following observations prompted us to hypothesize that a subset of specific genes/signaling pathways are responsible for the transition of breast tissue from normal/benign to malignant phenotype: 1. the rate of consanguinity is significantly high (52%); 2. a significantly increasing number of younger female patients (25−40 years) present to the clinic with advanced stages of BC; and 3. the transition from normal/benign to malignant phenotype of breast tissue requires the involvement of a subset of specific genes/signaling pathways. Therefore, the long-term objective of this study is to identify and validate the subset of genes that are responsible for this malignant transformation using functional genomic studies, by focusing on this young age group of patients attending BC clinic (sporadic and familial). RNA samples were isolated from 40 Breast Tumors and 40 Normal tissues and analyzed by Microarray Gene Expression Profiling. Among a number of genes that were up and down regulated, BRIP1, AREG, HOXB3 and MAGED1 were identified as potential genes that might underpin the transition to the malignant phenotype; these genes were validated by RTPCR using the same RNA samples that were examined by microarray. Pathway analysis was carried out to identify the major functional pathways connecting these genes. Ongoing sequencing experiments of these genes, using the same DNA samples used in the microarray experiments will ultimately identify any genetic alteration(s) that can affect the normal function of these genes. Functional validation assays aim to validate further the physiological relevance of these genes in tumor malignancy, and perhaps other novel genes specific to BC in the Omani population. Identification and validation of these genes will potentially guide the design of anti-cancer therapeutic strategies. 371 POSTER (Board P151) Selective targeting of head and neck cancer cells by perturbing reactive oxygen species homeostasis M. Kwon1 , J.W. Kim1 , E.H. Kim1 , J.Y. Park1 , S.Y. Kim1 , J.L. Roh1 . 1 Asan Medical Center University of Ulsan College of Medicine, Otolaryngology, Seoul, South Korea Background: Human malignancies, including head and neck cancer (HNC), often show inactivation of tumor suppressor genes and oncogene addiction associated with increased cellular stress. Adaptation to cellular stress is not a vital function of normal cells but is required of cancer cells, and as such might be a sensible target in cancer therapy. Piperlongumine is a naturally occurring small molecule selectively toxic to cancer cells but not normal cells. This study assesses the cytotoxicity of piperlongumine and its combination with cisplatin in HNC cells in vitro and in vivo. Materials and Methods: The effect of piperlongumine, alone and in combination with cisplatin, was assessed in human HNC cells and normal cells by measuring growth, death, cell cycle progression, reactive oxygen species (ROS) production and protein expression. Antitumor efficacy was assessed in preclinical tumor xenograft mouse models. Results: Piperlongumine killed HNC cells regardless of p53 mutational status but spared normal cells. It increased ROS accumulation in HNC cells, an effect that can be blocked by the antioxidant N-acetyl-L-cysteine. Piperlongumine induced effective and selective cell death in HNC cells by targeting the stress response to ROS, leading to the induction of death pathways involving JNK and PARP. Piperlongumine increased cisplatininduced cytotoxicity in HNC cells in a synergistic manner in vitro and in vivo. Conclusion: Piperlongumine might be a promising small molecule with which to selectively kill HNC cells and increase cisplatin antitumor activity by targeting the oxidative stress response.

120 Thursday 20 November 2014 372 POSTER (Board P152) Specific inhibition of hTERT expression by targeting common promoter mutations which cause quadruplex DNA instability D. Miller1 , A. Sokolova1 , S. Thomas1 , F. Rezzoug1 , J. Chaires1 , W. Dean1 , R. Buscaglia1 , J. Trent1 . 1 J.G. Brown Cancer Center U. of Louisville, Medicine, Louisville KY, USA Background: Telomerase reverse transcriptase (hTERT) is a catalytic subunit of the enzyme telomerase. It has recently been shown that the hTERT promoter is commonly mutated (>75%) in a malignant melanoma and glioblastoma. These mutations occur at four sites in a G-rich region which has previously been shown to form quadruplex DNA and to downregulate hTERT expression. We have tested the hypothesis that mutations in the quadruplex-forming region of the hTERT promoter destabilize quadruplex-formation resulting in increased hTERT expression and cellular proliferation. Materials and Methods: Quadruplex formation by the mutated and wild type hTERT promoter oligonucleotides was determined by circular dichroism. Analytical ultracentrifugation was used for sedimentation equilibrium analysis. Thermal denaturation was used to characterize the relative stability of the mutated and wild type oligonucleotides. Four cancer cell lines obtained from ATCC were used to characterize the growth inhibitory effect of mutated and wild type oligonucleotides (A549, Calu1, A375, T24). The MTT assay was used for the cell proliferation experiments. Results: In order to characterize the effects of the hTERT mutations, the biophysical properties of structures formed by wild-type and mutant TERT sequences were explored by several methods. Circular dichroism and thermal denaturation studies showed that all sequences formed quadruplex structures but that those formed by mutant sequences were less stable than the wild-type. Analytical ultracentrifugation showed that all sequences formed one major unimolecular folded form but that mutant sequences had a greater tendency to form misfolded aggregated species than the wildtype. Addition of the quadruplex binder TmPyP4 to the mutant sequences lessened that amount of such aggregates and resulted in sedimentation profiles that closely resembled the wild-type sequences. Treatment of cells with mutated hTERT promoter sequence with the mutated or wild type oligonucleotides resulted in significant growth inhibition that was time and concentration dependent. DNA crosslinking studies indicate that the G-rich oligonucleotide is binding to the C-rich strand by Watson-Crick base pairing. A similar growth inhibitory effect was seen in cells exposed to quadruplex stabilizing agents. Conclusions: The common mutations in the hTERT promoter destabilize quadruplex formation and likely prevent quadruplex-mediated transcriptional silencing. This instability can be overcome by quadruplex-binding drugs and/or oligonucleotides encoding this sequence. The growth of cell lines containing the hTERT promoter mutations is inhibited by oligonucleotides encoding the wild type and mutated sequences. We believe that this occurs by stabilization of the mutated quadruplex, resulting in transcriptional silencing. 373 POSTER (Board P153) AKT2 gene amplification is a marker for sensitivity to allosteric but not ATP-competitive AKT inhibitors T. Abe1 , K. Ichikawa1 , R. Fujita1 , M. Okada1 , K. Tanaka1 , M. Ohkubo1 , K. Yonekura1 , T. Shimomura1 , T. Utsugi1 . 1 Taiho Pharmaceutical Co. Ltd., Tsukuba Research Center, Tsukuba, Japan Background: AKT has a pivotal role as a regulator of the PI3K/AKT/mTOR signaling pathway and this signaling pathway has been found to be activated in various human tumors. Three isoforms of AKT (AKT1, 2, and 3) exist in mammalian cells, and have distinct functions. AKT2 gene amplification has been reported in several types of cancer, including a relatively high proportion of ovarian cancer (5−29%), and might contribute to tumorigenicity. However, it is not clear whether AKT2 gene amplification is correlated with sensitivity to AKT inhibitors. Therefore, we investigated the growth inhibitory effects of TAS-117, a novel potent and selective allosteric AKT kinase inhibitor, and three other AKT inhibitors (MK-2206, GDC-0068, and AZD5363) on cell lines with AKT2 gene amplification. Material and Methods: AKT enzymatic activity was examined using full length enzymes of AKT1, 2 and 3. Inhibition of AKT and its substrates by AKT inhibitors was assayed by immunoblot. Sensitivity to TAS-117 and other AKT inhibitors were tested in a cell proliferation assay. AKT2 gene copy number and mRNA expression information was obtained from cBioPortal for Cancer Genomics. Results: The growth inhibitory effects of TAS-117 and three other AKT inhibitors were evaluated using five human cancer cell lines, two of which have PTEN loss (A2780) or PIK3CA mutation (KPL-4), while the other three cell lines have AKT2 gene amplification (KLE, NCI-H2342, and NIH:OVCAR-3). All four AKT inhibitors potently inhibited growth of A2780

Poster Session – Molecular Targeted Agents I and KPL-4 cells. In the three cell lines with AKT2 amplification, allosteric AKT inhibitors (TAS-117 and MK-2206) showed stronger growth inhibitory activity than ATP-competitive AKT inhibitors (AZD5363 and GDC-0068). In addition, allosteric AKT inhibitors inhibited the phosphorylation of AKT and its downstream substrates at lower concentrations than ATP-competitive inhibitors in AKT2 gene amplified NIH:OVCAR-3 cells. Moreover, TAS-117 induced marked tumor growth inhibition with shrinkage in an NIH:OVCAR-3 xenograft model. Conclusions: These results indicate that cells with AKT2 amplification are more sensitive to allosteric AKT inhibitors than ATP-competitive AKT inhibitors, and AKT2 gene amplification could be a marker for sensitivity to allosteric AKT inhibitors. 374 POSTER (Board P154) Neuregulin 1 (NRG1) expression is a predictive biomarker for response to AV-203, an ERBB3 inhibitory antibody, in human tumor models K. Meetze1 , S. Vincent1 , S. Tyler1 , E. Mazsa1 , A. Delpero1 , S. Bottega1 , D. McIntosh1 , J. Gyuris1 , Z. Weng1 . 1 AVEO Pharmaceuticals, Drug Discovery, Cambridge, USA The v-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (ERBB3) receptor is a unique member of the ERBB family of receptor tyrosine kinases (RTKs) and is often overexpressed in a broad spectrum of human cancers including those of the head and neck, lung, breast, ovary, prostate, colon, pancreas, and gastrointestinal tract. Aberrant activation of ERBB3 signaling is associated with tumor pathogenesis and is often linked to poor prognosis. ERBB3 pathway activation is also implicated in therapeutic resistance to various anti-cancer agents. ERBB3 lacks significant tyrosine kinase activity and its activation requires its association with other RTK partners such as HER2, EGFR and MET. Neuregulin 1 (NRG1) is the predominant ligand for ERBB3 and can promote the heterodimerization of ERBB3 with other ERBB family members, resulting in the tyrosine phosphorylation of ERBB3 and activation of diverse downstream intracellular signaling networks. AV-203 is a humanized IgG1/k ERBB3 inhibitory antibody which has undergone a first-in-human Phase 1 clinical study in patients with advanced solid tumors. In preclinical studies, the tumor growth inhibitory activity of AV-203 was demonstrated in over two dozen human cancer xenograft models representing major human carcinomas, including breast, lung, kidney, colon and pancreatic cancers. To identify biomarkers that can predict response to AV-203, expression levels of ERBB3 and NRG1 were evaluated in these tumor models. A significant correlation was observed between the levels of NRG1 expression and tumor growth inhibition by AV-203. In contrast, there was no significant correlation of tumor growth inhibition with the expression levels of ERBB3. The correlation of response to ERBB3 inhibition by AV-203 and NRG1 expression in tumors was further validated using a collection of tumor explant models derived from patients with lung, ovarian, breast, head and neck or colon cancers. In conclusion, NRG1 is a promising biomarker that can predict sensitivity to ERBB3 inhibition by AV-203 in preclinical human cancer models. NRG1 warrants further evaluation and validation clinically as a potential predictive biomarker for response to AV203. Clinical Laboratory Improvements Amendment (CLIA) validation of an RT-PCR-based assay to detect NRG1 levels in human tumor samples has been completed; the assay can be used for patient selection in future AV203 clinical trials. 375 POSTER (Board P155) MER as a novel therapeutic target in colorectal cancer K. Wong1 , A.C. Tan1 , T. Pitts1 , P. Klauck1 , S. Earp2 , S. Frye3 , X. Wang3 , D.K. Graham4 , S.G. Eckhardt5 . 1 University of Colorado Anschutz Medical Campus, Medical Oncology, Aurora CO, USA; 2 University of North Carolina Lineberger Comprehensive Cancer Center, Medical Oncology, Chapel Hill, USA; 3 University of North Carolina, UNC Eshelman School of Pharmacy, Chapel Hill, USA; 4 University of Colorado Anschutz Medical Campus, Pediatrics, Aurora, USA; 5 University of Colorado Anschutz Medical Campus, Medical Oncology, Aurora, USA Background: Overall survival in metastatic colorectal cancer (CRC) remains limited with current regimens, and there is a need to identify new actionable pathways. MER and AXL are related tyrosine kinases (TK) of the same family implicated in the pathogenesis of several malignancies, and their inhibition has shown promising anti-tumor efficacy. However, their role in CRC has not been well described. We evaluated the significance of MER and AXL in CRC, their relationship to other oncogenic mutations, as well as the anti-proliferative effects of a novel MER-selective tyrosine kinase inhibitor (TKI). Methods: A tissue microarray (TMA) of metastatic tumors from 116 CRC patients was analyzed for MER/AXL expression by immunohistochemistry

Poster Session – Molecular Targeted Agents I (IHC). Relative RNA expression was assessed in 60 CRC and 26 normal colonic cell lines using RNA-Seq data, and correlated with known driver mutations. Gene and protein expression were confirmed by RT-PCR and Western blot, respectively. Clonogenic assays were performed to examine the effects of a novel MER TKI (UNC2025) on in vitro cell growth. Results: The CRC TMA demonstrated high relative MER and AXL protein expression in a subset of metastatic tumors. There was also great variability in the RNA levels of MER and AXL across CRC cell lines, with significant expression (RNA-Seq score >1.0) in 68% and 32% of models, respectively. By contrast, normal colonic cell lines exhibited lower MER (31%) and AXL (8%) expression. RT-PCR and Western blot corroborated with RNA-Seq data in the CRC cell lines analyzed. RAS mutations (KRAS exons 2 and 4, NRAS exons 2 and 3) occurred in 63% of cell lines with high MER TK, compared with 53% of those with low levels (p = 0.57). There were also no significant associations between BRAF (p = 0.17) or PIK3CA (p = 0.74) mutations with MER expression. The potent MER inhibitor UNC2025 dramatically impaired the clonogenicity of CRC cell lines (median IC50 0.08 (0.06 to 2.35) mM). Interestingly, MER expression did not correlate with response to UNC2025. Conclusions: The MER/AXL TKs are overexpressed in a subset of metastatic CRC tumors and cell lines at a higher frequency compared to normal colonic cells. There is no statistically significant correlation between known driver mutations and MER gene expression in CRC cells. The MERselective TKI UNC2025 demonstrates potent anti-tumor effects against CRC cell lines, and studies are ongoing to identify biomarkers of response and mechanisms of resistance. 376 POSTER (Board P156) Tumor-targeting Salmonella typhimurium A1-R enhances gemcitabine–bevacizumab efficacy on a patient-derived orthotopic xenograft (PDOX) pancreatic cancer nude mouse model Y. Hiroshima1 , M. Zhao1 , M.H.G. Katz2 , J.B. Fleming2 , S. Sato3 , T. Murakami3 , M. Yamamoto1 , F. Uehara1 , S. Miwa1 , S. Yano1 , M. Momiyama3 , Y. Zhang1 , A. Maawy4 , T. Chishima3 , K. Tanaka3 , M. Bouvet4 , I. Endo3 , R.M. Hoffman4 . 1 AntiCancer Inc., San Diego California, USA; 2 MD Anderson Cancer Center, Department of Surgery, Houston Texas, USA; 3 Yokohama City University, Department of Gastroenterological Surgery, Yokohama, Japan; 4 University of California San Diego, Department of Surgery, San Diego California, USA Background: Salmonella typhimurium A1-R (A1-R) selectively targets tumors and metastasis. The aim of this study was to examine the efficacy of A1-R treatment on VEGF-positive human pancreatic cancer in a patient-derived orthotopic xenograft (PDOX) model in combination with bevacizumab (BEV) and gemcitabine (GEM). Material and Methods: A VEGF-positive pancreatic cancer PDOX as well as a VEGF-positive human pancreatic cancer cell line (MiaPaCa-2-GFP) were orthotopically implanted in nude mice. Treatment groups included: (1) GEM (80 mg/kg, ip, weekly, 4 weeks); (2) GEM (80 mg/kg, ip, weekly, 4 weeks) + BEV (5 mg/kg, ip, twice a week, 4 weeks); (3) GEM (80 mg/kg, ip, weekly, 2 weeks) + BEV (5 mg/kg, ip, twice a week, 2 weeks) ? A1-R (1.5×108 CFU/body, ip, weekly, 2 weeks); and (4) saline (vehicle/control, ip, weekly, 4 weeks). Results: The mean tumor weight of each group in the MiaPaCa-2-GFP model was (1) GEM, 775.9±273.8 mg; (2) GEM/BEV, 413.5±108.3 mg; (3) GEM/BEV?A1-R, 257.5±57.1 mg; and (4) Control, 2655.4±583.9 mg. GEM/BEV?A1-R significantly reduced tumor weight compared to GEM/BEV treatment in the MiaPaCa-2-GFP model (p = 0.022). The tumor weight of each group in the PDOX model was (1) GEM, 263.1±129.1 mg; (2) GEM/BEV, 65.9±41.9 mg; (3) GEM/BEV?A1-R, 21.9±6.2 mg; and (4) Control, 998.8±377.7 mg. Conclusions: GEM/BEV?A1-R significantly reduced tumor weight compared to GEM/BEV treatment in the PDOX model (p = 0.029). These results demonstrate that in the pancreatic cancer PDOX and cell line models, S. typhimurium A1-R is effective in combination with anti-VEGF-GEM therapy. 377 POSTER (Board P157) Plucked hair as a platform for monitoring pharmacodynamic and mechanistic consequences of clinical exposure to the Wnt/beta-catenin inhibitor PRI-724 G. Miele1 , B. Reed1 , E. Harrison1 , T. Mefo1 , J. Read1 , T. Senba2 , T. Odagami3 . 1 Epistem Ltd, Pharmacogenomics Division, Manchester, United Kingdom; 2 Eisai Co. Ltd., Oncology HHC Unit, Tokyo, Japan; 3 PRISM Pharma Co. Ltd., Yokohama, Japan The Wnt/beta-catenin signaling pathway regulates cell morphology, motility and proliferation. Aberrant regulation of this pathway leads to neoplastic

Thursday 20 November 2014 121 proliferation. PRI-724 is potent, specific inhibitor of the canonical Wnt signaling pathway with potential antineoplastic activity. It specifically inhibits the recruitment of beta-catenin with its coactivator CBP and together with other transcription factors, beta-catenin/CBP binds to WRE (Wntresponsive element) and activates transcription of a wide range of target genes of Wnt/beta-catenin signaling. Blocking the interaction of CBP and beta-catenin by this agent prevents gene expression of many proteins necessary for growth, thereby potentially suppressing cancer cell growth. Monitoring target engagement is crucial to inform on early drug development decisions, and development of a peripheral tissue based gene expression signature of pathway inhibition could facilitate the continued clinical development of PRI-724. High vascularisation of the hair follicle, frequent epithelial origin of tumors, ease of sampling and high degree of congruence of expression in hair of pathways dysregulated in cancers, make the cellular bulb on plucked human scalp hair an excellent surrogate tissue for non-invasive monitoring of pharmacodynamic and mechanism of action (MOA) effects in clinical trials. We have sought to identify peripheral/surrogate molecular biomarkers of target engagement and pharmacodynamic (PD) activity that can be translated to clinical use during PRI-724 development. Using Epistem’s ex vivo plucked hair culture platform, plucked hairs from several healthy donors were exposed to varying doses of C82 (active metabolite of PRI724) over a 24 hour period. Total RNA was isolated from individual anagen hair bulbs post-culture and used to assess global transcriptional alterations in anagen hair resulting from C82 exposure. We observed biologically relevant alteration of the hair bulb transcriptome, ranging from −25-fold to +10-fold differential expression of target transcripts as a result of C82 exposure. Connectivity map analysis confirmed these responses to share relationships with compounds implicated in Wnt/betacatenin signaling, and a Wnt-dependent transcriptional signature was shown to be suppressed in the hair transcriptome. While the temporal and kinetic relationship between gene expression changes, toxicity, and clinical efficacy remains to be determined, the genes identified in this study may be used to provide further MOA information in clinical settings to monitor PD responses in plucked scalp hair obtained from patients exposed to PRI-724. 378 POSTER (Board P158) A first-in-human (FIH) safety and pharmacological study of SAR405838, a novel HDM2 antagonist, in patients with solid malignancies V. de Weger1 , M.P. Lolkema2,3 , M. Dickson4 , A. Le Cesne5 , A. Wagner6 , M. Merqui-Roelvink1 , A. Varga5 , W. Tap4 , G. Schwartz7 , G. Demetri6 , W. Zheng8 , G. Tuffal9 , S. Mace´ 10 , H. Miao8 , J.H.M. Schellens1 , M. de Jonge3 . 1 The Netherlands Cancer Institute, Amsterdam, Netherlands; 2 University Medical Center Utrecht, Utrecht, Netherlands; 3 Erasmus MC Cancer Institute, Rotterdam, Netherlands; 4 Memorial Sloan-Kettering Cancer Center, New York, USA; 5 Gustave Roussy, Villejuif, France; 6 Dana-Farber Cancer Institute, Boston, USA; 7 Columbia University Medical Center, New York, USA; 8 Sanofi Oncology, Cambridge, USA; 9 Sanofi R&D, DSAR, Montpellier, France; 10 Sanofi Oncology, Vitry-sur-Seine, France Background: SAR405838 is an oral spirooxindoles derivative antagonist of HDM2. Preclinical experiments showed robust p53 pathway activation, anti-proliferative, and pro-apoptotic effects. Treatment in HDM2-amplified xenograft models resulted in complete regression and prolonged survival. Material and Methods: A phase 1 FIH study in solid malignancy was conducted to determine the maximum tolerated dose (MTD), safety, pharmacokinetics (PK), and exploratory pharmacological activities of SAR405838. Oral administration schedules of once daily (QD) and once weekly (QW) were evaluated. An adaptive design was employed in dose escalation with overdose control, with at least 2 patients evaluable for doselimiting toxicity (DLT) required in each cohort and maximum dose escalation by 100%. The first cycle (3 weeks) was the primary DLT evaluation period, but late-onset DLT was also included in dose escalation decision. PK and biomarker (plasma MIC-1) samples were collected on Day 1 and Day 8 (for the QD schedule). A preliminary food effect was tested in Cycle 2. Results: In dose escalation, 24 patients received SAR405838 in the QD schedule (6 dose levels from 50 mg to 800 mg) and 23 patients were treated in the QW schedule (5 dose levels from 800 mg to 1800 mg). The most frequent adverse events were nausea, fatigue, vomiting, decreased appetite, and diarrhea, mostly grade 1−2 and manageable. Two patients at 400 mg QD experienced grade 4 thrombocytopenia as DLT. The MTD for the QD schedule was determined as 300 mg QD. SAR405838 exposure (AUC) was proportional to dose up to 400 mg, tmax ~4 hr, terminal t1/2 ~16 hr. Increase of plasma MIC-1 protein levels was correlated with SAR405838 blood concentration. No significant food effect was observed at the 300 mg QD dose level, but an increase in exposure was significant

122 Thursday 20 November 2014 at 1800 mg QW. No DLT has been observed so far in the QW regimen up to 1800 mg. Sustained disease stabilization has been observed mainly in soft tissue sarcoma patients. An expansion cohort was opened for the QD schedule, with 21 pts with dedifferentiated liposarcoma treated at 300 mg QD. Conclusions: p53 pathway activation and adequate safety in patients have been demonstrated with SAR405838, which supports the ongoing evaluation in monotherapy and combination therapies. The study is sponsored by Sanofi Oncology; NCT01636479. 379 POSTER (Board P159) Identification and rational in silico-based targeting of a novel mediator of metastatic breast cancer R. Clarkson1 , J. Soukupova1 , A. Wakefield1 , D. Turnham1 , W. Yang1 , C. Bordoni2 , A. Westwell2 , A. Brancale2 . 1 European Cancer Stem Cell Research Institute, Cardiff University School of Biosciences, Cardiff Wales, United Kingdom; 2 Cardiff University, School of Pharmacy, Cardiff Wales, United Kingdom The proto-oncogene Bcl-3 is a co-factor for the transcription factor NFkB, regulating NF-kB dependent gene expression through direct protein– protein interaction with the subunits, p50 and p52. Consequently it modulates both canonical and alternative NF-kB pathways responsible for a variety of cellular functions in normal and pathological settings. We recently reported an unexpected role for Bcl-3 in the spontaneous metastasis of mammary tumours in a mouse model of metastatic breast cancer. Loss of Bcl-3 expression resulted in an 80% reduction in metastatic tumour burden, with no overt detriment to normal tissue homeostasis. Suppression of Bcl3 interaction with p50/p52 using a protein-binding mutant of Bcl-3 replicated this phenotype in human breast cancer xenografts in vivo, an outcome linked to a reduction in cell motility in vitro. These properties make Bcl-3 an interesting drug target for metastatic breast cancer − however no pharmacological inhibitors of Bcl-3 had previously been reported and targeting of this co-factor would require unconventional molecular inhibition. Molecular modeling of Bcl-3 complexes, and a subsequent in silico compound screen of a putative pharmacophore on Bcl-3 resulted in the identification of a nM efficient inhibitor, JS6, that suppressed protein– protein interactions, modulated NF-kB signaling and reduced cell motility in vitro. Use of this experimental agent in mouse models of metastatic breast cancer was well tolerated and resulted in a dramatic reduction in metastatic tumour burden. These results suggest that JS6 and its analogues may be a useful tool to investigate further the molecular mechanisms of Bcl-3 action and show promise as a novel putative anticancer agent. 380 POSTER (Board P160) Induction of apoptosis with a novel dual cIAP1/XIAP antagonist in models of melanoma G. Ward1 , G. Chessari1 , C.N. Johnson1 , J. Lewis1 , S. Rich1 , N. Thompson1 . 1 Astex Pharmaceuticals, Cambridge, United Kingdom Background: Antagonists of the inhibitor of apoptosis proteins (IAP) are being tested in the clinic as cancer therapeutics, having the potential to switch pro-survival signalling pathways in cancer cells towards cell death, whilst maintaining a therapeutic window. We used fragment based drug discovery to develop a novel second generation IAP antagonist that is nonpeptidomimetic and does not contain an alanine as a warhead. Materials and Methods: The compound was evaluated using MDA-MB231 breast cancer, A375 and SK-MEL28 melanoma cell lines and xenograft models. Assays were set up to measure cIAP1 degradation in MDA-MB-231 cells and XIAP antagonism by immunoprecipitation in engineered HEK293 cells over-expressing XIAP and caspase-9. Apoptosis was measured by Western blotting or using cytometer-based fluorescent caspase-3 substrate assays. Results: The compound potently binds the BIR3 domains of both XIAP and cIAP1 in in vitro binding assays and these effects were confirmed in cells: cIAP1 is degraded and the XIAP/caspase-9 interaction is disrupted on treatment of cells with nM concentrations of compound. Our IAP antagonist has increased XIAP potency while maintaining similar cIAP1 potency compared to competitor alanine-containing compounds, and hence has a more balanced profile. The effects of our compound in melanoma cell lines were investigated. The compound inhibited the proliferation of A375 and SK-MEL-28 cells in the presence of TNFa and apoptosis was induced as demonstrated by increases in levels of cleaved PARP, caspase-3 and caspase-9. It significantly inhibited the tumour growth of MDA-MB-231 xenografts when dosed daily at 20 mg/kg p.o. Similar inhibition of tumour growth was also observed in melanoma models, including an A375 xenograft and a patient

Poster Session – Molecular Targeted Agents I derived xenograft (PDX), MEXF274. Degradation of cIAP1 and disruption of the XIAP-caspase-9 interaction was demonstrated in MDA-MB-231 and HEK293-XIAP-Caspase9 xenografts respectively, indicating antagonism of both cIAP1 and XIAP in vivo. Prolonged induction of apoptosis markers was also observed after a single 20 mg/kg p.o. dose of compound. Conclusions: We have a novel dual cIAP1/XIAP antagonist which exhibits potent induction of apoptosis in both in vitro and in vivo melanoma models, and has good drug-like properties. Our compound has the potential to be an efficacious therapeutic against cancers with an inflammatory phenotype. This compound is currently undergoing pre-clinical evaluation. 381 POSTER (Board P161) Efficacy of specific FGFR inhibitors against gatekeeper resistance mutations and shared mechanism of cell death in FGFR2-dependent endometrial cancer cell lines L. Packer1 , S. Byron2 , S. Stehbens1 , D. Loch1 , F. Dehkhoda1 , S. Stephenson1 , P. Pollock3 . 1 IHBI QUT, Brisbane, Australia; 2 TGen, Phoenix, USA; 3 IHBI QUT, Cancer Program, Brisbane, Australia Background: Our lab has now identified mutations in FGFR2 in 181/1531 (12%) of endometrioid endometrial cancers (ECs). Multiple FGFR inhibitors are in preclinical and clinical development. However, their efficacy against common resistance mutations, along with their mechanism of action is unknown. Materials and Methods: We have assessed the activity of BGJ398, AZD4547 and LY2874455 in multiple cell line models including a sensitive EC cell line transduced with the FGFR2 gatekeeper (GK) mutant (V565I), BaF3 cells transduced with various FGFR2 kinase domain mutations and the isogenic KMS11 (FGFR3 dependent) and KMS11-R (FGFR3 GK mutant V555M) myeloma cells. We also sought to determine whether these clinically relevant inhibitors induced cell death in FGFR2mutant EC cells via the novel mechanism previously identified with PD173074. Results: In all isogenic cell line systems LY2874455 was the most effective inhibitor against FGFR GK mutations. Treatment with all FGFR inhibitors resulted in mitochondrial depolarization as observed using the TMRE assay. Overexpression of Bcl-XL and BCL-2 blocked cell death, and knockdown of multiple ATG proteins failed to prevent cell death confirming that the cell death was mediated by mitochondria, rather than autophagy. The cell death in response to PD173074 as well as AZD4547 and BGJ398 was caspase-independent as well as independent of apoptosis-inducing factor (AIF) and Endonuclease G (EndoG), predominant mediators of caspase-independent apoptosis. Conclusions: LY2874455 shows superior activity against FGFR GK mutations. Clinically relevant specific FGFR inhibitors induce cell death via a novel caspase-independent mechanism in FGFR2-dependent EC cell lines. 382 POSTER (Board P162) Phase Ib study of oral dual-PI3K/mTOR inhibitor GDC-0980 in combination with capecitabine and mFOLFOX6 + bevacizumab in patients with advanced solid tumors and colorectal cancer L. Rosen1 , J. Goldman2 , J.M. Hubbard3 , M. Roos3 , J. Capdevila4 , J. Maynes4 , W. Lin5 , B. O’Keeffe5 , M. Lackner5 , J. Spoerke5 , J. Ware5 , B. Arnieri5 , E. Freas6 , S. Leong6 . 1 University of California Los Angeles, Medicine, Santa Monica CA, USA; 2 University of California Los Angeles, Division of Hematology Oncology, Santa Monica CA, USA; 3 Mayo Clinic, Department of Oncology, Rochester, USA; 4 Hospital Vall d’Hebron, Department of Medical Oncology, Barcelona, Spain; 5 Genentech Inc., Early Clinical Development, South San Francisco, USA; 6 University of Colorado Anschutz Medical Campus, Medical Oncology, Aurora, USA Background: The PI3K signaling pathway is dysregulated in a variety of cancers, including colorectal cancer. GDC-0980 is a potent oral PI3K/mTOR inhibitor and has demonstrated synergy with fluoropyrimidines and bevacizumab in preclinical models. Materials and Methods: In a Phase Ib dose escalation study of GDC-0980 in combination with capecitabine (Cap) (Arm A) or mFOLFOX6 + bevacizumab (Bev) (Arm B), the recommended Phase 2 doses (RP2D) were determined to be 40 mg GDC-0980 QD for 14 days in a 21-day cycle + 2000 mg/m2 Cap and 40 mg GDC-0980 QD for 7 days in a 14-day cycle + mFOLFOX6 + Bev (10 mg/kg) (ESMO 2012 #452P). GDC0980 40 mg with mFOLFOX6 + Bev was further evaluated in an expansion cohort of advanced CRC patients who had not received oxaliplatin within 1 year of starting study treatment. Patients enrolled during the dose escalation stage also continued to be monitored on study, and updated data for these patients are presented. Available archival tumor samples were analyzed to determine PIK3CA and KRAS mutation status as well as PTEN protein status.

Poster Session – Molecular Targeted Agents I Results: Forty-one patients were enrolled in the study (19 in Arm A; 22 in Arm B, including 11 in the CRC expansion cohort). The most common Grade 3 or higher adverse events (AEs), regardless of causality, at the RP2Ds were hyperglycemia (40%), stomatitis and hypophosphatemia (20% each) in Arm A (n = 10) and neutropenia (28%), hyperglycemia, aspartate aminotransferase increased, and neutrophil count decreased (17% each) in Arm B (n = 18). Notably, only one case of Grade 3 rash was observed (in Arm A at the RP2D). In Arm A, confirmed partial responses (cPR) were observed in one patient with head and neck squamous cell carcinoma (226 days on study) and one CRC patient with PIK3CA and KRAS mutations (915+ days on study). In Arm B, 3 patients in the escalation stage had a PR; with cholangiocarcinoma (unconfirmed [u]PR), anal cancer (cPR), and triple negative breast cancer (uPR). In the CRC expansion, 2 patients had a cPR, and 1 patient with a KRAS mutation had a uPR and sustained disease control for 462 days. The median time on study for CRC patients was 253 days. PI3K-related biomarker data will be presented for all patients with evaluable tissue. Conclusions: GDC-0980 40 mg in combination with Cap or FOLFOX6 + Bev is well-tolerated and has promising preliminary activity in advanced CRC. 383 POSTER (Board P163) Identification of potent and selective tankyrase 1/2 inhibitors with activity in a subset of APC mutant colorectal cancer S.M. Guichard1 , Y. Zhang1 , D. Ferguson1 , A. Mazzola1 , H. Wang1 , L. Bao1 , S. Grosskurth1 , J. Johannes1 , M. Wagoner2 , M. Zinda1 , S. Fawell1 , E.J.E. Pease1 , A. Schuller1 . 1 AstraZeneca R&D Boston, Oncology iMed, Waltham, USA; 2 AstraZeneca R&D Boston, Drug Safety & Metabolism, Waltham, USA Tankyrases (TNKS) 1 and 2 are Poly (ADP-ribose) polymerases targeting key proteins such as axin 2 for proteosomal degradation. Axin 2 is a key component of the b-catenin degradation complex and tankyrase 1/2 inhibition-mediated stabilization of axin 2 provides a druggable way to inhibit the b-catenin driven Wnt signaling pathway. Here we report the identification of potent inhibitors of TNKS1/2 (TNKS1 enz IC50 <10 nM) with up to 1000-fold selectivity against PARP1/2 with growth inhibitory activity in vitro and in vivo in a subset of APC mutant colorectal cancer models. TNKS inhibition was assessed by monitoring axin 2 protein levels by quantitative immunoblotting, modulation of Wnt target genes was determined using q-PCR (Taqman) and growth inhibition was determined by MTS assays. TNKSi stabilize axin 2 protein in a concentration- and time-dependent manner in several colorectal cancer cell lines including the COLO320 carrying a mutation in APC at S811 (this mutation leads to a truncation of APC after the armadillo domain). In COLO320, TNKSi modulated downstream Wnt target genes and induced growth inhibition (IC50 ~10−50 nM). Conversely, b-catenin mutant HCT-116 cells were insensitive to TNKSi confirming the selectivity of the inhibitors. In vivo, TNKSi modulated axin 2 and downstream Wnt target genes in a dose- and time-dependent manner: In vivo, free drug concentrations were maintained above the concentration required to elicit biomarker modulation in vitro for >24 h. Consistent with the PK, pharmacodynamic modulation of axin 2 and downstream Wnt target genes in COLO320 xenografts was dosedependent and persisted for >24 h. A quantitative PK/PD/efficacy model was established using both in vitro and in vivo data that links PK, axin 2 stabilization, Wnt target genes modulation and tumour growth inhibition. The antitumour activity observed with TNKSi at tolerated doses was consistent with prediction from the model. A survey of genetic aberrations in APC leading to a truncation similar to the one observed in COLO320 was carried out across 24 PDX models of CRC. TNKSi induces tumour growth inhibition at tolerated doses in one of the PDX models suggesting that a subset of CRC with short truncation of APC after the armadillo domain may be sensitive to TNKSi. To our knowledge, this is the first evidence of genetic aberrations in CRC associated with sensitivity to tankyrase inhibition in vivo. 384 POSTER (Board P164) REDX04988, a novel dual B-RAF/C-RAF inhibitor and a potential therapeutic for BRAF-mutant colorectal cancer J. Rainard1 , R. Testar2 , R. Poonawala1 , H. Mason1 , P. Smith1 , H. Brooke1 , V. Huart1 , S. Frith2 , J. Ahmet2 , J. Hall2 , A. Morrison3 , M.A. Campbell1 , M. Bingham2 , R. Armer2 . 1 Redx Oncology, Pharmacology, Liverpool, United Kingdom; 2 Redx Oncology, Medicinal Chemistry, Liverpool, United Kingdom; 3 Biocity Scotland, European Screening Centre, Newhouse, United Kingdom Redx Oncology has identified a novel dual B-RAF/C-RAF inhibitor with a unique and intriguing pharmacological profile. REDX04988 not only shows excellent efficacy against BRAFV600E melanoma cells but also prevents

Thursday 20 November 2014 123 paradoxical MAPK pathway activation in RAS-mutant, BRAFWT -melanoma cells. Moreover, REDX04988 induces sustained inhibition of the MAPK pathway in BRAFV600E colorectal cells by overcoming feedback reactivation of the pathway. Aberrant signalling via the MAPK pathway is commonly seen in cancer. RAF inhibitors such as dabrafenib and vemurafenib have been approved for the treatment of unresectable and metastatic BRAF-mutant melanoma, but these agents lack efficacy in BRAF-mutant colorectal cancer (CRC), partly because of EGFR-mediated feedback reactivation of the MAPK pathway. Furthermore, RAF inhibitor treatment of RAS-mutant, BRAFWT -melanomas has been associated with other skin cancers, such as cutaneous squamous cell carcinoma due to MAPK pathway paradoxical activation. There is therefore a clinical need for novel agents targeting the MAPK pathway that do not have these undesirable properties. Redx Oncology RAF inhibitors were screened for their ability to bind to RAF kinases and to inhibit the MAPK pathway in a variety of cellular contexts. REDX04988 was found to bind with high affinity to RAF kinases in vitro and to potently inhibit ERK phosphorylation and cellular proliferation in A375 (BRAFV600E ) melanoma cells. However, unlike dabrafenib and vemurafenib, REDX04988 does not induce paradoxical activation of the MAPK pathway in either IPC298 (NRASQ61L ) melanoma cells or HCT116 (KRASG13D ) CRC cells. Furthermore, REDX04988 shows sustained and potent inhibition of both pMEK and pERK in WiDr (BRAFV600E ) CRC cells over a 24 h period. Similar activity for REDX04988 has also been demonstrated in HCT116 (KRASG13D ) CRC cells. In contrast, dabrafenib, vemurafenib and the MEK inhibitors selumetinib and cobimetinib all failed to sustain inhibition at 24 h. Only the MEK inhibitor GDC-0623 demonstrated sustained inhibition similar to REDX04988. This inhibitory effect on pMEK and pERK levels was accompanied with antiproliferative activity in the CRC cell lines. Kinase profiling of REDX04988 suggests that it does not inhibit EGFR or MEK, but rather mediates its action through the blockade of RAF kinases. Thus REDX04988 has a unique pharmacological profile previously unseen with RAF inhibitors and as such presents a novel therapeutic opportunity in MAPK pathway-dependent cancers. 385 POSTER (Board P165) Noninvasive monitoring of acquired EGFR-T790M mutation and discovery of its heterogeneity in patients with advanced NSCLC treated with EGFR-TKI X. Ye1 , D. Zheng2 , M.Z. Zhang3 , Y. Sun1 , J.Y. Wang2 , J. Ni2 , H.P. Zhang2 , L. Zhang2 , J. Luo2 , J. Zhang4 , L. Tang4 , B. Su4 , G. Chen5 , G.S. Zhu1 , J.F. Xu2 , Y. Gu1 . 1 AstraZeneca, Translational Science AEM iMed, Shanghai, China; 2 Shanghai Pulmonary Hospital, Department of Oncology, Shanghai, China; 3 AstraZeneca, ISIT RDI, Shanghai, China; 4 Shanghai Pulmonary Hospital, Central Laboratory, Shanghai, China; 5 Shanghai Pulmonary Hospital, Department of Pathology, Shanghai, China Background: EGFR-T790M mutation, which is the valuable target for next generation of EGFR-TKI, accounts for about half of acquired resistance to current EGFR-TKI therapy in EGFR sensitive mutation positive NSCLC patients. Due to clinical challenge in obtaining tumor rebiopsies, noninvasive detection of EGFR-T790M in plasma circulating free DNA (cfDNA) has been proved to be valuable and feasible. However, questions remain about plasma testing: (1) What is the T790M mutation pick-up rate? (2) Is it possible to detect T790M earlier before clinical progression? (3) Does T790M always co-exist with a sensitive mutation in a tumor cell and what is its potential impact to chemotherapy following the EGFR-TKI resistance? Material and Methods: A highly sensitive droplet digital PCR (ddPCR) method for EGFR mutation testing in plasma was developed and its high sensitivity and specificity were validated in testing cfDNA from EGFR-TKIna¨ıve NSCLC patients. In this study, plasma samples were collected from 102 patients with advanced NSCLC who acquired resistance to EGFRTKI. EGFR-TKI treatment was continued after progressive disease (PD), with majority of patients receiving chemotherapy as well. ddPCR was used to detect the major EGFR mutations (19Dels, L858R and T790M) in plasma cfDNA. Results: Overall, EGFR-T790M was detected in 47 out of 102 postPD patients (46.1%). Among 21 of the 47 post-PD T790M+ patients, 10 (47.6%) were detected T790M+ during 0.8−4.2 months before PD (median: 2 months before PD). Interestingly, while T790M co-exists with a sensitive mutation in 43 of the 47 T790M+ patients (91.5%), T790M presents alone in 4 of the 47 patients (8.5%). Furthermore, dynamic quantitative changes of sensitive mutations (19Dels or L858R) and T790M in a few patients showed preliminary insight that, even in double mutant patients, T790M and sensitive mutant might not always come from the same tumor cell populations. Moreover, T790M status might affect outcome of chemotherapy following EGFR-TKI resistance.

124 Thursday 20 November 2014 Conclusions: Noninvasive monitoring of plasma cfDNA could detect T790M with a positive rate as high as that reported in tumor tissue in advanced NSCLC patients after acquiring EGFR-TKI resistance, and T790M could be detected in plasma with a median time of 2 months prior to clinical PD. T790M may present with or without EGFR sensitive mutation in tumor cells. Moreover, plasma T790M status could affect the outcome of chemotherapy following EGFR-TKI resistance. 386 POSTER (Board P166) PI3K and MEK inhibitor combination toxicities and relative dose intensity: Vall d’Hebron experience A. Azaro1 , D. Marino1 , A. Garrido-Castro1 , C. Cruz1 , M. Alsina1 , J. Perez1 , R. Dienstmann1 , G. Argiles1 , C. Hierro1 , M. Berzosa1 , B. Adamo1 , J. Tabernero1 , J. Rodon1 . 1 Vall d’Hebron Institute of Oncology, Molecular Therapeutics Research Unit of Cancer, Barcelona, Spain Introduction: Phosphatidylinositol 3-kinase ( PI3K) and mitogen-activated protein kinase (MAPK) pathways are highly networked together, and frequently altered in cancer. Preclinically, a simultaneous inhibition of both pathways leads to a more effective anti-tumor effect. However, combination studies of PI3K and MEK inhibitors have shown that fulldose, continuous treatment of drugs targeting both pathways do not seem feasible. Here we investigate the toxicities observed and their impact on dose recommendation and dose-intensity. Methods: We performed a retrospective analysis of the toxicities of patients treated with a PI3K inhibitor (2 PI3K/mTOR, 1 panPI3K, 1 PI3Ka inhibitor) combined with a MEK inhibitor (2 MEK1/2 inhibitors) in 5 phase I clinical trials. Safety was assessed by CTCAE v4. Toxicities possibly related to the study drugs, interruptions of the treatment related with toxicity and patient relative dose intensity (RDI) during cycle 1 (C1) and cycle 2 (C2) were recorded. Results: Sixty-six patients (35 males, 31 females, median age 58 y.o., range 34−77) with advanced solid tumors were included and treated in 5 clinical trials. RP2D in those trials was below the RP2D in monotherapy for all drugs in all trials. Twenty two percent of patients received <75% of the intended RDI in C1 and C2. Forty five percent of the patients interrupted treatment on C1 and 52% on C2 due to toxicity. Interruptions for >1week were 16% and 26% on C1 and C2 respectively (p = 0.052). Eighteen percent of patients abandoned the study on C2 due to toxicity. In C1 and C2, grade 3−4 related events included CPK elevation (9/13%), skin toxicity (3/8%), diarrhea (5/0%), fatigue (0/4%), transaminitis (3/0%), and lipase elevation (3/0%) respectively for each cycle. Subacute or chronic, grade 1−2 toxicities included diarrhea (65/45%), acneiform rash (65/61%), CPK elevation (50/39%), fatigue (30/37%), vomiting (27/32%), mucositis (25/28%) and hyperglicemia (20/37%). Conclusion: In comparison with described toxicities for each drug in monotherapy, combination studies showed an increased toxicity profile. Some on-target toxicities were higher than expected (based on the administered dose and dose intensity) such as skin toxicity and CPK elevation, and some unexpected, non-specific toxicities seem more frequent (transaminitis), suggesting a significant pharmacodynamic interaction. Acute, subacute and chronic toxicities observed with these combos do not allow a manageable RDI. 387 POSTER (Board P167) Combination of the ERK inhibitor GDC-0994 with the MEK inhibitor cobimetinib significantly enhances anti-tumor activity in KRAS and BRAF mutant tumor models M. Merchant1 , J. Chan1 , C. Orr1 , J. Cheng1 , X. Wang1 , T. Hunsaker1 , M.C. Wagle2 , S.A. Huang2 , J. Tremayne1 , H. Ngu3 , M. Solon3 , J. Eastham-Anderson3 , H. Koeppen3 , L. Friedman1 , M. Belvin4 , J. Moffat5 , M. Junttila1 . 1 Genentech, Translational Oncology, South San Francisco, USA; 2 Genentech, Early Stage Biomarker Discovery, South San Francisco, USA; 3 Genentech, Pathology, South San Francisco, USA; 4 Genentech, Tumor Immunology, South San Francisco, USA; 5 Genentech, Biochemical Pharmacology, South San Francisco, USA Background: Dysregulation of the mitogen-activated protein kinase (MAPK) pathway has been implicated in roughly 30% of all human cancers. Hyper-activation of MAPK signaling is initiated by mutations in the RAS or BRAF oncogenes or through upstream growth factor signaling. Accordingly, multiple inhibitors have been developed against pathway kinases, including BRAF, MEK and ERK. Single-agent kinase inhibitor regimens have demonstrated limited clinical benefit outside of metastatic melanoma. Combinations of inhibitors that target multiple nodes within the MAPK pathway may therefore be necessary in order to effectively suppress pathway signaling and achieve meaningful clinical benefit.

Poster Session – Molecular Targeted Agents I Materials and Methods: The ERK1/2 inhibitor GDC-0994 was tested alone and in combination with MEK and/or BRAF inhibitors. In vitro cell proliferation, apoptosis, cell cycle analysis and biochemical signaling analysis studies were performed. Combinations of GDC-0994 and cobimetinib (GDC-0973) and/or vemurafenib were tested in multiple human tumor xenograft models in mice. Genetically engineered mouse (GEM) models of pancreatic ductal adenocarcinoma (PDAC; KrasG12D ;p16/19FL/FL ;PdxCre) and non small-cell lung cancer (NSCLC; KrasG12D ;p53frt/frt ; AdenoviralCre) were also evaluated for tumor growth and survival in response to combination of GDC-0994 and cobimetinib. Results: We have developed a potent and selective ERK1/2 inhibitor, GDC0994, that is currently in PhI clinical trials. GDC-0994 has anti-tumor activity in KRAS and BRAF mutant tumor models. In time course experiments, inhibition of either the MEK or ERK nodes alone transiently suppressed the MAPK pathway due to feedback reactivation. In contrast, combination of MEK and ERK inhibitors more potently and durably suppressed the MAPK pathway output, leading to significantly improved inhibition of cell proliferation and increased apoptosis. In vivo, the combination was welltolerated and resulted in enhanced anti-tumor efficacy in multiple KRAS, NRAS and BRAF mutant tumor xenograft models. The combination of GDC-0994 and cobimetinib was highly effective in both the Kras-mutant PDAC and Kras-mutant NSCLC GEM models, leading to a greater number of tumor regressions (6 of 9 mice in PDAC model and 11 of 11 mice in NSCLC model), decreased tumor growth rates, and significantly increased survival that surpasses the chemotherapy control arms. Conclusions: The robust and dynamic nature of MAPK signaling, particularly in the context of therapeutic intervention, helps to explain the limited clinical success observed with single agent MAPK-targeting strategies tested to date. Our results elucidate a highly effective strategy that combines MEK and ERK inhibitors to more significantly suppress the MAPK pathway. These results add to the fundamental understanding of how the MAPK pathway mediates oncogenic signaling and offers a new combination approach to target MAPK dysregulated tumors. 388 POSTER (Board P168) An integrated molecular analysis of lung adenocarcinomas identifies potential therapeutic targets among TTF1-negative tumors R.J. Cardnell1 , C. Behrens1 , L. Diao2 , Y. Fan1 , X. Tang3 , J.D. Minna4 , G.B. Mills5 , J.V. Heymach1 , I.I. Wistuba3 , J. Wang2 , L.A. Byers1 . 1 MD Anderson Cancer Center, Thoracic/Head & Neck Medical Oncology, Houston TX, USA; 2 MD Anderson Cancer Center, Bioinformatics & Computational Biology, Houston TX, USA; 3 MD Anderson Cancer Center, Translational Molecular Pathology, Houston TX, USA; 4 UT Southwestern, Hamon Center for Therapeutic Oncology Research and the Simmons Comprehensive Cancer Center, Dallas TX, USA; 5 MD Anderson Cancer Center, Systems Biology, Houston TX, USA Background: Over the past ten years, major advances have been made in the treatment of lung adenocarcinoma (LUAD), including the identification of druggable driver genes in a subset of patients (e.g., EGFR). However, these drivers are uncommon in tumors lacking thyroid transcription factor 1 (TTF1), an immunohistochemical (IHC) marker routinely used in the diagnosis of LUAD (roughly 15−20%). Given the poor prognosis of TTF1negative tumors and the absence of a validated targeted therapy for this population, we performed an integrated molecular analysis to identify pathways or targets enriched in TTF1-negative tumors that could serve as potential drug targets. Material and Methods: We investigated the molecular profiles of tumors in two large, independent clinical LUAD cohorts (PROSPECT and The Cancer Genome Atlas (TCGA), n = 333 total) using reverse phase protein array profiling (RPPA) to measure key oncogenic pathways and targets (>127 total and/or phosphorylated proteins). The activity of targeted drugs and chemotherapies were then investigated for their in vitro activity in TTF1negative LUAD models. Results: Since TTF1 is measured in clinical practice by IHC, we first correlated IHC staining (using a 0–300 scale that reflects degree (0−3) and extent (0–100%) of cell staining) with TTF1 protein levels measured by RPPA. IHC and RPPA were strongly correlated (R = 0.57, p < 0.001), indicating that RPPA values were robust and highly correlated with the clinical standard. Density analysis of TTF1 scores in PROSPECT and TCGA revealed a bimodal distribution, creating an extended group of TTF1-negative that includes tumors with both no and very low TTF1. Confirming previous studies, the TTF1-negative LUAD had shorter overall survival (p < 0.001). Integrated molecular profiling revealed 3 potential targets (CyclinE1, Chk1 and Smad3) expressed at higher levels in TTF1 negative tumors in both LUAD cohorts. Using a proteomic ‘DNA repair score’ (Cardnell et al, 2013), TTF1-negative LUAD also showed an overall higher expression of DNA repair proteins. At the mutation level, in contrast to genes such as

Poster Session – Molecular Targeted Agents I EGFR, genomic analysis revealed an enrichment of KEAP1 mutations in TTF1-negative LUAD. Consistent with this finding, higher expression of Nrf2 (inhibited by KEAP1) was also observed in TTF1-negative tumors, indicating agents that target Nrf2 in these patients. Like patient tumors, LUAD cell lines also demonstrate a bimodal expression of TTF1 with TTF1-negative and positive groups. Consistent with their overexpression of DNA repair proteins, TTF1-negative cell lines had greater sensitivity to drugs with activity against DNA repair proteins. Conclusions: RPPA is a robust method to assay TTF1 expression. Molecular analysis indicates that TTF1-negative patient tumors overexpress multiple DNA repair proteins and Nrf2 and may respond preferentially to drugs against these targets. 389 POSTER (Board P169) A phase 1, dose-escalation, first-in-human study of ARQ 087, an oral pan-FGFR inhibitor, in adult subjects with advanced solid tumors K. Papadopoulos1 , A. Tolcher1 , M. Kittaneh2 , A. Patniak1 , D. Rasco1 , G. Chambers1 , G. Newth2 , R. Savage3 , T. Hall3 , B. Schwartz3 , J. Kazakin3 , P. LoRusso2 . 1 South Texas Accelerated Research Therapeutics, Medical Oncology, San Antonio TX, USA; 2 Karmanos Cancer Institute, Center for Translational Therapeutics, Detroit MI, USA; 3 ArQule Inc., Clinical Development, Woburn MA, USA Background: ARQ 087 is a novel, ATP competitive multi-kinase inhibitor with pan-Fibroblast Growth Factor Receptor (FGFR) activity against FGFR1−3 kinases. It demonstrated potent in vitro and in vivo inhibitory effects on a variety of human tumor cell lines and xenograft models. Dysregulation of FGFR signaling has been implicated in a number of developmental syndromes and in several human cancers. Study Design: We report preliminary results of a first-in-human study of oral ARQ 087 as a single agent in pts with advanced solid tumors. The primary and exploratory objectives include assessment of safety, tolerability, pharmacokinetics (PK), pharmacodynamic biomarkers and preliminary antitumor activity. The study design comprised 3−6 pts/cohort in dose escalation and a target selected expansion cohort (once Recommended Phase 2 Dose, RP2D, is established). Assessments include archival tumor molecular status, response by RECIST, v.1.1 every 8 wks, changes in plasma concentrations of FGF19, 21, 23, fasting plasma glucose and phosphorus. Results: To date, a total of 49 pts were enrolled and treated in 28-day cycles with an escalating dose of ARQ 087 (25–425 mg qod or qd). The majority of pts were white (84%), female (61%) with mean age 62 yrs (34−78). Three dose limiting toxicity events (reversible grade 3 AST increase) were reported, one at 250 mg and two at 425 mg qd dose levels. Common drugrelated AEs (mostly grade 1/2) were fatigue (53%), elevated LFTs (45%), nausea (45%), vomiting (20%) and diarrhea (20%). Best response in 32/40 evaluable pts was stable disease (80%). Eleven of 32 pts had stable disease for 16 wks [adrenocortical (2), renal cell, endometrial, ovarian carcinomas, choroidal melanoma, pleural mesothelioma, liposarcoma, osteosarcoma and chondrosarcoma]. Preliminary PK analyses revealed a long half-life (~1 wk) and accumulation with continuous dosing (~9 to 14 fold). Additionally, the Day 22 exposure (AUC and Cmax) increased in a dose proportional manner from 25 mg to 250 mg but plateaued at doses from 250 to 425 mg qd. Preliminary data showed that FGF19 levels generally increased in treated pts, with the increase more evident in pts who were on treatment for 16 wks. Additional biomarker and efficacy data will be presented. Conclusions: ARQ 087 was well tolerated with manageable, mostly grade 1/2 AEs. RP2D has not yet been established; further dose exploration is ongoing. 390 POSTER (Board P170) Inhibitors of EZH2 act synergistically with type 1 interferon to induce a potent interferon-stimulated gene response, triggering apoptosis in diffuse large B-cell lymphoma B. Bradley1 , F. Zhao1 , C.C. Yuan1 , S. Balasubramanian2 , P. Iyer2 , C. Hatton3 , B. Bryant3 , E. Normant2 , P. Trojer1 . 1 Constellation Pharmaceuticals, Biology, Cambridge, USA; 2 Constellation Pharmaceuticals, Pharmacology, Cambridge, USA; 3 Constellation Pharmaceuticals, Bioinformatics, Cambridge, USA The histone methyltransferase EZH2, a component of the PRC2 complex, is frequently mutated in diffuse large B-cell lymphoma (DLBCL), a non-Hodgkin’s lymphoma subtype. Activating mutations, such as point mutations in residue Y641 in the protein’s catalytic SET domain, lead to elevated levels of histone H3 trimethylation on residue K27 (H3K27me3). This altered chromatin state modulates the transcriptional program of

Thursday 20 November 2014 125 affected cells, likely playing a key role in pathogenesis. We have identified EZH2 small molecule inhibitors that abrogate proliferation of DLBCL cell lines in culture and in mouse xenograft models. Using gene expression profiling we identified a set of genes that is dramatically upregulated in response to EZH2 inhibition, including genes associated with a type 1 interferon (IFN) response. Interestingly, co-treatment of cells with EZH2 inhibitor and either IFN alpha or beta leads to a synergistic induction of this IFN-stimulated gene program, and concomitantly triggers a synergistic apoptotic response. This synergistic response not only affects cell lines already sensitive to EZH2 inhibitors, but also cell lines refractory to both EZH2 inhibition and IFN treatment. Accordingly, we show that EZH2 inhibition leads to a reduction in the repressive H3K27me3 mark on IFN response genes, leading to their expression, ultimately providing the mechanistic basis for the induction of apoptosis by EZH2 inhibitors alone, or in combination with IFN treatment in DLBCL. This mechanism, and combination treatment, has important implications for the treatment of DLBCL in the clinic. 391 POSTER (Board P171) LOXO-101, a pan TRK inhibitor, for the treatment of TRK-driven cancers S. Winski1 , B. Baer2 , D. Hartley2 , S. Rhodes2 , R. Wallace2 , S. Smith3 , N. Nanda3 , L. Kunkle4 , P. Lee1 , K. Bouhana1 . 1 Array BioPharma Inc., Pharmacology, Boulder, USA; 2 Array BioPharma Inc., Drug Metabolism, Boulder, USA; 3 Loxo Oncology, Development, Stamford, USA; 4 Loxo Oncology, Clinical, Stamford, USA Background: TRK family of neurotrophin receptors, TRKA, TRKB, and TRKC (encoded by NTRK1, NTRK2, and NTRK3 genes, respectively) and their neurotrophin ligands regulate growth, differentiation and survival of neurons. Translocations involving the NTRK kinase domain, mutations involving the TRK ligand-binding site, amplifications of NTRK , TRK splice variants, and autocrine/paracrine signaling are described in a diverse number of tumor types and may contribute to tumorigenesis. Recently NTRK1 fusions were described in a subset of adenocarcinoma lung cancer patients (Vaishnavi, 2013). LOXO-101 is a potent, ATP-competitive TRK inhibitor with IC50 s in low nanomolar range for inhibition of all TRK family members in binding and cellular assays, with 100× selectivity over other kinases. Methods: In vitro and in vivo evaluations including PK/PD correlations, pharmacokinetic (PK) and drug metabolism characterization, and nonclinical safety evaluation were conducted with standard procedures. Results: In rat and monkey studies LOXO-101 demonstrated 33–100% oral bioavailability and 60−65% plasma protein binding. It had low brain penetration, was well tolerated in 28 day (d) GLP toxicology studies and was neither an inducer nor inhibitor of human CYP3A4. In a constitutivelyactive TRKA murine tumor model developed to investigate activity of TRKinhibitory drugs, a single dose (30 mg/kg) of LOXO-101 reduced tyrosine phosphorylation of TRKA and downstream signal transduction (pERK) in the tumor >80%. LOXO-101 was well tolerated up to 200 mg/kg/day for 14 d in this model. Delays of tumor growth were observed with the lower dose LOXO-101, with sustained inhibition of tumor growth for up to 6 d after discontinuation of treatment with the higher doses. Crizotinib at 50 mg/kg was limited to 50% inhibition of pTRKA in tumor-bearing mice and was ineffective at blocking tumor growth. Conclusions: LOXO-101 is an orally bioavailable first-in-class kinase inhibitor developed specifically for TRK inhibition with favorable PK and drug metabolism properties in animals. LOXO-101 has demonstrated potent inhibition of TRKA in tumor bearing animals and was effective in inhibiting tumor growth in a TRKA driven malignancy. The ability to achieve concentrations above IC80 with this selective inhibitor may provide an improvement over multikinase therapy for TRK-driven malignancies. A Phase 1 clinical study has been initiated to determine its PK, PD, safety and anti-tumor activity in humans. 392 POSTER (Board P172) Using modelling & simulation to integrate mouse PK−PD-efficacy with preliminary human PK data to inform the Phase II doses and schedule for the experimental c-Met inhibitor AZD6094 (Volitinib) R. Jones1 , A. Cheung2 , T. Coleman2 , P. Ballard1 , C. D’Cruz3 , A. Schuller3 , M. Frigault3 , Y. Gu4 , Y. Sai4 , S. Weiguo4 , Y. Ren4 , W. Qing4 , L. Lindbom5 , K. Petersson5 . 1 AstraZeneca, Oncology IMED, Macclesfield, United Kingdom; 2 AstraZeneca, Early Clinical Development, Macclesfield, United Kingdom; 3 AstraZeneca, Oncology IMED, Waltham, USA; 4 Hutchison Medi Pharma, Oncology, Shanghai, China; 5 qPharmetra LCC, M&S, Ekero, Sweden MET is a transmembrane tyrosine kinase receptor that is deregulated across multiple cancer types. AZD6094 is a potent (IC50 4 nM) and selective

126 Thursday 20 November 2014 (>650 fold selectivity over 265 kinases), small molecule inhibitor of MET being co-developed with Hutchison MediPharma. AZD6094 is being developed as a monotherapy or combination therapy in multiple tumor indications including papillary renal cell carcinoma (PRCC) and gastric cancer. AZD6094 in MET sensitive mouse xenograft models (including RCC-43b & RCC-47 for PRCC; MKN-45, SNU-5 & Hs746T for gastric) demonstrates rapid, sustained and extensive inhibition of phosphorylated-MET (pMET) and tumour efficacy. The modelling objectives of this work were to build a generalised quantitative model that can be applied across cell-lines linking drug concentration to pMET to tumour growth inhibition (TGI). The model was then used to explore whether differences in sensitivities to AZD6094 observed across animal models is due to differences in the PK-pMET or pMET-TGI relationships. Additionally, the mouse pMET model was linked to the observed human PK model to simulate pMET inhibition to inform the Phase II dose selection in PRCC patients and select time-points to take tumour samples for pMET measurement. A population PK-PD-efficacy model was developed for mouse consisting of (1) a PK model connected to (2) an Emax model for the inhibition of pMET, driving (3) a variation on the Simeoni model for tumour growth inhibition. It was found that a direct response model for the inhibition of pMET with a single IC50 value best represented the data available across the different xenograft models. Conversely, it was necessary to fit individual EC50 values for each xenograft model for inhibition of the tumour growth. A human population PK model was established from emerging dose escalation Phase I study data. Using the human PK model to drive the mouse PD model simulations were run in order to explore the predicted degree and duration of pMET inhibition for different doses following once daily and twice daily dosing. At doses greater than 300 mg, the model predicts extensive (>80%) inhibition of pMET over the dosing interval with maximal inhibition being observed between 2 and 4 h. 393 POSTER (Board P173) Small modification of ceritinib enhances the activity against ALK C.H. Park1 , C.H. Kang1 , H.J. Jung2 , H.R. Kim2 , C.O. Lee1 , H.K. Lee1 , S.U. Choi1 . 1 Korea Research Institute of Chemical Technology, Research Center for Drug Discovery Technology, Daejeon, South Korea; 2 Korea Research Institute of Chemical Technology, Research Center for Medicinal Chemistry, Daejeon, South Korea Background: Ceritinib is an ALK inhibitor under phase II clinical trial, and it shows an impressive result in ALK fusion positive patients. To get the excellent ALK inhibitors, we synthesized several ceritinib derivatives. Materials and Methods: We made small modification in ceritinib expecially on piperidine and benzene ring. With the newly synthesized compounds, several enzyme assays, such as ALK wt, ALK L1196M, and Insulin Receptor, and cell cytotoxic assays using ALK fusion positive cell lines were done. To see the cytotoxic mechanism, we checked the ALK autophosphorylation, cell cycle and PARP-1 cleavage by western blot. We also did H3122 xenograft assay as in vivo mouse model and kinase profiling assay was done by Millipore.

Poster Session – Molecular Targeted Agents I Conclusion: We made a potent ALK inhibitor by small modification of ceritinib. The in vitro efficacy of KRCA-386 is as good as ceritinib, however, KRCA-386 is much better than ceritinib in vivo efficacy. 394 POSTER (Board P174) Sensitivity of acute myeloid leukemia cells to a urokinase-activated anthrax lethal toxin (PrAgU2/lf) is dependent on uPAR expression and phospho-MEK1/2 levels A. Bekdash1 , S.H. Liu2 , S.H. Leppla2 , A.E. Frankel3 , R. Abi-Habib1 . 1 Lebanese American University, Department of Natural Sciences, Beirut, Lebanon; 2 National Institutes of Health, National Institute of Allergy and Infectious Diseases, Bethesda MD, USA; 3 UT Southwestern Medical Center, Simmons Comprehensive Cancer Center, Dallas TX, USA In this study, we attempt to target AML cells using a urokinase-activated anthrax lethal toxin (PrAgU2/LF). Anthrax lethal toxin consists of protective antigen (PrAg), the cell binding and internalization moiety and lethal factor (LF) the catalytic moiety. PrAg binds cells through ubiquitously expressed anthrax toxin receptors (ANTXR), is cleaved by furin proteases, oligomerizes, binds 3 to 4 molecules of LF and undergoes endocytosis, releasing LF into the cytosol. LF is a metalloprotease that cleaves all mitogen-activated protein kinase (MAPK) kinases. We have replaced the furin activation site of PrAg with a urokinase activation site (PrAgU2), generating PrAgU2/LF, a dual selective toxin targeting both the urokinase plasminogen activation system (uPA/uPAR) and the MAPK pathway. Potency of PrAgU2/LF was tested on 11 human AML cell lines and on CD+34 progenitor bone marrow blasts using a proliferation inhibition assay. uPAR expression and phospho-MEK1/2 levels were determined by flow cytometry. Cell cycle analysis was carried out by propidium iodide (PI)staining and type of cell death was determined by Annexin V/PI and active caspase staining. PrAgU2/LF was cytotoxic to 5 out of 11 AML cell lines (IC50 = 12–151 pM and percent cell death >75%) and induced cell cycle arrest in the surviving fraction of 3 out of the 5 sensitive cell lines. AnnexinV/PI and caspase staining showed an increase in the percentage of cells stained with both annexin V and PI along with negative staining for active caspases indicating that treatment with PrAgU2/LF leads to caspase-independent, non-apoptotic cell death in AML cells. All five PrAgU2/LF-sensitive cell lines had high levels of phospho-MEK1/2 and uPAR expression while the six resistant cell lines either lacked phospho-MEK1/2 (four cell lines) or lacked uPAR expression (two cell lines). Inhibition of uPAR through coincubation with an anti-uPAR antibody or desensitization of cells to MEK1/2 inhibition through growth in the presence of U0126 (MEK1/2 inhibitor) blocked toxicity of PrAgU2/LF to sensitive AML cells indicating the absolute requirement for both uPAR expression and MAPK activation for the activity of this toxin. CD+34 progenitor bone marrow blasts lacked uPAR expression, MAPK activation and were resistant to PrAgU2/LF demonstrating the tumor selectivity of this approach. Results indicate that the urokinase plasminogen activator and the MAPK pathway can be simultaneously targeted in AML using the dual selective, urokinase-activated PrAgU2/LF with cell sensitivity being dependent on uPAR expression and phospho-MEK1/2 levels. 395 POSTER (Board P175) cMet: Proof-of-concept clinical trial with volitinib in patients with advanced papillary renal cell cancer (PRCC)

Figure: Structure of KRCA-386.

T.K. Choueiri1 , B. Escudier2 , S. Kumar Pal3 , E. Jonasch4 , D. Heng5 , T. Powles6 , H.T. Arkenau7 , E. Clark8 , C. D’Cruz8 , M. Frigault8 , A.F. Nash9 , S.R. Morgan9 . 1 Dana-Farber Cancer Institute, Kidney Cancer Center, Boston, USA; 2 Institute Gustave Roussy, Immunotherapy Unit, Villejuif, France; 3 City of Hope, Comprehensive Cancer Center Duarte, California, USA; 4 MD Anderson Cancer Centre, Houston, Texas, USA; 5 Tom Baker Cancer Center, Calgary, Alberta, Canada; 6 St Bartholomew’s Hospital, Medical Oncology Centre, London, United Kingdom; 7 Sarah Cannon Research Institute, Harley Street, London, United Kingdom; 8 AstraZeneca, Gatehouse Park, Boston, USA; 9 AstraZeneca, Alderley Park, Macclesfield, United Kingdom

Results: In ALK enzyme assay, cellular phosphorylation assay, and the cell cytotoxic assay in ALK fusion positive cells, the effect of KRCA-386 is as good as ceritinib. Cell cycle assay and western blot with PARP-1 antibody indicate that ALK inhibitors, ceritinib and KRCA-386, induce the G1/S arrest to result in the apoptosis. Surprisingly, in vivo xenograft model, KRCA386 is much better than ceritinib. The tumor suppression effect of KRCA386 25mpk is better than ceritinib 50mpk. Kinase profiling assay show that KRCA-386 inhibits various kinase which is not inhibited by ceritinib. We anticipate that this characteristic of KRCA-386 enhances the in vivo efficacy.

Met is a receptor tyrosine kinase that is deregulated across multiple cancer types, leading to uncontrolled tumor cell growth, invasion and survival. cMet is amplified in ~5−20% of GC. Met is amplified (~4%) and overexpressed (~50%) in newly diagnosed NSCLC adenocarcinomas. Activating cMet mutations are present in hereditary and a subset of sporadic PRCC cases (up to 21% of type I PRCC). Trisomy of chromosome 7 (containing both cMet and HGF genes) has been reported in 45−75% of sporadic PRCC cases; 81% of type I and 46% of type II PRCC have copy number alterations of cMet. PRCC represents 10−15% of RCC, and is the most common form of non-clear cell RCC.

Poster Session – Molecular Targeted Agents I AZD6094 (HMPL-504, Volitinib) is a potent, selective cMet inhibitor. Preclinically, AZD6094 inhibits in vitro growth of cMet amplified gastric and lung cell lines. In vivo AZD6094 induces regressions in PRCC explant models, cMet-amplified gastric xenografts, and inhibits the growth of cMetexpressing lung xenografts. In Phase I, AZD6094 was well tolerated most AEs are CTC AE grade 1/2 & showed good oral absorption with high exposures and low accumulation risk after multiple oral administrations. Promising anti-tumor activity in multiple PRCC patients receiving AZD6094 was demonstrated in Phase I, which triggered a Phase II trial. An open label, Phase II non-randomised multi-centre study to assess the efficacy of AZD6094 monotherapy in treatment naive and previously treated PRCC patients has been initiated. Eligibility includes PRCC histopathology, ECOG status 0 or 1, ability to comply with the collection of tumor samples, adequate haematological, liver & kidney function, and measurable disease (RECIST 1.1). Patients may be entered into the study based on local PRCC assessment, which is confirmed by a central laboratory to be included in the statistical analysis set. The study employs a 2-stage design with a total number of approximately 75 patients. The primary endpoint is ORR. Secondary endpoints include safety, PFS, OS and DoR. Multiple exploratory translational endpoints are also planned e.g. molecular profiling of tumor samples, circulating tumor DNA for predictive biomarkers, pharmacodynamic changes in total-cMet and phosphorylated-cMet in baseline and on-treatment biopsies. In addition Patient Reported Outcomes will be administered to assess treatment impact & clinical benefit. The study is being delivered in collaboration with Sarah Cannon Research Institute and will open ~20 selected US, Canadian & EU centres. This study is sponsored by AstraZeneca, clinical trial information: NCT02127710. 396 POSTER (Board P176) Gastrointestinal stromal tumor associated with neurofibromatosis type I T. Takahashi1 , T. Nishida2 , R. Nakatsuka1 , M. Kaneda3 , S. Hirota4 , Y. Miyazaki1 , Y. Kurokawa1 , M. Yamasaki1 , H. Miyata1 , K. Nakajima1 , S. Takiguchi1 , M. Mori1 , Y. Doki1 , S. Serada5 , T. Naka5 . 1 Osaka University graduate school of medicine, Department of Surgery, Suita, Japan; 2 National Cancer Center Hospital East, Department of Surgery, Kashiwa, Japan; 3 Osaka University graduate school of medicine, Department of Dematology, Suita, Japan; 4 Hyogo college of medicine, Department of surgical pathology, Nishinomiya, Japan; 5 National Institute of Biomedical Innovation, Laboratory of Immune Signal, Ibaraki, Japan Background: Most gastrointestinal stromal tumors (GIST) have either KIT (80−85%) or PDGFRA (10%) mutations, which are considered to play a key role in the pathogenesis and progression of the tumors. Nearly 10% of GISTs lack these mutations (Wild type GIST). Wild type GIST may be a heterogeneous entity. Neurofibromatosis 1 (NF1) is the most common inheritable disease with estimated birth incidence of 1:4,000 and autosomal dominant transmission). Loss of neurofibromin, a negative regulator of RAS signaling, is thought to be causative for this syndrome. Although NF1 is associated with an increased risk of GIST, which may have no mutation in the both genes, little is known about their pathogenesis and sensitivity to target agents such as imatinib. The purpose of this study is to elucidate frequency of GISTs in the NF1 patients by MDCT screening and to examine clinical and pathological features of NF-1-associated GISTs compared with conventional GISTs and also evaluate sensitivity to imatinib as well as its basic mechanisms. Material and Methods: Study 1 (MDCT screening program): We have recommended MDCT screening and endoscopic examinations for clinically diagnosed NF1 patients visiting Osaka University Hospital. Ninety-five NF1 patients (age median 45 years (17−80), 35 male and 60 female) received MDCT between 2003 and 2012 and prospectively collected data were retrospectively analyzed. Study 2 (retrospective cohort study): We collected 1,335 sporadic GISTs from community hospitals in around Osaka, Japan between 2001 and 2010 retrospectively, and found 26 primary NF1-GISTs (1.8% of total sporadic GIST) and 6 recurrences, of which clinicopathological features such as the sensitivity of molecular target agency were analyzed. Study 3 (biochemical study): Using frozen tumor specimens obtained at each surgery, we preliminary evaluated the intracelullar signal transduction system of NF1-GIST, KIT exon 9 mutated GIST, KIT exon 11 mutated GIST and PDGFRA mutated GIST by western blotting of each tyrosine as well as ser/thr kinases. Using GIST cell line, we evaluated the sensitivity of imatinib by suppressing neurofibromin. Results: 1. NF1-GIST accounts for 1−2% of total sporadic GIST and GIST may occur in 6% of NF1 patients. 2. NF1-GIST mainly located in the small intestine, showed indolent features with spindle cell type and lacked mutations in KIT and PDGFRA with ICC hyperplasia. 3. NF1-GIST has little phosphorylated KIT and appeared to be insensitive to imatinib and sunitinib.

Thursday 20 November 2014 127 4. The sensitivity of imatinib decreased by suppressing neurofibromin in vitro. Conclusions: NF1-associated GIST has distinctive features from conventional sporadic GISTs and KIT-targeted TKI appeared to be ineffective to recurrent and advanced NF1-GISTs. 397 POSTER (Board P177) Characterization of molecular targets of therapy in Non-Small Cell Lung Cancer (NSCLC) utilizing a liquid biopsy S. Greene1 , D. Lu1 , R. Krupa1 , M. Harvey1 , J. Louw1 , A. Jendrisak1 , N. Bales1 , D. Marrinucci1 , J. Gray2 , R. Dittamore1 . 1 Epic Sciences Inc., San Diego CA, USA; 2 Moffitt Cancer Center, Hematology/Oncology, Tampa FL, USA Background: Molecular subtyping of NSCLC has identified molecular drivers of disease progression and enabled an era of precision medicine through the matching of driver alterations with targeted therapy. Currently, testing for alterations to EGFR, KRAS and ALK via tumor biopsy have been incorporated into the standard of care. Further predictive candidate markers include MET, RET and ROS1. However, many NSCLC patients have insufficient tumor sample or have high co-morbidities preventing access to tissue for the molecular characterization of tumors. We sought to develop a platform to enable diagnostic testing to assess clinically actionable NSCLC biomarkers from a single tube of blood utilizing a combination of protein/molecular characterization of CTCs and cfDNA. Material and Methods: 21 samples from NSCLC patients with tissue confirmed EGFRm (9), KRASm (7) and ALK (5) rearrangements were recruited and blood specimens were collected and shipped to Epic Sciences. All nucleated cells were plated onto glass slides and subjected to IF staining and CTC identification by fluorescent scanners and algorithmic analysis. CTCs, defined as traditional (CK+, CD45−, intact DAPI nuclei and morphologically distinct), apoptotic (CK+, CD45−, non-intact nuclei) and CK− (CK−, CD45−, intact and distinct) were identified. Plasma was separated prior to CTC analysis. Biomarker assays were developed utilizing biomarker positive and negative cell lines spiked into blood and run through the Epic process. IF protein assays included: EGFRm, ALK, MET, RET and ROS1; FISH assays: MET , ALK , RET and ROS1. EGFRm and KRASm cfDNA assays were developed utilizing published qPCR techniques. Results: CTC assays were developed for EGFRm, ALK, MET, RET and ROS utilizing combinations of IF and FISH (where applicable). Specificity of the assays were confirmed utilizing appropriate positive and negative cell lines for each molecular target. 3/5 patients with confirmed ALK rearrangements had detectable CTCs, and 1/3 had ALK+ CTCs and ALK rearrangements by FISH. KRASm or EGFRm were detected by qPCR in cfDNA from patients with confirmed mutations, and were not detectable in healthy controls. Patients with higher levels of EGFRm cfDNA also possessed higher frequency of CTCs. ALK protein was observed in traditional, apoptotic, small and CK− CTCs. Conclusion: Characterization of NSCLC biomarkers are feasible utilizing a combination of protein and molecular characterization assays for CTCs and cfDNA from a single tube of blood. These tests demonstrate sensitivity and specificity and may aid in patient selection for standard of care therapies or clinical trials with targeted therapeutics. The identification of ALK positive CTC subtypes identifies unique tumor cell morphology and evidence of epithelial plasticity. Further testing to determine association of CTC subtypes and durability of therapy response is under investigation. 398 POSTER (Board P178) TAS-119 a selective inhibitor of Aurora A kinase, potentiates taxane therapy in breast and lung cancer models Y. Nakatsuru1 , A. Hashimoto1 , H. Sootome1 , K. Ito1 , M. Sakuragi1 , A. Miura1 , N. Oda1 , H. Hirai1 , T. Utsugi1 . 1 Taiho Pharmaceutical Co. Ltd., Tsukuba Research Center, Tsukuba, Japan Background: Aurora A kinase is a serine/threonine kinase that plays important roles in the regulation of the M phase of the mammalian cell cycle. Aberrant expression of Aurora A has been associated with spindle checkpoint dysfunction and increased resistance to the microtubulepolymerizing agents, paclitaxel and docetaxel. Moreover, Aurora A overexpression/gene amplification has been reported in various types of human cancers. We have identified a highly potent and selective Aurora A inhibitor, TAS-119, which enhances the antitumor activity of paclitaxel and docetaxel in multiple cell lines in vitro and in vivo. Previously, we have shown that TAS-119 enhanced tumor growth inhibition by taxane in subcutaneously transplanted cervical, lung and ovarian cancer xenograft models. In this report, we investigated antitumor efficacy of TAS-119 in combination with taxanes in lung and breast cancer xenograft models. Moreover, we tested survival benefit by TAS-119 in preclinical therapeutic models.

128 Thursday 20 November 2014 Material and Methods: For in vitro experiments, cells were exposed to paclitaxel and/or TAS-119. Cell viability was determined by measuring cellular ATP concentration. For in vivo experiments, antitumor efficacy was assessed in subcutaneous HCC-1806-luc (triple negative breast cancer) and NCI-H460-luc (non-small cell lung cancer) xenograft models. The potential of taxane therapy with TAS-119 was evaluated in non-small cell lung cancer orthotopic models of H460-luc tumor in nude mice. To monitor tumor burden, in vivo bioluminescence imaging in individual mice was monitored weekly. Results: TAS-119 selectively inhibited phosphorylation of Aurora A without affecting the activity of Aurora B and Aurora C in NCI-H460 cells. TAS119 showed synergistic cell growth inhibition in combination with paclitaxel in NCI-H460 and HCC1806 cells. TAS-119 clearly enhanced antitumor efficacy of docetaxel in NCI-H460-luc and paclitaxel in HCC-1806-luc nude mice with sc tumors. In the lung orthotopic models, TAS-119 with docetaxel improved median survival, with significant benefit of the combination (155% ILS) over docetaxel alone (67% ILS). Conclusions: TAS-119 has a potential as an enhancer of taxane therapy in NSCLC and TNBC cells both in vitro and in vivo. 399 POSTER (Board P179) Rho-GTPase, RAC1 and Cdc42 mediates Wnt−beta-catenin signals for metastasis associated phenotypes in TNBC: A proof of concept study 1

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N. Dey , P. De , B. Leyland-Jones . Avera Cancer Institute, Department of Molecular & Experimental Medicine, Sioux Falls SD, USA Background: We have observed an upregulation of Wnt-beta-catenin pathway (WP) in triple negative breast cancer (TNBC) and our earlier study demonstrated that Wnt signaling in TNBC is associated with the metastasis and poor prognosis (Dey et al., 2013, BMC Cancer; Dey et al., 2013, PLosOne; Dey et al., 24th EORTC-NCI-AACR Symposium; Dublin 2012; Abs #376). RAC1-GTPase regulates many cellular phenotypes. We studied the role of RAC1/2 in the regulation of a number of different phenotypes including integrin-directed migration (De P et al., 2009, Exp. Cell Res.; De P et al., 2003, JBC). Here we provide pharmacological and genetic evidence that RAC1/Cdc42 mediates WP signals in the context of metastasis-associated phenotypes in TNBC. Material and Methods: Using cBioPortal we found the percentage of alterations in RAC1 gene in Breast Invasive Carcinoma as well as in the PAM50 Basal Breast Invasive Carcinoma. We used two pharmacological tools, (1) WP modulators (CHIR99021, Wnt-C59, XAV939) and (2) RAC1 inhibitors (NSC23766) and one genetic tool (SiRNAs). We studied metastasis-associated phenotypes including, (1) proliferation, (2) clonogenic growth, (3) fibronectin-directed migration, (4) matrigel invasion and (5) vascular mimicry in the context of RAC1 activation in a panel of 3−4 TNBC cell lines. Results: RAC1 Gene is altered in 10% of all 1137 cases of Breast Invasive Carcinoma (TCGA, Provisional) and in 18.5% of all 81 cases of PAM50 Basal Breast Invasive Carcinoma (TCGA, Nature 2012). Transient transfection of beta-catenin SiRNA (1) decreased cellular levels of beta-catenin, inhibited proliferation, attenuated metastasis-associated phenotypes and blocked fibronectin-mediated RAC1 as well as Cdc42 activations. Both Wnt-antagonists and RAC1 inhibitor blocked fibronectin-induced RAC1 activation (pulldown assay) and inhibited various metastasis-associated phenotypes. Conclusions: We have identified a novel Wnt-beta-catenin-RAC1 signaling axis involving small-GTP-RAC1 and Cdc42 for the metastasis in TNBC. Our data shows an obligate role of RAC1-GTP-ase in control of WP mediated metastasis-associated phenotypes in TNBC. The functional link between WP, metastasis and the activation of RAC1 is being currently worked out using RAC1SiRNA and pharmacological inhibitor of RAC1 in brain metastasizing MDA-MB231BR cells the results of which will be presented at the meeting. 400 POSTER (Board P180) Optimization of novel pyrido[2,3-b]pyrazine based small molecule fibroblast growth factor receptor 1, 2, 3 & 4 (FGFR) inhibitors into a potential clinical candidate P. Angibaud1 , O. Querolle1 , V. Berdini2 , G. Saxty2 , A. Cleasby2 , H. Colombel1 , I. Csoka1 , N. Esser3 , R. Gilissen3 , L. Meerpoel3 , C. Paulussen3 , I. Pilatte1 , V. Poncelet1 , D.C. Rees2 , B. Roux1 , T. Verhulst3 , V. Tronel1 , B. Wroblowski3 , C.W. Murray2 , J. Vialard3 . 1 Janssen R&D, Medicinal Chemistry, Val-de-Reuil, France; 2 Astex Pharmaceuticals, Discovery, Cambridge, United Kingdom; 3 Janssen R&D, Oncology, Beerse, Belgium Fibroblast growth factors (FGFs) and their receptors (FGFR 1, 2, 3 and 4) regulate a variety of key cellular processes, including proliferation,

Poster Session – Molecular Targeted Agents I migration, survival, and differentiation. Aberrant activation of the FGF/FGFR pathway is strongly implicated in oncogenic signaling in many tumor types. This has stimulated the development of a number of FGFR inhibitors, with diverse kinase inhibition and pharmacological profiles that are currently being evaluated in clinical studies. We recently reported that a quinoxaline moiety can bind efficiently to the hinge region in the catalytic sites of all FGFR family members. In a continuation of our efforts to identify FGFR 1, 2, 3 and 4 inhibitors we have discovered that pyrido[2,3-b]pyrazine are also efficient FGFR1 hinge region binders. Furthermore, small heterocycles were used to build preferential interactions with amino-acid Asp-641 in the FGFR1 catalytic site without introducing highly basic substitutions. This enabled us to discover potential clinical candidate molecules with nanomolar affinity on FGFR1−4, favourable drug-like properties and anti-tumoral activity in a FGFR2-dependent SNU-16 human gastric carcinoma xenograft model. Here we disclose the structure–activity relationships and ADME properties of novel and orally bioavailable pyrido[2,3-b]pyrazine FGFR inhibitors. 401 POSTER (Board P181) TAS-121, a highly potent and mutant-specific EGFR inhibitor, overcomes T790M-acquired resistance with promising antitumor activity through specific inhibition of mutant EGFR signaling M. Kato1 , K. Miyadera1 , K. Ito1 , Y. Aoyagi1 , A. Hashimoto1 , K. Yonekura1 , Y. Iwasawa1 , T. Utsugi1 . 1 Taiho Pharmaceutical Co. Ltd., Tsukuba Research Center, Tsukuba, Japan Background: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as erlotinib, gefitinib and afatinib, are among the most successful molecular targeted drugs to date. However, a high rate of severe diarrhea and rash due to wild type EGFR inhibition are observed as EGFRTKI-related adverse effects. Moreover, similar to other molecular targeted drugs, acquired resistance arises in most cases, and approximately 60% of its cause was selection and outgrowth of cells with secondary gatekeeper T790M mutation of EGFR. Therefore, EGFR-TKIs that specifically inhibit EGFR harboring mutations including T790M without affecting wild-type EGFR represent a potentially more efficacious and less toxic clinical approach. Materials and Methods: Biochemical assay and cell growth assay were conducted using a microfluidic mobility shift assay and CellTiter-GloTM luminescent cell viability assay, respectively. Antitumor evaluation was analyzed in athymic nude mice and nude rats bearing with NSCLC cell lines, NCI-H1975 harboring EGFR T790M/L858R mutations. Results: TAS-121 was discovered as a mutant selective EGFR inhibitor with excellent oral bioavailability and in vivo antitumor potency. Enzymatic dissociation assay and ATP-competitive assay revealed that TAS-121 had a non-ATP competitive inhibition mode. In cell panel analysis, TAS-121 showed high selectivity to cell harboring activating and resistant mutation of EGFR among approximately one hundred cell lines and engineered Ba/F3-EGFR cell lines. Furthermore, TAS-121 showed a minimal effect on growth of normal human keratinocyte compared with other EGFR-TKIs which induced apoptotic cell death at high concentration. Once daily dosing of TAS-121 inhibited NCI-H1975 tumor growth not only in subcutaneously implantation model in nude mice and nude rats, but also in orthotopic lung implantation model and intra-cranial implantation model in nude mice. Conclusions: These findings suggest that TAS-121 specifically inhibits mutant EGFR, and it leads to high cellular selectivity and potent antitumor activity in various in vivo models. Therefore, TAS-121 may represent a novel therapeutic strategy in the treatment of NSCLC patients harboring activating and resistant mutation of EGFR. It is possible that the lower potency for TAS-121 against wild-type EGFR will translate lower skin and gastrointestinal side-effects. 402 POSTER (Board P182) Characterization of the oncogenic properties of mutant isocitrate dehydrogenase 1 (IDH1R132H) in human primary cells 1 M. Barradas1 , L. Diezma1 , C.M. Perez-Ferreiro ´ , A. Cerezo1 , E. Lospitao1 , 1 S. Peregrina1 , S. Jimenez ´ , N.A. Brooks2 , R. Torres3 , S. Geeganage2 , R. Gilmour2 , S. Velasco-Miguel1 . 1 Spanish National Cancer Research Centre (CNIO), Cell Signalling Therapies, Madrid, Spain; 2 Lilly Research Laboratories, Discovery Oncology, Indianapolis, USA; 3 Lilly Research Laboratories, Quantitative Biology, Alcobendas, Spain

Background: Mutations in the NADP-dependent enzymes isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) occur frequently in various types of human cancer. These mutations result in the inability of the enzyme to convert isocitrate to a-ketoglutarate, producing instead 2-hydroxyglutarate (2HG). 2HG works as an oncometabolite inhibiting a broad family of a-ketoglutarate-dependent dioxygenases. IDH mutations have been

Poster Session – Molecular Targeted Agents I described as an early event in the process of transformation. Our aim is to investigate the potential oncogenic effects of mutant IDH1 in human primary normal cells. Material and Methods: We have over-expressed in human primary fibroblasts a mutant form of IDH1 (IDH1R132H) and performed a weekly serial passage of these cells to study the effect of the mutation on proliferation rate and life span. We have also analyzed the metabolic changes produced by mutant IDH1 using a Seahorse XF Analyzer. Finally, we have carried out transformation assays of these primary cells in the presence of mutant IDH1 combined with the expression of additional oncogenes that affect different tumor suppressor pathways. Results: We have found that mutant IDH1 provokes oncogene-induced senescence (OIS) in normal human primary fibroblasts. This effect requires the accumulation of several population doublings and is the result of a premature accumulation of the tumor suppressor protein p16. IDH1R132Hinduced OIS can be prevented with the treatment of inhibitors of the mutant IDH1, indicating the mechanism is mediated through the effects of the oncometabolite 2HG. In Seahorse experiments, we have shown that the mutant IDH1 expressing cells have a higher metabolic rate, mimicking the effects of other known oncogenes such as H-RASV12. Finally, we have generated immortal cell lines derived from the IDH1-mutant transfected primary fibroblasts through introduction of several transforming hits in order to elucidate the pathways involved in the development of IDH1-mutant tumors. Conclusions: These datademonstrate that mutant IDH1R132H behaves as an oncogene in primary cells, reprogramming their metabolism and activating OIS, but unlike more potent oncogenes such as H-RASV12, this effect requires several population doublings and is likely dependent on downstream pathways regulated by a-ketoglutarate-dependent dioxygenases. 403 POSTER (Board P183) A novel dielectrophoretic microwell array system for detection and single cell analysis of circulating tumor cells from breast cancer patients T. Sawada1 , A. Morimoto2 , T. Mogami2 , K. Iijima2 , Y. Akiyama2 , K. Katayama2 , T. Futami2 , M. Yunokawa3 , K. Tamura3 , M. Watanabe4 , Y. Koh4 , F. Koizumi5 . 1 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Chemotherapy, Tokyo, Japan; 2 TOSOH Corporation, Tokyo Research Laboratory, Tokyo, Japan; 3 National Cancer Center Hospital, Breast Oncology and Medical Oncology, Tokyo, Japan; 4 Third Department of Internal Medicine, Wakayama Medical University, Wakayama, Japan; 5 Tokyo Metropolitan Cancer and Infectious Diseases Center Komagome Hospital, Laboratory Medicine, Tokyo, Japan Background: Molecular characterization of Circulating Tumor Cells (CTCs) at single cell is important for further understanding of tumor cell dissemination in cancer progression, however it remains a technical challenge. We have developed a cell capture system for molecular characterization of single CTCs based on a high-density dielectrophoretic microwell array technology. Material and Methods: 15 advanced breast cancer patients and 2 healthy donors were recruited at National Cancer Center Hospital. CTC enrichment was performed by density gradient centrifugation with RosetteSep® . After CTC enrichment, samples were loaded into the cell entrapment chamber, followed by application of 20 Vp-p AC voltage (1 MHz for 3 minutes), so as to entrap cells in the microwells using the dielectrophoretic force. Then samples were fixed and labeled with DAPI, FITC-anti-cytokeratin (CK), PE-anti-CD45 antibodies, and detected by image-based analysis using a fluorescence microscope. The results of the CTC enumeration were compared with those by CellSearch® system. The DAPI+, CK+/−, CD45− cells were picked up by micropipette, and isolated single cells were subjected to whole genome amplification followed by sequencing in 50 cancer associated-genes on the Ion Torrent PGM platform. Results: In 15 blood samples from patients, CK-positive (+) and CKnegative (−) CTCs (DAPI+, CD45−) detected by our system ranged from 1 to 38 CTCs, and 100% (15/15) of the samples were above the threshold level (1/3 mL). On the other hand, with CellSearch® system, 66.7% of the samples had a 2/7.5 mL threshold level. In 2 blood samples from the healthy donors, both systems detected 0 CTC. Single cell isolation was performed 5 of the 15 samples (17 cells). 16 cells presented at least one mutation in 50 cancer associated-genes (frequency 44.1–100%), indicating that the isolate single cells were positively CTCs. CK-positive CTCs showed hot spot mutations in PIK3CA, TP53, HRAS, and MLH1 (5/7 cells), while Kit, HRAS, MLH1, APC, and SMARCB1 hot spot mutations were found in CK-negative CTCs (7/9 cells). These results demonstrated that single cells isolated by our system are useful for further genomic analysis and may contribute to the discovery of new therapeutic targets.

Thursday 20 November 2014 129 Conclusions: We established an efficient and convenient platform for the capture and characterization of single CTCs. 404 POSTER (Board P184) Targeting PI3K somatic mutations reduces invasion and EMT in squamous cell carcinoma of the lung A. Cavazzoni1 , M. Bonelli1 , F. Saccani1 , S. La Monica1 , M. Galetti1 , C. Caffarra1 , D. Cretella1 , C. Fumarola1 , R. Alfieri1 , P.G. Petronini1 . 1 University of Parma, Department of Clinical and Experimental Medicine, Parma, Italy Background: Squamous cell lung cancer (SQCLC) represents 30−40% of all non-small-cell lung tumors and, to date, no targeted therapies are clinically available. A prominent role in the pathogenesis and metastasization of SQCLC has been attributed to aberrant activation of the phosphoinositide 3-kinase (PI3K) signaling pathway, due to amplification (25−40%) or oncogenic driver mutations E545K and H1047R of the PIK3CA (3−10%). Deregulation of PI3K/AKT/mTOR pathway is involved in lung tumorigenesis and it has been associated with high grade tumors (G3-G4) and advanced disease (stage III). The aim of this study was to analyse the effect of three inhibitors, NVP-BEZ235, NVP-BKM120 and NVP-BYL719, showing different specificity towards PI3K and mTOR proteins, in SQCLC cell lines harboring p110a subunit amplification or mutations. Material and Methods: SKMES-1 cells were stable transfected with plasmids containing wild type, or mutated E545K or H1047R p110a subunit of PI3K and clones showing high activity of PI3K/AKT/mTOR signalling were selected for further investigation. H596 and HCC2450 cells, carrying E545K and H1047R PI3K point mutation respectively, were also included in the study. Results: The PI3KCA mutated/amplified clones showed an increased growth rate both in 2D and 3D cultures but no increased inhibition of cell proliferation after drug treatments was observed with respect to SKMES-1. Since PI3K axis controls cell motility through the activation of RhoA/Rac1/CDC42 proteins, we analyzed the effect of PI3K inhibitors on the invasive phenotype and epithelial–mesenchymal transition (EMT). PI3KCA mutated/amplified clones showed increased migration and invasion properties associated with increased activity of RhoA, CDC42, Rac1 and MMP 2 and 9 proteins. PI3K inhibitors significantly reduced migration/invasion capability, MPPs production, RhoA family activity and EMT only in cells carrying PI3K gain of function. In vivo experiments confirmed that tumors from PI3K mutated clone retained the mesenchymal phenotype and proved the ability of BYL719 in reducing the expression of vimentin in cytokeratin 7 positive cells, whereas we do not observe differences in the change of tumor volume between xenografts from SKMES-1 and mutated clone after BYL719 treatment. Conclusion: The data presented confirm that the use of specific PI3K inhibitors reverted the invasive phenotype and inhibited the EMT in the presence of PI3K gene alterations. 405 POSTER (Board P185) Aurora kinases A and B are required for KRAS-induced lung cell oncogenicity 1 E.O. Ozorio dos Santos1 , M.N. Aoki1 , E. Levantini2 , D.S. Basseres ` . 1 ˜ Paulo, Department of Biochemistry − Institute of University of Sao ˜ Paulo, Brazil; 2 Harvard Medical School, Beth Israel Chemistry, Sao Deaconess Medical Center, Boston, USA

Background: Lung cancer is the leading cause of cancer deaths worldwide, and a significant proportion of lung cancers are associated with driver mutations in the KRAS proto-oncogene. Unfortunately, targeted therapies based on direct KRAS targeting have so far been unsuccessful and intense efforts are underway to identify KRAS targets that play crucial roles in oncogenesis, in order to develop targeted therapies for KRASinduced malignancies. Because serine-threonine mitotic kinases Aurora A (AURKA) and B (AURKB) have been shown to promote KRAS-induced transformation, we hypothesized (1) that AURKA and AURKB are important KRAS targets in lung cancer; and (2) that their inhibition would impair KRAS-mediated oncogenesis. Material and Methods: In order to verify whether oncogenic KRAS induces AURKA or AURKB expression, we first used three cell based-models: (1) an immortalized primary lung epithelial cell line and its isogenic KRAStransformed counterpart, (2) KRAS wildtype H1703 lung cancer cell line engineered to express oncogenic KRAS inducibly, and (3) KRAS positive lung cancer cell lines H358 and A549 stably expressing inducible shRNAs targeting KRAS. Results: In all cases, KRAS expression positively correlated with AURKA and AURKB mRNA and protein expression, indicating that AURKA and AURKB are transcriptional targets of KRAS in lung cancer cells. In order to investigate whether Aurora kinases are therapeutically relevant KRAS

130 Thursday 20 November 2014 targets in lung cancer, we used genetic and/or pharmacological approaches in the abovementioned cells to inactivate AURKA and/or AURKB. Inducible shRNA-mediated knockdown of AURKA or AURKB in KRAS-positive H358 and A549 cell lines, as well as treatment with a dual Aurora kinase inhibitor (AI II, Calbiochem), decreased growth, viability, migration, proliferation, and anchorage-independent growth. In addition, Aurora inhibition in both cell lines efficiently increased cell death by apoptosis. Interestingly, these effects were observed only in the presence of KRAS mutations, and Aurora inhibition had no effect on normal or tumorigenic cells without KRAS mutations or with inhibition of KRAS expression by RNA interference. This suggests that Aurora kinase inhibition therapy can specifically target KRAS transformed cells. Conclusions: In conclusion, our results support our hypothesis that Aurora kinases are important KRAS targets in lung cancer and suggest Aurora kinase inhibition as a novel approach for KRAS-induced lung cancer therapy. 406 POSTER (Board P186) Aflibercept (Zaltrap) directly attenuates the migration and invasion of colorectal cancer cells 1 ´ , M. Ayadi1 , V. Poindessous1 , M. Chiron2 , A. Bouygues1 , P. Mesange E. Dochy3 , T. Andre´ 4 , A. de Gramont4 , A.K. Larsen1 . 1 INSERM & ˆ Universite´ Pierre et Marie Curie Hopital Saint-Antoine, Cancer Biology & Therapeutics Saint-Antoine Research Center, Paris 12, France; 2 Sanofi France, Oncology, Vitry-sur-Seine, France; 3 Sanofi Belgium, Medical Development, Diegem, Belgium; 4 Hopital Saint-Antoine, Medical Oncology, Paris 12, France

Background: Colorectal cancer (CRC) cells express VEGF as well as functional VEGF receptors thereby mediating both paracrine and autocrine VEGF-signaling. Autocrine VEGF-signaling in CRC cells has been associated with resistance to 5-FU and hypoxia as well as with increased migration and invasion. Aflibercept (Zaltrap® also known as zivaflibercept in the United States) is a soluble recombinant protein approved for treatment of metastatic CRC that selectively neutralizes VEGF-A, VEGF-B and PlGF. Material and Methods: HCT-116 CRC cells and their 5-fluorouracil (5-FU) resistant variant were subject to ELISA analysis for the secretion of VEGF-A, VEGF-B and PlGF under normoxia and hypoxia. The Boyden chamber was used to establish the influence of aflibercept on CRC cell migration and invasion. The cytotoxic effects of 5-FU in the absence or presence of aflibercept was determined by the MTT viability assay. Results: The secretion of VEGF-A was significantly up-regulated in the 5-FU resistant cells compared to the parental cells under both normoxia and hypoxia (p < 0.01 and p < 0.001, respectively) as well as in the correspondent tumor xenografts (p < 0.01) whereas the expression of VEGF-B and PlGF was comparable. The increased VEGF-A levels were associated with increased migration of the 5-FU resistant cells, compared to the parental cells, under both normoxia and hypoxia and could be attenuated by aflibercept in both cell lines. Invasion was significantly increased for the 5-FU resistant cells under both normoxia and hypoxia and could be attenuated by aflibercept in the resistant cells under hypoxia. Aflibercept by itself had no detectable influence on the viability of parental or 5-FU resistant cells under normoxia or hypoxia, but was able to increase the sensitivity of the 5-FU resistant cells to 5-FU under hypoxia. Conclusions: Aflibercept directly attenuated the migration of CRC cells under all conditions examined without any influence on the viability. Cells with acquired 5-FU resistance showed up-regulation of VEGF-A associated with increased migration and invasion, especially under hypoxic conditions. Aflibercept inhibited these VEGF-A mediated functions. 407 POSTER (Board P187) Structural basis for inhibition of ligand-dependent and -independent ErbB3 activation by KTN3379 D. Alvarado1 , S. Lee2 , E. Greenlee2 , G.F. Ligon1 , J.S. Lillquist1 , E.J. Natoli1 , J. Amick2 , Y. Hadari1 , J. Schlessinger2 . 1 Kolltan Pharmaceuticals Inc., Department of Research, New Haven, USA; 2 Yale School of Medicine, Department of Pharmacology, New Haven, USA Background: The goal of this study is to understand the structural basis for inhibition of ligand-dependent and ligand-independent activation of ErbB3 by KTN3379, an antibody in phase 1 clinical development. Materials and Methods: X-ray crystallography was used to solve the structure of KTN3379 Fab in complex with the complete ErbB3 ectodomain. Results: Here, we report that the monoclonal antibody KTN3379 robustly inhibits both mechanisms of ErbB3 activation in different tumor model settings. The crystal structure of the KTN3379 Fab in complex with the full ErbB3 extracellular domain reveals that the antibody binds with very

Poster Session – Paediatric Oncology high affinity to a novel and unique epitope in the boundary of domains 2 and 3, and locks the receptor in an inactive conformation. Thus, KTN3379 interferes with the first step of ErbB3 activation, which is required for both ligand-dependent and ligand-independent signaling. Using structureguided mutations, we improved the affinity of the antibody-receptor complex even further by engineering additional contacts of the Fab to domain 2 in ErbB3. Conclusions: Our studies uncover the structural basis for the double mechanism of action of KTN3379 and define a unique epitope within ErbB3. The mechanism of inhibition described here should be also applicable to EGFR and ErbB4. 408 POSTER (Board P188) A potent and selective small molecule inhibitor of MCL-1 sensitizes DLBCL cell lines to the BCL-2 selective inhibitor ABT-199 D.C. Phillips1 , Y. Xiao2 , L. Lam1 , E. Litinovic3 , L. Roberts-Rapp1 , A.J. Souers1 , J.D. Leverson4 . 1 AbbVie Inc., Discovery Oncology, North Chicago, USA; 2 AbbVie Inc., Early Discovery Oncology, North Chicago, USA; 3 Abbott Molecular Inc., Des Plaines, USA; 4 AbbVie Inc., Oncology Development, North Chicago, USA Background: Aberrant expression and/or function of BCL-2 family proteins, which are essential regulators of apoptosis, contribute to the development of cancer. Diffuse Large B-cell lymphoma (DLBCL) represents the most common subtype of non-Hodgkin’s lymphoma (NHL). As a population, DLBCL cell lines positive for the t(14;18) translocation and/or possessing elevated BCL-2 copy number (BCL-2High ) are sensitive to navitoclax or the BCL-2 selective inhibitor ABT-199. Despite this, some BCL-2High cell lines remain resistant to either agent. Herein we utilize novel, selective and potent inhibitors of MCL-1 (A-1210477) or BCL-xL (A-1155463) to evaluate the role of these proteins in resistance to navitoclax and ABT-199 in DLBCL. Materials and Methods: We assessed the cytotoxicity of navitoclax, ABT-199, A-1155463 alone or in combination with the MCL-1 inhibitor A-1210477 or the CDK9 inhibitor flavopiridol in a panel of navitoclaxresistant DLBCL cell lines. Synergistic potential was subsequently determined via Bliss analysis. Additionally, expression of BCL-2 family members was determined by luminex and the mechanism of synergy determined using MesoScale ELISA. Results: We show that the MCL-1 specific inhibitor A-1210477 sensitizes navitoclax-resistant DLBCL cell lines to apoptosis. Chemical segregation of this synergy with the BCL-2 selective inhibitor ABT-199 or BCL-xL selective inhibitor A-1155463 indicated that MCL-1 and BCL-2 are the two key anti-apoptotic targets for sensitization. Synergy between navitoclax and A-1210477 in the majority of BCL-2Low DLBCL cell lines was BCL-xL driven. The CDK9 inhibitor flavopiridol down-regulated MCL-1 expression and synergized with ABT-199 in BCL-2High DLBCL cell lines, correlating with the synergy observed between ABT-199 and A-1210477. Conclusions: Collectively these data emphasize that BCL-2 status is predictive of ABT-199 efficacy in DLBCL not only as a single agent, but also in the adjuvant setting with anti-tumorigenic agents that modulate MCL-1 levels. These studies also potentially identify a patient population (BCL-2Low ) that would benefit from BCL-xL (navitoclax) driven combination therapy. Disclosures: DCP, YX, LL, LRR, AJS & JDL are AbbVie employees and are stock holders. The design, study conduct and financial support were provided by AbbVie. AbbVie participated in the data generation, interpretation of data, review and approval of this publication. EL is an employee of Abbott Molecular Inc.

Paediatric Oncology 409 POSTER (Board P189) Transcription factor activating protein 2 beta (TFAP2B) mediates neuronal differentiation in neuroblastoma F. Ikram1 , S. Ackermann1 , F. Roels1 , R. Volland1 , B. Hero1 , F. Hertwig1 , 3 H. Kocak1 , D. Dreidax2 , K.O. Henrich2 , F. Berthold1 , P. Nurnberg ¨ , F. Westermann2 , M. Fischer1 . 1 University Hospital of Cologne, Department of Pediatric Oncology, Cologne, Germany; 2 German Cancer Research Center, Department of Tumor Genetics, Heidelberg, Germany; 3 University of Cologne, Cologne Center for Genomics (CCG), Cologne, Germany Background: Induction of tumor cell differentiation by retinoic acid is an important part of current neuroblastoma treatment protocols. The molecular mechanisms underlying differentiation processes in neuroblastoma, however, are still poorly understood. Genes of transcription factors of

Poster Session – Paediatric Oncology the AP-2 family (TFAP2) play important roles in embryonic development and differentiation. We aimed at investigating the role of TFAP2B in neuroblastoma pathogenesis and differentiation. Methods: The association of TFAP2B expression with prognostic markers and outcome was analyzed in 649 primary neuroblastomas using microarray data. Methylation of CpG sites related to TFAP2B was investigated by using Illumina 450K arrays in 105 primary neuroblastomas. To evaluate the functional relevance of TFAP2B in neuroblastoma, TFAP2B was re-expressed in IMR-32 and SH-EP neuroblastoma cells, and knocked down in SH-SY5Y and BE(2)-C neuroblastoma cells. Results: Low expression of TFAP2B was associated with adverse patient outcome (p < 0.001) and unfavorable prognostic markers, e.g. stage 4 disease, age >18 months, MYCN amplification, and unfavourable gene expression-based classification (p < 0.001 each). In addition, low TFAP2B expression was strongly correlated with methylation of promoter regions of the TFAP2B gene in high-risk neuroblastoma. In IMR-32 cells, demethylation with 5-Aza-2 -deoxyctidine induced TFAP2B expression. Tetracycline inducible re-expression of TFAP2B in IMR-32 and SH-EP cells significantly impaired proliferation and led to G1-arrest (p < 0.01 each). Morphological signs of neuronal differentiation and senescence were observed after TFAP2B induction in IMR-32 and SH-EP cells, respectively. Knock-down of TFAP2B by lentiviral transduction of specific shRNAs effectively abrogated retinoic acid induced neuronal differentiation of SHSY5Y and BE(2)-C cells as indicated by microscopic examination and lack of up regulation of neuron related genes such as microtubule associated protein 2 (MAP2), neurofilament middle chain (NEFM) and synaptophysin (SYP). Conclusion: Our data suggest that TFAP2B expression is silenced in highrisk neuroblastoma by promoter methylation, and that TFAP2B expression might be critical for differentiation in neuroblastoma. 410 POSTER (Board P190) Pediatric Preclinical Testing Program (PPTP) Evaluation of the p53−MDM2 Antagonist MK-8242 M. Smith1 , M. Kang2 , P. Reynolds2 , R. Lock3 , H. Carol3 , R. Gorlick4 , A. Kolb5 , J. Maris6 , S. Keir7 , J. Wu8 , R. Kurmasheva9 , P. Houghton9 . 1 NCI, Cancer Therapy Evaluation Program, Bethesda, USA; 2 Texas Tech University Health Sciences Center, Cancer Center, Lubbock, USA; 3 Children’s Cancer Institute Australia, Leukemia Biology Program, Randwick, Australia; 4 Montefiore Medical Center, Department of Pediatrics, Bronx, USA; 5 A.I. duPont Hospital for Children, Department of Oncology, Wilmington, USA; 6 Children’s Hospital Philadelphia, Hematology-Oncology, Philadelphia, USA; 7 Duke University Medical Center, Department of Surgery, Durham, USA; 8 St. Jude Children’s Research Hospital, Biostatistics, Memphis, USA; 9 Nationwide Children’s Hospital, Center for Childhood Cancer, Columbus, USA Introduction: TP53 is a tumor suppressor frequently inactivated in cancer by gene mutations or defective signaling. Mutated p53 is uncommon in many childhood cancers and hence agents able to free p53 from inhibitory mechanisms, and induce apoptosis, may have therapeutic benefit in the pediatric setting. MK-8242 is a selective inhibitor of p53−MDM2 binding that frees p53 from negative control, activating the p53 pathway in cancer cells leading to cell cycle arrest and apoptosis. Methods: MK-8242 evaluated against the 23 cell lines of the PPTP in vitro panel using 96 hour exposure at concentrations from 1 nM to 10 mM. MK8242 was tested against the PPTP in vivo panel focusing on p53 wild type (WT) xenografts at a dose of 125 mg/kg (solid tumors) and 75 mg/kg (leukemias) administered orally days 1−5 and 15−19 followed by 23 days of observation. MDM2 and p53 expression for the PPTP models tested were available through Affymetrix U133 Plus 2.0 arrays (Clin Cancer Res 2008:14:4572−83). Results: In vitro, the median IC50 for MK-8242 was 0.07 mM for p53 WT cell lines versus >10 mM for p53 mutant cell lines. In vivo, MK-8242 was well tolerated, with 40 of 43 xenograft models evaluable for efficacy. MK-8242 induced a two-fold or greater delay in time to event in 9 of 21 (43%) p53 WT solid tumor xenografts, including: 1/1 rhabdoid tumor, 2/2 Wilms tumor, 2/3 Ewing, 3/6 rhabdomyosarcoma models, but no neuroblastoma (n = 4) or osteosarcoma (n = 6) models. Objective responses were observed in 7 solid tumor xenografts: maintained complete response (MCR) or complete response (CR) for a Wilms tumor, a Ewing sarcoma, an embryonal rhabdomyosarcoma, one medulloblastoma, and two ependymomas, and partial responses (PR) for a rhabdoid tumor xenograft. For the systemicdisease ALL panel, among 8 xenografts there were 2 CR and 6 PR. Two additional MLL-rearranged xenografts (MV4;11 and RS4;11) grown subcutaneously were also tested, with the former showing MCR and the latter tumor demonstrating PR. Solid tumor xenografts with mutant p53 (EW5, Rh10, and Rh30R) showed progressive disease (PD1, or PD2) to MK-8242, as expected. The osteosarcoma xenografts were p53 WT, but had very low p53 expression and low MDM2 expression, and they failed to

Thursday 20 November 2014 131 respond to MK-8242. The ALL xenografts expressed the highest levels of p53 and MDM2 among the PPTP panels and showed the most consistent responses to MK-8242. Conclusions: MK-8242 induced tumor regressions across multiple solid tumor histotypes and induced CRs or PRs for most ALL xenografts. This high level of activity supports prioritization of MK-8242 for further evaluation. 411 POSTER (Board P191) Druggability of p16 deleted pediatric leukemia: The novel cell line POETIC3 identifies potential agents and drug combinations for mechanism based targeted therapeutics J. Reimer1 , A. Kovulchuk2 , Y. Ruan1 , R. Shah1 , A. Jayanthan1 , M. Perinpanayagam1 , T. Truong1 , I. Auer-Grzesiak3 , J. Luider3 , O. Kovulchuk2 , T. Trippett4 , A. Narendran1 . 1 Alberta Children’s Hospital, Calgary Alberta, Canada; 2 University of Lethbridge, Department of Biological Sciences, Lethbridge Alberta, Canada; 3 Calgary Laboratory Services, Calgary Alberta, Canada; 4 Memorial Sloan-Kettering Cancer Center, New York NY, USA Background: Loss by homozygous deletion of the p16 gene (CDKN2) has been found in many human malignancies, including leukemia. Loss of p16 leads to loss of cell cycle inhibition, enabling the potential for unregulated malignant cell proliferation. Gene knockout experiments suggested that the loss of p16 leads to increased tumor development. The incidence and prognostic significance of p16 deletions in childhood ALL have been described in a number of recent studies but the molecular mechanisms the mediate these processes are currently unclear. Methods: A continuously growing cell line was established, following ethics board approval and written informed consent, from a 13 year-old female patient with blast populations that with mono- and bi-allelic deletion of CDKN2A. After cloning and expansion over more than 20 passages the cell line retained the molecular and cell surface markers of the parental cells. Using a p16 normal leukemia line and normal lymphocytes as control, these cells were then subjected to gene expression profiling and evaluation for the activation status of markers of cell cycle regulation and associated cellular functions such as proliferation, survival and DNA damage repair activity. An in vitro drug sensitivity screen was carried out using a panel of approximately 200 targeted therapeutics. Results: Gene expression profiling and gene ontology pathway analyses identified a number of significant alterations including the pathways of cell cycle control, spliceosome, DNA replication, purine pyrimidine synthesis and DNA mismatch repair and identified alterations in various nodes in the cell cycle regulatory network. Western blot analysis showed increased constitutive activity of ERK and expression of cyclin B and gH2AX compared to control. Drug screening studies identified a distinct group of effective targeted agents including aurora kinase inhibitors (VX680, AZD1152), CDK inhibitors (PD0332991, dinaciclib) and mTOR inhibitors (AZD8055, INK128 and deforolimus). Drug combination studies with the CDK inhibitor PD0332991 identified strong synergy with the conventional chemotherapeutic agents asparaginase and cytarabine, moderate synergy with methotrexate and additive activity with doxorubicin. Discussion: In this study, we report the establishment of a cell line derived from p16 deleted leukemia cells of a pediatric patient. The availability of this cell line provides means to identify the consequence of this molecular aberration with respect to altered growth regulatory pathways and allows screening of libraries of potential targeted therapeutic agents for future studies. 412 POSTER (Board P192) The Pediatric Preclinical Testing Program (PPTP): Analysis of the first 10 years in vivo testing P. Houghton1 , R. Lock2 , H. Carol2 , R. Gorlick3 , A. Kolb4 , J. Maris5 , S. Keir6 , J. Wu7 , M. Kang8 , P. Reynolds8 , R. Kurmasheva1 , M. Smith9 . 1 Nationwide Children’s Hospital, Center for Childhood Cancer, Columbus, USA; 2 Children’s Cancer Institute, Leukemia Biology Program, Randwick, Australia; 3 Montefiore Medical Center, Department of Pediatrics, Bronx, USA; 4 A.I. duPont Hospital for Children, Department of Oncology, Wilmington, USA; 5 Children’s Hospital Philadelphia, Hematology-Oncology, Philadelphia, USA; 6 Duke University Medical Center, Department of Surgery, Durham, USA; 7 St. Jude Children’s Research Hospital, Biostatistics, Memphis, USA; 8 Texas Tech University Health Sciences Center, Cancer Center, Lubbock, USA; 9 NCI, Cancer Therapy Evaluation Program, Bethesda, USA The PPTP is a consortium established by the NCI to evaluate novel therapeutics and drug combinations using in vitro and in vivo models of childhood cancers. Its primary objective is to identify agents that have

132 Thursday 20 November 2014 significant antitumor activity in preclinical models of childhood cancers that may be prioritized for clinical testing. The PPTP has characterized a panel of 23 cell lines and 57 xenograft models in the primary testing panel representing most of the more common pediatric solid malignancies and acute lymphoblastic leukemia subtypes (ALL). Additional cell line-derived and PDX models are available for expanded testing should an agent show activity against specific cancer types in the primary screen. Molecular characterization includes expression profiles, SNP analysis, and exome sequencing for all models. Agents tested include small molecule drugs, antibodies, antibody-conjugates, and replication competent viruses. The preclinical models accurately identified known clinically active drugs (vincristine, cyclophosphamide, cisplatin, topotecan), with an overall response rate of 40% (70/175 tests). In contrast, the overall response rate for 21 signaling inhibitors was ~2%, despite clear pharmacodynamic evidence showing target inhibition in several cases. For example, the MEK inhibitor selumetinib (AZD6244) was active against only a BRAF(V600E) mutated glioma xenograft among 45 models tested. Inhibitors of the IGF-1 receptor-TOR pathway induced regressions in 9 of 321 models tested (2.8%), with activity largely limited to sarcoma. Agents showing high activity at exposures in mice not achieved in patients at comparable schedules included the Aurora A kinase inhibitor alisertib, the Polo-like kinase inhibitor BI-6727, and the pre-pro-drug PR-104. At relevant patient exposures PR104 demonstrated activity only in T cell ALL models that have high AKR1C3 activity. The tubulin binding agent eribulin demonstrated broad activity against solid tumors and ALL models at dose levels relevant to clinical exposure, including 4 of 5 Ewing sarcoma models in which vincristine had low activity. Genomic explanations for ‘exceptional responders’ were identified for the MEK inhibitor selumetinib (see above) and for the PARP inhibitor BMN 673 (PALB2 biallelic mutations). Agents demonstrating high activity against leukemia models included the CD19-targeted antibody drug conjugate SAR-3419, the MDM2 inhibitor RG7112 and the Bcl-2 family inhibitor ABT-263. Agents (or combinations) that the PPTP identified as active that have advanced to clinical testing in children include: selumetinib in low-grade glioma (NCT01089101); alisertib in ALL and neuroblastoma (NCT01154816); NTX-010 (NCT01048892); temsirolimus + cyclophosphamide + vinorelbine for rhabdomyosarcoma (NCT01222715); BMN 673 + temozolomide (NCT02116777), and eribulin (NCT02082626). Supported by NO1-CM-42216 from the NCI. 413 POSTER (Board P193) Next-generation sequencing identifies the mechanism of tumourigenesis caused by loss of SMARCB1 in malignant rhabdoid tumours M.A. Finetti1 , M. Selby1 , A. del Carpio Pons1 , J. Wood1 , B. Skalkoyannis1 , A. Smith1 , S. Crosier1 , S. Bailey1 , S. Clifford1 , D. Williamson1 . 1 Newcastle University, NICR, Newcastle, United Kingdom Introduction: Malignant Rhabdoid Tumours (MRT) are unique malignancies caused by biallelic inactivation of a single gene (SMARCB1). SMARCB1 encodes for a protein that is part of the SWI/SNF chromatin remodelling complex, responsible for the regulation of hundreds of downstream genes/pathways. Despite the simple biology of these tumours, no studies have identified the critical pathway involved in tumourigenesis. The understanding of downstream effects is essential to identifying therapeutic targets that can improve the outcome of MRT patients. Methods: RNA-seq and 450k-methylation analyses have been performed in MRT human primary malignancies (n > 30) and in 4 MRT cell lines in which lentivirus was used to re-express SMARCB1 (G401, A204, CHLA266, and STA-WT1). The dynamics of chromatin binding and remodelling following SMARCB1 re-expression has been analysed by ChIP-seq. The MRT cell lines were treated with 5-aza-2 -deoxycytidine followed by global gene transcription analysis (RNA-seq and 450k-methylation) to investigate how changes in methylation lead to tumourigenesis. Results: We show that primary MRTs present a unique and distinct expression/methylation profile which confirms that MRT broadly constitute a single and different tumour type from other paediatric malignancies. However despite their common cause MRT can be can sub-grouped by location (i.e. CNS or kidney). We observe that re-expression of SMARCB1 in MRT cell lines determines activation/inactivation of specific downstream pathways such as IL-6/TGFbeta. We also observe a direct correlation between alterations in methylation and gene expression in CD44, GLI2, GLI3, p16, p21 and JARID after SMARB1 re-expression. Loss of SMARCB1 also promotes expression of aberrant isoforms and novel transcripts and causes genome-wide changes in SWI/SNF binding. Conclusion: The next generation transcriptome and methylome analysis in primary MRT and in functional model give us detailed downstream effect of SMARCB1 loss in MRTs. The integration of data from both primary and functional models has provided, for the first time, a genome-wide catalogue of SMARCB1 tumourigenic changes (validated using systems

Poster Session – Paediatric Oncology biology). Here we show how a single deletion of SMARCB1 is responsible for deregulation of expression, methylation status and binding at the promoter regions of potent tumour-suppressor genes. The genes, pathways and biological mechanisms indicated as key in tumour development may ultimately be targetable therapeutically and will lead to better treatments for what is currently one of the most lethal paediatric cancers. 414 POSTER (Board P194) Genomic profiling using a clinical next generation sequencing (NGS) assay reveals genomic alterations to guide targeted therapy in advanced neuroblastoma patients S. Ali1 , E.M. Sanford1 , M.J. Hawryluk1 , J. Chmielecki1 , K. Wang1 , G.A. Palmer1 , N.A. Palma1 , D. Morosini1 , R. Erlich2 , R. Yelensky1 , D. Lipson1 , J.S. Ross1 , Y. Mosse3 , P.J. Stephens1 , J.M. Maris3 , V.A. Miller1 . 1 Foundation Medicine Inc, Cambridge, USA; 2 Foundation Medicine Inc, Biomedical Informatics, Cambridge, USA; 3 Childrens Hospital of Philadelphia, Philadelphia, USA Introduction: High-risk neuroblastoma patients have a survival rate below 50% despite dose-intensive chemoradiotherapy. Treatment using molecularly targeted therapy could more effectively manage patients with less toxicity, but would be best deployed via rational guidance from identification of genomic alterations (GAs) that suggest responsiveness to such therapies. We reviewed the GAs in 143 advanced, high-risk neuroblastoma cases who underwent prospective genomic profiling by clinical next generation sequencing (NGS) using Foundation Medicine assays to identify actionable alterations that might allow successful trials of targeted therapy. Methods: Genomic profiling was performed on 154 tumors from 143 patients, with 8 patients repeatedly profiled on distinct specimens. Genomic profiling was performed using a clinical NGS assay (Foundation Medicine1) able to simultaneously characterize all classes of GAs (base substitutions, small insertions/deletions, copy number alterations, and rearrangements) on primary tumors or metastatic specimens, either pre- or post-treatment. For each specimen, a minimum of 182–236 cancer-related genes were sequenced to a minimum of 250× coverage. Clinically relevant GAs were defined as those for which there are FDA-approved agents and/or agents being evaluated in clinical trials. Results: Patient characteristics: median age 6 (range 0−71 yrs); 86 males, 57females. A minimum of 87 cases had metastatic disease. Specimens were sequenced to an average depth of greater than 500×, and GA were present in 76% (109/143) of cases. These 143 cases harbored 202 GAs (1.4 alterations per tumor; range 1 to 7). 84 cases (59%) percent of cases harbored at least one actionable GA, with a mean of 1.0 actionable GAs per tumor (range 1 to 4). GAs in MYCN (31 cases; 21.6%), ALK (27 cases; 18.9%, including 1 fusion gene) and ATRX (12 cases; 8.4%) were identified. Clinically relevant genomic alterations included CDK4 or CDK6 amplification (9 cases, 6.3%), RPTOR amplification (6 cases, 4.2%), HGF amplification (5 cases, 3.5%), FGFR1 base substitution (4 cases, 2.8%), and BRAF amplification (3 cases, 2.1%). One case harbored a novel BEND5-ALK fusion and the patient showed a response to crizotinib. Conclusions: Profiling the tumor genomes of 143 high-risk neuroblastoma cases with led to the identification of clinically relevant GAs not traditionally searched for in this patient group. In particular, alterations in ALK , CDK4/6, FGFR1, BRAF and mTOR pathway genes are linked to targeted therapies available for the treatment of relapsed and refractory neuroblastoma through agents on the market or in clinical trials that show potential to positively impact this devastating disease 415 POSTER (Board P195) Comprehensive next generation sequencing of solid tumors from 669 adolescents and young adults reveals a distinct spectrum of targetable genomic alterations D. Morosini1 , K. Wang2 , K. Wagner3 , B. Gershenhorn4 , R. Yelensky5 , D. Lipson6 , J. Chmielecki7 , S.M. Ali8 , J.S. Ross9 , P.J. Stephens10 , V.A. Miller11 . 1 Foundation Medicine Inc, VP of Clinical Development, Cambridge, USA; 2 Foundation Medicine Inc, Bioinformatics, Cambridge, USA; 3 Banner MD Anderson Cancer Center, Medical Oncology, Gilbert, USA; 4 CTCA, Medical Oncology, Chicago, USA; 5 Foundation Medicine Inc, Biomarker and Companion Diagnostic Development, Cambridge, USA; 6 Foundation Medicine Inc, Computational Biology, Cambridge, USA; 7 Foundation Medicine Inc, Cancer Genomics, Cambridge, USA; 8 Foundation Medicine Inc, Medical Affairs, Cambridge, USA; 9 Foundation Medicine Inc, Pathology, Cambridge, USA; 10 Foundation Medicine Inc, CSO, Cambridge, USA; 11 Foundation Medicine Inc, CMO, Cambridge, USA Background: Adolescents and young adult (AYA, age 15−39) cancers (CA) pose unique challenges for patients and clinicians, yet genomic alterations

Poster Session – Paediatric Oncology (GA) in AYA CA have not been well characterized. To identify potential therapeutic targets, we profiled 669 AYA solid tumors, and describe 2 cases highlighting the efficacy of matching GA to targeted therapy. Methods: Tumor DNA extracted from FFPE tissue underwent library construction and hybrid capture for all exons of 236 CA-related genes and 47 introns of 19 genes frequently rearranged in CA and was sequenced to an average depth of >700× (Foundation Medicine, Cambridge, MA). Sequence data were assessed for base substitutions, insertions/deletions, copy number alterations, and rearrangements. We sought to characterize GAs in AYA and compare those with historical estimates for non-AYA CA patients. Results: 26 AYA tumor types were profiled; the most common sites of origin were breast (BC), (16%), brain (13%), sarcoma (9%), colon (9%), gynecologic (7%) and lung (LC) (7%). An average of 3.4 GAs/sample was found (range 0−22). Potentially actionable GAs were identified in 71% of cases. ALK fusions were found at 8× the frequency in AYA LC vs. LC patients >39; FISH concordance and outcomes data were obtained in half of the ALK positive AYA LC patients. AYA brain CA patients with a diagnosis of GBM had a >15 fold increase in ATRX and IDH1 mutations compared to GBM patients >39; As GBMs in young adults typically arise from astrocytomas (vs. de novo in older adults) and increased ATRX/IDH1 mutations are a known feature of astrocytomas, our findings are consistent with previous literature. AYA BC had high concordance in general with BC patients >39, with relative decreases in GA frequency in PIK3CA, MYST3, and CDH1 in the AYA population. Although not the primary focus of this study, 2 patient vignettes were notable. In one case, a 19 y/o F with LC was admitted to hospice having failed SOC chemotherapy. Prior testing was negative for EGFR, ALK and ROS1. Genomic profiling revealed a novel ALK rearrangement and she began crizotinib therapy. Within 5 days she was discharged to home and scans at 1 and 2 months showed PR by RECIST. A 39 y/o F with a rectal neuroendocrine tumor and BRAF V600E mutations experienced an 8 month response to vemurafenib with relief of tumor-related symptoms that is ongoing. Conclusion: Comprehensive genomic profiling can detect all classes of GAs across AYA tumor types. These data show significant differences with literature based reports of GA in adult CA and demonstrate the feasibility and relevance of identifying GAs that can potentially guide targeted treatment decisions in a high proportion of patients. 416 POSTER (Board P196) Results of phase I study of bolus 5-fluorouracil in children and young adults with recurrent ependymoma K.D. Wright1 , D.C. Turner2 , K.M. Haddock2 , M.O. Jacus2 , K.E. Harstead2 , S.L. Throm2 , V.M. Daryani2 , G.W. Robinson1 , G.T. Armstrong3 , A. Onar-Thomas4 , C.F. Stewart2 , A. Gajjar1 . 1 St Jude Children’s Research Hospital, Oncology, Memphis Tennessee, USA; 2 St Jude Children’s Research Hospital, Pharmaceutical Sciences, Memphis Tennessee, USA; 3 St Jude Children’s Research Hospital, Epidemiology and Cancer Control, Memphis Tennessee, USA; 4 St Jude Children’s Research Hospital, Biostatistics, Memphis Tennessee, USA Background: Established antineoplastic antimetabolite, 5-Fluorouracil (5-FU) possesses antitumor activity in ependymoma tumor-bearing mice administered as an intravenous (IV) bolus but not as a continuous infusion. We investigated the safety of weekly bolus 5-FU in children with recurrent ependymoma. Secondary objectives included documentation of toxicities and preliminary antitumor activity. Materials and Methods: We initiated a phase I dose escalation study of weekly IV bolus 5-FU in children and young adults with recurrent ependymoma every 4 weeks followed by a 2 week rest period, constituting once cycle. The starting 5-FU dosage was 500 mg/m2 . We treated a maximum of 3 cohorts of patients at 400 (n = 6), 500 (n = 15) and 650 (n = 5) mg/m2 , de-escalating due to toxicity. The first 6 weeks of therapy comprised the dose-limiting toxicity (DLT) evaluation period. During the study, there appeared to be an association between hematologic toxicity and prior number of therapies. Heavily pre-treated patients defined as those with more than 2 prior chemotherapy regimens (range 3−7) and all of whom had been re-irradiated did not tolerate the 500 mg/m2 dose. Further analysis revealed a significant relationship (p = 0.03) between prior number of chemotherapeutic regimens and toxicity. As such, the dose was re-escalated to 500 mg/m2 in a less heavily pretreated cohort (n = 6) of patients. Results: Twenty-three of twenty-six patients with recurrent anaplastic ependymoma enrolled were evaluable. Eight patients with stable disease or partial response remained on study a median of 3 courses (range 2−9), one of whom elected to come off study after 6 courses due to compliance issues. Five patients experienced grade 4 neutropenia, two at 650 mg/m2 and the other 3 at 500 mg/m2 , all of whom were heavily pretreated. One patient experienced grade 3 diarrhea. Interestingly, complete and partial

Thursday 20 November 2014 133 responses within metastatic lesions were noted in certain patients whose index lesion at the primary site progressed. At 500 mg/m2 , the median 5-FU Cmax , AUC0−tlast , and a t1/2 were 848 mM, 195 mM×hr, and 8.0 min, respectively. Conclusions: Bolus 5-FU is well tolerated in less heavily pretreated patients and possesses anti-tumor activity. The maximum tolerated dose for this cohort of patients is 500 mg/m2 given as an IV bolus once weekly for 4 consecutive weeks of a 6 week period. 417 POSTER (Board P197) Evaluating the activity of the p53−MDM2 inhibitor NDD0005 in Ewing sarcoma J. Pecqueur1 , B. Vormoor1 , Y. Zhao1 , H. Newell1 . 1 Northern Institute for Cancer Research, Newcastle upon Tyne, United Kingdom Background: Ewing sarcoma (ES) is the second most common paediatric bone malignancy. 90% of ES tumours retain wild-type p53. Small molecule inhibitors of the p53−MDM2 interaction have been developed which prevent binding of the MDM2 regulatory protein to p53, enabling wild-type p53 to promote growth inhibition and apoptosis in malignant cells. Nutlin-3a, a cisimidazoline derivative, has been shown to induce selective cytotoxicity in ES cell lines with wild-type p53. Investigation of other compound series, such as the spiro-oxindoles (MI-63) and the isoindolinones (NDD0005), is required to extend these findings. We aimed to assess p53 pathway activation by NDD0005 and evaluate the growth inhibitory and cytotoxic effects of NDD0005 compared with nutlin3a and MI-63 in CADO-ES1 (p53 wild-type) and TC71 (p53 mutant) ES cell lines. NDD0010 was also examined as the less active enantiomer of NDD0005 and served as a control compound. Methods: Protein expression following NDD0005 treatment was analysed by Western blotting. Growth inhibition of CADO-ES1 and TC71 cells by NDD0005, nutlin-3a and MI-63 was assessed in XTT studies. Cytotoxicity of these inhibitors was assessed by clonogenic assays.

Growth inhibition (XTT assay), mean GI50 ±SD (mM) NDD0005 NDD0010 MI-63 Nutlin-3a

Cytotoxicity (clonogenic assay), mean LD50 ±SD (mM)

CADO-ES1

TC71

p-value

CADO-ES1

TC71

p-value

2.5±0.60 15±2 1.5±0.25 1.5±0.61

12±3.2 14±2.6 18±1.5 18±5.3

0.0067 0.63 <0.0001 0.0058

9.7±2.1 1.9±1.5 2.0±0.92 3.0±2.2

19±1.5 19±1.5 21±1.5 >30

0.0031 0.62 <0.0001 <0.0001

Results: NDD0005 induced an increase in p53 and p53 target genes in CADO-ES1 cells. As expected, growth inhibition (XTT assays) for single agent NDD0005, Nutlin-3a and MI-63 occurred at concentrations 5- to 12-fold lower in the p53 wild type CADO-ES1 cells compared to the p53-mutated TC71 cells. The cytotoxicity as assessed by clonogenic assays in CADO-ES1 cells was equivalent to growth inhibition for MI-63 and Nutlin-3a treatment. NDD0005 was found to be less potent in cytotoxicity assays as compared to growth inhibition assays, with a 4 fold higher LD50 than in growth inhibition assays. TC71 cells remained viable at high inhibitor concentrations. Conclusions: p53−MDM2 inhibitors stabilised p53 in wild-type p53 ES cells. NDD0005 produced selective growth inhibition and cytotoxicity in CADO-ES1 cells, as did Nutlin-3a and MI-63. The potent and specific activity of p53−MDM2 inhibitors in wild-type p53 cells supports the potential future application of p53−MDM2 inhibitors as ES therapeutics. 418 POSTER (Board P198) Population pharmacokinetics of intravenous bolus 5-fluorouracil in a phase I trial for children and young adults with recurrent ependymoma D.C. Turner1 , K.M. Haddock1 , M.O. Jacus1 , K.E. Harstead1 , S.L. Throm1 , V.M. Daryani1 , C.F. Stewart1 , K.D. Wright2 . 1 St Jude Children’s Research Hospital, Pharmaceutical Sciences, Memphis Tennessee, USA; 2 St Jude Children’s Research Hospital, Oncology, Memphis Tennessee, USA Background: 5-Fluorouracil (5-FU), a pyrimidine analog, possesses antitumor activity in ependymoma tumor-bearing mice administered by intravenous (IV) bolus. (Atkinson, Cancer Cell, 2012). Materials and Methods: We initiated a phase I study of IV bolus 5-FU in children and young adults with recurrent ependymoma to investigate the pharmacokinetics (PK) of weekly bolus 5-FU at 3 dosage levels (400, 500, and 650 mg/m2 ) and compare patients’ 5-FU systemic exposures with those observed in the preclinical mouse model. Due to a lack of published

134 Thursday 20 November 2014 PK data in children, dosage selection was guided by preclinical doseexposure relationships. Plasma PK studies were conducted in consenting patients on days 1, 8, and 22 of 5-FU administration of course 1 and day 1 of course 2. Serial plasma samples were collected before and at the end of the 5-minute infusion (EOI), then 15, 30, 60, 90, 120 (±10), and 180 minutes (±10) after the EOI. Plasma samples were analyzed via a validated, high performance liquid chromatographic assay. Plasma concentration-time data were fit with a 2-compartment nonlinear mixed effect model to estimate population pharmacokinetic parameters, including inter- and intra-subject variability. Estimated pharmacokinetic parameters included central compartment volume (Vc ), total systemic clearance (Clt ), and intercompartmental rate constants (Kcp , Kpc ). Results: Population estimates for Clt and Vc were 8.3 L/hr/m2 and 3.8 L/m2 , respectively, with large interindividual variability (%CV) of 60% for Clt and 80% for Vc . The distributional decay of 5-FU was rapid with a median posthoc alpha half-life t1/2,alpha of 7.9 minutes. Clt was similar during Course 1 and also between courses 1 and 2. Typical plasma AUC values in our population at the lowest dosage (400 mg/m2 ) exceeded those observed in the preclinical ependymoma mouse model (123 mM·hr) suggesting efficacious exposures were likely achieved in the tumors of children receiving bolus 5-FU. Conclusions: Our data provide initial pharmacokinetic parameters to describe the disposition of 5-FU given as an intravenous bolus to children with ependymoma. Furthermore, PK modeling and simulation using accurate murine models in preclinical testing can be applied to derive starting pediatric drug dosages for clinical study. 419 POSTER (Board P199) Targeted inhibition of casein kinase II (CK2) produces a strong therapeutic effect in pediatric leukemia S. Dovat1 , C. Song1 , C. Gowda1 , K.J. Payne2 . 1 Penn State College of Medicine, Pediatrics, Hershey PA, USA; 2 Loma Linda University, Anatomy, Loma Linda CA, USA Background: High-risk acute lymphoblastic leukemia (ALL) is characterized by an aggressive clinical course and high mortality. Previous studies established that the function of the Ikaros tumor suppressor is impaired in this type of leukemia. The Ikaros (IKZF1) gene encodes a DNAbinding protein that regulates transcription of its target genes via chromatin remodeling. Casein kinase II (CK2) is a tumor-promoting enzyme that is essential for cellular proliferation. CK2-mediated phosphorylation of Ikaros in vivo interferes with its DNA-binding activity and subcellular localization. We hypothesize that the targeted inhibition of CK2 will restore Ikaros function and have an anti-leukemia effect. Materials and Methods: CK2 activity in primary B-ALL cells was determined by kinase assay. CK2 inhibition was achieved by shRNA or by a specific inhibitor. In vivo DNA binding was determined by quantitative chromatin immunoprecipitation (qChIP). Gene expression was analyzed by quantitative real-time PCR (qRT-PCR). Cellular proliferation was assayed by trypan blue and MTT test. Primary B-cell ALL and B-ALL human-mouse xenografts were used to test the in vivo therapeutic effect of CK2 inhibitors. Results: Kinase assays show that CK2 activity is over 5-fold increased in primary B-ALL cells as compared to normal bone marrow cells. Treatment of B-ALL cell lines and primary B-ALL cells with CK2 inhibitors results in impaired cellular proliferation, suggesting that elevated CK2 activity is essential for proliferation of B-ALL cells. Molecular inhibition (by shRNA) and pharmacological CK2 inhibition (with TBB) is associated with increased DNA-binding of Ikaros to promoter regions of its target genes, as evidenced by qChIP. Ikaros target genes included a large set of genes that are essential for cell cycle progression (e.g. CDC2, CDC16, CDC25A). Increased Ikaros binding following CK2 inhibition results in epigenetic changes. These are consistent with the formation of the repressive chromatin around sites of Ikaros binding at target genes and transcriptional repression of cell cycle-promoting genes leading to cell cycle arrest. In vivo treatment of human-mouse xenografts with a specific CK2 inhibitor resulted in a strong therapeutic, anti-leukemia effect with prolonged survival of the treated mice as compared to controls. Analysis of the in vivotreated leukemia cells from xenografts showed strong Ikaros binding and repression of cell cycle-promoting genes via chromatin remodeling. Conclusions: In summary, our results identified CK2 as a novel therapeutic target in B-cell ALL and suggest that CK2 inhibition can enhance the tumor suppressor function of Ikaros resulting in transcriptional repression of cell cycle-promoting genes and an anti-leukemia effect. Supported by National Institutes of Health R01 HL095120, and the Four Diamonds Fund Endowment.

Poster Session – Paediatric Oncology 420 POSTER (Board P200) Analysis of genomic alterations in Ewing sarcoma (German cohort) reveals cooperating mutations and novel therapy targets G.H.S. Richter1 , K. Agelopoulos2 , E. Schmidt2 , K. von Heyking1 , B. Moser2 , H.U. Klein3 , U. Kontny4 , M. Dugas3 , K. Poos5 , E. Korsching5 , 7 8 8 T. Buch6 , G. Kohler ¨ , C. Rossig ¨ , D. Baumhoer9 , H. Jurgens ¨ , 10 S. Burdach1 , W.E. Berdel2 , C. Muller-Tidow ¨ , U. Dirksen8 . 1 Klinikum ¨ Munchen, rechts der Isar Technische Universitat ¨ Children’s Cancer Research Center and Department of Pediatrics, Munchen, ¨ Germany; 2 Westfalian Wilhelms University of Muenster, Medicine A, Munster, ¨ 3 Germany; Westfalian Wilhelms University of Muenster, Institute of Medical Informatics, Munster, ¨ Germany; 4 Medical Faculty RWTH Aachen University, Pediatrics, Aachen, Germany; 5 Westfalian Wilhelms University of Muenster, Institute of Bioinformatics, Munster, ¨ Germany; 6 Technische ¨ Munchen, Universitat ¨ Institute for Medical Microbiology Immunology and Hygiene, Munchen, ¨ Germany; 7 Hospital Fulda, Institute for Pathology, 8 Fulda, Germany; Westfalian Wilhelms University of Muenster, Pediatric Hematology and Oncology, Munster, ¨ Germany; 9 Bone Tumor Reference Center at the Institute of Pathology, Pathology, Basel, Switzerland; 10 ¨ University Hospital Halle, Klinik fur ¨ Innere Medizin IV Hamatologie und Onkologie Landeszentrum fur ¨ Zell- und Gentherapie, Halle, Germany Ewing sarcoma (ES) is a bone and soft tissue malignancy that arises predominantly in the pelvis and long bones in children, adolescents and young adults with early metastasis to lung and bone. Genetically, ES is defined, in the majority of patients, by balanced chromosomal EWS/ETS translocations, which give rise to oncogenic chimeric proteins (EWS−ETS). EWS−FLI1 is the most common fusion protein and the result of a balanced t(11;22)(q24;q12) translocation. Further contributing somatic mutations involved in disease development have only been observed at low frequency. A low mutation rate appears to be a general feature of pediatric cancers, in particular in fusion driven tumors. However, little is known about cooperating events that affect oncogenesis and/or progression of ES. Material and Methods: Patient material was obtained from clinical studies of the Cooperative Ewing Sarcoma Study Group (CESS). We analyzed the genetic landscape of ES by next generation sequencing. We performed whole exome sequencing on 52 individual ES as well as whole genome sequencing and transcriptome analysis on a subset of samples. Exoms were sequenced as 100 bp paired-end runs on HiSeq2000/2500 systems (Illumina, San Diego, CA, USA). WGS was performed at Complete Genomics using 10 mg of high molecular grade DNA. Sequences were mapped to the National Center for Biotechnology Information (NCBI) reference genome build 37. To discover relevant somatic missense mutations, the data were filtered. Significance of discovered mutations was analyzed using the MuSig algorythm. FGFR-expression was determined on 42 ES samples by immunohistochemistry, amplification of FGFR was analyzed by qRT PCR in 41 patients. For FGFR knock down, ES cell lines were lentivirally transduced with the vector pLKO-GFP containing 2 different shRNA against human FGFR1. In vivo tumor growth was assessed using Rag2−/−gC−/− mice on a BALB/c background were obtained from the Central Institute for Experimental Animals (Kawasaki, Japan). Results: Mutation counts were generally low but relapsed tumors consistently showed a 2−3-fold increased number of mutations. We identified a number of genes recurrently mutated in ES including coiledcoil domain containing protein 19 (CCDC19, at 11.5%), stromal antigen 2 (STAG2, at 11.5%), schlafen family member 11 (SLFN11; at 7.7%), fibroblast growth factor receptor (FGFR) 1 at 2%, and others. Their overall low mutation frequency argues against additional recurrent driver mutations for this disease. However, an activating point mutation on chr 8 in the FGFR1 locus in one patient, frequent amplification of the FGFR1 locus (31.7%) in primary tumors and widespread high level expression suggested an important role for FGFR1 in this disease. RNA-Interference mediated knock down of FGFR1 expression in ES cell lines strongly inhibited proliferation and completely suppressed tumor growth in a xenograft model. Conclusion: Taken together, our data provide evidence that integration of sequencing data with gene amplification, expression and functional analysis are crucial to identify oncogenic drivers in cancers with low rates of recurrent mutations. FGFR1 constitutes a relevant target for novel therapeutic approaches in Ewing sarcoma.

Poster Session – Paediatric Oncology 421 POSTER (Board P201) Molecular profiling for factors predicting sensitivity or resistance to therapy in relapsed child cancer F. Saletta1 , C. Wadham2 , J. Byrne1 , D. Ziegler3 , G. McCowage4 , M. Haber5 , G. Marshall3 , M. Norris2 . 1 The Children Hospital at Westmead, Children Cancer Research Unit, Westmead, Australia; 2 Children’s Cancer Institute, Molecular Diagnostics, Randwick, Australia; 3 Sydney Children’s Hospital, Kids Cancer Centre, Randwick, Australia; 4 The Children Hospital at Westmead, Oncology department, Westmead, Australia; 5 Children’s Cancer Institute, Experimental Therapeutics, Randwick, Australia Background: Despite the overall success achieved in treating childhood cancers, a major challenge is to find alternative treatment options for those children who suffer relapse. This requires more detailed understanding of the molecular pathways responsible for maintaining and promoting an aggressive drug resistant phenotype, as well as the successful targeting of these pathways. Material and Methods: We implemented a molecular profiling approach of individual tumors, which includes low-density expression microarray analysis (a panel of 96 marker genes), next-generation sequencing (Ion Proton sequencing hotspot panel, 90–100 genes) and automated immunohistochemistry (IHC, 30 antibodies to 25 proteins/phosphoproteins). Results: Preliminary testing has been conducted on 12 cell lines representing the major childhood cancer subtypes, as well as 8 patientderived primary cultures including 4 inflammatory myofibroblastic tumors, 2 diffuse intrinsic pontine gliomas and 2 acute lymphoblastic leukemias (ALL). This revealed tumor-specific expression patterns (i.e. EGFR and BCL2 gene expression in neuroblastoma) and, in some cases, significant positive associations between gene mutations and expression levels (e.g. increased cKIT levels in association with cKIT N822K in Kasumi acute myeloid leukemia cells and overexpression of R110L TP53 in neuroblastoma SKN-DZ cells). A retrospective study is ongoing using diagnostic frozen and formalin fixed paraffin embedded samples from 55 pediatric cancer patients who subsequently experienced relapse, plus 25 ALL patientderived mouse xenograft cell samples. We will determine the frequency of specific molecular targets within these populations, and use IHC staining to confirm expression profiling and sequencing results, where relevant. To do this, we are constructing cell line, diagnostic tumor and ALL xenograft tissue microarrays. Mice bearing patient-derived ALL xenografts will allow in vivo testing of the efficacy of molecularly targeted drugs. Conclusion: We have implemented a screening approach to identify molecular targets in pediatric cancer patients who have failed the best current treatments available, and for whom treatment options following relapse are extremely limited. The ultimate goal is to undertake a multicentre prospective clinical trial in paediatric patients with recurrent or refractory solid tumors or leukaemias for whom targeted drug therapy will be identified using molecular profiling. 422 POSTER (Board P202) CBL0137, a novel NFkB suppressor and p53 activator, is highly effective in pre-clinical models of neuroblastoma M. Haber1 , J. Murray2 , L. Gamble2 , A. Carnegie-Clark1 , H. Webber1 , M. Ruhle1 , D. Carter3 , A. Oberthur4 , M. Fischer4 , D. Ziegler1 , G.M. Marshall3 , K. Gurova5 , C. Burkhart6 , A. Purmal6 , A.V. Gudkov5 , M.D. Norris2 . 1 Children’s Cancer Institute Australia, Experimental Therapeutics, Sydney, Australia; 2 Children’s Cancer Institute Australia, Molecular Diagnostics, Sydney, Australia; 3 Children’s Cancer Institute Australia, Molecular Carcinogenesis, Sydney, Australia; 4 University of Cologne, Children’s Hospital Cologne, Cologne, Germany; 5 Roswell Park Cancer Institute, Department of Cell Stress Biology, Buffalo, USA; 6 Cleveland BioLabs Inc., Oncology Efficacy Evaluation, Buffalo, USA Background: CBL0137 is a carbazole-based anti-cancer agent with a unique mechanism of action. It is an indirect inhibitor of the chromatin remodeling complex FACT (Facilitates Chromatin Transcription). Inhibition of FACT by CBL0137 modulates the activity of several transcription factors involved in cancer: NF-kB and HSF1 are suppressed, while p53 is activated (Science Transl. Med, 2011). We have examined FACT expression in neuroblastoma as well as the efficacy of CBL0137 in preclinical models of this disease. Material and Methods: Expression profiles of650 primary untreated neuroblastomas were analyzed for the expression of two FACT subunits, SSRP1 and SPT16, and related to clinical outcome. Colony-forming assays were used to study the effect of CBL0137, either alone or combined with chemotherapeutic drugs in human MYCN-amplified neuroblastoma cell lines. Cohorts of homozygous TH-MYCN mice with small palpable tumours, or BE(2)-C xenografted nude mice, were treated with CBL0137, alone or combined with chemotherapeutic drugs.

Thursday 20 November 2014 135 Results: High levels of two FACT subunits were associated with MYCN amplification, and were strongly predictive of poor neuroblastoma outcome (p < 0.0001). As a single agent, CBL0137 administered iv had remarkable anti-tumour activity in both mouse models. CBL0137 synergized strongly with a variety of chemotherapeutic agents, including vincristine, cisplatin, etoposide and cyclophosphamide, both in vitro in colony forming assays and in vivo, in TH-MYCN tumour-bearing mice. Most dramatic results were observed when CBL0137 was combined with cyclophosphamide/topotecan, a highly active therapy for relapsed neuroblastoma that is currently in clinical trial for newly diagnosed patients. Cylophosphamide/topotecan plus CBL0137 resulted in cure of 90% of tumour-bearing MYCN-transgenic mice and a doubling of the lifespan of BE(2)-C xenografted nude mice. CBL0137 also synergized strongly with the alternate relapse backbone, irinotecan/temozolomide. Conclusion: These results are superior to any combination chemotherapy regimens we have tested in these models, and a Phase I COG trial of CBL0137 in refractory pediatric cancer patients is currently being planned. 423 POSTER (Board P203) RNA helicase A is essential for 1p36 gene KIF1Bb tumor suppression in neuroblastomas Z.X. Chen1 , K. Wallis2 , S.M. Fell2 , V.R. Sobrado2 , M.C. Hemmer2 , D. Ramskold ¨ 2 , Z. Choo1 , U. Hellman3 , R. Sandberg2 , R.S. Kenchappa4 , T. Martinsson5 , J.I. Johnsen6 , P. Kogner6 , S. Schlisio2 . 1 National University of Singapore, Department of Physiology, Singapore, Singapore; 2 Ludwig Cancer Research, Ludwig Cancer Research, Stockholm, Sweden; 3 Ludwig Cancer Research, Ludwig Cancer Research, Uppsala, Sweden; 4 Moffitt Cancer Center, Neuro-Oncology Program, Tampa, USA; 5 ¨ University of Gothenburg, Department of Clinical Genetics, Goteborg, Sweden; 6 Karolinska University Hospital, Department of Women’s and Children’s Health, Stockholm, Sweden Background: Developmental apoptosis of neuronal precursors is crucial in determining the final number of terminally differentiated cells. During neural development, cells undergo apoptosis as growth factors such as NGF becomes limiting. Abnormal NGF signaling or aberrant developmental apoptosis is implicated in pediatric nervous system tumors. Several genes act upon a developmental apoptotic pathway that is activated when NGF becomes limiting for neuronal progenitors and requires KIF1Bb. KIF1Bb is necessary and sufficient for neuronal apoptosis during NGF withdrawal. KIF1Bb maps to 1p36.2, a region that is frequently deleted in neural crestderived tumors including neuroblastomas. Methods: Large-scale immunoprecipitation followed by mass spectrometry, cloning and mutagenesis studies, apoptosis assays, immunofluorescence, lentiviral expression or shRNA-based studies, siRNA silencing, RNA-SEQ, RT-PCR, immunohistochemistry, NGF withdrawal experiments, patient studies and mouse developmental/conditional knockout models are the key methods used. Results: We identified a transcriptional basis for KIF1Bb-induced apoptosis, which requires a RNA/DNA helicase known as RNA helicase A (DHX9). KIF1Bb interacts with DHX9 to promote translocation of cytoplasmic DHX9 into the nucleus, resulting in transcription of apoptotic XIAPassociated factor 1 (XAF1). Transcription-impaired or nuclear localizationimpaired DHX9 is unable to potentiate KIF1Bb-induced cell death. Knockdown of DHX9 also protects from KIF1Bb-induced cell death whereas negative KIF1Bb domain or patient-associated point mutants are unable to translocate cytoplasmic DHX9 into the nucleus. Furthermore, XAF1 silencing protects from KIF1Bb-induced apoptosis whereas XAF1 overexpression is able to induce apoptosis under low KIF1Bb condition. Conditional knockout of KIF1Bb also specifically ablates XAF1 expression, suggesting that KIF1Bb and XAF1 act along the same pathway. Conclusion: Recent literature strongly pointed to KIF1Bb as a bonafide tumor suppressor. Our findings provide a mechanistic understanding of this role, whereby KIF1Bb interacts with cytoplasmic DHX9 leading to its accumulation in the nucleus to initiate a unique transcriptional signature that includes apoptotic effectors such as XAF1.

136 Thursday 20 November 2014

Poster Session – Toxicology

Toxicology 424 POSTER (Board P204) Nonclinical safety assessment of a humanized anti-OX40 agonist antibody, MOXR0916 R. Prell1 , W. Halpern1 , J. Beyer1 , J. Tarrant1 , S. Sukumaran1 , M. Huseni1 , R. Kaiser2 , D. Wilkins2 , S. Karanth2 , H. Chiu1 , J. Ruppel1 , C. Zhang1 , K. Lin1 , L. Damico-Beyer1 , J. Kim1 , H. Taylor1 . 1 Genentech, Safety Assessment, South San Francisco CA, USA; 2 Charles River Laboratories, Preclinical Services, Reno NV, USA Background: Cancer immunotherapy (CIT) has the potential to be a transformative approach for oncology patients. Inhibition of immune checkpoints, such as PD-1, PD-L1 and CTLA-4, has been the primary approach to date. Agonizing OX40, a co-stimulatory TNF superfamily receptor, presents an alternative CIT target. Here we describe the results of the nonclinical safety studies in cynomolgus monkeys to enable initial clinical studies of an OX40 agonist antibody, MOXR0916, in patients with advanced or metastatic solid tumors. Methods: The in vivo effects of MOXR0916 were initially assessed in a 4-week repeat dose pilot toxicity, toxicokinetics (TK), and pharmacodynamic (PD) study in cynomolgus monkeys at dose levels of 0, 0.01, 0.3 or 10 mg/kg. This was followed by a GLP 6-week repeat dose toxicity, TK, PD, and cardiovascular (CV) safety pharmacology study in cynomolgus monkeys at dose levels of 0, 0.5, 5, or 30 mg/kg. In each study, MOXR0916 was administered via IV bolus injection every 2 weeks for a total of 3 or 4 doses, respectively. Endpoints evaluated included body weights, food consumption, neurological evaluations, physiological parameters, pulse oximetry, respiration rates, continuous CV and hemodynamic parameters, clinical pathology, urinalysis, serum cytokines, peripheral immune cell populations, peripheral receptor occupancy, TK, anti-therapeutic antibodies (ATA), organ weights, gross and microscopic pathology. Results: Administration of MOXR0916 every other week for up to 6 weeks (total of four doses) was generally well tolerated in cynomolgus monkeys at doses up to 30 mg/kg. Findings in the pilot and GLP studies were generally consistent between both studies and included acute post dose reactions and transient increases in acute phase proteins and select chemokines/cytokines in a subset of ATA positive animals starting primarily after the second MOXR0916 administration. These changes were limited to monkeys below the 10 mg/kg dose level and may be directly related to the development of ATAs. There was no indication of persistent T, B, or NK cell activation in the periphery or secondary lymphoid tissues, and no evidence of excessive cytokine release in either study. Conclusion: MOXR0916 was generally well tolerated in cynomolgus monkeys at doses up to 30 mg/kg when administered every other week for 6 weeks and suggests that a Phase I clinical trial testing MOXR0916 could be initiated safely in patients with refractory solid tumors. 425 POSTER (Board P205) Evaluation of drug reactions to anti-neoplastic agents in Phase I clinical trials 1

2

3

4

1

1

M. Bupathi , J. Hajjar , K. Hess , S. Bean , D. Karp , F. Meric-Bernstam , A. Naing1 . 1 MD Anderson Cancer Center, Investigational Cancer Therapeutics, Houston TX, USA; 2 Baylor College of Medicine, Department of Immunology Allergy and Rheumatology, Houston TX, USA; 3 MD Anderson Cancer Center, Department of Biostatistics, Houston TX, USA; 4 MD Anderson Cancer Center, Department of Pharmacy, Houston TX, USA Background: Anti-neoplastic drug reactions can be divided into two categories: IgE mediated hypersensitivity or systemic infusion reactions due to cytokine release. Methods: We conducted a retrospective study evaluating all patients who received anti-neoplastic therapy by the Phase I Investigational Cancer Therapeutics group from January 1, 2013 to November 1, 2013 in the outpatient treatment center. Anti-neoplastic therapy in the form of targeted therapy {monoclonal antibody & small molecules}, immunotherapy, and conventional chemotherapy were given IV or PO. Demographics, type of cancer, symptoms at time of reaction, white blood cell count around time of infusion, severity of reaction, duration of infusion prior to reaction, medications given at time of the reaction, number of infusions post reaction, re-occurrence with re-challenge, and total number of reactions were evaluated. Results: 597 patients received anti-neoplastic agents during this interval, of which 9 patients (4 male and 5 female) had drug reactions, none were IgE medicated anaphylactic reactions. All 9 patients had an ECOG of 0 or 1. The average WBC count around the infusion time was 6.4 (K/uL). The cancer diagnosis of those with reactions were gastric (N = 2), colorectal

(N = 1), pancreatic (N = 1), uterine (N = 1), cervical (N = 1), head and neck (N = 1), non-small cell lung cancer (N = 1), and adnexal carcinoma (N = 1). Five patients were treated with monoclonal antibody, three patients treated with conventional chemotherapy and one was patient treated with a small molecule inhibitor. On average reactions occurred 58 minutes (range 44– 120) after initiating therapy. Severity of the reaction per CTCEA v 4.0 ranged from grade 1 to 2. Four of the 8 patients who were re-challenged with the same therapy developed a similar reaction. Conclusion: Our results indicate that drug reactions to anti-neoplastic agents are rare and most of the reactions are not IgE related anaphylactic reactions. Most of the systemic infusion reactions were due to treatment with monoclonal antibodies. 426 POSTER (Board P206) Serum levels of CCL22 and CCL25 might predict skin rash induction the commonest adverse event by bendamustine in the treatment of malignant lymphoma Y. Terui1 , R. Kuniyoshi1 , Y. Mishima1 , K. Hatake1 . 1 Japanese Foundation for Cancer Research, Clinical Chemotherapy, Tokyo, Japan Introduction: Bendamustine has been recently approved for the treatment of low-grade malignant lymphoma. In spite of the better efficacy, skin rash is the commonest adverse event by bendamustine in the treatment of malignant lymphoma. To understand the mechanism of the skin rash, we prospectively examine the relationship between skin rash and chemokines. Methods: After informed consent of the patients who would received bendamustine (B) or bendamustine + rituximab (BR), the sera were collected before and after the treatment of B or BR. Cytokine/Chemokine array was performed by Bio-Plex system. Results: 20 patients with FL (n = 18), MCL (n = 1), and low-grade B-cell lymphoma (n = 1) were enrolled in this study. All patients were treated with B or BR regimen. 9 patients showed skin rash after the treatment of bendamustine. In Cytokine/Chemokine array, serum levels of CCL22 and CCL25 before the treatment were significantly higher in skin rash group, and serum levels of CXCL10 and CXCL11 after skin rash were higher significantly. Conclusion: Serum levels of CCL22 and CCL25 might predict skin rash induction − the commonest adverse event by bendamustine − and CXCL10 and CXCL11 might contribute to induction of skin rash by bendamustine. 427 POSTER (Board P207) Hematotoxicity potential of new drug candidates measured in hematopoietic progenitors in bone marrow samples J. Ballesteros1 , D. Primo2 , P. Hernandez1 , A. Robles1 , A.B. Espinosa2 , E. Arroyo2 , V. Garcia-Navas1 , J. Sanchez-Fenoy1 , M. Jimenez1 , M. Gaspar1 , J.L. Rojas1 , J. Martinez-Lopez3 , J. Gorrochategui1 . 1 Vivia Biotech, Tres Cantos-Madrid, Spain; 2 Vivia Biotech, Salamanca, Spain; 3 Hospital 12 de Octubre, Hematology, Madrid, Spain Background: Hematotoxicity is a major toxicity concern of oncology drug candidates. Bone marrow failure contributes significantly to morbidity and mortality by inducing severe infections and bleedings. Hematotoxicity of drug candidates is not investigated until expensive preclinical studies in dogs. There is a need to estimate human hematotoxicity in early drug development. The aim of this study is to measure depletion analysis of hematopoietic stem cells (HSCs) that could reflect the degree of drug’s induced hematotoxicity for oncology candidates, using our flow cytometrybased automated Exvitech© platform. Material and Methods: 10 Normal Bone Marrow (NBM) samples at diagnosis from lymphoma patients prior to any therapeutic intervention with confirmed absence of BM infiltration by flow cytometry were included. For a first approach, we have selected two known and related nucleoside cytotoxic drugs (cytarabine and clofarabine). A multiple staining (CD45v450/Anexin-FITC/CD117-PE/CD34PerCP/CD38APC/CD19APCya7) was capable to identify and distinguish the most immature population (CD34+ /CD45dim /CD38+ or CD38− ), B-precursors (CD34+ /CD45dim /CD19+ ) from the more mature B-(CD45+ /CD19+ /SSClo ), or T-(CD45+ /CD19− /SSClo ) lymphocytes. Drug response was evaluated as a depletion survival index of each cell population relative to the average of 6 control wells in each plate. Results: As expected, both drugs induce hematotoxicity in most of the studied persons’ samples, but not all. Using the same drug concentration for each drug for all the patient samples, results reflect that cytarabine has similar activity as clofarabine in terms of efficacy (Emax: 28% vs 27%) but with 33-fold less potency (EC50 : 7mM vs 0.21mM) in the immature population. This reflects a lower hematological toxicity which is consistent with clinical practice. Interestingly, for both drugs there is a large range of interpatient variability inside this population in terms of efficacy (cytarabine,

Poster Session – Toxicology range Emax: 2−76% and clofarabine, range Emax: 12−42%) and potency (cytarabine, range EC50 : 3−14mM and clofarabine, range EC50 : 0.01−2mM) suggesting that in a subsets of vulnerable patients, drug doses could be tailored. Conclusion: Vivia ExviTech© platform is able to measure HSCs depletion in addition to other cell populations for novel drugs or before patient’s treatment that could contribute to a more selective drug development or a better clinical management of patients. This approach can be applied to small molecule or biologics, and combinations of said drugs that form the basis of treatments. This approach can be applied early in discovery to select among hit candidates, or in development to identify combinations with synergistic hematotoxicity.

Thursday 20 November 2014 137

th

26 EORTC–NCI–AACR Symposium on Molecular Targets and Cancer Therapeutics

Friday 21 November 2014

Poster Session – Drug Synthesis

Friday 21 November 2014

Friday 21 November 2014 141

11:00–13:00

PLENARY SESSION 8

Targeting RAS and Other Driver Oncogenes 428 ORAL PRESENTATION Clinical acquired resistance to combined RAF/EGFR or RAF/MEK inhibition in BRAF mutant colorectal cancer (CRC) patients through MAPK pathway alterations R. Corcoran1 , E.M. Coffee1 , E. Van Allen2 , L.G. Ahronian1 , N. Wagle2 , E.L. Kwak1 , J.E. Faris1 , A.J. Iafrate1 , L.A. Garraway2 , J.A. Engelman1 . 1 Massachusetts General Hospital, Cancer Center, Boston Massachusetts, USA; 2 Dana Farber Cancer Institute, Cancer Center, Boston Massachusetts, USA Background: BRAF mutations occur in ~10% of CRC and confer poor prognosis. While RAF inhibitor monotherapy leads to response rates of 60−80% in BRAF mutant (BRAFm) melanoma, response rates in BRAFm CRC are poor (~5%). Promising recent studies with RAF inhibitor-based combinations, including combined RAF/EGFR or RAF/MEK inhibition, have produced higher response rates in BRAFm CRC, but a better understanding of acquired resistance mechanisms will be critical to improving therapy. Methods: Mechanisms of acquired resistance to RAF/EGFR or RAF/MEK inhibition were evaluated in BRAFm CRC cell lines and in clinical biopsies obtained from BRAFm CRC patients following disease progression after initial response or prolonged stable disease to either therapeutic combination. BRAFm CRC cell lines were cultured in the presence of combined RAF/EGFR or RAF/MEK inhibitors until resistant clones emerged. Candidate resistance mutations were identified through exome sequencing. Matched pre-treament, post-progression, and normal DNA from BRAFm CRC patients treated with either RAF/EGFR or RAF/MEK combinations were analyzed by whole exome sequencing to identify clinical acquired resistance mechanisms. Results: In resistant clones generated from BRAFm CRC cell lines selected with either RAF/EGFR or RAF/MEK inhibitor combinations, KRAS exon 2 mutations were identified. KRAS mutation led to sustained MAPK pathway activity and cross-resistance to either RAF/EGFR or RAF/MEK inhibitor combinations. Interestingly, the triple combination of RAF/EGFR/MEK inhibition was able to suppress MAPK activity and overcome resistance. In a BRAFm CRC patient with prolonged stable disease on a RAF/EGFR inhibitor combination, whole exome sequencing identified the presence of KRAS amplification in a progressing lesion. RNA sequencing of the same lesion confirmed KRAS transcript overexpression, and ~25-fold amplification of KRAS in the progressing lesion and absence of amplification in the pre-treatment biopsy was confirmed by FISH. In one patient with a minor response to a RAF/MEK inhibitor combination, whole exome sequencing identified amplification of the c-mer oncogene (MERTK) receptor tyrosine kinase as the likely acquired resistance mechanism. In another patient with a minor response to a RAF/MEK inhibitor combination, whole exome sequencing identified subclonal ARAF and MEK1 mutations in the post-progression biopsy, suggesting the presence of heterogeneous acquired resistance mechanisms in this progressing lesion. Conclusions: The identification of alterations affecting the MAPK pathway in BRAFm CRC patients who have developed clinical acquired resistance to RAF/EGFR or RAF/MEK inhibitor regimens underscores the importance of the MAPK pathway in this cancer. Understanding the mechanisms of acquired resistance can lead to novel combination strategies to overcome or delay resistance.

Friday 21 November 2014

Poster Sessions Drug Synthesis 429 POSTER (Board P001) Development of extracellular signal-regulated kinase 5 (ERK5) inhibitors for anti-cancer therapy S. Myers1 , N. Martin1 , R. Bawn1 , T. Blackburn1 , L. Barrett1 , T. Reuillon1 , B. Golding1 , R. Griffin1 , T. Hammonds2 , I. Hardcastle1 , H. Leung3 , D. Newell1 , L. Rigoreau2 , A. Wong2 , C. Cano1 . 1 Northern Institute For Cancer Research, Newcastle Upon Tyne, United Kingdom; 2 Cancer Research Technologies, London, United Kingdom; 3 The Beatson Institute for Cancer Research, Glasgow, United Kingdom Background: Extracellular signal-regulated kinase 5 (ERK5) is a member of the protein kinase superfamily, which plays an essential role in the transduction of extracellular signals to intracellular effectors. Activation of the ERK5 signalling pathway is associated with cell survival, proliferation, and differentiation, and thus ERK5 over-expression may have implications in carcinogenesis. High levels of ERK5 are associated with poor patient prognosis and the presence of bony metastases in advanced prostate cancer. It has recently been discovered that ERK5 is also involved in the development and progression of hepatocellular carcinoma (HCC). Therefore, the discovery and development of small molecule inhibitors of ERK5 is a desirable therapeutic area.

Method: Two potent inhibitors (1 and 2) have been described in the literature and provide insight into the therapeutic effect caused by ERK5 inhibition and further guide our structure activity relationship (SAR) studies. High throughput screening (HTS) of chemical libraries conducted by Cancer Research Technology identified a number of hit compounds showing ERK5 inhibitory activity. Results: Benzo[d]thiazole (3) and 3-cyanopyridine (4) based inhibitors were identified and chosen for validation by re-synthesis. Interestingly, the compounds showed good to moderate activity against ERK5. Conclusion: ERK5 inhibition was achieved by two different chemotypes. The syntheses of these small libraries and their biological evaluation will be discussed. 430 POSTER (Board P002) Design and structure–activity relationships of highly potent and bioavailable imidazolinone FASN KR domain inhibitors G. Bignan1 , R. Alexander1 , J. Bischoff1 , P. Connolly1 , M. Cummings1 , S. De Breucker2 , N. Esser2 , E. Fraiponts2 , R. Gilissen2 , B. Grasberger1 , B. Janssens2 , T. Lu1 , D. Ludovici1 , L. Meerpoel2 , C. Meyer3 , M. Parker1 , D. Peeters2 , C. Rocaboy4 , C. Schubert1 , K. Smans2 . 1 Janssen, Oncology, Spring House PA, USA; 2 Janssen, Oncology, Beerse, Belgium; 3 Janssen, Oncology, Val de Reuil, France; 4 VillaPharma Research, Chemistry, Murcia, Spain Fatty Acid Synthase (FASN) is a complex dimeric protein with seven catalytic domains that converts acetyl-CoA and malonyl-CoA into palmitic acid in mammalian cells. While FASN is expressed at low levels in most normal tissue (except liver and adipose tissue), FASN is up-regulated in cancer cells, providing fatty acid building blocks for rapid cell growth and cell division. The role of FASN cancer therapy has also been well documented over the past decade. Increased FASN expression is correlated with disease progression and poor prognosis in many cancers including prostate, breast, ovary, colon, and lung. Additionally, FASN is

142 Friday 21 November 2014 thought to play an important role in carcinogenesis by protecting cells from apoptosis. Recent publications disclosing FASN inhibitors suggest a pharmacophore common to many chemical series, wherein an aromatic group and an acylated cyclic amine are attached to a central scaffold. We postulated that imidazolinone would be an acceptable and drug-like scaffold, inspired by the precedent of irbesartan, an approved antihypertensive drug with a spirocyclopentyl-imidazolinone core. This hypothesis led to the discovery of a new series of imidazolinone-based FASN inhibitors. Extensive structure–activity relationship work in our laboratories resulted in numerous molecules with potent enzyme and cell activity, selectivity, and oral bioavailability. One such compound is a potent inhibitor of human FASN enzymatic activity (IC50 = 28 nM) and also potently inhibits proliferation of A2780 ovarian cells in lipid-reduced medium (IC50 = 13 nM). Addition of palmitate to the medium rescues the cells from FASN inhibition, demonstrating on-target effects. Potent activity is also seen in other cancer cells such as PC3M (IC50 = 25 nM) and LnCaP-Vancouver prostate cells (IC50 = 66 nM). This lead compound is highly bioavailable (F 61%) in mice and shows good plasma and tumor exposures after oral dosing. In a pharmacodynamics (PD) model in H460 lung xenograft-bearing mice, oral treatment resulted in elevated tumor levels of malonyl-CoA and decreased tumor levels of palmitate. This compound and other members of the imidazolinone series potently inhibit the FASN KR domain (IC50 = 54 nM); specific binding to KR was confirmed by co-crystal structures of several examples. In summary, we discovered a new series of FASN inhibitors built upon an imidazolinone scaffold that are potent in enzyme and in cell proliferation assays, are highly bioavailable, and bind to KR domain. Moreover, rescue of lipid-reduced cellular activity upon addition of palmitate suggests selectivity, and PD studies in tumor-bearing mice confirm target engagement.

Molecular Targeted Agents II 431 POSTER (Board P003) Real-time pharmacokinetic (PK) results from an ongoing randomized, parallel-dose phase 1 study of onapristone in patients (pts) with progesterone receptor (PR)-expressing cancers F. Lokiec1 , J. Bonneterre2 , A. Italiano3 , A. Varga4 , M. Campone5 , T. LeSimple6 , A. Leary7 , V. Dieras8 , K. Rezai9 , S. Giacchetti10 , S. Proniuk11 , A. Bexon12 , E. Gilles13 , J. Bisaha14 , A. Zukiwski15 , ˆ Rene´ Huguenin, Radio-pharmacology, P. Cottu16 . 1 Institut Curie-Hopital St Cloud, France; 2 Centre Oscar Lambret, Breast Cancer Unit, ´ Early Phase Trials and Sarcoma Lille, France; 3 Institut Bergonie, Units, Bordeaux, France; 4 Gustave Roussy, Early Phase Trials Unit, 5 ´ Villejuif, France; Institut de Cancerologie de l’Ouest, Medical Oncology, ` Saint-Herblain-Nantes, France; 6 Center Eugene Marquis, Medical Oncology, Rennes, France; 7 Gustave Roussy, Gynecologic Oncology, Villejuif, France; 8 Institut Curie, Clinical Investigations, Paris, France; 9 ˆ Institut Curie-Hopital Rene´ Huguenin, Radio-pharmacology, Paris, ˆ France; 10 Hopital Saint-Louis, Medical Oncology, Paris, France; 11 Arno Therapeutics, Preclinical, Flemington, USA; 12 Bexon Clinical Consulting, Clinical Development, Montclair, USA; 13 Invivis Pharmaceuticals, Clinical Development, Bridgewater, USA; 14 Arno Therapeutics, Clinical Operations, Flemington, USA; 15 Arno Therapeutics, Clinical Development, Flemington, USA; 16 Institut Curie, Medical Oncology, Paris, France Background: Onapristone is a type I PR antagonist, which prevents PR-induced DNA transcription. Presence of transcriptionally activated PR (APR), could indicate potential for onapristone anticancer activity and is being developed as an immunohistochemistry companion diagnostic. Onapristone anti-cancer activity is documented in multiple pre-clinical models and clinical studies in pts with hormone therapy-na¨ıve or tamoxifenresistant breast cancer. An extended-release (ER) tablet formulation of onapristone was designed to address the liver function test (LFT) elevations seen with immediate-release (IR) onapristone, by reducing the Cmax . Prior data indicate a T1/2 of 1−4 hours for IR onapristone. Materials and Methods: This is a multi-center, open-label, randomized, parallel-group, 2-stage phase 1 study (total n ~60; NCT02052128) ongoing in 7 centers in France with ANSM and ethics committee approval obtained in 2013. Female pts 18 years with tumors expressing PR (including endometrial, ovarian, breast) are eligible. The primary endpoint is the recommended phase 2 dose (RP2D) of ER onapristone, with a 57-day observation period for dose-limiting toxicity (DLT); secondary endpoints include: safety, efficacy, and real-time PK. Six pts per cohort receive onapristone ER tablets 10, 20, 30, 40 or 50 mg BID, or onapristone IR tablets 100 mg QD until progressive disease or intolerability in Stage 1. A

Poster Session – Molecular Targeted Agents II data review committee (DRC) monitors/reviews safety signals, DLTs, PK and efficacy data. This abstract focuses on the results of the ongoing realtime PK analyses. Results: as of 17 June 2014, 18 pts, 3 per cohort, have validated PK data and 10 pending PK results (28 enrolled). Onapristone AUC and Cmax are dose-proportional across all dose levels including 100 mg IR, with coefficients of determination (r2 ) of 0.90 and 0.71, respectively. Steady state is consistently attained at approximately 200 hours (~8 days) in all patients, with T1/2 at 8−12 hours proving substantially longer than previously published in single-dose onapristone studies. PK was drawn in 3 pts who had LFT elevations concurrent with progressive hepatic disease (PD). Exposure at the time of the LFT elevations did appear increased in these pts, but results are inconclusive due to concurrent liver PD. Conclusions: AUC and Cmax data support the use of the ER administration as a strategy to potentially mitigate LFT elevations. After repeated dose administration, the terminal t1/2 differed substantially from single dose administration. 11 C-onapristone PET/PK studies are planned to evaluate the tumor residence time and drug uptake. Real-time data collection and PK analysis is ongoing with recruitment ~10 pts/month. 432 POSTER (Board P004) ODM-203, a novel, selective and balanced FGFR and VEGFR inhibitor with strong anti-tumor activity in FGFR- and VEGFR-dependent cancer models T. Holmstrom ¨ 1 , A. Moilanen1 , T. Linnanen1 , G. Wohlfahrt1 , S. Karlsson1 , 1 ¨ , S. Samajadar2 , S. Rajagopalan2 , R. Oksala1 , T. Korjamo1 , M. Bjorkman S. Chelur2 , K. Narayan2 , R. Ramachandra2 , T. Anthony2 , S. Ds2 , M. Ramachandra2 , P. Kallio2 . 1 Orion Pharma, R&D Oncology Research, Turku, Finland; 2 Aurigene Discovery Technologies Limited, Research, Bangalore, India Background: Genomic alterations in fibroblast growth factor receptors (FGFR) and upregulation of vascular endothelial growth factor receptors (VEGFR) are often found in the same cancer types such as gastric, lung and breast and these alterations correlate with patient survival and disease progression. In addition, both FGFR and VEGFR signaling promote tumor angiogenesis. Activation of FGFR signaling has also been described to function as a compensatory angiogenic signal following development of resistance to VEGF inhibition. Methods and Results: ODM-203 is a novel and selective inhibitor of the FGFR and VEGFR families exhibiting equal potency (IC50 6−35 nM) on FGFR 1−4 and VEGFR 1−3. ODM-203 potently suppresses FGFR signaling (IC50 20–100 nM) and cell proliferation (IC50 50–150 nM) in tumor cell lines with deregulated FGFRs. It also inhibits VEGF-induced endothelial cell (HUVEC) proliferation (IC50 33 nM) with similar potency as it inhibits proliferation in FGFR-dependent cell lines. In vivo, ODM-203 shows strong anti-tumor activity in several FGFR-dependent xenograft models at welltolerated oral doses. This in vivo anti-tumor activity is associated with suppression of phospho-FGFR. ODM-203 also shows strong anti-tumor activity in angiogenic orthotopic xenograft models. Lack of activity in a FGFR and VEGFR independent tumor model further supports the selective mode of action of ODM-203. Oral administration of ODM-203 was well tolerated with only FGFR or VEGFR inhibition related physiological events observed in vivo in rat and dog. Conclusion: Based on our findings, ODM-203 has a unique profile with equal potency for both FGFR and VEGFR kinase families which correlates with strong anti-tumor activity in both FGFR-dependent and angiogenic tumor models. Taken together, these results warrant exploring the activity of ODM-203 in FGFR-dependent angiogenic tumors in man. 433 POSTER (Board P005) Genomic predictors of therapeutic sensitivity to TAS-119, a selective inhibitor of Aurora-A kinase H. Sootome1 , N. Fujita1 , A. Miura1 , T. Suzuki1 , H. Fukushima1 , S. Mizuarai1 , H. Hirai1 , T. Utsugi1 . 1 Taiho Pharmaceutical Co. Ltd., Tsukuba Research Center, Ibaraki, Japan Background: Identification of molecular or genomic biomarkers to predict therapeutic efficacy of molecularly targeted agents is a desirable aspect of drug development when starting clinical trials. We previously reported TAS-119, a highly potent and selective Aurora-A inhibitor, showed synergistic enhancement of antiproliferative effects of taxanes in multiple cell lines. Here we demonstrate that TAS-119 as a single agent exhibits dramatic tumor growth inhibition on certain types of cell lines, and found that sensitivity was linked to the genetic alterations with either gene amplification of MYC oncogene or gene mutation of beta-catenin. Identification, validation and characterization of these predictive markers were presented in this report.

Poster Session – Molecular Targeted Agents II Material and Methods: Cell proliferation of a panel of 240 human tumorderived cell lines was measured by the signal intensity of the incorporated nuclear dye after 72 hour exposure to TAS-119. Using the IC50 values in both sensitive and resistant cell lines, an analysis of the correlation between the single gene abnormality and the sensitivity of TAS-119 was performed. Information of gene abnormalities were gathered by referring to the public information within the following databases; Cancer Cell Line Encyclopedia (CCLE), Sanger Cancer Genome Project (CGP) and Catalogue of Somatic Mutations in Cancer (COSMIC). Results: Among the 10 most sensitive cell lines selected by IC50 values, 4 cell lines (IMR-32, NCI-H69, CHP-212 and MC-IXC) had amplification N-Myc or c-Myc, and another 4 cell lines (SW48, AGS, HCT-116 and A427) had CTNNB1 (beta-catenin) mutations. Oral administration of TAS119 showed substantial tumor growth reduction of tumors possessing these gene mutations in xenograft models. TAS-119 treatment down-regulated Myc protein expression in N-Myc-amplified neuroblastoma cell lines, which is suggested as one potential mechanism of growth inhibition. Conclusions: Two gene abnormalities, Myc amplification and/or CTNNB1 mutation, were identified as predictive markers for TAS-119 sensitivity as a single agent. Antitumor efficacy by TAS-119 monotherapy will be evaluated in upcoming clinical trials of this compound. 434 POSTER (Board P006) Bipolar androgen therapy for men with castration sensitive and castration resistant prostate cancer: Reversing resistance and maintaining sensitivity to androgen ablative therapies S.R. Denmeade1 , E.S. Antonarakis1 , M.A. Eisenberger1 , M.A. Carducci1 , H. Wang1 , C.J. Paller1 , J.T. Isaacs1 , M.T. Schweizer1 . 1 Johns Hopkins University, Oncology, Baltimore MD, USA Targeting androgen receptor (AR)-axis signaling through disruption of androgen−AR interaction remains the primary treatment for metastatic prostate cancer. Unfortunately, all men develop resistance to primary castrating therapy and secondary androgen deprivation therapies (ADT). Resistance develops as castrate-resistant prostate cancer (CRPC) cells adaptively upregulate AR levels through overexpression, amplification, mutation and expression of ligand-independent variants in response to chronic exposure to a low testosterone (T) environment. However, preclinical models suggest that AR overexpression represents a therapeutic liability that can be exploited via exposure to supraphysiologic T leading to prostate cancer cell death via multiple mechanisms. Preclinical studies supported pilot studies in men with castration sensitive and castration resistant prostate cancer. In the first study, 16 asymptomatic CRPC patients with low to moderate metastatic burden were treated with T cypionate (400 mg intramuscular; day 1 of 28) and etoposide (100 mg oral daily; days 1−14 of 28). After 3 cycles, those with a declining PSA continued on T monotherapy. Castrating androgen deprivation therapy (ADT) was continued to suppress endogenous T production thus allowing for the rapid cycling from supraphysiologic to near-castrate serum T levels, a strategy termed bipolar androgen therapy (BAT). BAT resulted in high rates of PSA (7/14 evaluable patients) as well as radiographic responses (5/10 evaluable patients). Four men remained on BAT for 1 year. All patients (10/10) demonstrated PSA reductions upon receiving androgen ablative therapies post-BAT, with 8/8 men receiving abiraterone or enzalutamide having >50% PSA reduction, suggesting BAT may also restore sensitivity to these agents. In summary, in this study BAT showed promise as treatment for CRPC and is now being further evaluated in larger clinical trials. In the second pilot study, 31 asymptomatic men with progressive prostate cancer with low to moderate metastatic burden received 6 months of androgen deprivation therapy followed by 3 months of intramuscular T-cypionate (400 mg) followed by 3 months of ADT for two cycles. The endpoint of this ongoing study is the percent of patients with PSA <4 ng/ml after 18 months of therapy. Overall, BAT was well-tolerated in both studies with lower extremity edema as the most common side effect. No worsening of pain was observed in either study. BAT also produced subjective improvement in well-being, activity level and sexual function.

Friday 21 November 2014 143 435 POSTER (Board P007) A pan-cancer tumor-derived epithelial-to–mesenchymal transition (EMT) signature determines patterns of drug sensitivity and enrichment in immune target expression following EMT M.P. Mak1 , P. Tong2 , L. Diao2 , P.K.S. Ng3 , Y. Fan4 , R.J. Cardnell4 , D.L. Gibbons4 , W.N. William4 , J.V. Heymach4 , K.R. Coombes2 , ˜ L.A. Byers4 , J. Wang5 . 1 Instituto do Cancer do Estado de Sao ˜ Paulo, Brazil; 2 MD Anderson Cancer Paulo, Medical Oncology, Sao Center, Bioinformatics & Computational Biology, Houston TX, USA; 3 MD Anderson Cancer Center, Systems Biology, Houston TX, USA; 4 MD Anderson Cancer Center, Thoracic/Head & Neck Medical Oncology, Houston TX, USA; 5 MD Anderson Cancer Center, Bioinformatics and Computational Biology, Houston TX, USA Background: We previously demonstrated the EMT association with drug response in lung cancer using a robust, platform-independent EMT signature derived in lung cancer cell lines. Given the contribution of tumor microenvironments, EMT markers may vary in patient tumors (vs. cell lines) and across tumor types. To better understand the common molecular features of EMT, we extended our investigation of the lung EMT signature to 11 tumors types from TCGA (n = 1934) to develop a pan-cancer tumorderived EMT signature, for which we used tumor data and cell lines to evaluate clinical relevance. Material and Methods: Candidate EMT genes were selected from top mRNAs correlated with four established EMT markers (CDH1, VIM, FN1 and CDH2) across tumor types. The pan-cancer EMT signature, defined with a pathway-guided approach, consists of 86 genes regulating 21 core pathways based on the candidate genes. The pan-cancer EMT signature was correlated with the mutational landscape, copy number alterations (CNAs), mRNA, micro-RNA (miR), and protein expression to identify EMTassociated events. To evaluate the signature’s therapeutic relevance, the relationship between EMT score and drug sensitivity was evaluated in public cell line databases. Results: The introduced signature encompasses a set of core EMT genes that strongly correlate with known EMT markers across different tumor types. Clinically, high-grade renal clear cell carcinoma and head and neck cancers had higher EMT scores (p < 0.009), although EMT was not associated with a statistically significant difference in survival. Overall mutation and CNA burden were not correlated with EMT status. However, TP53 mutation was more frequently associated with epithelial status. Potential therapeutic targets commonly over-expressed in mesenchymal tumors included PDGFRB, MMP2 (r > 0.6, >10 tumor types) and Axl (r > 0.6, n > 9 tumor types). Further pathway analysis revealed an enrichment of immune-related pathways in tumors that had undergone EMT, with higher expression of immune targets (e.g., OX40L, PDL2, PD1, CTLA4) in mesenchymal tumors (r > 0.3). At the protein level, E-cadherin and Her3 were the highest expressed among epithelial tumors, while fibronectin1 and p21 were highest in mesenchymal tumors (r > 0.25, n  8 tumor types). Besides the association of miR expression and EMT score, validated miR targets were also highly correlated with EMT (p < 0.001). In vitro, mesenchymal cell lines were overall more resistant to EGFR and ERBB2 inhibitors, and more sensitive to FGFR, PDGFR and GSK3 inhibitors. Conclusions: A novel tumor-driven EMT signature identifies mesenchymal status across different tumor types. This pan-cancer signature identifies PDGFR, Axl, and immune checkpoints (e.g., CTLA4) as potential therapeutic targets in mesenchymal cancers. 436 POSTER (Board P008) Monitoring activity of RXDX-101 in Phase 1/2 patients using a pharmacodynamic assay for TrkA activation D. Murphy1 , H. Ely1 , R. Patel2 , G. Wei2 , A. Diliberto1 , R. Shoemaker3 , J. Christiansen1 . 1 Ignyta, Diagnostic Development, San Diego, USA; 2 Ignyta, Biology, San Diego, USA; 3 Ignyta, Bioinformatics, San Diego, USA Introduction: RXDX-101 is a tyrosine kinase inhibitor currently in Phase 1 clinical trials with activity against TrkA as well as TrkB, TrkC, ALK and ROS1 proteins (encoded by NTRK1, NTRK2, NTRK3, ALK and ROS1 genes, respectively). RXDX-101 is indicated for patients with advanced, refractory, relapsed or metastatic solid tumors who have exhausted standard treatment options and who exhibit molecular alterations in any of the target genes. Due to multiple potential metabolic and physiological effects, the amount and effectiveness of circulating RXDX101 and its active metabolites must be measured. Due to challenges in monitoring TrkA activation directly in liquid biopsy samples, we developed a pharmacodynamic (PD) test that measures the effect of treated patient plasma containing RXDX-101, and its active metabolites, on the activation of TrkA (phospho-TrkA/p-TrkA) in a human lymphoma cell line.

144 Friday 21 November 2014

Poster Session – Molecular Targeted Agents II

Methods: Karpas 299 lymphoma cells were mixed with plasma from patients treated with RXDX-101 and stimulated with human nerve growth factor (hNGF) to induce activation of TrkA. Plasma effects were investigated using both pooled and multiple individual donor plasma with varying levels of RXDX-101 spiked-in. p-TrkA levels are measured by ELISA in the presence and absence of drug and normalized to baseline hNGF stimulated conditions to assess percent change in presence of RXDX101. To evaluate assay robustness, plasma from patients receiving RXDX-101 was tested for decreased p-TrkA levels. Results: The IC50 of p-TrkA expression in response to RXDX-101 treatment in human plasma was ~6nM, with a lower limit of detection of 3nM. Reproducibility studies (n = 5) support that at the IC50 value, there was a 50±13% reduction in p-TrkA levels in 7nM treated plasma (CV of 26%). This was highly correlative when plasma from different patients was used (n = 10) (48% reduction in p-TrkA +/− 13% with a CV of 26%). Treated patients were evaluated for active RXDX-101 capable of targeted inhibition. We intend to present results from patients enrolled in phase 1 studies. Conclusions: We have developed a PD assay to indicate the activity of RXDX-101 active forms in the plasma of treated patients that is robust and quantitative. This assay will be used to monitor the drug availability in patients enrolled in Phase 1 and 2 clinical trials for RXDX-101. The quantitative nature will allow analysis to be performed across patients as well as monitoring longitudinal impact of RXDX-101.

improved by introducing a second binding site that recognizes a highly expressed antigen on the tumor cell surface. Guided by this prediction, we engineered MM-131, a bispecific antibody designed to block HGF binding to c-Met with high potency through concurrent binding to epithelial cell adhesion molecule (EpCAM). We assessed the potency and activity of MM-131 in models of c-Met-driven cancer at different levels of EpCAM expression. Results: Our model predicted that the anti-c-Met activity of MM-131 should increase with higher levels of EpCAM, as binding to EpCAM enables MM131 to pre-concentrate at the cell surface and effectively compete with HGF binding to c-Met. We validated this prediction across a panel of cell lines, observing dose-dependent inhibition of HGF-mediated pathway activation, cell viability, and migration, as well as inhibition of tumor growth in vivo. Additionally, we found that MM-131 downregulates c-Met, but not EpCAM, in tumor cells with high levels of c-Met and EpCAM expression, providing ligand-independent anti-tumor activity. Importantly, MM-131 did not exhibit agonistic activity in a wide range of experimental systems. Quantitative assays measuring EpCAM, HGF, and c-Met in tumor samples showed that EpCAM is expressed at high levels in a wide range of c-Met-driven tumor types, suggesting a broad opportunity for clinical development of MM-131. Conclusions: In our preclinical studies, MM-131 inhibited both liganddependent and ligand-independent c-Met signaling with no associated agonistic activity. MM-131 is expected to enter clinical trials in 2015.

437 POSTER (Board P009) PIM kinase inhibitor AZD1208 sensitises SCLC to BH3 mimetic AZD4320

439 POSTER (Board P011) PI3K/mTOR inhibitor VS-5584 targets cancer stem cells and prevents tumor regrowth after chemotherapy in preclinical models of small cell lung cancer

R. Sloane1 , B. Bola1 , M. Lancashire1 , C. Hodgkinson1 , C. Morrow1 , K. Simpson1 , C. Dive1 . 1 Cancer Research UK Manchester Institute, Clinical and Experimental Pharmacology, Manchester, United Kingdom Small cell lung cancer (SCLC) is an aggressive disease characterised by rapid growth, early metastasis and poor prognosis. Despite initial responses to the standard of care of cisplatin and etoposide, the majority of SCLC patients undergo drug resistant relapse. Numerous trials of new chemotherapeutic agents in recent decades have shown minimal improvement in patient prognosis, highlighting the need for novel treatment strategies. AZD4320 is a BH3 mimetic targeting BCL2 and BCLXL. This class of drug interacts with the BH3 domain of anti-apoptotic BCL2 family proteins to induce apoptosis. Although BH3 mimetics demonstrated potent activity against SCLC cell lines in vitro and in vivo this was not recapitulated in Phase II clinical trials. However, the anti-apoptotic BCL-2 family member MCL1 to which AZD4320 does not bind provides a resistance mechanism for this class of drug and we hypothesised that targeting MCL1 would sensitise SCLC to AZD4320 providing an effective, rational combination treatment. MCL1 down-regulation by PIM inhibition has been shown to occur via global down regulation of protein translation with significant effects on protein levels of the short lived MCL1 protein. AZD1208 is an ATP competitive inhibitor of PIM 1, 2 and 3 and has in vitro and in vivo activity in prostate and haematological cancer. PIM kinases are able to protect cancer cells from apoptosis by phosphorylating BAD, freeing the anti-apoptotic BCL2 protein. We have shown that AZD1208 treatment down-regulates MCL1, reduces levels of phosphorylated BAD and sensitises 6/6 SCLC cell lines tested to AZD4320 in vitro, resulting in IC50s in the nano molar range. We are testing the efficacy of this combination in vivo using traditional SCLC xenografts and unique CTC derived mouse models and investigating the proposed molecular mechanism underlying this sensitisation. 438 POSTER (Board P010) MM-131: A bispecific antibody that inhibits c-Met signaling through avid binding to the EpCAM tumor antigen B.D. Harms1 , A. Lugovskoy1 , A. Abu-Yousif1 , A. Fulgham1 , M. Geddie1 , S.V. Su1 , N. Kohli1 , B. Johnson1 , K. Masson1 , U.B. Nielsen1 , B. Schoeberl1 , G. MacBeath1 . 1 Merrimack Pharmaceuticals, Discovery Division, Cambridge, USA Background: The c-Met receptor tyrosine kinase, along with its ligand hepatocyte growth factor (HGF), is a driver of proliferative and migratory signaling in tumor cells and is thought to be a key mediator of drug resistance and metastasis. Development of bivalent antibodies targeting c-Met is challenging, however, because they mimic HGF binding and cause pathway activation. Solutions to this problem include engineering monovalent antibodies or selecting bivalent antibodies that work by downregulating c-Met. To date, these strategies have resulted in c-Met therapies with either reduced potency or partial agonistic activity. Material and Methods: Through computational modeling, we found that the potency of a monovalent antibody targeting c-Met could be dramatically

V. Kolev1 , M. Padval1 , Q. Wright1 , J. Ricono2 , D. Weaver1 , J. Pachter1 , Q. Xu1 . 1 Verastem Inc, Cambridge MA, USA; 2 Molecular Response, San Diego CA, USA Small cell lung cancer (SCLC) is a highly aggressive malignancy with a 5-year overall survival rate of only 5−10%. Most patients with SCLC initially respond to chemotherapy but subsequently experience aggressive tumor recurrence, which may be attributed to the presence of a subpopulation of tumor initiating cancer stem cells (CSCs). We have previously demonstrated that the orally bioavailable dual PI3K/mTOR inhibitor VS5584, which is currently in phase 1 clinical development, preferentially targets CSCs in breast and ovarian cancer preclinical models. Here we report that VS-5584 inhibits proliferation and induces apoptosis in SCLC cell lines in vitro and exhibits antitumor activity in SCLC xenograft models in vivo. VS-5584 is also synergistic with standard of care chemotherapy cisplatin and etoposide in reducing the viability of NCI-H69 SCLC cells. Importantly, VS-5584 is more potent against CSCs than bulk tumor cells and thus reduces the proportion of CSCs in preclinical models of SCLC. A drug resistant population of small cell lung cancer CSCs (side population) can be monitored by enhanced ability to exclude Hoechst 33342 dye. In the H69 SCLC cell line, VS5584 effectively eliminated the side population CSCs, while cisplatin and etoposide enriched this CSC population. In vivo, VS-5584 caused significant tumor growth inhibition and reduced the proportion of CSCs in NCI-H841 SCLC tumors as evidenced by a significant decrease in the percentage of side population cells and approximately 70-fold reduction in tumor-initiating capability when tumor cells from VS-5584-treated animals were injected into secondary immunodeficient mice. Consistent with the notion that CSCs are responsible for cancer relapse after chemotherapy, VS-5584 significantly reduced tumor regrowth following cessation of cisplatin treatment in the H69 SCLC xenograft model and in a SCLC patient-derived xenograft (PDX) model. The preferential targeting of CSCs by VS-5584 in preclinical models of SCLC provides the rationale for clinical development of VS-5584 either as a single agent or in combination with chemotherapeutic agents to potentially extend time to relapse and improve outcome for patients with small cell lung cancer. 440 POSTER (Board P012) Phenotypic alteration in a highly metastatic variant of the MDA-MB-231 cell line: role of Annexin A1 Y. Tu1 , E. Fietz1 , J. Cameron2 , A. Stewart1 . 1 University of Melbourne, Pharmacology and Therapeutics, Melbourne Victoria, Australia; 2 Peter MacCallum Cancer Centre, Cancer Metastasis Laboratory, Melbourne Victoria, Australia Annexin A1 (ANXA1) is a multifunctional protein known to regulate the innate and adaptive immune responses. It has also been implicated in the progression of various cancers. In most types of breast cancer there is a significant loss of ANXA1 expression in the primary tumour. However, increased expression of ANXA1 in patients with invasive breast tumour

Poster Session – Molecular Targeted Agents II correlates with poor outcomes and survival (Yom et al., 2011). Similarly, breast cancer cell lines with a more metastatic phenotype show higher levels of ANXA1. We therefore hypothesised that ANXA1 contributes to the metastatic processes of breast cancer. Using the breast adenocarcinoma MDA-MB-231 cell line and a highly metastatic (MDA-MB-231HM) variant cell line derived from six cycles of pulmonary metastasis to the mammary fat pad (Li et al., 2006), we investigated the influence of ANXA1 on migration and proliferation. MDA-MB-231HM.LNm5 cells were isolated from an axillary lymph node metastasis from Balb-SCID mice inoculated intramammary fat pad with MDA-MB-231 HM cells (Cancer Metastasis Laboratory, Peter MacCallum Cancer Centre). Compared to MDA-MB-231 cells, MDA-MB-231HM.LNm5 cell line have significantly higher levels of ANXA1 (P < 0.05) and appear to have increased migratory response to FCS (1%v/v) in the modified Boyden Chamber (167±26% P < 0.05). Stable knock-down of ANXA1 in the MDAMB-231HM.LNm5 reduces cell migration to levels shown by the parent MDA-MB-231 cells. Proliferation after 48 h was significantly lower in the MDA-MB-231HM.LNm5 cells compared with MDA-MB-231 cells (59±8% P < 0.05). This slowly proliferating phenotype was also reverted when ANXA1 level was stably decreased. In contrast, knocking down ANXA1 affected neither the migration nor the proliferation of the parent MDA-MB231 cells. Our results demonstrated that decreasing ANXA1 levels could reverse aspects of the phenotype MDA-MB-231 cells acquired after cycles of metastasis and re-implantation, suggesting a contribution of Annexin A1 to metastasis of breast cancer. 441 POSTER (Board P013) The role of methylation in metastasis of oral squamous cell carcinoma: understanding the OSCC methylome M. Clausen1 , L.J. Melchers1 , T. De Meyer2 , S. Denil2 , W. Criekinge2 , G.B. Wisman3 , J.L.N. Roodenburg1 , E. Schuuring4 . 1 University Medical Center Groningen, Oral and Maxillofacial Surgery, Groningen, Netherlands; 2 Ghent University, Mathematical Modeling Statistics and Bioinformatics, Ghent, Belgium; 3 University Medical Center Groningen, Gynecologic Oncology, Groningen, Netherlands; 4 University Medical Center Groningen, Pathology, Groningen, Netherlands Background: Oral Squamous Cell Carcinoma (OSCC) is characterized by an increasing incidence and a 60% 5-year survival. The most important prognostic factor for OSCC is the presence of lymph node (LN) metastases but the detection of LN metastases in the clinic by palpation and imaging is inaccurate resulting in under and overtreatment of patients. To improve LN diagnosis new detection methods are needed. DNA methylation studies can be used to identify novel biomarkers, as epigenetics have been established as an important regulator of metastatic potential. The aim of this study is to identify new DNA methylation markers that predict LN metastasis in OSCC. Materials and Methods: Genome-wide methylation patterns of metastatic (n = 6) and non-metastatic OSCC (n = 6) were assessed using MethylCapSeq. Subsequently, analysis was performed on the most differentially methylated loci to identify pathways and genomic loci associated with LN metastasis. Additionally, the methylation data were combined with expression data of 222 OSCC patients acquired by an expression microarray with a 696 gene panel associated with N-status in OSCC. Finally, we validated these findings on the OSCC samples (n = 174) in The Cancer Genome Atlas (TCGA). Results: We found that genes on chromosome 7 are the most hypermethylated in metastatic OSCC in comparison to non-metastatic OSCC, more specifically a four million bp loci around the EGFR gene. In total 26 genes were found to be differentially methylated by MethylCapSeq as well as differentially expressed by microarray. In the OSCC TCGA validation cohort, all 26 genes are significantly differentially expressed between metastatic and non-metastatic OSCC. For these 26 differentially expressed genes 88 probes were found in the TCGA Infinium 450k methylation data that are both significantly differentially methylated between the two groups and correlated with mRNA levels. Finally, six of these probes overlapped with the genomic regions annotated by MethylCap-Seq, representing five genes. Conclusion: We identified chromosomal loci, pathways and gene promoters associated with LN metastasis in OSCC patients. For 10 genes the differential methylation, expression and correlation between methylation and expression was validated on an independent OSCC cohort from the TCGA database. Increased understanding of the metastatic OSCC methylome will contribute to the identification of DNA methylation markers, pathways and potential therapeutic targets in OSCC primary tumors that will improve chances of providing the proper lymph node treatment and may result in improvement of survival and quality of life. This research was supported by the Center for Translational Molecular Medicine (project AIRFORCE).

Friday 21 November 2014 145 442 POSTER (Board P014) Targeting urokinase plasminogen activator for radioimmunotherapy using an antagonistic internalizing human antibody A. LeBeau1 , H.F. VanBrocklin2 . 1 University of Minnesota Medical School, Pharmacology, Minneapolis, USA; 2 University of California San Francisco, Radiology and Biomedical Imaging, San Francisco, USA Background: The plasminogen activation system (PAS) − consisting of the secreted serine protease urokinase plasminogen activator (uPA), the receptor of uPA (uPAR) and the endogenous inhibitor of uPA (PAI-1) − is overexpressed in a number of adenocarcinomas with poor clinical outcome. Enzymatically active uPA is known to cleave substrates that promote tumor growth and metastasis. Active uPA, therefore, represents a promising target for therapeutic intervention. Detailed here is the development of a novel uPA-targeted human antibody for radioimmunotherapy (RIT). Material and Methods: A human antibody specific for secreted and uPAR bound forms of active uPA was discovered using a human Fab phage display library. This antibody, U33 IgG, was labeled with 111 In for in vitro characterization studies in prostate cancer cell lines and for in vivo SPECT/CT imaging and biodistribution studies in PC3 xenograft mice. The therapeutic efficacy of 177 Lu labeled U33 IgG (177 Lu-U33 IgG) was evaluated in vitro and in vivo in an RIT study using a single dose of 100 mCi in PC3, DU145, and LNCaP xenografts. Results: The in vitro characterization studies in prostate cancer cell lines found that the U33 IgG mimicked the function of PAI-1 resulting in internalization of the uPA-U33 complex when bound to uPAR. This mechanism of internalization resulted in a sensitive SPECT/CT imaging probe with a high tumor uptake of 43.2% injected dose per gram (%ID/g) in PC3 xenografts. Subsequently, 177 Lu-U33 IgG was found to be effective in vitro at selectively killing the uPA expressing cell lines PC3 and DU145, but not the uPA null LNCaP. As an RIT agent, 177 Lu-U33 IgG had no effect on the LNCaP tumors, but dramatically reduced the tumor burden in the PC3 and DU145 xenografts resulting in complete tumor eradication with no regrowth by day 75. Conclusions: U33 IgG is a unique antibody that activates internalization of the U33/uPA/uPAR complex through a complex mechanism. Because of this novel internalization mechanism, U33 IgG could be sequestered inside cancer cells resulting in a potent RIT agent when coupled to 177 Lu. These data lay the foundation for further studies to evaluate the utility of U33 IgG as an RIT agent and potential translation into humans.

443 POSTER (Board P015) Notch3-targeted antibody drug conjugates have superior preclinical efficacy to Notch signaling inhibitors K.G. Geles1 , Y. Gao2 , L. Sridharan1 , A. Giannakou1 , T.T. Yamin1 , J. Golas1 , M. Charati1 , J. Lucas1 , K. Wang3 , S. Pirie-Shepherd3 , M. Roy4 , M. Follettie1 , A. Maderna5 , X. Li6 , L. Tchistiakova2 , H.P. Gerber1 , P. Sapra1 . 1 Pfizer, Oncology Research Unit, Pearl River NY, USA; 2 Pfizer, Global Biotherapeutic Technologies, Cambridge MA, USA; 3 Pfizer, Precision Medicine, La Jolla CA, USA; 4 Pfizer, Investigative Pathology, Groton CT, USA; 5 Pfizer, Medicinal Chemistry, Groton CT, USA; 6 Pfizer, Drug Safety R&D, Groton CT, USA Background: Therapeutic compounds that inhibit aberrant Notch signaling are currently being tested in clinical trials for cancer indications, but their utility may be limited to tumors with constitutive pathway activation and addiction to Notch signaling. Of the Notch receptors, Notch3 has a restricted normal tissue distribution and a non-lethal knockout phenotype in mice. Constitutive activation of Notch3 signaling is oncogenic in many contexts and can induce tumors in genetic mouse models. Methods and Materials: To broaden the spectrum of patients potentially sensitive to Notch pathway interference, we have developed antibody– drug conjugates (ADCs) that target the Notch3 receptor by combining the specificity of high affinity anti-Notch3 antibodies with the cytotoxicity of microtubule inhibitors. Results: Notch3 is over-expressed or amplified in a variety of human tumors, especially in breast, lung and ovarian cancers. We examined the efficacy and safety of ADCs generated from both inhibitory and non-inhibitory antibodies in order to select a lead candidate. Our data demonstrate that Notch3-ADCs induce prolonged tumor regressions in preclinical models regardless of their ability to block signaling. These ADCs had enhanced efficacy compared to other Notch inhibitors and also allowed targeting of tumors that over-expressed Notch3 but were not necessarily addicted to its signaling. Conclusion: The robust preclinical efficacy and safety profile demonstrate that a Notch3-targeted ADC merits clinical testing.

146 Friday 21 November 2014 444 POSTER (Board P016) Debio 1143 in combination with carboplatin and paclitaxel in patients with non-small cell lung cancer (NSCLC), triple-negative breast cancer (TNBC) and platinum-refractory epithelial ovarian cancer (EOC). Preliminary results of a Phase I dose-escalation study C. Le Tourneau1 , I. Ray-Coquard2 , N. Isambert3 , C.A. Gomez-Roca4 , P. Cassier2 , M.P. Sablin1 , E. Ruits5 , B. Gavillet5 , C. Zanna5 , ´ d’oncologie P. Fumuleau3 , J.P. Delord4 . 1 Institut Curie, Departement ´ ´ ´ ´ medicale, Paris, France; 2 Centre Leon Berard, Departement de ´ medecine carcinologique, Lyon, France; 3 Centre Georges-Fran¸cois ´ Leclerc, Service d’oncologie medicale, Dijon, France; 4 Centre Claudius ´ ´ Regaud, Oncologie Clinique Departement Medecine, Toulouse, France; 5 Debiopharm International S.A., Medical Affairs, Lausanne, Switzerland Inhibitors of apoptosis proteins (IAPs) modulate multiple processes, including caspases activation and NF-úB signaling. Expression or overexpression of IAPs has been reported for a variety of tumor types and are correlated with tumor growth and resistance to apoptosis induced by standard chemo and radiation therapies. The small molecule Debio 1143 is a potent orally-active IAP antagonist able to promote apoptosis in tumor cells by restoring caspase activity, and modulating NF-úB signaling and TNFa effects. As a single agent, Debio 1143 inhibits cell growth and induces apoptosis in a subset of human cancer cell lines and in multiple xenograft models of human cancer, including TNBC, NSCLC and EOC. Furthermore, Debio 1143 works synergistically with conventional chemotherapeutic (such as platins and taxanes), targeted agents, and radiotherapy in nonclinical tumor models. The use of Debio 1143 is of great interest in EOC where carboplatin resistance is induced by an activation of IAPs by the tumor microenvironment. Debio 1143 monotherapy was well tolerated in cancer patients up to 900 mg QD and achieves exposures that were previously shown to be active in animal models. Methods: This is a phase I, multicenter, open-label, non-randomized study comprising a dose-escalation part (A) and an expansion part (B) that enrolls treatment-na¨ıve or previously treated patients with squamous NSCLC, basal-like/claudin low TNBC and platinum-refractory EOC. In Part A, the MTD of Debio 1143 combined with paclitaxel and carboplatin is estimated via an adaptive dose-escalation design using a modified continual reassessment method. Debio 1143 is administered once daily on Days 1−5 of every 21-day cycle. The starting dose of Debio 1143 was 200 mg QD on Days 1−5 of every 21-day cycle. Carboplatin (AUC 6) and paclitaxel (175 mg/m2 ) were administered on Day 1 of every 21-day cycle. PK, PD and pharmacogenomic assessments are included. Part B is an expansion phase in which 12 patients per each indication are enrolled to better evaluate safety and signals of efficacy at the recommended dose. Results: Enrolment to cohort DL1 began in January 2013. Haematological DLT was observed in cohorts 1 and 2 despite the reduction of the Debio 1143 dose to 100 mg. Therefore, the study protocol was amended reducing the doses of carboplatin to AUC 5 and paclitaxel to 135 mg/m2 . The enrolment restarted using Debio 1143 doses of 100, 125, 150, 175, and 200 mg. Afterwards no DLT was observed up to 175 mg dose. Exposure to Debio 1143 was variable between patients, but it was in the range of exposure expected to be active, based on pre-clinical pharmacological models. Hints of activity was observed in patients with TNBC and EOC. The pharmacodynamic effect of Debio 1143 on cIAP1 degradation in PBMCs was confirmed at doses starting from 100 mg/day. The dose escalation phase is ongoing with 16 patients enrolled as of June 2014. Pharmacogenomic assessments are ongoing. 445 POSTER (Board P017) MEK inhibition enhances gemcitabine efficacy by increasing MDM2-mediated ubiquitination and degradation of RRM1 F. Vena1 , E. Li Causi2 , T. Hagemann2 , J.A. Hartley1 , S. Goodstal3 , D. Hochhauser1 . 1 University College of London, UCL Cancer Institute, London, United Kingdom; 2 Barts Cancer Institute, Cancer and Inflammation, London, United Kingdom; 3 EMD Serono, Research Institute, Billerica, USA Background: Gemcitabine, a nucleoside analogue, is an important treatment for metastatic and locally advanced pancreatic ductal adenocarcinoma (PDAC), but provides only modest benefit. Targeting downstream effectors of the RAS/ERK signaling pathway by direct inhibition of the MEK1/2 protein kinase is considered a promising therapeutic strategy for PDAC since aberrant activation of this pathway or mutations of the oncogene genes K-RAS is frequently found in PDAC. Ribonucleotide Reductase (RR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides, which are essential for both DNA replication and repair. There is evidence that RR large subunit one (RRM1) levels can predict survival, since high expression of RRM1 is associated with resistance to gemcitabine.

Poster Session – Molecular Targeted Agents II Material and Methods: The human pancreatic cancer cell lines BxPC-3 and Panc-1 were used for in vitro analysis; cell survival and apoptosis was assessed by MTT and Caspase 3/7 Glo assays. Western blot was performed to determine protein expression. The in vivo sensitivity of pancreatic cancer to gemcitabine with pimasertib was determined in xenograft models. Results: Combination treatment of gemcitabine with pimasertib, an orally bioavailable small molecule inhibitor that binds to an allosteric site close to the adenosine triphosphate binding site of MEK1/2, induced synergistic effects on proliferation and enhanced gemcitabine-induced apoptosis in BxPC-3 and Panc-1 cells compared to single drugs alone. In addition, significant tumor growth reduction in pimasertib combined with gemcitabine − vs. single agent gemcitabine-treated mice bearing TB32048 pancreatic xenograft tumors was observed. Western blot analysis showed effective abrogation of P-ERK and downregulation of RRM1 protein expression by pimasertib. Gemcitabine sensitivity was significantly increased after knockdown of RRM1 with specific siRNA confirming its role in gemcitabine resistance. Next we showed that mRNA levels of RRM1 were not reduced upon pimasertib treatment, however, pre-treatment with the proteasome inhibitor MG132 impaired its downregulation, suggesting that RRM1 is degraded via the ubiquitin–proteasome system. Immunoprecipitation analysis indicated that pimasertib induces RRM1 ubiquitination through K48 linkage. siRNA analysis demonstrated that the E3 ligase MDM2, physically interacts with and ubiquitinates RRM1 and this effect is mediated by AKT. Pre-treatment with the PI3 kinase inhibitor GDC-0941 impaired RRM1 downregulation induced by pimasertib. Finally, co-treatment of pimasertib with the MDM2 antagonist Nutlin-3 prevented RRM1 from being degraded confirming the role of MDM2 on RRM1 degradation. Conclusions: These results indicate that the combination of MEK inhibitors with gemcitabine could represent a good therapeutic strategy for the treatment of patients with advanced PDAC. 446 POSTER (Board P018) The cancer stem cell inhibitors VS-6063 (defactinib) and VS-5584 exhibit synergistic anticancer activity in preclinical models of mesothelioma Q. Xu1 , W.F. Tam1 , C.M. Vidal1 , V.N. Kolev1 , Y. Kadariya2 , C.W. Menges2 , J.R. Testa2 , J.A. Pachter2 . 1 Verastem Inc, Cambridge, USA; 2 Fox Chase Cancer Center, Cancer Biology Program, Philadelphia, USA Malignant pleural mesothelioma is an aggressive cancer of the lining of the lung in which cancer stem cells may drive resistance to current chemotherapy. There is only one treatment regimen approved for use, pemetrexed plus a platinum agent, which is used as front-line therapy and results in median overall survival of only 12 months. Unfortunately, there are no approved second line options for patients with actively progressing disease after front-line therapy. VS-6063 (defactinib) is an oral small molecule that targets cancer stem cells through the inhibition of focal adhesion kinase (FAK). FAK is a cytoplasmic tyrosine kinase which orchestrates cell signaling through integrins and growth factor receptors and has been shown to be critical for tumor initiation and cancer stem cell function (Luo et al., Cancer Res, 2009; Shibue et al., Cancer Disc, 2012; Kolev et al., Cancer Res, 2013, 73(8 Suppl): 236). VS6063 has demonstrated tolerability, target inhibition, and preliminary signs of clinical activity as a single agent and in combination with paclitaxel in Phase 1 clinical trials (Jones et al., J Clin Oncol 29: 2011 (suppl; abstr 3002); Patel et al., J Clin Oncol 32:5s, 2014 (suppl; abstr 5521)). Based on these preclinical and clinical data, VS-6063 is being tested in a registrationdirected, randomized, double-blind, placebo-controlled trial in malignant pleural mesothelioma immediately following front-line therapy (COMMAND Trial, NCT01870609). In an effort to expand the mesothelioma patient population that may potentially benefit from drugs targeting cancer stem cells, we sought to identify agents that show synergistic anticancer activity with VS-6063. An in vitro combination screen was carried out evaluating VS-6063 in combination with 20 anticancer agents including both cytotoxic drugs and targeted agents. Several anticancer agents, including the dual PI3K/mTOR inhibitor VS-5584, showed synergistic activity with VS-6063. We report here characterization of the combination activity of VS-6063 and VS-5584 in mesothelioma models in vitro and in vivo. VS-6063 and VS-5584, both alone and in combination, reduced the proportion of mesothelioma cancer stem cells. In addition, single agent treatment with VS-6063 or VS-5584 reduced the viability of mesothelioma cells cultured in 3D matrigel, and the combination of VS-6063 and VS5584 displayed synergistic reduction in cell viability based on multiple combination analysis models. When tested in vivo for reduction of mesothelioma tumor growth, VS-6063 and VS-5584 were each active as single agents. In combination, VS-5584 further enhanced the antitumor efficacy of VS-6063 in this model. The combination of VS-6063 and VS-5584 represents a novel therapeutic approach and these data support the clinical evaluation of the combination

Poster Session – Molecular Targeted Agents II of VS-6063 and VS-5584 in patients with actively progressing mesothelioma following front-line therapy. 447 POSTER (Board P019) Novel, quantitative in vivo shRNA screening approach identifies new molecular targets to block cancer metastasis L. Willetts1 , R. Paproski1 . 1 University of Alberta, Oncology, Edmonton Alberta, Canada Background: The identification of novel therapeutic targets to block cancer metastasis is a key step towards the reduction of cancer-related patient deaths. For prostate cancer, the most commonly-diagnosed cancer in men, the 5 year survival rate for those with metastatic disease is less than 10%. We have recently established that intravasation, a critical step of metastasis, is dependent upon production cell migration. In this study, we have utilized a quantitative in vivo screening approach that relies on the assessment of in vivo cell migration as a primary readout. Methods: Our screen approach utilizes an ex ovo avian embryo platform combined with real-time, high-resolution 3D visualization of human cancer metastasis. When injected intravenously, fluorescent human cancer cells form hundreds of individual metastatic colonies that can be simultaneously visualized in vivo. A customized Matlab-based analysis software was developed to interpret 3D images from this model to generate a metastatic colony invasiveness index (C.I.). As a proof of principle, we showed that previously described inhibitors of cell migration and metastasis significantly reduces the C.I., which closely correlates with their antimetastatic potential. In the full screen, human head and neck cancer cells (Hep3) and prostate cancer cells (PC3) were transduced with a whole genome shRNA and injected intravenously into the CAM. Metastatic colonies with C.I. below that of control cells were excised and expanded. shRNA identities were determined by high-throughput sequencing. Results: More than fifty novel genetic regulators of metastasis were identified that have not been previously described using other approaches. Of these, there were eleven druggable targets that included cancer cell invasion and cell cycle regulating protein kinases, cell surface and nuclear receptors and cytoskeleton modulating enzymes. shRNA-mediated knockdown of these genes blocked human cancer cell metastasis in both experimental and spontaneous mouse models of metastasis. Lead hit serine-threonine kinase SRPK1 was significantly associated with prostate cancer invasion and metastasis using publicly-available gene expression data. We demonstrate that SRPK1 directly modulates the splicing isoform profile of several key cell adhesion molecules, impacting cell migration in vitro and in vivo. Inhibition of SRPK1 decreases the ability of prostate cancer cells to metastasize in vivo. Conclusion: We developed a novel and robust in vivo screening platform that provides a rapid and quantitative assessment of therapeutic targets of metastasis. Using this platform, we have identified numerous promising therapeutic targets that that are functionally involved in cancer invasion and metastasis. 448 POSTER (Board P020) Exposure to EGFR inhibitors influences release of extracellular vesicles by tumor cells R. van der Meel1 , S.M. van Dommelen1 , P. de Corte1 , M. Coimbra2 , W.W. van Solinge1 , P. Vader1 , R.M. Schiffelers1 . 1 University Medical Center Utrecht, Laboratory of Clinical Chemistry and Haematology, Utrecht, Netherlands; 2 New York University School of Medicine, Leon H. Charney Division of Cardiology and the Marc and Ruti Bell Program in Vascular Biology Department of Medicine, New York, USA Background: Inhibition of epidermal growth factor receptor (EGFR) signaling using molecular inhibitors has proven to be an effective strategy to inhibit tumor growth of several types of cancer in patients. EGFRexpressing tumor cells release extracellular vesicles (EV) that promote angiogenesis and tumor development via the transfer of functional EGFR to nearby endothelial cells. We have studied the effects of EGFR inhibitors on the release of EVs from tumor cells for monitoring of disease progress. Material and Methods: EGFR-expressing tumor cells (A431, MDAMB-468) were exposed for 48 h to the monoclonal antibody cetuximab (Erbitux® ) and the kinase inhibitor erlotinib (Tarceva® ) after which cell viability was determined by MTS assay. Tumor cells were cultured in serum-free medium in the presence of EGF and EGFR inhibitors for 48 h followed by EV isolation from the culture medium using centrifugation. EV size distribution and concentration was determined using nanoparticle tracking analysis. Western Blotting was performed to determine the levels of (phosphorylated) EGFR in the isolated EVs. Endothelial cells (HUVEC) were exposed to isolated EVs released by treated tumor cells to determine effects on cell signaling using Western Blotting.

Friday 21 November 2014 147 Results: Cetuximab and erlotinib dose-dependently inhibit EGFRexpressing tumor cell viability. Isolated EVs released by tumor cells cultured in the presence of EGF are enriched in phosphorylated EGFR. Exposure to EGFR inhibitors affects the number of EVs released by tumor cells. Tumor cells cultured in the presence of EGF and inhibitors release a different subtype of EVs containing altered (phosphorylated) EGFR expression levels compared to untreated tumor cells. Treatment of HUVECs with Isolated EVs from treated tumor cells influences Akt and MAPK signaling. Conclusions: Cetuximab and erlotinib treatment alters the number of EVs released by tumor cells, their subtype, and EGFR expression levels. The effects of EGFR-inhibition on EVs released by tumor cells may be a mechanism by which molecular inhibitors influence angiogenesis, tumor development or metastasis. The expression level signature of EVs released by tumor cells may be a method to monitor disease progression and therapy. 449 POSTER (Board P021) Cytokine induces MIR-424 expression and modulates SOCS2/STAT5 signaling pathway in oral cancer S.G. Shiah1 , H.Y. Peng1 , S.L.C. Jin2 , J.Y. Chang3 , C.C. Kuo4 . 1 National Health Research Institutes, National Institute of Cancer Research, Zhunan Miaoli County, Taiwan; 2 National Central University, Department of Life Sciences, Taoyuan, Taiwan; 3 National Cheng Kung University Hospital, Department of Internal Medicine, Tainan, Taiwan; 4 National Health Research Institutes, Institute of Biotechnology and Pharmaceutical Research, Zhunan Miaoli County, Taiwan SOCS (Suppressor of Cytokine-induced Signaling) proteins consist of eight members and share a central src homology 2 domain (SH2), a C-terminal SOCS box and unique N-termini. Members of the SOCS family are negative regulators of STAT signaling pathways. Recent years, the SOCS family is found to play important roles in cancer, but there are still many functions and mechanisms need to be further explored. The down-expression of SOCS2 has been found in solid organ malignancies, such as colorectal cancer, breast cancer, lung cancer and liver cancer. Our preliminary microarray data showed that 31 (77.5%) of 40 oral cancer patients have lower SOCS2 expression profile. Meanwhile, miRNAs are 21−24 bases non-coding RNA that regulate gene expression either by degrading target mRNAs or by inhibiting their translation. We use microRNA.org and miRNAmap database to predict the putative miRNAs that can target SOCS2. We found that miR-424−5p is the only one of overlapping miRNA predicted from these two databases. And we confirmed this finding by transfection of miRNA mimic and by 3 UTR reporter assay. We also found miR-424−5p inhibitor could increase expression of SOCS2 protein and inhibits STAT5 activation in oral cancer cells. In addition, we found that overexpression of miR-424−5p could promote cell migration, invasion and STAT5 activation via downregulation of SOCS2. Taken together, our results identify a novel mechanism for mir-424−5p-mediated progression of oral cancer and establish a functional link between mir-424−5p, SOCS2, and STAT5 signaling pathway. Furthermore, we found that IL8 increased miR424−5p expression, which activated STAT5 pathways through suppressing SOCS2 expression. 450 POSTER (Board P022) Precise gene editing of mutant NRAS using CRISPR to determine sensitivity to trametinib C. Hose1 , N.D. Fer1 , M. Burkett1 , J. Connelly1 , E. Harris1 , J. Lih2 , M. Williams2 , D. Evans1 , T. Silvers1 , A. Monks1 , R. Parchment3 , B.A. Teicher4 , J.H. Doroshow5 , A. Rapisarda1 . 1 Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc. DTP MPB, Frederick MD, USA; 2 Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc. DCTD CDP, Frederick MD, USA; 3 Frederick National Laboratory for Cancer Research, Leidos Biomedical Research Inc. DCTD LSS, Frederick MD, USA; 4 National Cancer Institute, DCTD DTP MPB, Rockville MD, USA; 5 National Cancer Institute, DCTD, Rockville MD, USA Sarcomas represent a heterogeneous group of cancers with significant unmet medical needs. We have examined the response of 64 sarcoma cell lines to treatment with 103 Approved Oncology Drugs (available from The NCI/DTP Open Chemical Repository) and 420 agents in the Investigational Agents library, using inhibition of proliferation as an endpoint. The fibrosarcoma HT1080 is exquisitely sensitive to trametinib (IC50 = 0.023mM), while the other 2 fibrosarcoma in the panel were either resistant (Hs913T, IC50 > 10 mM) or moderately sensitive (SW694, IC50 = 0.35mM) to this agent. Sequencing of these three lines for the identification of actionable mutations of interest (aMOI), as identified in the NCI/DCTD MPACT trial, showed that Hs913T has a BRAF gain of

148 Friday 21 November 2014 function aMOI and a NF1 loss of function aMOI, while SW694 has a NF1 loss of function aMOI, but these mutations do not correlate with sensitivity to trametinib. Interestingly, HT1080 has a heterozygous mutation in NRAS (Q61K) and mutations in NRAS have been shown to correlate with sensitivity to trametinib in cell lines with BRAF wild type background. To validate whether NRAS Q61K determines sensitivity to trametinib in HT1080, we used the CRISPR/Cas9 system to repair NRAS Q61K by homologous recombination. The system utilizes the type II prokaryotic CRISPR/Cas9 adaptive immune system and targets the Cas9 nuclease by a 20 nucleotides guide sequence cloned upstream of a 50-NGG “protospacer adjacent motif” (PAM). The induced site-specific strand breaks are repaired either by nonhomologous end-joining (NHEJ) to yield indels or by homologous recombination (HR) if homologous donor templates are available, thereby enhancing the efficiency of HR-based gene targeting. We designed several different small guide RNA sequences and we verified that all sequences were able to introduce indels in the NRAS gene using a control cell line. We therefore used either wild type Cas9 or Cas9D10A (nickase) with donor DNAs to perform NRAS gene repair. Clones are actively being screened for NRAS repair. Results for these experiments will be presented in the poster. The combination of drug sensitivity data, together with the mutation sequencing and precise gene editing using the CRISPR/Cas9 system may lead to precise targeting of genomic errors and to the identification of more effective therapeutic strategies for sarcoma. Funded by NCI Contract No. HHSN261200800001E. This research was supported [in part] by the Developmental Therapeutics Program in the Division of Cancer Treatment and Diagnosis of the National Cancer Institute. 451 POSTER (Board P023) Correlative and updated clinical endpoint analysis of a multicenter phase II trial of selumetinib (AZD6244) plus erlotinib in chemotherapyrefractory advanced pancreatic adenocarcinoma (PDAC) A.H. Ko1 , A.H. Tempero1 , T.B. Bekaii-Saab2 , P. Kuhn3 , R. Courtin1 , S. Ziyeh1 , S. Tahiri1 , R.K. Kelley1 , E. Dito1 , A. Ong1 , R. Linetskaya1 , A. Talasaz4 , A.P. Venook1 , W. Korn5 . 1 University of California San Francisco, Hematology/Oncology, San Francisco CA, USA; 2 The Ohio State University, Comprehensive Cancer Center, Columbus OH, USA; 3 Scripps Research Institute, Department of Cell Biology, La Jolla CA, USA; 4 Guardant Health Inc., Oncology Services, Redwood City CA, USA; 5 University of California San Francisco, Gastroenterology/Medical Oncology, San Francisco CA, USA Background: Pharmacologic inhibition of MEK leads to enhanced signaling through EGFR with hyperactivation of a parallel RAS effector pathway (PI3K), supporting a therapeutic strategy of combined target inhibition in PDAC to overcome this negative feedback loop. Based on preclinical evidence of synergistic activity between EGFR and MEK inhibitors, we conducted a non-randomized phase II trial of erlotinib plus AZD6244, a selective, ATP uncompetitive inhibitor of MEK1/2, in patients with PDAC who had received exactly one prior line of chemotherapy. Methods: 46 patients were enrolled into this study at two study centers. Treatment consisted of erlotinib 100 mg + AZD6244 100 mg daily in 3-week cycles, with tumor evaluation by CT scan every 2 cycles. Primary objective was overall survival (OS). Correlative endpoints included enumeration of circulating tumor cells, expression of EMT-related proteins, and nextgeneration sequencing analysis of cell-free (cf) DNA. Results: 46 patients enrolled at 2 sites between 1/2011 and 1/2013 (median age 67 y.o. [range 40−84]; ECOG PS (0/1/2): 30/14/2; prior gemcitabine-based vs. FOLFIRINOX vs. other 34/10/2. Patients received a median of 2 cycles (range, 1−7). Disease control rate was 58% (0 PR; 19 with stable disease (SD) >6 weeks, 12 with SD >12 weeks, including 12 (26%) minor responses). 13/34 patients (38%) demonstrated CA19−9 decline 50%. Estimated median PFS and OS by Kaplan–Meier are 1.9 and 7.3 months, respectively. 58% of patients were alive at 1 year from starting study treatment. Grade 3/4 AEs possibly/likely attributable to study treatment include rash (10 patients), hypertension (6), diarrhea (6), anemia (5), and nausea/vomiting (4). No deaths have been attributable to study treatment. Correlative analyses included assessment of E-cadherin expression in tumor material, demonstrating E-cadherin expression was heterogeneous and high expression of the protein was associated with greater likelihood of CA19−9 decline. Analysis of cfDNA is ongoing and results will be presented at the meeting. Conclusions: Dual targeting of EGFR/MEK signaling shows antitumor activity in PDAC in a subset of patients, in particular those with high levels of E-cadherin expression, which is in agreement with our preclinical findings. Our data warrant further evaluation of this drug combination, possibly in comparison to cytotoxic treatment, in this salvage setting where no standard of care currently exists. Supported by CTEP and NIH R21 CA149939.

Poster Session – Molecular Targeted Agents II 452 POSTER (Board P024) eIF2alpha phosphorylation determines the adaptation of tuberous sclerosis complex mutant cells to stress and their response to anti-tumor therapies A. Koromilas1 , C. Tenkerian1 , J. Krishnamoorthy1 , R. Kamindla1 , U. Kazimierczak1 , S. Wang1 . 1 McGill University, Lady Davis Institute for Medical Research, Montreal − Quebec, Canada Background: Phosphorylation of the alpha (a) subunit of the eukaryotic translation initiation factor eIF2 at serine 51 (eIF2aP) is an important mechanism of cell adaptation to various stress forms. Induction of eIF2aP is mediated by four kinases namely PKR, PERK, GCN2 and HRI, each of which becomes activated by different stimuli. Increased eIF2aP can act as either an inducer of cell death or a promoter of cell survival in response to distinct forms of stress. Specifically, activation ofthe PKReIF2aP arm is associated with induction of cell death whereas activation of the PERK-eIF2aP and/or GCN2-eIF2aP arm(s) with cell survival, increased tumor growth and resistance to chemotherapeutic drugs. We previously demonstrated that genetic disruption or pharmacological inhibition of the PI3K-Akt pathway leads to the activation of the PERK-eIF2aP arm to counteract the loss of Akt and promotecell survival. In this study, we have investigated the molecular events utilized by Akt and eIF2aP to control the survival of tuberous sclerosis complex (TSC)-deficient cells under stress caused by chemotherapeutic drugs. Materials and Methods: We employed MEFs and human fibrosarcoma HT1080 cells in which mTOR, Rictor or Raptor was inactivated by genetic means. We also utilized TSC-mutant MEFs and human TSC-deficient lymphangioleiomyomatosis (LAM) cells engineered to be impaired in eIF2aP. Cells were treated with pharmacological inhibitors of mTOR as well as drugs that induce either endoplasmic reticulum (ER) stress (e.g. thapsigargin, bortezomib) or oxidative stress (e.g. H2 O2 , phenylarsine oxide). The drug effects were examined in vitro and in vivo in SCID mice. Results: Herein, we demonstrate that genetic or pharmacological inhibition of mTORC2 but not of mTORC1 induces eIF2aP through PERK activation. mTORC2 deficiency increases PERK activity owing to Akt inactivation, which impairs PERK by phosphorylation. The biological significance of the data was addressed in TSC-deficient cells, which contain low mTORC2Akt and high mTORC1 levels. TSC-deficient cells exposed to ER stress exhibited increased levels of the PERK-eIF2aP arm, which substituted for the loss of Akt to facilitate survival. However, TSC-mutant cells exposed to oxidative stress downregulated the PERK-eIF2aP arm but upregulated the PKR-eIF2aP arm through an mTORC1-mediated mechanism to promote death. Conclusions: eIF2aP is a master regulator of TSC-deficient cell fate in response to distinct forms of stress caused by chemotherapeutic drugs. Given that pharmacological inhibitors of eIF2aP have started to emerge, inhibition of eIF2aP may be a suitable means to treat TSC-deficient tumors in combinational therapies with drugs that induce ER stress. Alternatively, increased eIF2aP may be the ‘Achilles heel’ of TSC-mutant cells for the implementation of pro-oxidant therapies that rely on the pro-apoptotic effects of eIF2aP. 453 POSTER (Board P025) Plasma metabolomic signature of novel signal transduction inhibitors from preclinical identification to clinical validation J.E. Ang1 , R. Pandher1 , Y. Asad1 , D.J. Skene2 , P. Workman1 , S. Eccles1 , J. De Bono1 , S. Kaye1 , U. Banerji1 , S. Davies2 , F.I. Raynaud1 . 1 The Institute of Cancer Research, Division of Cancer Therapeutics, Sutton, United Kingdom; 2 The University of Surrey, Centre for Chronobiology, Guildford, United Kingdom Background: Modern anticancer drug development is increasingly reliant on the assessment of biomarkers. The limited tumour sampling possible in a clinical study has shifted the matrix studied towards surrogate tissues and circulating biomarkers. Method: We assessed the utility of plasma metabolomics by LC-MS as potential pharmacodynamic biomarkers of novel signal transduction inhibitors in preclinical models and following treatment with PI3K and MEK inhibitors. Descriminatory signatures were identified that were tested in early clinical trials. Results: Consistent changes in plasma metabolite levels with modulation of the PI3K pathway were identified from preclinical models by comparing genetically engineered PTEN+/− mice with wild-type littermate controls; athymic mice with/without PTEN−/− human tumour xenografts and changes following treatment using a PI3K inhibitor. Target/pathway-specificity was evaluated by comparing these changes with those following treatment with carmustine, a chemotherapeutic agent, or a MEK inhibitor. The implicated plasma metabolites include amino acids, carnitine derivatives and lipids. In the clinical setting, the impact of time-of-day and food intake on the

Poster Session – Molecular Targeted Agents II biomarker candidates was evaluated. Despite physiological variations, consistent specific changes in the relevant biomarker candidates could be observed following treatment with either a PI3K or MEK inhibitor in plasma from human tumour xenograft-bearing mice and patients with advanced solid cancers. These data suggest that plasma metabolomics is a valid strategy for monitoring pharmacodynamic effects of novel targeted anticancer agents. Conclusions: Unique changes in plasma metabolite profiles were identified in preclinical tumour models with systemic modulation of the PI3K or RAS-RAF-MEK pathway that were validated in human subjects with advanced solid tumours. 454 POSTER (Board P026) Monitoring therapy response and resistance mutations in circulating RNA and DNA of plasma from patients with malignant melanoma D. Enderle1 , K. Brinkmann1 , T. Koestler1 , S. Bentink1 , K.T. Flaherty2 , J. Skog3 , M. Noerholm1 . 1 Exosome Diagnostics GmbH, Martinsried/Munich, Germany; 2 Massachusetts General Hospital Cancer Center, Boston, USA; 3 Exosome Diagnostics Inc., Cambridge, USA Background: Activating BRAF-mutations (V600E) are frequent events in malignant melanoma, occuring in about 60% of all patients. Mutated BRAF is an important drug target for specific inhibitors, (e.g. Vemurafenib), and monitoring the BRAF mutation status in patients can be useful for informed treatment decisions. Performing repeated tissue biopsies is expensive and an invasive procedure for the patient and does not assess the status of the complete tumor burden including metastases. Isolation of the tumor’s genetic material from blood samples in a ‘liquid biopsy’ is an actively researched option. Blood plasma contains at least two sources of cell-free nucleic acids: circulating cell-free DNA (cfDNA), generated by apoptotic or necrotic cells, and RNA enclosed in extracellular vesicles including exosomes (exoRNA), actively secreted by cells in the body − including the cancerous tissue. Combining both sources of nucleic acids does not only maximize the yield of the limited amount of genetic material circulating in plasma, it also allows to monitor both, biologically different, sources for treatment response and the early occurrence of resistance mutations. Materials and Methods: We applied a novel, spin-column based method (EXO52) to co-isolate all exoRNA and cfDNA out of blood plasma samples from longitudinal clinical samples of patients with malignant melanoma. Purification of high quality nucleic acids and subsequent reverse transcription allows the analysis of mutations present in both fractions of nucleic acids by ultra deep sequencing (UDS) of a gene panel covering 15 mutation hotspots from 8 genes with NGS. The UDS data was generated using a custom library preparation method and analyzed with a novel bioinformatics pipeline to efficiently call the rare mutations. Results: Somatic mutations from melanoma tumors can be readily detected on both exoRNA and cfDNA from plasma samples and was used here to study the mutations over the course of up to 8 months of treatment. Combining both exoRNA and cfDNA is beneficial due to the limited amount of genetic material present in the sample material, especially in patients with lower amounts of circulating mutations. Differential isolation of either RNA or DNA were used to define sets of mutations that are detected earlier in one or the other source since they are generated by different biological mechanisms. Conclusions: The co-isolation of exoRNA and DNA with EXO52 spincolumns is well suited to monitor somatic mutations in melanoma patients over the course of treatment. Combining the information from both mutation sources in the EXO52 eluate and the downstream analysis allows for a more complete picture of the treatment response and recurrence. 455 POSTER (Board P027) Role of MDM2 as therapeutic target in gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) F. Briest1 , I. Grass2 , F. Christen1 , F. Lewens1 , H. Freitag1 , D. Kaemmerer3 , J. Saenger4 , M. Hummel5 , B. Siegmund1 , P. Grabowski1 . 1 ¨ Charite´ − Universitatsmedizin CBF, Gastroenterology, Berlin, Germany; 2 ¨ Berlin, Dept. of Chemistry and Biochemistry, Berlin, Freie Universitat 3 Germany; Zentralklinik Bad Berka GmbH, Dept. of General and Visceral Surgery, Bad Berka, Germany; 4 Institute of Pathology, Bad ¨ Institute of Pathology Berka, Germany; 5 Charite´ − Universitatsmedizin, CBF, Berlin, Germany Background: The p53 pathway is altered in gastroenteropancreatic neuroendocrine tumors (GEP-NENs) although mutations in the TP53 gene are rare events. However, aberrant activation of its negative regulators, caused e.g. by gene amplification of the MDM2 gene are frequently observed. Therefore, we assessed MDM2 expression in a series of GEPNEN fresh frozen material and studied the effects of MDM2 inhibition in human GEP-NEN cell lines.

Friday 21 November 2014 149 Materials and Methods: Fresh frozen tumor material of 35 gastroenteropancreatic neuroendocrine tumors was analysed for MDM2 and p53 expression. Furthermore, the GEP-NEN cell lines BON, QGP-1, KRJ-1 and LCC18 were treated with Nutlin-3, a small molecule inhibitor of MDM2−p53 interaction. Treated cells were analyzed by WST-1 proliferation and apoptosis assays. In Western Blot analyses, fractioned lysates of nuclear and cytoplasmic proteins, as well as whole cell lysates were analyzed for p53 pathway protein expression and activation after Nutlin-3 treatment. TP53 Mutation analysis of the cell lines was performed to verify the druggability with Nutlin-3 in vitro. Results: MDM2 is strongly expressed in GEP-NENs patients and cell lines. MDM2 inhibition leads to a decrease in cellular proliferation in vitro in p53 wild type cell lines. Further data concerning the association of MDM2 expression with clinical parameters, primary tumor localization and target gene expression will be presented on the meeting. Conclusion: MDM2 is involved in aberrant GEP-NEN signal transduction and a druggable target in p53 wild type cells, which merits further investigation. 456 POSTER (Board P028) SF3B1 mutations induce disease relevant aberrant mRNA splicing in cancer and confer sensitivity to spliceosome inhibition S. Buonamici1 , K. Lim2 , J. Feala2 , R. Darman1 , K. Myint3 , E. Park4 , D. Aird5 , B. Chan4 , P. Fekkes4 , R. Furman6 , G. Keaney7 , P. Kumar3 , K. Kunii1 , X. Puyang5 , M. Thomas1 , Y. Mizui1 , M. Warmuth8 , P. Zhu5 , L. Yu2 , P. Smith1 . 1 H3 Biomedicine, Drug Discovery Biology, Cambridge, USA; 2 H3 Biomedicine, Bioinformatics, Cambridge, USA; 3 Eisai, Biomarkers and Personalized Medicine, Andover, USA; 4 H3 Biomedicine, Target Compound Profiling, Cambridge, USA; 5 H3 Biomedicine, Target Genomics, Cambridge, USA; 6 Weill Cornell Medical College, Medical Oncology, New York, USA; 7 H3 Biomedicine, Medicinal Chemistry, Cambridge, USA; 8 H3 Biomedicine, CEO, Cambridge, USA Hotspot mutations in SF3B1 have been identified in chronic lymphocytic leukemia (CLL), myelodysplastic syndromes and several solid tumors. SF3B1 is part of the U2 snRNP complex which binds to the pre-mRNA branch point site and is involved in recognition and stabilization of the spliceosome at the 3 splice site. We applied an analysis pipeline capable of detecting both annotated and novel splice junctions to RNAseq profiles from tumor samples with SF3B1 hotspot mutations (SF3B1MUT ) or wild-type SF3B1 (SF3B1WT ) in breast cancer, melanoma and CLL. This analysis revealed 626 aberrant splice junctions to be significantly upregulated in SF3B1MUT compared to SF3B1WT of which 246 are novel events. The most common splicing change (~80%) observed in SF3B1MUT tumors was selection of alternative 3 splice sites. A shared set of aberrantly spliced mRNAs was observed across different hotspot mutations and lineages; however, unique splicing patterns were also observed suggesting lineage specific effects. Analysis of sequence features around alternative 3 splice sites suggests the recognition and usage of a cryptic splice site by SF3B1MUT containing U2 complexes. A common outcome of these splicing defects was introduction of a premature stop codon in exons downstream of the alternative splice junction leading to nonsense mediated decay (NMD) of aberrant transcripts. Proteomic analysis of isogenic NALM-6 SF3B1WT and SF3B1K700E cells confirmed decreased expression of proteins whose mis-spliced mRNA is subjected to NMD e.g. the NFKB pathway component TAK1. To generate insights into disease mechanisms, gene set enrichment analysis using both aberrantly spliced and differentially expressed genes in SF3B1MUT CLL samples was performed. This analysis revealed defects in pathways related to stem cell and hematopoietic cell differentiation as well as de-regulation of polychrome repressive complex 2 target genes among others. These data indicate SF3B1 mutations offer a mechanism to deregulate gene expression and change protein expression in pathways relevant to the disease phenotype of SF3B1 mutated CLL. We confirmed that at least 10 of the reported hot spot SF3B1 mutations induce aberrant splicing following expression in 293T cells indicating they share a common neomorphic activity. In addition, we demonstrate that a modulator of wild-type and SF3B1MUT associated splicing induces apoptosis in cell lines with hotspot mutations in SF3B1 but not in a cell line containing an SF3B1 mutation that does not induce aberrant splicing. Finally, in a xenograft model of NALM-6 SF3B1K700E cells we demonstrate dose- and time-dependent modulation of splicing and increased overall survival suggesting SF3B1 modulation as a promising approach to treat SF3B1 mutated cancers.

150 Friday 21 November 2014 457 POSTER (Board P029) Preclinical pharmacology of AZD5312, a generation 2.5 antisense oligonucletotide targeting the androgen receptor with differentiated activity from enzalutamide B.R. Davies1 , A. Thomason2 , R. Ellston2 , H. Campbell2 , C. D’Cruz3 , A.M. Mazzola3 , T. Zhou4 , J. Schmidt4 , M. Jo4 , Y. Kim4 , A.R. McLeod4 , D. Blakey2 , N. Brooks2 . 1 AstraZeneca, Oncology iMED, Macclesfield Cheshire, United Kingdom; 2 AstraZeneca, Macclesfield Cheshire, United Kingdom; 3 AstraZeneca, Boston MA, USA; 4 ISIS Pharmaceuticals, Carlsbad CA, USA The androgen receptor is a clinically validated target in prostate cancer; signalling through the androgen receptor remains an important driver of disease progression even in castration resistant disease, as shown by the recent successful phase 3 trials of second generation androgen receptor antagonist enzalutamide and CYP17 inhibitor abiraterone. However, resistance to these agents inevitably emerges, due to a variety of mechanisms including mutations in the receptor and expression of spliced variants that lack the ligand binding domain but still drive AR signalling. Down-regulation of AR using an antisense oligonucleotide has the potential to overcome these resistance mechanisms. AZD5312 causes dosedependent down-regulation of full length AR mRNA and protein, inhibits AR-dependent gene expression, and induces apoptosis in prostate cancer cell lines including LNCaP and VCaP. In 22Rv1 cells, AZD5312 also downregulates the spliced variant AR-v7, and shows a differential effect on a 48gene AR transcriptome signature to enzalutamide, whereas this response is similar in LNCaP cells that only express full length AR. Moreover, AZD5312 inhibits proliferation of 22Rv1 cells, whereas enzalutamide does not. In vivo, AZD5312 down-regulates full length and spliced variant forms of AR, and inhibits PSA protein expression, in the LuCAP86.2 transplantable prostate cancer xenograft model. AZD5312 also reduces AR expression and inhibits the growth of AR-expressing MDA-MB-453 breast cancer xenografts in nude mice supplemented with dihydrotestosterone. In conclusion, AZD5312 is a potent antisense oligonuleotide targeting AR that is therapeutically active preclinically, with differentiated activity from enzalutamide. 458 POSTER (Board P030) Combinational genome and proteome survey of therapeutic targets of hepatocellular carcinoma T. Yamada1 . 1 National Cancer Center Research Institute, Division of Chemotherapy and Clinical Research, Tokyo, Japan Background: A multi-kinase inhibitor, sorafenib (Bayer Pharma AG), has been shown to significantly improve the outcome of patients with unresectable hepatocellular carcinoma (HCC), but the period of survival elongation is limited and only a small proportion of the patients achieved the anticipated therapeutic benefits. It would be necessary to explore the mechanisms of drug resistance and develop new therapeutics. Derangements in the phosphorylation of signalling molecules are hallmarks of HCC. We searched for potential therapeutic target molecules through the comprehensive profiling of kinase genes and proteins. Material and Methods: We sequenced the entire coding regions of 511 protein kinase genes in 20 HCC cell lines using a so-called next generation sequencer. The phosphorylation status of 180 signalling molecules in the 120 pathways registered on the NCI-Nature curated database was determined in 23 HCC cell lines using high-density fluorescence reversephase protein arrays (RPPA). Results: We identified 8 non-synomymous alterations in the 4 mTOR and MAPK pathway genes (RPS6KA1, RPS6KA6, RPS6KB2, and BRAF). We also found that the increased levels of phosphorylated S6 ribosomal protein and c-Raf were associated with the sensitivity of the HCC cell lines to sorafenib. An mTOR inhibitor, AZD8055 (AstraZeneca), exerted growthinhibitory activity against sorafenib-resistant HCC cell lines. Chou–Talalay median dose effect analysis revealed the synergistic effect of mTOR and MAPK inhibitors. Conclusions: Sorafenib is a multi-kinase inhibitor that blocks tumour cell proliferation and angiogenesis through inhibition of c-RAF and b-RAF as well as many receptor tyrosine kinases. The present results indicate crosstalk between the mTOR and MAPK signalling pathways in HCC cell lines and the resistance of HCC to sorafenib is likely attributable to the alternative activation of mTOR signalling. Furthermore, the aberrant mTOR activation was mediated by the genetic and post-translational alterations of the pathway molecules. Use of a combination of mTOR and MAPK inhibitors may be effective for the treatment of HCC patients.

Poster Session – Molecular Targeted Agents II 459 POSTER (Board P031) Preclinical characterization of CC-115, a novel inhibitor of DNA-PK and mTOR kinase currently under clinical investigation D.S. Mortensen1 , K.E. Fultz2 , W. Xu2 , T. Tsuji2 , M. Hickman3 , M. Abbasian3 , G. Khambatta3 , B. Cathers3 , P. Worland4 , M.F. Moghaddam5 , J. Apuy5 , S. Richardson5 , J. Elsner1 , G. Shevlin1 , S. Perrin-Ninkovic1 , S. Canan1 , H.K. Raymon6 , R.K. Narla6 , S. Peng6 , S. Sankar2 . 1 Celgene, Medicinal Chemistry, San Diego CA, USA; 2 Celgene, Oncology Research, San Diego CA, USA; 3 Celgene, Biochemistry, San Diego CA, USA; 4 Celgene, Integrative Research Development, San Diego CA, USA; 5 Celgene, DMPK, San Diego CA, USA; 6 Celgene, Pharmacology, San Diego CA, USA The DNA-dependent protein kinase (DNA-PK) and the mammalian target of rapamycin (mTOR) are both members of PI3K-related kinase (PIKK) family of serine/threonine kinases. mTOR kinase (TORK) exists within two distinct multiprotein complexes, mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) and regulates cell growth, metabolism, proliferation and survival by integrating growth factor signaling with cellular nutritional status and energy use. mTORC1 and mTORC2 are critical mediators of the PI3K/AKT pathway, which is frequently mutated in many cancers, leading to hyperactivation of mTOR signaling. DNA-PK is involved in the repair of DNA double strand breaks. In addition, DNA-PK has been shown to phosphorylate AKT in response to DNA damage suggesting that the mTOR and DNA-PK pathways are linked through the regulation of AKT. Studies have shown that AKT signaling is important for cell survival following DNA damage; thus the dual inhibition of AKT through TORK and DNAPK inhibition would be expected to have added clinical benefit. Here we describe the preclinical characterization of CC-115, a NCE currently under clinical trial investigation. CC-115 is a potent, selective and orally bioavailable dual inhibitor of DNA-PK and TORK, demonstrating inhibition of mTORC1 (pS6), mTORC2 (pAkt-S473) and DNA-PK (pDNA-PK-S2056) in cellular systems. Antiproliferative activity was demonstrated in a panel of tumor cell lines and induction apoptosis was observed in a number of the cell lines tested. Excellent PK properties and demonstrated cellular efficacy of CC-115 allowed for in vivo investigations. Treatment of tumor bearing mice orally with CC-115 afforded inhibition of pathway biomarkers in tumors. CC-115 exhibited dose-dependent tumor growth inhibition in multiple solid tumor xenografts. In summary, CC-115, a selective and potent dual inhibitor of DNA-PK and TORK, has demonstrated anti-proliferative activities and induction of apoptosis in multiple cell lines. CC-115 has further shown, in vitro and in vivo, potent inhibition of both mTORC1 (pS6), mTORC2 (pAkts473) and DNA-PK (pDNA-PK-S2056) and has exhibited dose-dependent efficacy in tumor xenograft models. 460 POSTER (Board P032) Molecular biomarkers of inflammatory signature in melanoma S. Ekmekcioglu1 , M. Shin-Sim2 , K. Tanese3 , V.G. Prieto3 , D.S. Hoon2 , E.A. Grimm3 . 1 University of Texas MD Anderson Cancer Center, Melanoma Medical Oncology, Houston Texas, USA; 2 John Wayne Cancer Institute, Saint John’s Health Center, Santa Monica CA, USA; 3 University of Texas MD Anderson Cancer Center, Melanoma Medical Oncology, Houston TX, USA Features of chronic inflammation are accepted as contributing to progression of many cancers, including melanoma. The purpose of this research was to identify inflammation-related proteins in human melanoma tumor specimens for association with patient outcome associated with survival, for patients with stage III melanoma. Using tissue microarrays, we retrospectively analyzed samples from 158 patients with Stage III melanoma to investigate a panel of 15 inflammatory protein markers by immunohistochemistry. Two assessments were made as percentage of positively stained cells and intensity of staining. For the analyses, four time-to-event outcomes were considered: overall survival (OS), disease specific survival (DSS), recurrence-free survival (RFS), and time to recurrence (TTR). The Kaplan–Meier method was used to estimate the distribution of survival times, and the log-rank test was used to compare distributions. This analysis presents results from several models of interest for RFS and OS and indicates that there is a significant relationship between RFS and OS with combination of CD74 expression and absence of iNOS and MIF expression in melanoma (P < 0.001 and P = 0.007, respectively). Combination of CD74 expression and absence of iNOS and MIF expression in melanoma was associated with better survival in stage III patients. These findings indicate the need for further research into inflammatory proteins’ role in the tumor and microenvironment in melanoma.

Poster Session – Molecular Targeted Agents II 461 POSTER (Board P033) A phase I trial of dabrafenib (BRAF inhibitor) and pazopanib in BRAF mutated advanced malignancies S. Haraldsdottir1 , F. Janku2 , C. Timmers3 , S. Geyer1 , L.J. Schaaf1 , J. Sexton1 , J. Thurmond1 , V. Velez-Bravo2 , V.M. Stepanek2 , E. Bertino1 , K. Kendra1 , A. Mortazavi1 , V. Subbiah2 , M. Villalona-Calero1 , M. Poi4 , M. Phelps4 , M.H. Shah1 . 1 The Ohio State University, Medical Oncology, Columbus, USA; 2 University of Texas M.D. Anderson Cancer Center, Medical Oncology, Houston, USA; 3 The Ohio State University, Solid Tumor Translational Science Resource, Columbus, USA; 4 The Ohio State University, Pharmacology, Columbus, USA Background: BRAF inhibitor therapy is associated with an impressive albeit short-lived response rate in the treatment of malignant melanoma with BRAF V600 mutation. Mechanisms of resistance include upregulation of the MAPK pathway and upregulation of PDGFR-b. Combining therapies targeting VEGF and MAPK pathways could enhance therapy effects and target potential pathways of resistance. We report a NCCN-sponsored phase I trial combining dabrafenib with pazopanib in the treatment of advanced BRAF mutated malignancies. Material and Methods: This phase I dose-escalation trial included patients with any advanced BRAF mutated malignancy who had failed standard therapies. Prior use of dabrafenib or pazopanib was not allowed. Dose level (DL) 1 started with dabrafenib 50 mg bid and pazopanib 400 mg qd, escalating to 150 mg bid and 800 mg qd on DL 5. Pharmacokinetics were measured and efficacy was evaluated by RECIST 1.1 and tumor markers every 8 weeks. Results: Fifteen patients have enrolled on the first 4 DLs with a median age of 59 years and ECOG performance status of 0 (14%), 1 (79%) or 2 (7%). Tumor types include melanoma, thyroid cancer, ovarian cancer, lung cancer, appendiceal adenocarcinoma, bladder cancer, colon neuroendocrine cancer (NEC) and parotid cancer. A dose-limiting toxicity (grade (G) 3 bowel perforation) was seen on DL 3 in a patient with colonic high-grade NEC after 4 days on study drugs. The drug related adverse events include nausea (40%), vomiting (27%), skin lesions (27% including one melanoma), fatigue (20%), diarrhea (13%), fever (13%), weight loss (13%), decreased white blood cell count (13%) and hand-foot syndrome (13%). Serious adverse events leading to hospital admission include a G3 and G4 pulmonary emboli, G3 pleural effusion, G3 abdominal infection and G2 fever. Fourteen patients were evaluable for response. Two patients with low-grade serous ovarian cancer had a partial response (14%, 1 unconfirmed), one patient with appendiceal adenocarcinoma had stable disease for 9 months and 1 patient with thyroid cancer has stable disease ongoing for 4 months. Two patients on DL 4 have not had restaging scans. DL 5 is now accruing. Pharmacokinetics for the first 6 patients on study suggests induction of pazopanib metabolism on C2D1. Conclusions: The combination of dabrafenib with pazopanib was well tolerated on the first 4 dose levels with 1 dose-limiting toxicity seen. Clinical activity was seen in patients with ovarian cancer, appendiceal adenocarcinoma and thyroid cancer who were all heavily pretreated. This study was approved and funded by the National Comprehensive Cancer Network (NCCN) Oncology Research Program from general research support provided by GlaxoSmithKline, LLC. 462 POSTER (Board P034) Clinical sequencing of cancer in real-time by digital sequencing of cell-free DNA for tailoring targeted therapy in refractory cancer patients A. Talasaz1 , S. Mortimer1 , B.J. Schiller1 , G. Mei1 , S. Huang2 , D.S.B. Hoon2 , H. Eltoukhy1 . 1 Guardant Health Inc., Redwood City CA, USA; 2 Dept Molecular Oncology, John Wayne Cancer Institute, Santa Monica CA, USA Introduction: Genomic alterations of metastatic cancers may be discordant with alterations in primary tumors due to continuous evolution of disease and tumor heterogeneity. Genomic profiling of cell-free DNA (cfDNA) through a simple blood test can provide insights into cancer genomics when biopsies of residual disease are not an option. Methods: We used single-molecule digital sequencing technology, Guardant360 for high-fidelity and comprehensive profiling of 80,000 bases across 54 actionable genes in cfDNA. Somatic mutations and gene amplifications have been concurrently analyzed and the concordance of alterations in cfDNA and matched biopsies were studied in patients with advanced cancers. The discordance mutations in patients who developed resistance to targeted therapies have been studied. Results: The concordance of tumor and cfDNA mutations across 150 patients was 92% when tumor biopsies were taken concurrently with the blood draw. Previously reported and some novel resistance-related alterations were found in cfDNA of refractory patients. Across more than 500 patients, the overall detection rate of somatic alterations in cfDNA

Friday 21 November 2014 151 approached 90% for all indications. We also showed the promise of cfDNA analysis in monitoring the response to targeted therapies in the select patients with longitudinal blood samples. Conclusions: Comprehensive sequencing of patient’s cancer through simple blood test enables monitoring the genomic evolution of the residual disease in real time and can be utilized for tailoring targeted therapies for patients with refractory disease. 463 POSTER (Board P035) PTPN11 is a central node in intrinsic and acquired resistance to targeted cancer drugs A. Prahallad1 . 1 Netherlands Cancer Institute, Molecular Carcinogenesis, Amsterdam, Netherlands Background: Resistance to targeted therapies is a major clinical problem. Inhibitors that target oncogenic proteins in cancer-relevant pathways often yield responses of limited duration in patients. Tumors that harbor driver mutations in for instance BRAF or KRAS can be treated with BRAF or MEK inhibitors. However, these drugs often are ineffective due to feedback activation of Receptor Tyrosine Kinases (RTKs). This feedback activation restores the pathway activity and neutralizes the effect of the inhibitors. One such example is the inefficacy of BRAF inhibitors in BRAF mutant CRC. However, simultaneous inhibition of BRAF and EGFR can overcome feedback mediated resistance to BRAF inhibitor monotherapy in BRAF mutant CRC, showing the potential of drug combinations to battle intrinsic resistance to targeted cancer drugs. Materials and Methods: We performed a shRNA-based loss-offunction genetic screen centered around the phosphatome to search for phosphatases that are synthetically lethal with BRAF inhibition in the BRAF mutant CRC cell lines Widr and Vaco, which are both refractory to BRAF inhibitor monotherapy. Results: We found that inhibition of PTPN11 is synthetic lethal with BRAF inhibition is BRAF mutant colon cancer. We show that both knockdown and knockout of PTPN11 blocks the signaling downstream of RTKs, and prevents feedback mediated re-activation of the MAPK pathway upon BRAF inhibition. We extend this observation to KRAS mutant colon, lung and pancreatic cancer cell lines, all of which are refractory to MEK inhibitors. Furthermore, loss of PTPN11 also reversed growth factor mediated resistance in BRAF mutant melanoma. In vivo, we find that BRAF mutant melanoma patients that have become resistant to BRAF inhibitors due to RTK activation engage PTPN11 for signaling. Conversely, a patient with an acquired secondary mutation in the BRAF protein that causes the BRAF inhibitor resistance does not engage PTPN11. This makes activated PTPN11 a suitable biomarker to monitor RTK mediated resistance. Conclusions: Our data indicate that simultaneous inhibition of an oncogenic driver mutation and PTPN11 can have strong anti-proliferative effects and this could be a strategy to treat tumors that do not respond to monotherapies or have become resistant to them due to RTK activation. In addition, activation of PTPN11 can serve as a biomarker to assess MAPK inhibitor treatment efficacy. 464 POSTER (Board P036) Influence of warm and cold ischemia on molecular patterns in clinical biospecimen F.T. Unger1 , N. Lange2 , P.C. Uhlig3 , H. Juhl4 , K.A. David4 . 1 Indivumed GmbH, Proteomics/Genomics/Drug Testing, Hamburg, Germany; 2 Indivumed GmbH, Genomics, Hamburg, Germany; 3 Indivumed GmbH, Immunohistochemistry, Hamburg, Germany; 4 Indivumed GmbH, Indivumed, Hamburg, Germany Background: The development of personalized medicine in oncology is fostered by high-throughput analysis of molecular biomarkers in human cancer biospecimens. Tissue quality strongly depends on the pre-analytical conditions in which it was acquired. Therefore, warm and cold ischemia times that tissues are exposed to during collection are of critical importance. Insufficient quality of such tissues may lead to spurious results and data misinterpretation resulting in biased stratification of patients. The present study was conducted to gain a better understanding of the effects of warm and cold ischemia on the molecular composition of tissue specimens and especially on critical clinical biomarkers whose expression and activity level inform targeted therapy evaluation in cancer. Material and Methods: Normal and cancer tissue from patients undergoing CRC (n = 50) surgery were analyzed comparing samples taken by endoscopy at the beginning of surgery with postsurgical samples fixed within 10, 20 and 45 minutes postsurgery. In addition, we collected from patients undergoing liver surgery (n = 43) normal liver tissue before and 10 minutes after clamping of the hepatic artery and compared those samples together with normal and metastatic cancer tissue fixed 10, 20 and 45 minutes after completion of resection. Tissue samples were analyzed using

152 Friday 21 November 2014 a whole genome gene expression array as well as different technologies for protein expression and protein phosphorylation with an emphasis on the epidermal growth factor receptor (EGFR)-pathway. Results: In this study, we demonstrated that surgery, vascular clamping and subsequent processing had a significant impact on the expression of genes and the expression and the phosphorylation status of proteins. The gene expression profiling showed a high variability between patients. Significant changes of expression in individual patients occurred in up to 690 genes in normal tissue (mean 118 genes) and in up to 4,116 genes in primary cancer tissue (mean 1,553 genes) comparing presurgery and ischemia time points postsurgery. Clinically highly relevant was the finding that expression of EGFR-pathway proteins and, in particular, their phosphorylation status is affected by surgery and postsurgical tissue processing. For example, the expression level of EGFR changed more then 2-fold in cancer tissue of 30% of the patients. Conclusion: An understanding of tissue data variability in relation to processing techniques during and postsurgery is mandatory when testing surgical specimens in the context of clinical diagnostics, drug development, or identification of predictive biomarkers. To obtain reliable expression data, tissue processing for research and diagnostic purposes needs to be highly standardized. Alterations in molecular patterns due to ischemia, identified in this study have to be analyzed with caution in research and development programs. 465 POSTER (Board P037) Biomarker analysis in Phase 1b study of anti-cancer stem cell antibody Tarextumab (TAR) in combination with nab-paclitaxel and gemcitabine (Nab-P+Gem) demonstrates pharmacodynamic (PD) modulation of the Notch pathway in patients (pts) with untreated metastatic pancreatic cancer (mPC) A. Kapoun1 , E. O’Reilly2 , A. Cohn3 , J.C. Bendell4 , L. Smith5 , J.H. Strickler6 , W. Gluck7 , Y.W. Liu1 , B. Wallace1 , R. Tam1 , B. Cancilla1 , A. Brunner1 , D. Hill1 , L. Zhou1 , J. Dupont1 , C. Zhang1 , M. Wang1 . 1 OncoMed Pharmaceuticals, California, USA; 2 Memorial Sloan-Kettering Cancer Center, NY, USA; 3 Rocky Mountain Cancer Center, Denver CO, USA; 4 Sarah Canon Research Institute, Nashville TN, USA; 5 START, San Antonio TX, USA; 6 Duke University, Durham NC, USA; 7 Greenville Hospital System, Greenville SC, USA Background: The Notch pathway plays a central role in embryonic development, the regulation of stem and progenitor cells, and is implicated in many human cancers. TAR is a fully human IgG2 which inhibits the signaling of both Notch2 and Notch3 receptors. Tumor regression was seen in Notch3 expressing patient-derived pancreatic cancer xenografts when TAR was combined with nab-paclitaxel and gemcitabine (Nab-P+Gem). The maximum tolerated dose (MTD) of single agent TAR was 7.5 mg/kg every other week (Smith, EORTC 2012); the main dose limiting toxicity (DLT) was Grade 3 diarrhea. We sought to determine the PD effects of various doses of TAR combined with Nab-P+Gem on Notch signaling, stem cell pathways, and other aspects of its mechanism of action by examining surrogate tissues (e.g. hair follicle and blood) and also serial tumor biopsies from Phase Ib patients. Additionally this study examines Notch 3 gene expression as a predictive biomarker and its correlation with clinical response. Methods and Results: Biomarker analysis of surrogate tissues and tumors was performed in the Phase 1b study of the anti-cancer stem cell antibody TAR in combination with Nab-P+Gem in metastatic pancreatic cancer. Forty patients enrolled in 7 dose-escalation cohorts were analyzed for biomarkers. Notch pathway-related genes including HEYL, HES2, and NOTCH2 and cancer stem cell markers were found to be regulated in hair follicles at TAR doses of 7.5 mg/kg and above in combination with NabP+Gem. Plasma and blood RNA biomarkers were also modulated by TAR plus Nab-P+Gem. Conclusions: The PD effects of TAR plus Nab-P+Gem on Notch targets, stem cell pathways in surrogate tissues were clearly established. Detailed PD and predictive biomarker results will be presented in surrogate and serial tumor tissues. Updated efficacy data and its correlation to Notch3 expression levels will also be presented. The Phase 2 component of the ALPINE study is initiating in 2014.

Poster Session – Molecular Targeted Agents II 466 POSTER (Board P038) IGF2 drives IGF oncogenic signaling in HCC and emerges as a potential target for therapies I.M. Quetglas1 , R. Pinyol1 , D. Dauch2 , A. Portela3 , A. Villanueva4 , J. Peix1 , M. Higuera1 , A. Moeini1 , J. Zucman-Rossi5 , M. Esteller6 , V. Mazzaferro7 , L. Zender2 , J.M. Llovet1 . 1 IDIBAPS, HCC Translational Research Laboratory Barcelona Clinic Liver Cancer Group (BCLC) Liver Unit Hospital Cl´ınic CIBEREHD University of Barcelona, Barcelona, Spain; 2 University of Tuebingen, Division of Translational Gastrointestinal Oncology Department of Internal Medicine I, Tuebingen, Germany; 3 IDIBELL, Cancer Epigenetics and Biology Programme, Barcelona, Spain; 4 King’s College Hospital, Institute of Liver Studies, London, United ´ fonctionnelle des tumeurs solides, Paris, Kingdom; 5 INSERM, Genomique France; 6 IDIBELL, 3Cancer Epigenetics and Biology Programm, Paris, France; 7 INT, Fondazione IRCCS Istituto Nazionale dei tumori, Milan, Italy Background: Hepatocellular carcinoma (HCC) is the 16th absolute cause of death world-wide and accounts for 80% of all liver cancers. IGF signaling has a relevant role in the pathogenesis of HCC and elucidation of its key molecular drivers is important to overcome the poor therapeutic results obtained so far by targeting this pathway in HCC. We aimed to explore the oncogenic potential of IGF2 and IGF1R in genetically modified animal models and to assess the efficacy of molecular therapies against these targets. Material and Methods: A cohort of 228 HCCs was characterized analyzing gene expression, exomic mutations, DNA copy number, methylation status and kinome profiling with a focus on the IGF pathway. Activation of IGF2 fetal promoters (P3-P4) were additionally assessed by qRT-PCR. In order to evaluate the role of the IGF pathway components in HCC, a mouse model was generated using tail vein injection of plasmid DNA, and transposonmediated genomic integration of IGF2 or IGF1R along with c-myc and Akt. IGF2 and IGF1R levels were assessed in the liver tumors by qRT-PCR, and IGF1R activation was evaluated via Western Blot. Therapeutic potential of the monoclonal antibody BI836845 (Boehringer Ingelheim), selective for IGF ligands, was studied in vitro in 2 HCC cell lines overexpressing IGF2 (Hep3B, Huh7) and 2 with normal expression (PLC5, SNU449) by analyzing cell survival, proliferation and pathway activation. Results: Surrogates of IGF pathway activation such as IGF1R kinase activation (62/228;28%), 2-fold change IGF2 overexpression (28/228;12%), pIGF1R signature activation (95/228;43%) and hypomethylation of IGF2 fetal promoters (21/54 vs 7/171, p < 0.0001) were identified in the whole cohort, and enriched in the Proliferation molecular subclass (p < 0.001 in all cases). Overexpression of IGF2 and IGF pathway activation in the HCC mouse model was confirmed. Only mice overexpressing IGF2 showed increased number of tumors and significantly reduced survival (p = 0.02) when compared to those with c-myc/Akt alone. The BI836845 monoclonal antibody against IGF ligands significantly reduced cell viability and proliferation (40% decrease, p = 0.002) and inhibited IGF pathway activation (abrogation of pIGF1R, pAkt, pErk), specifically in HCC cell lines overexpressing IGF2. Conclusions: IGF2 demonstrated to be a key driver of IGF axis in aHCC GEMM model and has emerged as a potential target in a subgroup of HCC patients showing IGF pathway activation. IGF2 blockage by a novel anti-IGF therapy (BI836845) results in a decrease of cell viability and proliferation. This study provides the rationale for exploring IGF2 as target of therapies in pre-clinical and early clinical studies in HCC. 467 POSTER (Board P039) Two in one − Delivery of apoptotic signal into cancer cells by new class of TRAIL derived, fusion protein S.D. Pawlak1 , B.M. Zerek1 , P.K. Rozga1 , K. Bukato1 , M. Szymanik1 , M. Teska-Kaminska1 , A. Pieczykolan1 , A. Jaworski1 , M. Galazka1 , K. Poleszak1 , W. Strozek1 , J.S. Pieczykolan1 . 1 Adamed Sp. z.o.o., Drug Discovery, Warsaw, Poland The tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that initiate apoptosis of tumor cells through the activation of their death receptors. The ability of TRAIL to selectively induce apoptosis of tumor cells without normal cells makes it an attractive agent for cancer therapy. However, in most cases it wasn’t potent enough to become the effective single therapy agent. The antimicrobial, peptide ‘KLA’ (KLAKLAK)is also well known as powerful apoptosis-inducing agent that can disrupt the mitochondrial membrane. However its antitumor effect is limited as peptide has to be targeted and internalized. To overcome the deficiencies of TRAIL and peptide ‘KLA’ we have merged them creating a novel molecule AD-O56.9, which comprised features of both components. The soluble domain of TRAIL (carrier and in some cases also an effector) has been fused with a peptide (KLAKLAK)2 (effector).

Poster Session – Molecular Targeted Agents II The peptide was additionally linked with poly-arginine cell penetrating domain to increase efficiency of its internalization. To allow separation of TRAIL/Apo2L domain and the effector peptide after reaching the tumor, we linked these two domains with a sequence motif recognized by MMPs and uPa proteases. Targeted effector should induce cancer cell death by triggering mitochondrial membrane permeabilization and swelling, resulting in the induction of apoptosis. AD-O56.9 protein was produced using E. coli as expression system, and purified from insoluble inclusion bodies. The obtained molecule was characterized biophysically. MTT cell viability assay was used to assess AD-O56.9 mediated killing of carcinoma cells. Flow cytometric analysis were used to evaluate influence of the AD-O56.9 on mitochondrial membrane integrity, and other apoptosis biomarkers. The SPR was used to confirm interaction with cellular targets − death receptors. The tumoricidal activity of AD-O56.9 was evaluated in NOD/SCID mice bearing different types of human cancers xenografts. AD-O56.9 exhibits cytotoxic effect on various cancer cell lines (established and primary and TRAIL-sensitive and TRAIL-resistant), but showed no toxic effect on normal cells. The ‘KLA’ peptide that enables to overcome resistance to TRAIL is effective only being a part of AD-O56.9. Analyzing apoptosis markers as PARP cleavage and caspases activity, on sensitive cell line (NCI-H460) and TRAIL-resistant cell line (A549) we showed that AD-O56.9 induced apoptosis in these cells. AD-O56.9 also caused strong depolarization of mitochondrial membrane that is a cellular target for ‘KLA peptide’. Importantly, AD-O56.9 administration caused significant regression on mice xenograft models of TRAIL sensitive human pancreatic carcinoma MIA PaCa-2, human oesophageal adenocarcinoma OE19, TRAIL-resistant human hepatocellular carcinoma HepG2 and human uterine sarcoma MES\erhyphen;SA/Dx5. Our novel fusion protein AD-O56.9 is able to induce apoptosis in many cancer cell lines, that are TRAIL resistant and causes tumor regression in mice bearing human cancer cells. The obtained results show that selection and linking two anticancer molecules that displays some therapeutic deficiency but with complementary properties could be the promising strategy in development of new therapeutics. 468 POSTER (Board P040) Clinicopathologic implication of c-MYC gene copy number gain and overexpression in colorectal cancer K. Lee1 , Y. Kwak2 , G. Choe1 , W. Kim2 , D. Kim3 , S. Kang3 , H. Lee1 . 1 Seoul National University Bundang Hospital, Pathology, Seongnam-si, South Korea; 2 Seoul National University Hospital, Pathology, Seoul, South Korea; 3 Seoul National University Bundang Hospital, Surgery, Seongnam-si, South Korea Background: The aim of this study was to determine the incidence and clinical implications of c-MYC gene copy number (GCN) gain and overexpression in the patients with primary colorectal cancer (CRC). Materials and Methods: c-MYC status was determined by performing dual-color silver in-situ hybridisation (SISH), mRNA in-situ hybridisation (ISH), and immunohistochemistry (IHC) in a retrospective cohort of 367 consecutive CRC patients. In addition, b-catenin expression was investigated by IHC and microsatellite instability (MSI) was analysed with 5 microsatellite markers. Results: c-MYC amplification, defined as a c-MYC:CEP8 ratio  2.0, was observed in 31 patients (8.4%) and GCN gain, defined as a c-MYC copies/nucleus  4.0, was found in 63 patients (17.2%). c-MYC GCN gain was more frequently observed in CRCs located in the sigmoid colon and rectum than other location (P = 0.034). c-MYC GCN gain was associated with large tumour size (P = 0.041) and microsatellite stable or MSI-low (P = 0.029). Although c-MYC amplification did not predict patients’ prognosis (P = 0.142), c-MYC GCN gain was significantly associated with worse patients’ survival (P = 0.015). By multivariate Cox regression analysis, the hazard ratio of c-MYC GCN gain was 2.350 (95% CI, 1.453 to 3.802; P < 0.001). In a subgroup of stage II and III, c-MYC GCN gain was also significantly associated with worse prognosis by univariate (P = 0.034) and multivariate (P = 0.040) survival analysis. The incidence of c-MYC protein overexpression was approximately 201 of 367 patients (54.8%). c-MYC protein overexpression was more correlated with mRNA overtranscription [correlation coefficient (CC) = 0.479] and b-catenin nuclear expression (CC = 0.282) than c-MYC/CEP ratio (CC = 0.195) and c-MYC GCN gain (CC = 0.211). In contrast to c-MYC GCN gain, c-MYC protein overexpression, mRNA overtranscription, and b-catenin nuclear expression were significantly associated with better prognosis by univariate survival analysis (P = 0.010, P = 0.042, and P = 0.011, respectively), but not significant by multivariate analysis. Conclusions: c-MYC GCN gain was an independent prognostic factor in both consecutive primary CRC patients and in the subgroup of stage II and III CRC patients. However, there was low correlation between c-MYC protein overexpression and c-MYC GCN gain or amplification. Our findings

Friday 21 November 2014 153 may serve as a basis for future studies on predicting poor outcome in CRC patients and on selecting candidates for c-MYC targeted therapy. 469 POSTER (Board P041) OTX015, a novel BET-bromodomain (BET-BRD) inhibitor, is a promising anticancer agent for human glioblastoma L. Ouafik1 , C. Berenguer1 , M. Cayol1 , L. Astorgues-Xerri2 , M. Bekradda3 , E. Odore4 , K. Rezai5 , M.E. Riveiro3 , E. Cvitkovic6 . 1 UMR911-CRO2, Team 4, Marseille Cedex 05, France; 2 Oncology Therapeutic ˆ Development, Hopital Beaujon Department of Medical Oncology, Clichy Cedex, France; 3 Oncology Therapeutic Development, Department of Medical Oncology, Clichy Cedex, France; 4 Oncology Therapeutic Development, Institut Rene´ Huguenin Hospital, Saint Cloud, France; 5 Institute Curie, Rene´ Huguenin Hospital, Saint Cloud, France; 6 Oncology Therapeutic Development, Head of Consulting, Clichy Cedex, France Background: BRD2 and BRD4, members of the BET family of bromodomains, were recently described to be significantly increased in glioblastoma multiforme (GBM), the most common primary adult brain cancer. Small molecules antagonizing BET proteins binding to acetylated histones are thus candidate therapies for GBM. We report in vitro and in vivo antitumoral effects of OTX015 (OncoEthix SA, Switzerland), a potent BET-inhibitor, in U87MG cells, a p53 wild-type human glioblastoma-astrocytoma model with an epithelial-like phenotype, sensitive to temozolomide (TMZ). Material and Methods: OTX015 GI50 was determined with MTT assays in human U87MG cells after 72 h. GBM cells were also treated with OTX015 500nM for 4 h and 24 h and RNA levels of 9 genes were evaluated (C-MYC; N-MYC; MTHFD1L; HEXIM1; HIST2H2BE; HIST1H2BJ; SESN3; HIST1H2BK; HIST2H2BF, HIST2H4A). qRT-PCR were performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. 105 U87MG cells were injected in the frontal lobe of male athymic NMRI nude mice and 5 days later mice were randomized to 4 groups (6 mice/group); vehicle (PBS, once daily, ip, continuous), OTX015 (25 or 50 mg/kg/bidaily, gavage, continuous), or TMZ (100 mg/kg/daily, ip, days 6−10). Animals were sacrificed upon apparent symptoms. Median survival was determined by Kaplan–Meier method, with a log-rank Mantel-Cox test. Results: OTX015 and JQ1, the benchmark compound, displayed antiproliferative effects in U87MG after 72 h with GI50 values of 0.9 and 2.1 mM, respectively. C-MYC mRNA levels remained unchanged up to 24 hexposure to OTX015, while HIST2H2BE, HIST1H2BK, HIST2H2BJ and HIST2H4A were upregulated. OTX015 significantly increased survival in U87MG-bearing mice with median survival of 27 and 29 days for 25 and 50 mg/kg OTX015, respectively vs 21 days in control mice (p < 0.05). No major side effects were seen during OTX015 treatment, whereas TMZ led to pronounced weight loss over 5 days treatment. Conclusion: C-MYC appears to play a minor role in mediating OTX015 antitumor effects while p21 and HEXIM-related genes coding for cell cycle regulation and histones are upregulated, indicating chromatin remodeling. Oral OTX015 significantly slowed progression in an orthotopic human glioblastoma mouse model, highlighting the therapeutic potential of OTX015 in GBM. 470 POSTER (Board P042) Transcriptional regulation of cancer stem cells marker CD133 by p53 E.K. Park1 , S.Y. Bang1 , S.A. Yi1 , J.W. Han1 . 1 Research Center for Epigenome Regulation (RCER) School of pharmacy Sungkyunkwan University, Department of Biochemistry and Molecular Biology, Suwon Gyeunggi-do, Korea Background: Cancer stem cells (CSCs) are known as a rare population of tumor cells that are responsible for tumor initiation and self-renewal capacity. Based on these characteristics, CSCs have been considered as a potential target for cancer therapy. CD133 is currently one of the bestcharacterized CSCs markers in various tumor lines. However, the precise function and regulation mechanism of CD133 in CSC are still unclear. In this study, we investigated the role of p53 in transcriptional regulation of CD133. Methods: We treated doxorubicin to the various cancer cell lines and examined expression level of CD133 by Immunocytochemistry, qRT-PCR and Western blot analysis. Luciferase reporter gene assay and Chromatin Immunoprecipitation assay was performed for analyzing CD133 promoter activity and p53 promoter binding sites in the CD133 promoter. The functions of CD133 were measured using cell counting, colony formation assay and Brdu Incorporation assay. Results: We found opposite expression pattern of CD133 and tumor suppressor p53 in several cancer cell lines. CD133 expression is decreased by either induction or overexpression of p53, indicating that p53 acts as a

154 Friday 21 November 2014 negative regulator of CD133. In addition, p53 directly binds on the CD133 promoter and suppresses its promoter activity, showing that CD133 could be a novel target gene of p53. Furthermore, CD133 is necessary for tumor formation and growth of cancer cells, and these functions of CD133 are obstructed by overexpression of p53. Conclusions: These results suggest that p53 is a key modulator for CD133 expression in CSC, and provide novel perspective on targeting CSCs for the cancer therapy. 471 POSTER (Board P043) Screening of Champions predictive TumorGraft platform guides the clinical development of the selective dual BRAF-EGFR inhibitor CEP-32496 B. Ruggeri1 , M. Wabler2 , E. Bruckheimer2 , B. Wilkinson2 , B. Dorsey1 , S. Trusko1 , J. Friedman2 . 1 Teva Pharmaceuticals, Discovery Research, Baltimore MD, USA; 2 Champions Oncology, Baltimore MD, USA Mutations in the BRAF gene have been identified in approximately 7% of cancers, including 60−70% of melanomas and 4−16% colorectal cancers. CEP-32496 (Teva Pharmaceuticals) is an orally active dual inhibitor of wild type and V600E-mutant BRAF kinase and EGFR, as well as other oncogenic protein kinases, including BCR/ABL and RET. Champions Oncology has developed a predictive platform that utilizes the implantation of patient derived xenografts (PDX) in immune-deficient mice that preserves the biological properties of the original human tumor and provides an accelerated and focused translational path into clinical trials. A screening study using a panel of Champions TumorGraft® models of human colorectal cancer and melanoma possessing either the BRAF mutant or BRAF wild type genotype was conducted to compare the therapeutic activity of CEP-32496 versus a clinically approved BRAF inhibitor, vemurafenib, as well as two standard of care agents (irinotecan or temozolomide). This screening study demonstrated an overall comparable or superior response (p < 0.05) of CEP-32496 compared to vemurafenib in 13 out of 15 tumorgraft models of melanoma. Notably, CEP-32496 displayed robust therapeutic activity (p < 0.05) over vemurafenib in 7 tumorgraft models of BRAF-mutated colorectal cancer. Given these initial results in colorectal cancer, we further explored the anti-tumor activity of CEP-32496 and several standard of care agents for colorectal cancer (irinotecan, oxaliplatin, 5-FU, and cetuximab) given as monotherapy or in combination in two tumorgraft models of BRAF-mutated colorectal cancer. In both colorectal tumorgraft models, the combinations of sub-optimal oral doses of CEP-32496 and optimal doses of each standard of care agent achieved significantly improved anti-tumor activity (p < 0.05) relative to standard of care agents and in many cases vehicle-treated animals. The combination treatment regimens were generally well tolerated exhibiting no body weight loss or mortality. Based on this dataset, CEP-32496 is currently in clinical trials in cancer patients. 472 POSTER (Board P044) Do we hit the target? Phospho-reactome measurements determine efficacy of targeted therapies L. van ’t Veer1 , M. Moro2 , Z. Chen3 , P.R.E. Lee1 , B. Pan4 , D. Brunen5 , A. Prahalled5 , R. Bernards5 , J.P. Coppe1 . 1 University California San Francisco, Cancer Center, San Francisco, USA; 2 Showa University, Breast Surgical Oncology, Tokyo, Japan; 3 Kinogea, Bioinformatics, Shanghai, China; 4 Peking Union Medical College, Breast Surgery, Beijing, China; 5 Netherlands Cancer Institute, Molecular Carcinogenesis, Amsterdam, Netherlands Background: Treating cancer increasingly relies on targeting kinases. Knowing which kinases or kinase networks circumvent therapeutic interventions, are today’s biomedical challenges. Measuring the activity of kinase enzymes, and potentially monitoring the functionality of the entire human phospho-reactome at once, remains largely unexplored. We developed a new assay to monitor the phospho-catalytic activity of multitude kinase enzymes. We used this assay to identify oncogenic phospho-signatures prevalent in breast cancer, and drug-sensitivity profiles in models of kinase-targeted therapies. Methods: We developed the high throughput kinase activity-mapping (HTKAM) assay to quantify the catalytic state of kinases. 228 phosphosensing probes derived from biological target sites of kinase enzymes, were developed into an ATP-consumption screen to identify the activity signatures of EGFR, MAPK, AKT, ABL and SRC family kinases. We used breast cancer cell lines (MDA-MB-231, MDA-MD-436, HCC70, AU565, MCF7, T47D), and a series of cancer cell lines treated with one or combinations of kinase-targeting agents (vemurafanib, gefitinib). Functional phospho-signatures were derived from HT-KAM measurements. Results: In cancer cell extracts, the HT-KAM phospho-sensing assay revealed the heterogeneity of EGFR, AKT, MEK, ERK, and SRC kinase

Poster Session – Molecular Targeted Agents II activity networks for recombinant kinases and in lysates from breast, colorectal, lung, and melanoma cancer cells. The HT-KAM assay identified that MEK/ERK activity was shut down after BRAFV600E inhibition. We successfully identified that Vemurfanib-resistant cells were dependent on hyper-activated feedback modulator kinases EGFR and AKT, and their downstream substrates, including mTOR. Combination therapy inhibiting both BRAFV600E and a parallel kinase driving resistance, successfully led to cancer cell growth inhibition or death. Conclusion: This unique strategy and resources allows to comprehensively measure the catalytic activity of multitude kinases at once, ultimately representing a new molecular dimension to characterize biological samples. Our new phospho-reactome profiling system defined how therapeutic interventions lead to reprogramming of phospho-circuits, revealing previously unknown circuits. 473 POSTER (Board P045) FDG PET/CT as imaging biomarker in the era of molecular targeting therapies: sequential FDG PET/CT demonstrated biological response and acquisition of resistance to tyrosine kinase inhibitor therapy for renal cell carcinoma N. Nakaigawa1 , K. Namura1 , D. Ueno1 , U. Tateishi2 , T. Inoue2 , M. Yao1 . 1 Yokohama City University Graduate School of Medicine, Department of Urology, Yokohama, Japan; 2 Yokohama City University Graduate School of Medicine, Department of Radiology, Yokohama, Japan Background: Tyrosine kinase inhibitors (TKIs) targeting vascular endothelial growth factor receptor and suppressing biological activity of renal cell carcinoma (RCC) improved the prognosis of patients with advanced RCC. It has been desired a novel index assessing the biological response to TKIs. FDG PET/CT is a non-invasive tool to evaluate glucose metabolic status, which can be the index of biological activity of cancer. We investigated the sequential FDG accumulation change during TKI treatment. Material and Methods: We studied retrospectively the 38 patients treated with TKIs (19 sorafenib and 19 sunitinib) and evaluated by FDG PET/CT sequentially until their RCC demonstrated progression disease (PD) defined by RECIST criteria version 1.1. The standardized uptake value (SUV), which is a semiquantitative simplified measurement of the tissue FDG accumulation rate, was measured and investigated the sequential changes of maximum SUV (SUVmax), which was the highest SUV in the each RCC lesion, and maximum SUVmax (max SUVmax), which was highest SUV in the individual patients during TKI treatment. Results: The max SUVmax in 28 cases (74%) of total 38 cases was decreased during TKI treatment and the median progression-free survival (PFS) of them was 203 days, and max SUVmax in 10 cases was increased and that of them was 110 days. There was statistic difference (P = 0.005). In the 28 cases with decreasing max SUVmax, the median date when SUVmax reached to nadir was 61 days. The max SUVmax increased after nadir in 23 cases of the 28 cases (82%) showing decrease in the max SUVmax. Totally, the max SUVmax in 33 (87%) cases increased before or at the same time cancer showed PD defined by RECIST criteria. The max SUVmax at PD was higher than that before TKI treatment in 19 cases (50%). Conclusions: The RCC which FDG accumulation was decreased by TKI treatment showed long PFS. Additionally, the increase in FDG accumulation during TKI treatment suggested the acquisition of resistance to TKI. FDG PET/CT had potency as an imaging biomarker to assess the biological response of RCC to TKI treatment. 474 POSTER (Board P046) Potentiation of oxaliplatin in colon cancer by JNK inhibition I. Vasilevskaya1 , M. Selvakumaran1 , L. Cabal-Hierro1 , P. O’Dwyer1 . 1 University of Pennsylvania, Abramson Cancer Center, Philadelphia, USA Background: We have shown that modulation of hypoxia-induced signaling through MKK4/MKK7 exerts opposing effects on oxaliplatin cytotoxicity in the HT29 colon adenocarcinoma cell line, resulting in higher sensitivity when MKK4 function is impaired and higher resistance when MKK7 is down-regulated. Higher sensitivity to oxaliplatin in hypoxic HT29 cells was also observed upon down-regulation of JNK1, but not JNK2, implying differential involvement of JNK iso-forms in mediating cytotoxic effect of the drug. We wished further investigate how modulation of signaling through JNK would influence oxaliplatin cytotoxicity in a panel of colon cancer cell lines of different genetic backgrounds, with an aim to confirm JNK1 as a possible molecular target to enhance efficacy of chemotherapy. Materials and Methods: Induction of JNK pathways by hypoxia and sensitivity to oxaliplatin were assessed in 12 colon cancer cell lines. Pharmacological and molecular approaches to inhibit JNK signaling were used (by small molecule CC-401 and by dominant negative constructs for

Poster Session – Molecular Targeted Agents II JNK1 and JNK2, respectively) to assess the role of JNK and its iso-forms in hypoxic resistance of colon cancer cell lines to oxaliplatin. Combination indices for oxaliplatin and CC-401 were calculated based on MTT assays. Induction of cell death and DNA damage were studied using flow cytometry. Studies involving stable introduction of dominant negative JNK1 or JNK2 were conducted both on monoclonal cell lines (HT29-derived) and pooled cells (SW620, HCT116). Results: Our data demonstrate that stress signaling through JNK is induced by hypoxia in the majority of colon cancer cell lines assessed. Hypoxic resistance to oxaliplatin varies significantly, implying cell-specific effects of JNK on sensitivity. Pharmacological inhibition of JNK augments the cytotoxicity of oxaliplatin in multiple colon cancer cell lines. We also show that colon cancer cell lines differ in their responses to oxaliplatin when JNK1 or JNK2 are down-regulated, from no effect (HCT116) to increased sensitivity (HT29, SW620). Neither pharmacological, nor molecular inhibition of JNK affects significantly hypoxia- or oxaliplatininduced DNA damage. In hypoxic HT29 cells, CC-401 shows synergism with oxaliplatin, SN-38 and 5-FU with CI50 of 0.42, 0.6 and 0.63, respectively. Finally, we confirm enhancement of oxaliplatin efficacy by CC401 in vivo, in HT29-derived mouse xenografts. Conclusions: These findings in vitro and in vivo support the involvement of JNK1 in resistance to cytotoxic therapy in colorectal cancer models. Inhibition of this pathway may be a rational therapeutic approach. 475 POSTER (Board P047) The synergistic anti-proliferative effect of combining the FGFR inhibitor, ARQ 087 with the AKT inhibitor, ARQ 092 in human cancer cell lines and PDX models E. Marchlik1 , Y. Yu1 , E. Chiesa1 , D. Dransfield1 . 1 ArQule Inc., Biology, Woburn, USA Cancer cells utilize multiple signaling pathways to sustain their growth and proliferation. Two major pathways that have been reported to regulate cancer cell growth are the FGFR/FRS2a/Erk and the PI3K/AKT/mTOR pathways. In order to identify cancer subtypes that are sensitive to the combined targeting of these pathways, we performed 2-dimensional (2D) proliferation assays with a panel of cancer cell lines and 3-dimensional (3D) patient derived xenograft (PDX) clonogenic assays treated with the combination of the FGFR inhibitor, ARQ 087 and the AKT inhibitor, ARQ 092. The combinatorial index (CI) was calculated using the Chou– Talalay method and a synergistic effect was reported in cell lines with CI value <0.85 and models with CI values <0.8. In a panel of 45 cancer cell lines, 24% (11/45) had a synergistic effect, 62% (28/45) had an additive effect, and 13% (6/45) had an antagonistic effect in response to combinatorial treatment of ARQ 087 and ARQ 092. Synergism with ARQ 087 and ARQ 092 was observed in 38% (3/8) of ovarian and 44% (4/9) of endometrial cancer cell lines. All 11 cancer cell lines that showed synergism contain PIK3CA/R1, suggesting that mutations in these genes may correlate with an increased sensitivity to this combination. Analysis of the 3D PDX model revealed that 7/100 3D PDX tumor models showed a synergistic response. These seven tumor models were derived from colon, breast, ovary, pancreas, pleuramesothelioma, renal, and sarcoma origin. Interestingly, the ovarian cancer model that had a synergistic response harbored a PIK3CA mutation, supporting the in vitro 2D data suggesting the role of PI3K mutations in predicting synergism in this gynecologic cancer. An additional 14 tumor models showed slightly enhanced efficacy in response to this combination. Together these studies of cancer cell lines and PDX models suggest that female gynecological tumors harboring PIK3CA/R1 mutations have increased sensitivity to combined FGFR and AKT pathway inhibition. Future studies will be focused at understanding the molecular mechanism(s) that contribute to this response. 476 POSTER (Board P048) FGFR2 targeting with allosteric inhibitor RPT835 I. Tsimafeyeu1 , F. Daeyaert2 , W. Yin3 , J. Ludes-Meyers3 , M. Byakhov4 , S. Tjulandin5 . 1 Russian Pharmaceutical Technologies, Moscow, Russian Federation; 2 FD Computing − Molmo Services, Beerse, Belgium; 3 Altogen Labs, Austin, USA; 4 Moscow Clinical Research Center, Moscow, Russian Federation; 5 N.N. Blokhin Russian Cancer Research Center, Moscow, Russian Federation Background: Existing strategies for inhibiting FGFR aim to inhibit the kinase activity by binding at intracellular part of receptor. In contrast, attempts to regulate FGFR signaling through an extracellular mechanism are less documented. Methods: Based upon crystal structures of the FGFR2 with its native ligand and knowledge of both peptide and small-molecule agents, we have developed a computational protocol of inhibitor RPT835 that predicts the putative binding of a FGF to the extracellular domains of the

Friday 21 November 2014 155 FGFR2. To identify the effects of RPT835 on FGF2 binding, induced intracellular signaling pathways cell lines expressing endogenous FGFR1 (MDA MB 134) and FGFR2 (KATO III) were treated with RPT835. The phosphorylation status of signal transduction proteins was analyzed using Western blotting. To determine whether RPT835 inhibits FGF2 interaction with FGFR2 an enzyme linked assay to measure FGF2:FGFR2 binding was developed. To control for possible non-specific effects by RPT835 on FGF2 binding we performed the binding assay with anti-FGF2 antibody coated wells. To identify RPT835 effects on specific FGFR subtypes (FGFR1 and FGFR2) each receptor was exogenously expressed by cell transfection and FGF2 induced receptor phosphorylation was analyzed. Results: In computational and de novo studies the inhibitor RPT835 binding at the extracellular part of FGFR2 with molecular weight of 413 kDa was identified. FGF2 stimulation of KATO III cells resulted in a robust activation of FGFR2 (4.3-fold), FRS2a (11.4-fold), p44/42(MAPK, 12-fold), AKT (4-fold) and p70S6 (1.2-fold). Stimulation of MDA MB 134 cells primarily resulted in activation of p44/42(MAPK, 6-fold). RPT835 had a dramatic inhibitory effect with IC50 <10 nM on FGF2 induced phoshphorylation of FRS2a in KATO III cells. However, RPT835 had no direct effect on FGF2-dependent FGFR1/FGFR2 and intracellular kinases phosphorylation levels in either cell lines. In a non-radioactive highthroughput binding assay and in a binding assay with anti-FGF2 antibody coated wells there were no effects of RPT835 on FGF2-FGFR2 binding. RPT835 did not impact FGF2 induced FGFR1 and FGFR2 phosphorylation and significantly inhibited phospho-FRS2a in an FGFR2-model in cells transfected with exogenous receptors. In vitro and in vivo antitumor activity of RPT835 was documented and reported (AACR Annual Meeting 2014 (abstr. 1737). Conclusions: We have discovered RPT835, an allosteric small molecular weight inhibitor of the extracellular part of FGFR2. 477 POSTER (Board P049) Jab1/Csn5: a new player driving the resistance to HER2-targeted therapies for breast cancer F. Claret1 , T. Vu1 , T.J. Shackleford1 , J.L. Allensworth1 , Q. Zhang1 , F.J. Esteva1 , E. Drakos1 , R. Zhang1 , L. Tian1 , G.Z. Rassidakis2 . 1 University of Texas MD Anderson Cancer Center, Molecular Therapeutics Unit 950, Houston, USA; 2 University of Texas MD Anderson Cancer Center, Hematopathology, Houston, USA Trastuzumab, a successful ErbB2 (HER2)-targeted therapy for breast cancer, blocks HER2/neu signaling and suppresses the PI3K/Akt pathway, thereby increasing the cell cycle inhibitor p27Kip1 , which causes cell arrest and/or apoptosis. However, trastuzumab resistance is a major obstacle in clinical management of HER2-positive breast cancer. A deeper understanding of the signaling pathways underlying trastuzumab resistance is needed to develop more effective therapy. In addition, because predictors of tumor response to trastuzumab (except HER2 positivity) are lacking, clinicians cannot predict which patients with HER2-positive breast cancer will respond to trastuzumab. We identified Jab1/CSN5 as a novel oncogene. Jab1 is amplified and overexpressed in >50% of breast tumors, and aberrant Jab1 expression correlates with poor prognosis. Jab1 negatively regulates p27, mediates p27 nuclear-to-cytoplasmic export and degradation and contributes to the loss of p27 that is seen in >50% of breast tumors and that correlates with poor clinical outcome. Jab1 plays an important role in the pathogenesis of breast cancer and is an ideal target for therapeutic intervention. We found that JAB1 DNA copy number and expression are increased in many breast cancer patients, that Jab1 overexpression in HER2-amplified breast cancer cells overcame trastuzumab-mediated p27 accumulation, and that silencing Jab1 improved response to trastuzumab. We analyzed the effect of silencing Jab1 on the PI3K/Akt pathway and on protein stability after trastuzumab treatment. We also investigated the association between Jab1 and p27 expression in 33 patients uniformly treated with trastuzumab and chemotherapy and assessed whether Jab1 expression level is a novel predictor of trastuzumab resistance. We found that Jab1 overexpression significantly correlated with poor response to trastuzumab in patients with HER2-positive breast cancer and that Jab1 contributed to trastuzumab resistance by negatively regulating tumor suppressor gene, p27. Therefore, targeting Jab1 oncogenic function in combination with trastuzumab therapy is a promising clinical application to overcome trastuzumab resistance.

156 Friday 21 November 2014 478 POSTER (Board P050) Pharmacological disruption of the Astrocytic Elevated Gene-1 (AEG1) in anticancer intervention: PB0412_3 (PB03) as a first-in-class AEG1 interacting agent J. Jimeno1 , G. Acosta2 , C. Teixido1 , C. Olbiol3 , N. Karachaliou1 , ˜ 3 , M. Sanchez-Ronco4 , J. Bertran1 , M.A. Molina1 , O. Villacanas A. Gimenez-Capitan1 , J.C. Monasterio1 , M. Taron1 , R. Rosell1 , F. Albericio2 . 1 Pangaea Biotech SL, Medical Oncology Service and Laboratory, Barcelona, Spain; 2 IRB, Barcelona, Spain; 3 Intelligent Pharma, Barcelona, Spain; 4 UAM, Madrid, Spain Background: AEG1 is highly expressed in most solid tumors conferring multidrug resistance, increased proliferation, angiogenesis, metastatic potential and poor outcome. AEG1 activates multiple signal transduction pathways such as PIK3/AKT, NFKB, MAPK, WNT and is a rational target for therapeutic intervention in cancer. Our previous data in advanced EGFR mutated and EGFR wild type lung cancer patients confirmed that AEG1 overexpression is indicative of poor outcome to targeted and pleiotropic therapy. In addition, AEG1 functionality is instrumental in the natural history of brain tumors. Material and Methods: We designed an in silico model based on inhibition of the NFkB (p65) binding domain of AEG1, identifying a possible interacting interface between the two proteins that could be modeled as a 3-dimensional structure. This interface was reduced and transformed into a 3-point pharmacophore for virtual screeningcomparing both molecular interaction fields and the pharmacophore. A total of 217,000 chemical entities (CEs) were tested in this model. Each CE was scored using the Hercules® affinity ranking. The top sixty CEs amongst the high scores were selected, and thus 4 compounds were proposed for in vitro screening. Twenty analogs were then synthesized, and PB03, a small molecular weight polyheterocyclic compound, was selected as the CE lead. Results: PB03 displays antitumor activity at low mM and nM concentrations. PB03-induced growth inhibition appears to be independent of EGFR, KRAS, BRAF, PIK3CA and p53 mutational status, Her2 amplification, and BRCA1 and AEG1 mRNA expression. PB03 clusters in vitro molar potency (MP) of Glial (Gliobastoma, GBM) and Neural Crest (Neuroblastoma (NBM) and Melanoma) -derived tumors as compared with PB03 profile in other solid tumors and in non-tumoral cells (p < 0.0001). A lack of impact of MGMT methylation status on PB03 (MP) sensitivity has been observed. PB03 is between 500 and 1000 fold more potent than temozolomide and MTIC in GBM and NBM (p < 0.0001). The mean PB03 IC50s in human non-tumoral cell lines is 2.08mM versus 1.22mM in the solid tumor panel vs 0.40mM in the Glial and Neural Crest-derived panel vs 0.11mM in GBM+NBM. Preliminary data in PC9 shows downregulation of AEG1 mRNA expression upon exposure to PB03. Expansion of the Glial and Neural Crest-derived tumor panel, cell kinetics, drug-induced cell death and in vivo/PK-PD studies in in vivo models is ongoing. PB03 has a negative score when screened in the SCIfinder® chemical data base confirming PB03 as a first-in-class CE. Conclusions: This is the first successful attempt at the pharmacological intervention against the AEG1 pathway. The observed clustering of Glial and Neural Crest-derived malignancies is unique and a tool for rational development. 479 POSTER (Board P051) The discovery and development of potent and specific anti-SialylTn antibodies for the treatment of solid tumors K. Meetze1 , D. Ghaderi1 , M. Zhang1 , I. Purnajo1 , J. Hermann1 , J. Fett1 , J. Behrens1 , A. Paula Galvao da Silva1 . 1 Siamab Therapeutics, Newton, USA Differentiation between normal and malignant tumor cells represents the gold standard for the development of highly specific cancer diagnostics and therapeutics. Aberrant glycosylation forms have been described in numerous human cancers, identifying glycans as an entire class of cancerspecific cell surface molecules suitable for tumor targeting. Expression of tumor-associated carbohydrate antigens (TACAs) frequently correlates with invasiveness, metastasis and tumor grade. Further, TACAs play important functional roles in immune suppression, tumor migration, cell adhesion, and metastasis; therefore, therapeutic antibodies targeting TACAs have the potential to not only kill cancer cells but also inhibit critical biological functions in cancer progression. While the presence of the SialylTn (STn) carbohydrate antigen in normal adult human tissues is rare, STn is present in many human cancers, including ovarian, bladder, breast, cervical, colon, and lung cancer, among others. STn expression in cancers is correlated with metastatic disease, poor prognosis, decreased survival, and lack of response to chemotherapy. De novo expression of STn can modulate carcinoma cells, suppress local immune function, change the malignant phenotype, and lead to more aggressive cell behaviors. As

Poster Session – Molecular Targeted Agents II such, STn is not only an interesting biomarker and cancer target, but interfering with STn function offers the intriguing potential to have significant immunologic and anti-metastatic therapeutic benefits. Siamab Therapeutics has developed a glycan array containing a large number of chemically synthesized and well-defined glycans. Using the glycan array, antibodies binding specifically to sialic acids can be unequivocally identified with high precision. Highly specific monoclonal antibodies that target the STn glycan have been generated from mice. The targeted epitopes are within the glycan itself, not a particular glycopeptide or carrier protein, which should offer the broadest potential to bind to multiple STn-glycosylated proteins on cancer cell surfaces. These antibodies have been shown to bind with high affinity as well as demonstrate protein internalization in MDA-MB231-STn overexpressing cells. In an in vivo study using a STn-expressing tumor model, these antibodies were able to significantly inhibit the growth of tumors. These, as well as other STn-specific antibodies, are currently being developed for the treatment of solid tumors. 480 POSTER (Board P052) Identification of novel small molecules as selective PAK4 allosteric modulators (PAMs) by stable isotope labeling of amino acids in cells (SILAC) W. Senapedis1 , Y. Landesman1 , M. Schenone2 , B. Karger3 , S. Wu3 , S. Shacham1 , E. Baloglu1 . 1 Karyopharm Therapeutics Inc., Natick, USA; 2 Broad Institute, Cambridge, USA; 3 Northeastern University, Barnett Institute, Boston, USA Background: P21-Activated Kinase 4 (PAK4) is a member of the PAK family of proteins that regulate cell survival, cell division and apoptosis. The six members of the PAK family are divided into two groups; Group I (PAK1, 2, 3) and Group II (PAK4, 5, 6), based upon their sequence homology and regulatory mechanisms. PAK4 is a member of the group II family of PAKs and is amplified or mutated in many cancer types. PAK4 is also a key downstream effector of the K-Ras pathway. Material and Methods: Stable isotope labeling by amino acids (SILAC) is a tool to encode cell populations metabolically for quantification of proteins by mass spectrometry. We labeled HeLa and U2OS cells with heavy arginine and lysine and purified small molecule interacting proteins using compound-tagged resin. Mass spectrometry analysis was used to identify target interactors. Standard molecular biology and biochemistry tools were used to verify target proteins. Results: After pull down of proteins from SILAC labeled lysates and mass spectrometry analysis, we have identified PAK4 as the target of our novel benzodihydrofuranyl derivatives. We confirmed interaction through isothermal calorimetry, thermal stability shift assay, and surface plasmon resonance. PAK4 interaction was preferential; however, there was weak binding to PAK5 and PAK6 and no binding to Group I PAKs. Treatment of tissue culture cells with the small molecules inhibited PAK4 signaling pathways, repressed proliferation, and induced apoptosis. These compounds inactivated PAK4 by directly inducing PAK4 destabilization. Our novel orally bioavailable small molecules showed efficacy in tumor xenograft models in mice with minimal toxicity. Conclusions: Here we present results of pre-clinical developmental studies on our lead PAK4 allosteric modulators (PAMs). PAK4 represents a novel anti-cancer target as a major downstream effector of Ras oncogene. We have identified selective, orally-bioavailable small molecule PAK4 allosteric modulators with anti-tumor activity both in vitro and in vivo. This represents a novel mechanism of the protein kinase inactivation involving degradation of PAK4 rather than direct inhibition of the kinase activity. Moreover, these allosteric modulators induce tumor cell growth arrest and apoptosis. Based on the in vitro and in vivo activity, these PAK4 allosteric modulators show promising results for the treatment of a wide variety of cancers. 481 POSTER (Board P053) In vitro and vivo evaluation of the pan FGFR inhibitor ARQ 087 and selective pan AKT inhibitor ARQ 092 in endometrial cancer: potential for combination therapy J. Meade1 , M.J. Wick1 , T. Vaught1 , R. Chavez1 , M. Rundle1 , K. Stanfield1 , B. Quattrochi1 , K.P. Papadopoulos1 , D.T. Dransfield2 , Y. Yu2 , E. Marchlik2 , E. Chiesa2 , A.W. Tolcher1 . 1 South Texas Accelerated Research Therapeutics, (START), San Antonio TX, USA; 2 ArQule, Woburn MA, USA Background: Cancer therapies targeting aberrant signaling through the fibroblast growth factor (FGFR) family of receptors or the PI3K/AKT pathway have shown clinical efficacy in certain tumor types. The importance of one or both targets in endometrial cancer subtypes is unclear. To test this possibility we screened ARQ 087, a potent multi-kinase inhibitor with pan-FGFR inhibitor and the AKT kinase inhibitor ARQ 092 in a panel of endometrial cancer cell lines. We subsequently evaluated

Poster Session – Molecular Targeted Agents II these agents alone in a panel of patient-derived xenograft (PDX) models of endometrial cancer. Methods: For in vitro studies, cell lines were cultured in serum-containing media and treated with single agent and concurrent combinations at various concentrations for 72 hours followed by the MTS assay. PDX uterine models were established in immune-deficient mice from primary or metastatic patient tissue and linked with patient treatment and outcome data and characterized using RNA and DNA-based analysis. We evaluated the in vivo efficacy of the pan-FGFR inhibitor ARQ 087 and a selective pan-AKT inhibitor ARQ 092, administered alone on clinically relevant schedules with efficacy compared between control and treatment groups (n = 1−3). Results were reported at day 28 after final treatment and tumor volume endpoint including comparative T/C values and tumor regressions were calculated. Results: Nine endometrial cancer cell lines were tested in vitro with 78% (7/9) reporting GI50 values between 1 and 5 mM in response to ARQ 087 and 33% (3/9) sensitive to ARQ 092 (GI50 <1mM). For in vivo studies, twentythree models were evaluated with six reporting statistically significant (p < 0.05) activity to ARQ 087 and eleven to ARQ 092. Sensitivity towards both agents overlapped in five of these models (ST259, ST413, ST953, ST1385 and ST1529), suggesting potential for additive or combination activity in a subset of endometrial cancers. Conclusion: We evaluated ARQ 087 and ARQ 092 in cancer cell lines and an endometrial PDX panel and identified a subset of models sensitive to either and both agents. Additional preclinical studies are underway to determine combination benefit for these agents and the potential for clinical combination.

Friday 21 November 2014 157 utility of FGFR inhibitors for the treatment of FGFR-driven cancers. Challenges remain to identify FGFR inhibitors that do not have offtarget inhibition, e.g. of VEGFR2, and that allow for strategies that translate into significant reduction of FGFR activity to improve clinical responses in patients while minimizing on-target toxicity. Here, we show the pharmacologic and antitumor activity profile of an irreversible covalent pan-FGFR inhibitor that was designed using a tailored covalency approach to target the cysteine residue within the kinase domain. Covalency imparts long lasting pharmacodynamics inhibition and tumor regression. Preclinical dosing schedule models demonstrated complete tumor regression while significantly sparing FGFR on-target biomarker changes. Materials and Methods: FGFR1−4 and VEGFR2 enzymatic activities were measured using the Caliper microfluidics Labchip system. FGFR2amplified (SNU16) and FGFR3-fusion (RT4) xenograft tumor models were used to investigate pharmacodynamics and efficacy. For tumor inoculation, cancer cells were implanted into the rear flank of immunocompromised mice. Once tumor volume reached a mean average of 175 mm3 , mice were randomized and treated with FGFR inhibitor. Cmpd ID

Biochemical IC50 (nM)

Cell proliferation IC50 (nM) FGFR1 FGFR2 FGFR3 FGFR4 VEGFR2 SNU16 RT4 HCT116

Ponatinib AZD-4547 BGJ-398 PRN-1109

8 1 1 1

2 0.4 0.6 2

52 5 2 6

18 23 27 40

2 37 141 163

83 10 7 0.9

960 230 180 55

1110 21000 14500 44000

482 POSTER (Board P054) Repurposing the antihelminthic mebendazole as a hedgehog inhibitor A.R. Larsen1 , R.Y. Bai2 , J.H. Chung2 , A. Borodovsky2 , G.J. Riggins2 , F. Bunz1 . 1 Johns Hopkins University, Radiation Oncology, Baltimore MD, USA; 2 Johns Hopkins University, Neurosurgery, Baltimore MD, USA Background: The antihelminthic agent mebendazole, a benzamidazole with a long history of safe use against nematode infestations and hydatid disease, has been shown to exhibit anticancer effects in cultured cells, experimental tumors and in human cancer patients. This study was designed to explore the effects of mebendazole and related compounds on hedgehog (Hh) signaling, a developmental pathway that is activated in many types of cancer. Materials and Methods: The effects of medbendazole on the canonical activation of GLI-mediated transcription were examined in cultured cells and in xenograft tumors. Transcription factor activity and gene expression were quantitatively assessed in vitro and in vivo. The primary cilium, a cellular organelle required for robust Hh pathway activity, was assessed by immunofluorescence. Results: Mebendazole potently inhibited Hh signaling and slowed the growth of Hh-driven human medulloblastoma cells at clinically attainable concentrations. The suppression of the Hh pathway by mebendazole could be counteracted by overexpression of downstream pathway components, but not by the overexpression of SMO, the apical signaling molecule that functions in the primary cilium. An analysis of the primary cilium revealed that mebendazole impaired ciliogenesis. Notably, inhibition of the primary cilium by mebendazole was able to suppress pathway activation by mutant forms of SMO that arise during treatment with vismodegib, a a SMO inhibitor that has recently been approved for use as an anticancer agent. Conclusions: This study demonstrates how a safe and widely used drug can be used to suppress Hh signaling. The anticancer activities of mebendazole and related compounds had been discovered years ago. These results suggest that inhibition of Hh signaling may be an important component of this anticancer effect. Mebendazole can be safely administered at high doses over extended time periods. We accordingly propose that mebendazole could clinically tested as a prospective therapeutic agent for many tumors that are dependent on Hh signaling. Because mebendazole is generally well tolerated, it used be used alone or in combination with agents such as vismodegib to improve the durability of responses. 483 POSTER (Board P055) Irreversible covalent pan-FGFR inhibitors are highly efficacious against FGFR-dependent cancers V.T. Phan1 , E. Verner1 , M. Gerritsen1 , J.M. Bradshaw1 , D.M. Goldstein1 , R.J. Hill1 , D. Karr1 , J. LaStant1 , P. Nunn1 , D. Tam1 , J. Shu1 , J.O. Funk1 , K. Brameld1 . 1 Principia Biopharma, South San Francisco CA 94080, USA Background: Deregulation of the FGF/FGFR signaling pathway has been associated with tumor progression in a variety of solid and hematological tumors. Recently, phase 1 studies have demonstrated an encouraging

Figure: PRN-1109 chemical structure. Results: PRN1109 is a representative molecule from the lead series and is a potent FGFR1−4 inhibitor with biochemical and cellular IC50 < 40 nM (Table 1). Biochemical and pathway activation assays demonstrated that PRN1109 is highly selective for FGFR1−3 over VEGFR2 (100–1000×) and does not inhibit other kinases that share the same cysteine residue. In RT4 (FGFR3:TACC3 fusion) bladder cancer and SNU16 (FGFR2-amplified) gastric cancer models, rapid tumor regression is observed following daily oral dosing of the FGFR inhibitor. Following 5 days of dosing in an FGFR2driven tumor model, prolonged tumor regression was maintained after the compound was withdrawn. Plasma phosphate increases and soft-tissue calcification can be FGFR associated on-target toxicity effects. Here, we uncovered a dosing schedule strategy that maintains tumor regression while largely sparing on-target toxicity effects in preclinical models. Conclusions: A tailored covalency approach to the design and optimization of a pan-FGFR lead series has generated small molecule irreversible covalent inhibitors with high cellular potency and profound efficacy. Efficacy is maintained even with an intermittent dosing schedule providing preclinical evidence of the potential to separate antitumor activity from on-target toxicities. Selected molecules from this series are advancing into clinical development for the treatment of stratified cancers.

158 Friday 21 November 2014 484 POSTER (Board P056) Treatment of advanced solid tumors with golvatinib (E7050) in combination with lenvatinib (E7080) E.L. Kwak1 , D. Juric1 , J.M. Cleary2 , G. Cote1 , J.F. Hilton2 , K.T. Flaherty1 , K. Wood3 , C. Rance4 , S. Barrett4 , G.I. Shapiro2 . 1 Massachusetts General Hospital, Medicine, Boston MA, USA; 2 Dana-Farber Cancer Institute, Medicine, Boston MA, USA; 3 Eisai Europe Ltd, Hatfield, United Kingdom; 4 Eisai Inc., Clinical, Woodcliff Lake NJ, USA Background: Lenvatinib (L) is an oral multi-targeted tyrosine kinase inhibitor of VEGFR 1−3, FGFR 1−4, PDGFRa, RET and KIT (Phase 1 single-agent MTD 24 mg QD). Golvatinib (G) is a highly potent, small molecule ATP-competitive inhibitor of the MET receptor tyrosine kinase and multiple members of the Eph receptor family as well as KIT and RON, based on isolated kinase assays (Phase 1 single-agent MTD 400 mg QD). Combination therapy with agents that block MET, VEGF, FGF, PDGFa and ephrin signaling constitutes a promising approach to circumvent adaptive or pre-existing tumor resistance pathways, and preclinical models indicate that the combination of L and G could overcome some intrinsic resistance to each agent. This Phase 1b study assessed safety, MTD, and preliminary antitumor activity of L plus G in patients (pts) with advanced solid tumors. Methods: Pts with advanced solid tumors, ECOG PS 0−1, 18 years (yrs) and adequate organ function were eligible. Starting dose was 12 mg/day L and 200 mg/day G once daily administered continuously in 28-day cycles in a conventional 3+3 dose escalation design (dose range 12−20 mg/day L and 200–400 mg/day G). An expansion cohort confirmed the MTD and established the recommended Phase 2 (RP2) dose. Results: 28 pts (M/F: 15/13; median age 61.5 yrs [range 34−75]) received combination dosing (median exposure 2.5 cycles [range 1−13], 8 pts ongoing at data cut) of L plus G in escalation cohorts (12 mg + 200 mg [n = 3]; 20 mg + 200 mg [n = 3]; 20 mg +300 mg [n = 6]; 20 mg + 400 mg [n = 8]) and in the expansion cohort 20 mg +300 mg [n = 8]. Two DLTs (at 20 mg + 400 mg) were observed during dose escalation: elevated AST and hyponatremia (1 pt) and inability to achieve at least 75% of planned dose in cycle 1 due to fatigue. The MTD/RP2 dose was determined to be 20 mg L + 300 mg G (4 DLTs in 14 evaluable pts [6 pts escalation cohort and 8 pts expansion cohort] at this dosing level). Frequently (>30%) occurring AEs of any grade and irrespective of relationship were fatigue, diarrhoea, nausea, vomiting, decreased appetite and dehydration. One fatal event of dyspnea, 21 days after discontinuing study drug for progressive disease, was observed. There was 1 possibly related G4 event of bowel perforation in the expansion cohort. Confirmed partial responses were seen in 5 (28%) pts [prostate (2 pts), endometrial (2 pts) and nasopharyngeal cancer]. Stable disease for at least 8 weeks was seen in an additional 13 pts. Conclusions: The MTD and RP2 dose was lenvatinib 20 mg combined with golvatinib 300 mg. This dose combination toxicity was consistent with the individual agents’ toxicity profiles, and there were no new safety signals identified. The observed activity warrants further evaluation of the combination in patients with advanced solid tumors.

Poster Session – Molecular Targeted Agents II patients) and found to associate with poor clinical outcome. To explore the role that GSTMs play in breast cancer we transiently modulated the expression of all 5 isoforms in vitro. GSTMs downregulation by siRNA significantly increased cell viability, intracellular reactive-oxygen species (ROS) levels and invasion potential. Interestingly, these changes were mainly observed in ER+ cell lines carrying a PIK3CA activating mutation, reinforcing the hypothesis that the GSTM-associated phenotype acts through the PI3K signaling pathway. We next profiled gene expression after the modulation of GSTMs and again linked GSTM levels to PI3K pathway activity. Remarkably, downregulation of GSTMs was correlated with a significant increase in the activation of MAPK, AKT and the transcription factor STAT5. Transcription factor motif analysis shows that STAT5 is significantly enriched in the promoter region of genes belonging to the GSTM signature. Conclusions: Our findings indicate that GSTMs expression directly limits the aggressive behavior of the large subclass of ER+ breast cancer cells that have constitutively activated PI3K/AKT pathway. 486 POSTER (Board P058) Development of small molecule activators of protein phosphatase 2A for the treatment of lung cancer J. Sangodkar1 , S. Mazhar2 , D. Kastrinsky3 , M. Ohlmeyer3 , G. Narla2 . 1 Mount Sinai School of Medicine, Medicine, New York, USA; 2 Case Western Reserve University, Medicine, Cleveland, USA; 3 Mount Sinai School of Medicine, Structural Biology, New York, USA Background: KRAS is the most common recurrent oncogenomic mutations driving the growth of NSCLC and accounting for ~25% of patients with advanced NSCLC. Patients with KRAS mutations respond poorly to current therapies driving the pursuit of new treatment strategies to improve the lives of patients suffering from KRAS driven lung cancers. While oncogenic kinases have proven to be successful targets for cancer treatment, the therapeutic targeting of phosphatases, the key negative regulators of these same pathways, has remained largely unexplored. Through reverse engineering of tricyclic neuroleptic drugs, we developed a first-in-class series of molecules, as represented by TRC-794 and DT-1154, that have favorable pharmaceutic properties, directly bind and activate the serine/threonine phosphatase 2A (PP2A). A critical role for PP2A as a tumor suppressor has previously been established, and inhibition and lossof-function changes in PP2A occur in human lung cancers, and its dominant targets are protein kinases and oncogenic proteins including ERK and AKT. Methods: A panel of lung cancer cell lines was used to understand the functional and biological effects of TRC-794 and DT-1154. To understand the effects of these small molecule activators of PP2A (SMAPs) on cell viability and survival, we used MTT and colony formation assays. Apoptosis was evaluated through annexin V staining and cell cycle profile analysis. Additionally, global phosphoproteomic profiling was performed using TiO2 enriched chromatography coupled with MS/MS analysis. Effects of TRC794 and DT-1154 in vivo was assessed using A549 and H358 xenograft and Kras transgenic mouse models.

485 POSTER (Board P057) Glutathione S-transferases M1−5 reduce the aggressive behaviour in breast cancer by modulating the PI3K/AKT pathway A. Bergamaschi1 , S. Levy1 , A. Scott1 , J. Jeong1 , M. Kiefer1 , E. Beasley1 , J. Baker1 . 1 Genomic Health Inc., R&D, Redwood City CA, USA Background: The human GSTM (GSTm) gene family encodes five closely related enzymes, GSTM1–GSTM5 (GSTMs) that glutathionylate and thereby detoxify electrophilic compounds including carcinogens, environmental toxins and products of oxidative stress. Further, GSTMs regulate a number of protein kinases by either direct binding or by glutathionylation. Remarkably, six many independent gene expression profiling studies have demonstrated that increased GSTM expression strongly correlates with favorable clinical outcome of primary ER+ breast cancers. To understand whether this correlation is a causal one, we have explored this question via breast cancer genomic data analyses and in vitro modulation of these enzymes. Methods: RNA-Seq analysis of 136 breast cancer patients was used to identify a gene signature associated with GSTM levels. In vitro up or down modulation of GSTMs was conducted in a panel of 10 breast cancer cell lines. Cell viability was measured using Cell-titer GLO and invasion assay by Boyden chamber. Protein expression of GSTMs and newly discovered targets were investigated by protein arrays, ELISA and Western blotting. Results: Functional classification of the gene signature associated with GSTM RNA levels found enrichment of genes involved in the PI3K/AKT pathway, indicating an inverse correlation between PI3K/AKT pathway activation and GSTM expression. This GSTM signature was independently validated in publicly available genome-wide expression datasets (~1000

Results: Treatment of lung cancer cell lines with SMAPs resulted in decreased cell viability, decreased colony formation, and an increase in apoptosis. Global phosphoproteomic analysis of TRC-794 treated KRAS lung cancer cell lines revealed ERK signaling as the only commonly perturbed pathway in drug treated cell lines which was confirmed by western blotting. Single agent TRC-794 and DT-1154 treatment of KRAS GEMM and xenograft mouse models of lung cancer resulted in tumor stasis, induction of tumor cell apoptosis and cell cycle arrest to comparable levels seen with a combination of AKT and MEK inhibitors. SMAP treatment was associated with significant AKT and ERK dephosphorylation in vivo. Additionally, the compounds demonstrate favorable pharmacokinetics and show no overt toxicity in mice or rats.

Poster Session – Molecular Targeted Agents II Conclusion: Taken together, these findings point to therapeutic activation of PP2A as a novel strategy for the treatment of advanced KRAS-mutant NSCLC. Activation of tumor suppressor proteins such as PP2A could offer the opportunity to identify novel synergistic strategies for the treatment of a number of cancer types. Nevertheless, translation of a PP2A activation strategy into clinical medicine has required pharmaceutically tractable agents for development. Our studies represent a first step into that new territory and highlight the potential for the development of small molecule activators of other protein phosphatases and tumor suppressor proteins. 487 POSTER (Board P059) EC-70124, a multi-kinase inhibitor, blocks NF-kB and STAT3 dependent signaling in prostate cancer stem cells G. Civenni1 , P. Costales2 , C. Garc´ıa-Inclan ´ 3 , D. Albino1 , N. Longoni1 , L.E. Nunez ˜ 2 , F. Moris2 , G.M. Carbone1 , C.V. Catapano1 . 1 Institute of Oncology Research (IOR), Tumor Biology and Experimental Therapeutics, Bellinzona, Switzerland; 2 Entrechem, Oviedo, Spain; 3 Hospital Universitario Central de Asturias, Dept. Otolaryngology, Oviedo, Spain EC-70124 is a novel multi-kinase inhibitor generated by combinatorial engineering of the biosynthetic pathways of glycosylated indolocarbazoles, like staurosporine and rebeccamycin. Biochemical kinase assays showed that EC-70124 has a unique spectrum of activity against multiple kinases and with degree of inhibition comparable or better than other clinically approved kinase inhibitors. Furthermore, in vitro testing suggested that NF-kappa B and STAT3 could be important targets of EC-70124 through the inhibition of the upstream activating kinases (e.g., IKK and JAK) of these key oncogenic transcription factors. Both NF-kappa B and STAT3 signaling pathways are over-active in diverse types of tumors, including prostate cancer, and there is considerable interest in developing inhibitors of these pathways. In this study, we analyzed the activity of EC70124 in prostate cancer cell lines, including androgen dependent and independent cell models. EC-70124 showed activity (IC50: 100–400 nM) in cell proliferation/viability assays both in androgen receptor (AR) positive (LNCaP, VCaP and 22RV1) and negative (DU145 and PC3) cell lines. Furthermore, EC-70124 was a potent inhibitor of CSC component in the in vitro prostate-sphere and self-renewal assays (IC50: 100–300 nM). Treatment with EC-70124 reduced STAT3 and IkB phosphorylation, STAT3 and NF-kappa B reporter activity, and expression their target genes in prostate cancer cells. Moreover, STAT3 and IkB alpha phosphorylation and expression of specific target genes were greatly reduced in isolated prostate CSCs in vitro. Importantly, EC-70124 (40 mg/kg, PO, daily for 2 weeks) inhibited growth of prostate tumor xenografts in immunedeficient mice. Tumor xenografts from EC-70124 derived mice showed marked reduction of IkB alpha phophorylation and target gene expression. Importantly, this was associated with a marked recuction in the CSC content in EC-70124 treated tumor xenografts determined by ex vivo sphere formation assay. These results indicate that EC-70124 acts as a dual inhibitor of STAT3 and NF-kappa B signaling pathways and is a potent inhibitor of tumor growth blocking maintenance and self-renewal of the prostate CSC component. EC-70124 may provide the basis for new therapeutic approaches in the treatment of prostate cancer. 488 POSTER (Board P060) Development of a publicly accessible knowledgebase to facilitate decision support for clinical cancer genomics reporting K. Shaw1 , J. Zeng1 , A.M. Bailey1 , A. Johnson1 , V. Holla1 , E.V. Bernstam2 , J. Butts3 , J. Mendelsohn1 , G.B. Mills1 , F. Meric-Bernstam1 . 1 MD Anderson Cancer Center, Khalifa Institute for Personalized Cancer Therapy, Houston, USA; 2 The University of Texas Health Science Center, School of Biomedical Informatics, Houston, USA; 3 MD Anderson Cancer Center, Internet Services, Houston, USA Background: Public efforts characterizing thousands of human tumors along with normal germline information have confirmed that any number and combination of alterations may drive tumorigenesis. With several hundred genes now on frequently recurring mutation lists and potentially actionable gene lists in human cancer, it is nearly impossible for any individual to maintain a comprehensive understanding of the alterations in those genes that may be clinically actionable. With groups reporting that fewer than 10% of patients with actionable alterations are receiving targeted treatment, a resource that distills potential therapeutic opportunities and increases entry on to trials would have wide utility. Materials and Methods: Data for each mutation, relevant therapeutic agents and corresponding clinical trials were annotated. Each variant was annotated for the level of evidence that associated a specific alteration in a potentially actionable cancer gene with a potential therapeutic opportunity with appropriate references. Specific mutations, copy number variants

Friday 21 November 2014 159 and fusions were linked to targeted agents, clinical trials, and functional data. Data were distributed via a publicly accessible website, reports and proactive clinical trial alert notifications. Results: Our group has developed a growing, public resource that launched in April 2014. At the time of abstract submission http:// www.personalizedcancertherapy.org/ includes detailed descriptions of 13 actionable genes and more than 200 individual somatic and cancerrelevant germline mutations annotated for functional significance. Due to different alterations in the same gene having distinct functional effects and therefore distinct therapeutic liabilities, each variant was annotated for its functional significance. This provides a level of evidence for actionability at a variant level instead of at the gene level. Clinical trial alerts and proactive reports with decision support annotations are designed to improve clinician recognition of molecular data in the patient record, increase patient accrual onto targeted trials and the development of novel trials at the institution. Conclusions: Proactive notification of therapeutic opportunities and detailed functional annotations of tumor molecular data has a goal of improving patient assignment to genomically-driven and -informed clinical trials. Sharing of the underlying data with the broader community is supported and the underlying infrastructure will be discussed in hopes of synergizing with others and developing this as a robust public resource available to patients and physicians around the world. 489 POSTER (Board P061) Development and clinical validation of a quantitative mass spectrometric assay for PD-L1 protein in FFPE NSCLC samples E. An1 , W. Liao1 , S. Thyparambil1 , J. Rodriguez2 , R. Salgia3 , I. Wistuba2 , J. Burrows1 , T. Hembrough1 . 1 OncoPlex Diagnostics, Rockville MD, USA; 2 MD Anderson Cancer Center, Houston TX, USA; 3 University of Chicago, Medicine, Chicago IL, USA Background: Binding of PD-L1 expressed on tumor cells to the PD1 on T lymphocytes transduces immuno-inhibitory signals which cripples the T cell’s ability to combat the tumor. Several anti-PD-L1 and anti-PD1 agents are in clinical trials and both regimens have reported promising preliminary results in NSCLC patients suggesting that tumor expression of PD-L1 is associated with a response to either anti-PD-L1 and anti-PD1 treatment. IHC is the current method to assess PD-L1 expression in FFPE tissue; however, PD-L1 IHC has yielded mixed results; some studies showed high false positive by IHC while another study showed that 13% of the PD-L1 negative patients responded to treatment. Moreover, IHC is low throughput and assessing multiple druggable targets by IHC is tissue consuming. As such, there is an urgent need to develop quantitative and multiplexed tests to assess biomarker expression. We have developed and clinically validated a quantitative mass spectrometric assay to measure PD-L1 expression in FFPE tissue. Method: We used trypsin digestion mapping of rPD-L1 to identify optimal quantitative peptides. Stable isotope-labeled peptides were synthesized as internal standards, and standard curves were generated in pyrococcus complex matrix to determine LOD, LLOQ, accuracy, precision and linearity of the assay. The PD-L1 assay was pre-clinically validated on 14 cell lines with known expression levels of PD-L1. The assay was run on archived FFPE sections from 9 normal lung tissues, 21 early staged and 4 advanced staged NSCLC patients. The multiplex assays also successfully subclassify NSCLC samples to adenocarcinoma or squamous cell carcinoma. Results: PD-L1 SRM assay had a LOD of 75 amol and LOQ of 100 amol. PD-L1 protein expression was detected in 7 cell lines (7/14) ranging from 76–375 amol/mg tumor protein. The regression analysis between SRM and mRNA analysis demonstrated excellent correlation (R2 = 0.8894). For the clinical analysis of NSCLC tissue, while no normal lung tissue expresses detectable level of PD-L1, ~24% of early stage NSCLC (5/21) and 50% of advanced stage NSCLC (2/4) express measurable PD-L1 protein. Interestingly, in this initial cohort, all of the PD-L1 positive early staged NSCLC were squamous cell carcinoma while in a small set of advanced staged NSCLC, PD-L1 expression was seen in both squamous cell carcinoma (1/3) and adenocarcinoma (1/1). Characterization of larger cohorts of NSCLC tissue is currently underway. Conclusions: We develop and clinical validate a quantitative proteomic PD-L1 assay to accurately measure PD-L1 expression in FFPE tissue. Additional quantitative assays for both lymphocyte (CD3, CD8, CD68) and immunotargets (PD1, B7-H3) are under development. This proteomic assay is a critical component of our multiplexed biomarker analysis, and will allow more accurate identification of potential candidates for PD-L1 or PD1 targeted therapies.

160 Friday 21 November 2014 490 POSTER (Board P062) Potent and selective inhibitors of the KRAS-signaling nanocluster protein, CNKSR1, block oncogenic KRAS signaling and mut-KRAS cell growth L. Kirkpatrick1 , G. Triana-Baltzer1 , M. Indarte1 , M. Scott1 , R. Lemos2 , G. Powis2 . 1 PHusis Therapeutics Inc, San Diego, USA; 2 Sanford Burnham Medical Research Institute, San Diego, USA Introduction: KRAS, the predominant form of mutated RAS (mutKRAS), is found in ~25% of patient tumors across many cancer types and plays a critical role in driving tumor growth and resistance to therapy. CNKSR1 (connector enhancer of kinase suppressor of Ras 1) has been validated as a protein associated with KRAS in the RAS membrane signaling nanocluster and we have shown is critical for mutKRAS but not wild type (wt)-KRAS signaling. CNKSR1 is a multi-domain protein with a pleckstrin homology (PH)-domain, which we have exploited to discover inhibitors that selectively affect mut-KRAS tumor growth. Proprietary modeling algorithms and state of-the-art commercial drug discovery software and ligand-based approaches were used to screen an in silico library of over 3 million compounds, providing first generation pharmacophores that bound to the expressed PH-domain of CNKSR1, and that specifically inhibited mut- but not wt-KRAS cell line growth. Through iterative modeling, structural design and experimental evaluation we have now identified novel second generation inhibitors of CNKSR1 that potently and selectively inhibit mut-KRAS NSCLC cell line growth. Material and Methods: in silico molecular docking and modeling in concert with BiaCore surface plasmon resonance spectroscopy for PH-domain binding were used to evaluate novel chemical compounds as CNKSR1 inhibitors. Growth inhibition studies used a panel of 23 NSCLC lines with a variety of KRAS mutations. The top inhibitors were further evaluated for effects on KRAS signaling. In vivo analyses of was undertaken to evaluate pharmacokinetic, pharmacodynamic, maximally tolerated dose and antitumor activity. Results: Iterative rounds of modeling and evaluation have provided a number of potent and selective CNKSR1 inhibitors with SAR around three pharmacophores. The IC50 for cytotoxicity was found to correlate with in silico binding predications. The most active compounds, PHT-7390 and 7391, inhibited mut-KRAS signaling and the growth of mut-KRAS NSCLC cell lines at IC50 0.5 to 5 mM. Cassette pharmacokinetic studies on the four lead agents showed t1/2 of up to 3 hr and peak plasma levels beyond in vitro IC50 when administered at 5 mg/kg. The agents were well tolerated when delivered daily for 10 days at 75 mg/kg. In vivo antitumor analyses of lead agents will be presented. Conclusion: Iterative rounds of structural optimization have provided potent inhibitors of CNKSR1 that selectively inhibit the growth of mutKRAS xenografts demonstrating that the PH-domain of CNKSR1 can be targeted by small molecule inhibitors. Two lead agents have nanomolar growth inhibition with activity across a large number of mut-KRAS lines not limited by mutation. These agents may have therapeutic potential for patients with mut-KRAS for which there is currently no effective therapy. 491 POSTER (Board P063) Development and characteristics of resistance to the HER family tyrosine kinase inhibitor AZD8931 V.G. Brunton1 , H. Creedon1 , M. Muir1 , T. Klinowska2 , K. McLeod1 , A. Byron1 . 1 University of Edinburgh, Edinburgh Cancer Research Centre, Edinburgh, United Kingdom; 2 AstraZeneca, Oncology iMed, Macclesfield, United Kingdom Background: HER2 is a member of the epidermal growth factor receptor family consisting of EGFR (HER1), HER2, HER3 and HER4. HER2 overexpression or gene amplification is observed in approximately 15% of all invasive breast carcinomas, where it is predictive of poor outcomes. Existing HER2 directed therapies have revolutionized the management of HER2 positive breast cancer but inherent and acquired drug resistance are frequently encountered clinical problems. Emerging evidence from clinical trials suggests that inhibition of multiple targets in the EGFR family is more effective than single target inhibition which led to the development of AZD8931, a pan HER family tyrosine kinase inhibitor. Materials and Methods: In vitro, human resistant cell lines were generated by incubating the HER2 positive cell line SKBR3 with escalating doses of AZD8931, while in vivo resistance was generated in the HER2 driven MMTV-NIC mouse model of mammary cancer. Analysis of the resistant phenotypes was carried out using western blotting and reverse phase protein arrays (RPPA). A global proteomic and phosphoproteomic analysis of the parental and AZD8931 resistant SKBR3 cells was carried out using label-free quantitative mass spectrometry. Results: AZD8931 inhibited tumour growth in the MMTV-NIC mouse. However, successive cycles of AZD8931 resulted in the eventual generation

Poster Session – Molecular Targeted Agents II of resistant tumours. By crossing MMTV-NIC mice with a strain containing a floxed PTEN allele we generated progeny with mammary specific active HER2 expression and PTEN loss (PTEN+/− ). Loss of PTEN conferred resistance to AZD8931. Western blotting and RPPA demonstrated that acquired resistance to AZD8931 both in vitro and in vivo was associated with aberrant EGFR family signalling and loss of PTEN. Furthermore, proteomic analysis revealed a number of changes in the resistant cells associated with induction of an epithelial to mesenchymal (EMT) transition that were confirmed by western blotting and by immunohistochemistry in the mouse tumours. Conclusions: Multiple mechanisms are contributing to AZD8931 resistance and identification of changes in the total and phosphoproteome using an unbiased proteomics approach has helped to define key nodes that are driving the resistant phenotype. A greater understanding of the pathways leading to resistance of HER2 targeted therapies is required before effective treatment strategies can be implemented. 492 POSTER (Board P064) Quantification of exportin-1 (XPO1) occupancy by selective inhibitors of nuclear export (SINE) M. Crochiere1 , B. Klebanov1 , E. Baloglu1 , O. Kalid1 , T. Kashyap1 , W. Senapedis1 , D. del Alamo1 , S. Tamir1 , D. McCauley1 , R. Carlson1 , M. Kauffman1 , S. Shacham1 , Y. Landesman1 . 1 Karyopharm Therapeutics, Natick MA, USA Background: SINE are a family of small-molecules that selectively inhibit nuclear export through covalent binding to Cysteine 528 (Cys528) in the cargo binding pocket of Exportin 1 (XPO1/CRM1). Such binding leads to forced nuclear retention and activation of major tumor suppressor proteins (TSPs) such as p53, FOXO, pRB and IkB and results in specific cancer cell death. Selinexor, an orally available SINE compound currently in human phase I and II clinical trials for advanced cancers (Clinicaltrials.gov), forms a slowly reversible covalent bond with XPO1. Oral selinexor demonstrates maximal pharmacokinetic exposure at 1−2 hours and increases in pharmacodynamic markers of XPO1 inhibition at 4 hours that last for 48 hours. The goal of this study was to develop a binding assay that would enable quantification of XPO1 occupancy in patients’ PBMC and tumor cells following oral administration of selinexor. Methods: To measure the binding of SINE to XPO1, biotinylated leptomycin B (LMB) was utilized. Biotinylated LMB binds covalently and irreversibly to Cys528 in the cargo-binding site of free XPO1 and its activity was confirmed to be similar to that of unmodified LMB in cytotoxicity assays. To measure SINE binding to XPO1 in vitro, cancer cell lines and PBMCs from normal human donors were treated with SINE compounds prior to treatment with biotinylated LMB. In in-vivo studies, mice were given oral selinexor 0−10 mg/kg, then PBMCs were isolated and treated with biotinylated LMB. After incubation with biotinylated LMB, cells were harvested, lysed, and protein lysates were subjected to pull down experiments with streptavidin-conjugated beads followed by immunoanalysis of XPO1. Results: To evaluate selinexor–XPO1 binding kinetics in vitro, MM.1s cells were treated with serial dilutions (0−10 mM) of SINE compounds and unbound XPO1 was pulled down from cell lysates treated with biotinylated LMB. Immunoanalysis showed that 50% of XPO1 occupancy with selinexor was achieved at 0.07 mM (i.e., EC50 70nM). Selinexor–XPO1 occupancy experiments using human PBMCs isolated from donor whole blood showed 50% XPO1 occupancy at 0.05 mM. XPO1 occupancy in vivo was measured in mice following oral administration of selinexor at 0.25−10 mg/kg for 4 hours resulting in XPO1 occupancy in PBMCs of 1.2 mg/kg. To measure the in vivo pharmacokinetic occupancy of selinexor over time, mice were treated with a single dose of selinexor ranging from 1.5 to 10 mg/kg and competition was evaluated in PBMCs isolated from these mice at time points ranging from 4 to 96 hours. The results showed a dosedependent competition of XPO1 binding and significant XPO1 occupancy in vivo up to 72 hours post-treatment. Conclusions: This new XPO1 occupancy assay can be used to evaluate drug exposure following treatment with oral selinexor. XPO1 occupancy will be determined in the ongoing clinical studies and its utility to predict response to selinexor will be evaluated.

Poster Session – Molecular Targeted Agents II 493 POSTER (Board P065) Preclinical pharmacokinetic (PK)/pharmacodynamic (PD)/Efficacy modeling for MLN2480, an investigational pan-RAF kinase inhibitor, in A375 and SKMEL-2 human melanoma xenografts M. Patel1 , J. Chouitar2 , J. Mettetal1 , E. Gangolli3 , S. Balani1 , P. Shimoga1 , K. Galvin2 , W.C. Shyu1 , A. Chakravarty1 , C.J. Zopf1 . 1 Takeda Pharmaceuticals International Co., DMPK, Cambridge, USA; 2 Takeda Pharmaceuticals International Co., Cancer Pharmacology, Cambridge, USA; 3 Takeda Pharmaceuticals International Co., Translational Medicine Oncology, Cambridge, USA Background: MLN2480 is an orally administered, investigational small molecule pan-RAF kinase inhibitor currently in Phase 1 clinical development. Here, we built integrated PK/PD/efficacy models to understand the relationship between MAPK pathway inhibition and efficacy as well as the effect of dose schedule in two melanoma xenograft models, A375 and SKMEL-2. Materials and Methods: We fit a one-compartment PK model to MLN2480 plasma concentration time profiles following a single dose in mice. Tumor pERK levels in xenograft-bearing mice, measured by Western blotting following single (A375) or multiple (SKMEL-2) doses of MLN2480 were fit to a direct or indirect inhibitory Emax model, respectively, to describe the PK/PD relationship between the plasma concentration and %pERK inhibition. Activity in each xenograft was evaluated as the percent growth rate inhibition (%GRI), the percent change between treated and control exponential tumor volume growth rates over the treatment cycle. To connect PK, PD, and efficacy for each xenograft, we simulated the PK and PD profiles over the treatment cycle for each dose group, and estimated the averages of plasma concentration (Cavg ) and %pERK inhibition, respectively, using non-compartmental analysis. Results: From a multiple linear regression, schedule-related parameters are not predictive for drug effect demonstrating schedule-independent activity. For a variety of dose schedules, the relationship between total dose and SKMEL-2 xenograft growth rate is approximately linear (R2 = 0.90, p < 0.0001). A sigmoidal PK/efficacy model captures the relationship between Cavg and %GRI for both xenografts. A375 exhibits greater sensitivity than SKMEL-2 to the same Cavg of MLN2480, with the models predicting tumor regression (Emax = 172% GRI) and stasis (Emax = 102% GRI) as the maximal effects, respectively. Both xenografts have steep, sigmoidal PD/efficacy relationships, which show the same pERK inhibition is associated with greater %GRI in A375, and %GRI saturates with residual pERK (EC90 = 38% in A375, 22% in SKMEL-2). Conclusions: Integrated PK/PD and PK/efficacy modeling for MLN2480 response in A375 and SKMEL-2 xenografts shows a strong relationship between pERK inhibition and preclinical activity, and that maximal effect is achieved without complete inhibition of the MAPK pathway. The findings of pERK inhibition-dependent but not schedule-dependent activity provide translational guidance to clinical dose and schedule selection. 494 POSTER (Board P066) LPA6 promotes growth and tumorigenicity of hepatocellular carcinoma via activation of PIM-3 proto-oncogene kinase C. Lopane1 , V. Goffredo1 , F. Dituri1 , F. De Santis2 , A. Filannino3 , R.C. Betz4 , Y.Y. Li5 , N. Mukaida6 , P. Winter7 , C. Tortorella8 , G. Giannelli9 , C. Sabba` 8 , A. Mazzocca8 . 1 University of Bari School of Medicine, Interdisciplinary Department of Medicine, Bari, Italy; 2 Institute Curie, Centre de Recherche, Paris, France; 3 Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy; 4 Department of Emergency and Organ Transplantation, University of Bonn, Bonn, Germany; 5 Cancer Research Institute, Fudan University Shanghai Cancer Center, Shanghai, China; 6 Division of Molecular Bioregulation, Kanazawa University, Kanazawa, Japan; 7 GenXPro GmbH, Kanazawa University, Frankfurt Main, Germany; 8 Interdisciplinary Department of Medicine, University of Bari School of Medicine, Bari, Italy; 9 Department of Emergency and Organ Transplantation, University of Bari School of Medicine, Bari, Italy Background: Aberrations occurring in hepatocytes during hepatocarcinogenesis confer the tumorigenic properties needed for hepatocellular carcinoma (HCC) development. Overexpression of certain receptors is common among these alterations and recent evidence suggests that the expression of lysophosphatidic acid (LPA) receptors is dysregulated in HCC. However, the contribution of these receptors to the pathogenesis of HCC has not previously been evaluated. We investigated the role of LPA receptor-6 (LPA6) in HCC tumorigenesis and growth and its mechanisms. Material and Methods: We used a gain- and loss-of-function approach to elucidate how LPA6 sustains the HCC tumorigenic process. Additionally, deletion mutants were employed to corroborate the involvement of LPA6 in this process. We also performed an extensive gene profiling analysis

Friday 21 November 2014 161 by MACE to gain insight into genes under the control of LPA6. Finally, we analyzed LPA6 expression in HCC tissues and its correlation with patient survival. Results: We found that RNA-interference-mediated knockdown of LPA6 impaired tumorigenicity in human HCC xenografts and the expression profile of several genes, including proto-oncogene Pim-3 regulated by LPA6 at gene-promoter level. Conversely, overexpression of LPA6 in nontumorigenic HCC cells led to Pim-3 upregulation and the acquisition of a tumorigenic phenotype. Moreover, our experiments with deleted mutants indicate that the STAT3 binding site is important for the transcriptional activation of Pim-3 by LPA6. Lastly, we found that LPA6 overexpression in HCC patients is correlated with high proliferation rates, elevated Pim-3 levels and a worse clinical outcome. Conclusions: Taken together, our data demonstrate a novel tumorigenic mechanism and provide a rationale for therapeutic targeting of the LPA6−Pim-3 axis in HCC. 495 POSTER (Board P067) Divergent androgen regulation of UPR pathways drives prostate cancer Y.J. Arnoldussen1 , M. Storm1 , X. Sheng1 , M. Tesikova1 , Y. Jin1 , H.Z. Nenseth1 , S. Zhao1 , I.G. Mills2 , L. Fazli3 , P. Rennie3 , B. Risberg4 , H. Wæhre4 , H.E. Danielsen4 , G.S. Hotamisligil5 , F. Saatcioglu1 . 1 University of Oslo, Department of Biosciences, Oslo, Norway; 2 University of Oslo, Centre for Molecular Medicine Norway, Oslo, Norway; 3 The Vancouver Prostate Centre, Department of Urologic Sciences, Vancouver, Canada; 4 Oslo University Hospital, Institute for Cancer Genetics and Informatics, Oslo, Norway; 5 Harvard University, Department of Genetics and Complex Diseases, Boston, USA Background: Endoplasmic reticulum (ER) stress resulting from protein misfolding is a common feature of malignant cells. The unfolded protein response (UPR) counteracts ER stress which can profoundly affect the cancer cell phenotype. Methods: Androgen receptor (AR) and UPR gene signatures were compared in a prostate cancer (PCa) cohort. Western analysis and quantitative PCR was used to evaluate the effect of androgens on UPR pathway activation in PCa cells in vitro and in vivo. ChIP assays were used to investigate AR binding to UPR related gene promoters in vivo. IRE1a and XBP-1 knockdown PCa cell lines were generated and used in xenograft experiments. A small molecule inhibitor of IRE1a was used to evaluate its effect on tumor growth in two independent preclinical models in vivo. XBP1 expression in human prostate cancer specimens was determined by immunohistochemistry. Results: The canonical UPR pathways in PCa cells were directly and divergently regulated by androgens which are critical for PCa survival. AR bound to gene regulatory sites and activated the IRE1a branch of UPR, but simultaneously inhibited PERK signaling. IRE1a inhibition, or its target XBP1, profoundly inhibited PCa cell growth in vitro as well as in preclinical models in vivo. Consistently, IRE1a expression decreased in xenografts upon androgen withdrawal and regression. Furthermore, AR and UPR gene expression were positively correlated in human PCa cohorts. Conclusion: Androgens regulate a genetic switch that impacts UPR in PCa cells which suggests that targeting IRE1a signaling may be a novel therapeutic approach in PCa. 496 POSTER (Board P068) Prevalence of MET amplification, MET expression, and MET-related genomic alterations in non-small cell lung cancer (NSCLC) A.L. Ang1 , H. Yang2 , A.A. Anderson1 , R. Tang2 , M.A. Damore1 , R.D. Loberg1 . 1 Amgen Inc., Molecular Sciences & Computational Biology, Thousand Oaks CA, USA; 2 Amgen Inc., Biostatistics − Medical Sciences, Thousand Oaks CA, USA Background: Signaling through the MET receptor tyrosine kinase regulates cellular functions including proliferation, survival, migration, and morphogenesis. The frequency of genomic alterations in MET varies widely among solid tumors and has been reported to be in the range of 2−8% in NSCLC patients who have not developed resistance to tyrosine kinase inhibitors (Cappuzzo et al. 2009). The relationship between MET amplification, MET expression, and additional genomic alterations known to be important in NSCLC biology (e.g., EGFR and PI3K mutations, ALK fusion genes) has not been well established. Materials and Methods: Formalin-fixed, paraffin-embedded (FFPE) NSCLC whole tissue sections (N = 181) were collected at 7 sites in the Eastern United States from patients of various ethnic backgrounds (White or Caucasian, n = 151; Black or African American, n = 28; Asian, n = 1; missing, n = 1). Samples were mostly early-stage primary resections

162 Friday 21 November 2014 (stage I, n = 139; stage II, n = 25; stage III, n = 15; stage IV, n = 2) and were histologically diverse (adenocarcinoma, n = 124; squamous cell carcinoma, n = 53; carcinoma, n = 4). Samples were analyzed by fluorescence in situ hybridization (FISH) for MET gene amplification using an automated MET/CEN-7 IQFISH assay from Dako and by immunohistochemistry (IHC) for MET protein expression using an automated MET IHC assay (Ab clone MET4) from Dako. Samples were profiled for a panel of gene mutations using the Ion AmpliSeq™ Cancer HotSpot Panel v2 on an Ion Torrent™ Ion PGM™ Sequencer. In addition, copy number variations were determined using the NanoString nCounter® Copy Number Variation CodeSet, and transcript analysis was performed using the NanoString nCounter® PanCancer Pathways Panel with 30 custom transcripts added. Results: In FISH analyses, MET gene copy number variation ( 5 copies) was foundin 14 of 181 samples (7.7%) and MET gene amplification (based on the standard definition of MET/CEN-7 2.0) was found in 4 of 181 samples (2.2%). Two of the 4 MET gene amplified samples had  5 copies of MET . In the 180 evaluable whole tissue IHC samples, the median (25th percentile, 75th percentile) percent positive for membrane MET expression was 60% (40%, 90%). Median (25th percentile, 75th percentile) total MET expression (membrane plus cytoplasm) was 80% (60%, 95%). All samples that were gene amplified for MET or had high MET copy number also stained for MET protein by IHC. Correlations between MET amplification, MET expression, and additional genomic alterations are ongoing and will be presented. Conclusions: Our preliminary results indicate that while MET gene amplification is relatively rare in early-stage NSCLC, the prevalence of MET expression is quite high. All focally amplified tumors expressed MET.

497 POSTER (Board P069) Preclinical evaluation of dimeric IAP proteins inhibitor APG-1387, in triple negative breast cancer both in vitro and in vivo G. Wang1 , P. Min1 , H. Wang1 , Z. Du1 , M. Wu1 , H. Dong1 , D. McEachern2 , L. Liu2 , Y. Lin3 , X. Lin3 , J. Wen3 , Y. Zhang3 , L. Gu1 , M. Guo3 , Y. Zhai1 , S. Wang2 , D. Yang1 . 1 Ascentage Pharma Group, Biological science department, Hong Kong and Taizhou Jiangsu Province, China; 2 University of Michigan, Comprehensive Cancer Center and Departments of Internal Medicine Pharmacology and Medicinal Chemistry, Ann Arbor Michigan, USA; 3 Ascentage Pharma Group, Pharmaceutical science department, Hong Kong and Taizhou Jiangsu Province, China Background: Inhibitor of apoptosis proteins (IAPs) are key components of the complex cascade of protein signaling that activates caspases to initiate apoptosis of the cancer cell. IAPs play critical roles in the development of cancer and in resistance to therapy. APG-1387 is a novel small molecule which mimetic dimeric form of Smac by binding to both the BIR2 and BIR3 domains of IAPs at low nanomolar affinities, thus inhibits their function and promotes apoptosis. Preclinical studies were conducted to evaluate its antitumor activity, PK and safety profile. Methods and Results: APG-1387 binds to XIAP and cIAP-1/2, promoting apoptosis through activation of caspase-9/-3 and caspase-8. APG-1387 potently inhibits cell growth and induces apoptosis in a subset of human cancer cell lines and in multiple in vivo tumor models in a dose-dependent manner. APG-1387 was found to be very active in vitro in triple negative breast cancer (TNBC) MDA-MB-231 and MDA-MB-468 with IC50 between 0.03 to 0.86mM. The antitumor effect of APG-1387 as single agent or in combination with chemotherapies or targeted drugs was evaluated in TNBC xenograft models. APG-1387 at 1, 3 and 10 mg/kg, qd×5 days/week or once weekly, consecutive for 3 weeks, were well tolerated and achieved complete tumor regression or partial regression in the TNBC model even at a weekly dose schedule. In PD studies with tumors bearing mice, APG1387 was found to induce rapid degradation of cIAP-1, robust cleavage of PARP and Caspase 3 activation in MDA-MB-231 xenograft tumor tissues. PK/PD study in tumor bearing mice demonstrated that APG-1387 shown dose and time dependent degradation of cIAP-1 and XIAP in tumor tissues, with the half life in tumor tissues longer than 72 hr. In similar weeklydosing experiments with APG-1387 in combination with TRAIL, cytotoxic chemotherapy, or targeted anti-cancer agents, enhanced and effective tumor growth inhibition was demonstrated at doses that were well tolerated in mice. Thus, intermittent dosing schedules were as effective as daily schedules, which suggested a less frequency dosing schedule in clinic. Preclinical studies of ADME and PK profiles of the iv administration were well defined in Cynomolgus monkeys and in rats, biphasic plasma elimination curves were observed in the plasma. APG-1387 has been evaluated in single and repeat daily dose toxicity studies of up to 4 weeks duration in male and female rats and monkeys. In addition, safety pharmacology and genotoxicity studies have been conducted. APG1387 does not present a genotoxic hazard, and there were no safety pharmacology findings of concern for clinical use.

Poster Session – Molecular Targeted Agents II Conclusion: The data provides guidance to the future clinical applications of APG-1387 and suggests a broad use in oncology, and APG-1387 has entered phase I clinical study in Australia. 498 POSTER (Board P070) CDK4/6 inhibitor (LY2835219) exhibits potent anti-tumor activity in human lung cancer cell lines with intact retinoblastoma J.W. Goldman1 , E.B. Garon1 , D. Conklin1 , D.J.L. Wong1 , B. Wolf1 , D.D. Silveira1 , N. Kamranpour1 , S. Pitts1 , R.S. Finn1 , D.J. Slamon1 . 1 David Geffen School of Medicine at UCLA, Department of Medicine Division of Hematology/Oncology, Los Angeles CA, USA Background: Dysregulation of the cell cycle is a hallmark of cancer and can be an important contributor to carcinogenesis. LY2835219 is a potent, orally available, highly selective inhibitor of the cyclin D kinases (CDK) 4 and 6, blocking phosphorylation of retinoblastoma (Rb) at low nanomolar concentrations. The role of CDK inhibitors for lung cancer, either alone or in combination with other pathway inhibitors, is not clearly defined. We performed a series of pre-clinical studies to determine the in vitro sensitivity to LY2835219 across a panel of 53 molecularly characterized human nonsmall cell lung cancer (NSCLC) and 10 small cell lung cancer (SCLC) cell lines. Methods: The 50% inhibitory concentration (IC50 ) of LY2835219 was determined in vitro. Sensitivity to LY2835219 was also evaluated in combination with erlotinib, assessed by serial dilutions of both agents, and serial dilutions of LY2835219 with erlotinib fixed at 1mM in the NSCLC lines. Rb status was assessed by comparative genomic hybridization, and confirmed with data from the CCLE. Results: Sensitivity to LY2835219 (defined as an IC50  500nM) was seen in 42/53 (79%) of the NSCLC cell lines and 3/10 (30%) of the SCLC lines. Intact Rb appeared to be required for activity, and inactivated Rb was the primary determinant of resistance. 40/45 (89%) of Rb intact lines were sensitive compared to 1/11 (9%) of lines with Rb loss (p < 0.001). The correlation with intact Rb remained high when only the NSCLC cell lines were evaluated. The greater sensitivity when compared to the breast cancer panel is of particular interest, in light of the known clinical activity in that setting. Although synergy was seen with some of the lines treated with erlotinib and LY2835219, in general, it did not appear that the combination, when evaluated in vitro, added much beyond what was seen with the agents individually. Conclusion: Our results demonstrate potent growth inhibition of human lung cancer cell lines with LY2835219 in the majority of cell lines tested. Rb loss was the primary determinant of resistance. Use along with erlotinib did not demonstrate high degrees of synergy, indicating that LY2835219 could be effective as a single agent in NSCLC. Supported by K23CA149079 and the Wolfen Family Lung Cancer Research Program. 499 POSTER (Board P071) Phase 2a study of copanlisib, a novel phosphatidylinositol-3-kinase (PI3K) inhibitor, in patients with relapsed/refractory, indolent or aggressive lymphoma M. Dreyling1 , P.L. Zinzani2 , K. Bouabdallah3 , D. Bron4 , D. Cunningham5 , K. Linton6 , C. Thieblemont7 , E. Van den Neste8 , U. Vitolo9 , J. Grunert10 , M. Giurescu11 , S. Mappa12 , B.H. Childs13 , F. Morschhauser14 . 1 ¨ Munchen-Großhadern, Klinikum der Universitat ¨ Medizinische Klinik und Poliklinik III Department of Medicine, Munchen, ¨ Germany; 2 Policlinico S. Orsola-Malpighi, Department of Hematology and Oncology, Bologna, ´ Italy; 3 University Hospital of Bordeaux, Service d’Hematologie et ´ de Therapie Cellulaire, Pessac, France; 4 Jules Bordet Institute (Free University of Brussels − ULB), Department of Clinical and Experimental Hematology, Brussels, Belgium; 5 The Royal Marsden Hospital, GI & Lymphoma Unit Department of Medicine, Sutton, United Kingdom; 6 The Christie NHS Foundation Trust, Department of Haemato-oncology, ˆ Saint-Louis, Department of Manchester, United Kingdom; 7 APHP-Hopital Hemato-oncology, Paris, France; 8 Cliniques universitaires UCL Saint-Luc, Hematology Department, Brussels, Belgium; 9 Citta` della Salute e della Scienza di Torino, Department of Oncology and Hematology, Torino, Italy; 10 Bayer Pharma AG, Clinical Statistics, Wuppertal, Germany; 11 Bayer Pharma AG, Global Clinical Development, Berlin, Germany; 12 Bayer S.p.A., Global Development, Milan, Italy; 13 Bayer HealthCare Pharmaceuticals, Global Clinical Development, Whippany NJ, USA; 14 ˆ CHRU − Hopital Claude Huriez, Hematology Department, Lille, France Background: Copanlisib (BAY 80-6946) is a selective pan-class I PI3K inhibitor predominantly targeting d- and a-isoforms in preclinical studies. Preliminary efficacy (via investigator assessment) and safety of copanlisib

Poster Session – Molecular Targeted Agents II

Friday 21 November 2014 163

in relapsed/refractory non-Hodgkin lymphoma (NHL) or chronic lymphocytic leukemia (CLL) have been reported (Dreyling et al, ASH 2013). We report here updated primary efficacy results per independent radiologic reviews. Methods: Patients with histologically confirmed indolent or aggressive lymphoma or CLL, relapsed or refractory to 2 prior lines of treatment were enrolled. Copanlisib (0.8 mg/kg, iv) was administered on days 1, 8 and 15 of a 28-day cycle until disease progression or unacceptable toxicity. The primary endpoint was objective response rate (ORR). Results: A total of 33 patients with indolent NHL (iNHL) or CLL and 34 with aggressive NHL were treated (Table); median age 68 years; M/F = 32/35; median number of previous lines of treatment = 3; previous exposure to rituximab = 59. At the time of this analysis, 14 patients remain on treatment; median cycles administered = 4. In the indolent group, the ORR was 47% (90% CI 32−63) overall and 53% [90% CI, 32−73] in iNHL. In the aggressive NHL group, the ORR was 29% (90% CI, 17−45). Complete responses (CR) were observed in FL, MCL, and PTCL (Table). The most common adverse events (AEs) of all grades (G) were hyperglycemia (63%), hypertension (61%), fatigue (45%) and diarrhea (40%). G3−4 AEs occurring in >10% of patients included: hypertension (43%), neutropenia (27%), hyperglycemia (25%), and anemia (13%). AEs leading to dose modifications or permanent discontinuation occurred in 55% and 25% of patients, respectively. There were 3 drug-related grade 5 events: lung infection, meningitis and respiratory failure. Summary/Conclusions: Copanlisib is active as a single agent in heavily pretreated patients with relapsed/refractory indolent or aggressive lymphoma, with CRs demonstrated in FL, MCL, and PTCL. An acceptable toxicity profile was observed. Additional studies in patients with iNHL and selective aggressive lymphoma are ongoing. Response

CR/CRu PR SD PD NE/NA

Indolent*

Aggressive

FL (n = 15)

MZL (n = 3)

SLL (n = 1)

CLL (n = 13)

DLBCL MCL (n = 15) (n = 7)

2 5 6

2 1

1

5 6 1 1

1 6 4 4

2

2 3 1 1

Trans. PTCL Media. (n = 6) (n = 4) (n = 1)

FL G3b (n = 1)

2 2 3

1 1

1 1

Fl, follicular lymphoma; MZL, marginal zone lymphoma; SLL, small lymphocytic lymphoma; DLBCL, diffuse large B-cell lymphoma; MCL, mantle cell lymphoma; Trans., transformed indolent FL; PTCL, peripheral T-cell lymphoma; Media., mediastinal large B-cell lymphoma; PR, partial response; SD, stable disease; PD, progressive disease; NE/NA, not evaluable/not available. *1 patient ineligible.

500 POSTER (Board P072) Triple blockade with LEE011, everolimus, and exemestane in women with ER+/HER2− advanced/metastatic breast cancer: results from a Phase Ib clinical trial A. Bardia1 , C. Chavez-MacGregor2 , S. Modi3 , M. Campone4 , B. Ma5 , M. Kittaneh6 , L. Dirix7 , M. Motta8 , V. Zhang8 , S. Bhansali9 , M.L. Fjaellskog8 , M. Oliveira10 . 1 Massachusetts General Hospital Cancer Center, Boston MA, USA; 2 The University of Texas MD Anderson Cancer Center, Oncology, Houston TX, USA; 3 Memorial Sloan Kettering Cancer Center, Oncology, New York NY, USA; 4 Institut de ´ Cancerologie de l’Ouest/Rene´ Gauducheau, Oncology, Saint-Herblain, France; 5 The Chinese University of Hong Kong, Phase 1 Clinical Trial Centre Department of Clinical Oncology, Hong Kong, China; 6 Barbara Ann Karmanos Cancer Institute, Oncology, Detroit MI, USA; 7 Sint Augustinus Hospital, Oncology, Antwerp, Belgium; 8 Novartis Institute for Biomedical Research, Oncology, Cambridge MA, USA; 9 Novartis Pharmaceuticals, Oncology, East Hanover NJ, USA; 10 Vall d’Hebron University Hospital/VHIO, Oncology, Barcelona, Spain Background: The PI3K/AKT/mTOR and cyclin D–CDK4/6–INK4−Rb pathways have been implicated in resistance to aromatase inhibitor (AI) therapy. Combined therapy with exemestane (EXE) and the mTOR inhibitor everolimus (EVE) improves progression-free survival of patients (pts) with non-steroidal AI (NSAI)-resistant ER+/HER2− advanced breast cancer (aBC; BOLERO-2), but eventually pts progress. In preclinical BC models, LEE011 (LEE; CDK4/6 inhibitor) + EVE + EXE has enhanced antitumor activity over each agent alone. Here we report the Ph Ib part of a Ph Ib/II clinical trial of LEE + EXE ± EVE in pts with ER+/HER2− aBC. Materials and Methods: Postmenopausal pts with NSAI-resistant ER+/HER2− aBC received escalating doses of LEE (3 wks on/1 wk off), EVE (continuous), and fixed dose EXE (25 mg/d; continuous; triplet combination); or a safety run-in of LEE (600 mg/d; 3 wks on/1 wk off) and EXE (25 mg/d; continuous; doublet combination). Primary objective: MTD/RP2D determination in the triplet combination. Dose escalation is guided by a Bayesian Logistic Regression Model using escalation with overdose control principle and real-time PK. Safety, efficacy, and predictive biomarkers are also assessed.

Results: As of Mar 21, 2014, 16 pts were treated: 13 with the triplet combination of LEE (200 mg [n = 6]; 300 mg [n = 6]; 250 mg [n = 1]) + EVE 2.5 mg/d + EXE, and 3 with the doublet combination. Three DLTs were observed with LEE 300 mg + EVE 2.5 mg + EXE: 1 G3 febrile neutropenia and 2 G3 ALT elevations. Most common (>20%) G3/4 study drug-related AEs across both combinations were neutropenia (50%) and leukopenia (31%). Preliminary PK data suggest LEE exposure in the presence of EVE is similar to that of single agent (SA), while EVE exposure is 1.5- to 3-fold higher than that of SA. One pt had a PR (LEE 300 mg + EVE 2.5 mg + EXE; prior therapy for aBC: anastrozole and fulvestrant), 5 pts had SD (LEE 200 mg [n = 2]/LEE 300 mg [n = 3] + EVE 2.5 mg + EXE), and 1 pt had non-CR/non-PD (LEE 300 mg + EVE 2.5 mg + EXE). Nextgeneration sequencing (NGS) of tumor samples revealed alterations in the PI3K/AKT/mTOR pathway, including PTEN/PIK3R2 deletions and PIK3CA mutations, and in the cyclin D–CDK4/6–INK4−Rb pathway, including CCND1 (cyclin D1) amplification. The PR observed was in a pt with a PTEN deletion, and SD 6 cycles was observed in another pt with a CDKN2A (p16) deletion and CCND1/IGFR1 amplification. Two pts in the doublet combination had SD; as yet, no tumor samples for this cohort are available for NGS; however updated results will be provided. Conclusions: The Ph Ib study suggests triple blockade with LEE + EXE + EVE is feasible. Preliminary evidence of clinical activity was observed and dose escalation continues. Ph II of the study will compare LEE + EXE ± EVE with EXE + EVE. Alterations consistent with PI3K/AKT/mTOR, cyclin D–CDK4/6–INK4−Rb pathway activation, or both, were identified in tumors, and support the biologic rationale for the triple combination in BC. 501 POSTER (Board P073) Immunological STAT3 knockdown associated with anti-tumor activity in pre-clinical models translates to clinical samples, suggesting immune modulation contributes to the clinical activity of AZD9150, a therapeutic STAT3 ASO P. McCoon1 , R. Woessner1 , R. DuPont1 , K. Bell1 , M. Collins1 , L. Pablo1 , D. Lawson1 , P. Nadella1 , V. Jacobs2 , C. Womack2 , C. Reimer1 , D. Hong3 , J. Nemunaitis4 , Y.K. Kang5 , T.Y. Kim6 , H.Y. Lim7 , T. Okusaka8 , S. Nadano9 , C.C. Lin10 , P. Lyne1 . 1 AstraZeneca Pharmaceuticals, Oncology IMED, Waltham MA, USA; 2 AstraZeneca Pharmaceuticals, Oncology IMED, Alderley Park, United Kingdom; 3 MD Anderson Cancer Center, Oncology, Houston TX, USA; 4 Mary Crowley Cancer Research Centers, Dallas TX, USA; 5 Asan Medical Center, Oncology, Seoul, Korea; 6 Seoul National University Hospital, Oncology, Seoul, Korea; 7 Samsung Medical Center, Oncology, Seoul, Korea; 8 National Cancer Center Hospital, Oncology, Tokyo, Japan; 9 National Hospital Organization, Ehime, Japan; 10 National Taiwan University Hospital, Tainan, Taiwan Background: JAK-STAT3 signaling has an established role in tumor biology and is becoming clinically validated in oncology. The main focus of small molecule JAK inhibitors has been on blocking intrinsic tumor cell signaling associated with tumor growth and proliferation, rather than the immunosuppressive tumor microenvironment. Here we present data that demonstrate the efficacy of targeting STAT3 in either tumor cells or stromal and immune cells using an antisense oligonucleotide (ASO) approach in pre-clinical models, and translate these findings to patient samples from Phase I clinical studies of the therapeutic STAT3 ASO, AZD9150. Methods: The species specificity of human and mouse STAT3 ASOs and differential uptake patterns in the PC9 human xenograft and mouse CT26 syngeneic tumor models were exploited in these studies. Tumors were implanted subcutaneously in SCID (PC9) or immune competent Balb/c (CT26) mice, monitored for growth, and analyzed by flow cytometry, western blot, RNA expression, and immunohistochemistry (IHC) at designated time points. Results: STAT3 ASO treatment resulted in dose-dependent tumor growth inhibition (TGI) in both models, with 80% TGI in the CT-26 model and 90% TGI in the PC-9 model, both at well tolerated doses. Consistent with TGI in the PC9 xenograft model, IHC staining showed ASO uptake and up to 70% STAT3 protein knockdown in tumor cells. In the CT26 syngeneic model, STAT3 ASO uptake was primarily in stromal cells, including endothelium and immune cells. Characterization of tumors by flow cytometry showed a two-fold increase in CD45+ and CD8+ infiltrating cells, despite lack of STAT3 knockdown in tumor cells, indicating the effects resulted from stromal/immune cell STAT3 knockdown. In addition, stromal STAT3 knockdown resulted in decreased CD31 staining by IHC, consistent with decreased angiogenesis. Analysis of phase I patient samples demonstrated AZD9150 uptake by myeloid and erythroid cells in the bone marrow by IHC, and up to 80% STAT3 protein knockdown in all circulating leukocytes by flow cytometry in samples analyzed to date. Additional translational biomarker studies are underway to more fully characterize these results and an update will be presented.

164 Friday 21 November 2014 Conclusion: STAT3 ASO effects in immune and stromal cells in preclinical models and patient samples suggest that immune modulatory effects may contribute to the activity associated with early clinical responses. These data provide additional clinical hypotheses for AZD9150. 502 POSTER (Board P074) MI130004, an antibody–drug conjugate including a novel payload of marine origin: Evidences of in vivo activity 1 P.M. Aviles1 , M.J. Guillen1 , J.M. Dominguez1 , M.J. Munoz-Alonso ˜ , L.F. Garcia-Fernandez1 , M. Garranzo1 , V. Martinez1 , A. Francesch1 , 1 1 1 1 S. Munt , C.M. Galmarini , C. Cuevas . PharmaMar S.A., Preclinical, Colmenar Viejo (Madrid), Spain

Background: Antibody–drug conjugates (‘ADCs’) have emerged as powerful tools for the treatment of cancer, combining the potency of cytotoxic molecules with the selectivity of antibodies targeted towards specific antigens exclusively present in cancer cells. The identification of such potent molecules is one of the most critical bottlenecks in the generation of ADCs. In this regard marine compounds represent an interesting opportunity worth exploring as they possess the requirements needed to be considered promising payloads. This poster presents data on the potency and selectivity of an ADC (MI130004) conjugating trastuzumab with a compound of marine origin. Materials and Methods: PM050489, a marine compound belonging to the PharmaMar collection, was modified to include a suitable linker containing a maleimide group to enable conjugation to Cys residues. Conjugation to trastuzumab was performed after reduction of the antibody with a phosphine reagent and the resulting conjugate (MI130004) was purified by size exclusion chromatography. The purified species was tested for its cytotoxic potential against selected cell lines with high (HCC-1954 and SKBR-3) or null (MCF-7 and MBA-MD-231) HER2 expression. To evaluate MI130004 in vivo 5−7 weeks old SCID female mice were subcutaneously implanted with a suspension of 5×106 JIMT-1 cells in 100 ml of cell culture medium. Tumor (ca. 115 mm3 ) bearing animals (N = 10/group) were randomly allocated to receive MI130004 (10 mg/kg) or appropriated control treatments (placebo included). Treatments were administered weekly for 5 consecutive weeks. Antitumor effect was calculated using DT/DC (%), defined as a percentage of the change in tumor size for treated (T) and placebo (C) groups during the placebo-treated survival time (D). Complete tumor regression (CR) was defined when tumor volume <63 mm3 for 2 or more consecutive measurements. Results: MI130004 showed a remarkable selectivity towards cell lines with high HER2 expression (IC50s 0.282 and 0.182 nM against HCC-1954 and SK-BR-3 cell lines respectively) whilst HER2 null cells were unaffected in the range of concentrations tested (up to 300 nM). In vivo, MI130004 also demonstrated efficiency in causing an outstanding tumor reduction, with complete regressions in most of the animals that received the treatment. Conclusions: MI130004 has demonstrated selective and significant in vitro activity in HER2 overexpressing cell lines. In agreement, the treatment with MI130004 demonstrated significant in vivo antitumor activity in mice bearing JIMT-1 xenografts. These results confirm the remarkable potential of marine compounds for the design of new ADCs with therapeutic anticancer properties. 503 POSTER (Board P075) UNC2025: A small molecule inhibitor of merTK with efficacy in mouse melanoma models S. Earp1 , D. Darr2 , A. Holtzhausen2 , A. Zimmermann2 , K. Clark2 , L. Hunter2 , N. Sharpless2 , X. Wang3 , S. Frye3 , D. Graham4 . 1 UNC Lineberger Comprehensive Cancer Center, Chapel Hill, USA; 2 The University of North Carolina at Chapel Hill, Lineberger Comprehensive Cancer Center, Chapel Hill, USA; 3 The University of North Carolina at Chapel Hill, Center for Integrative Chemical Biology and Drug Discovery, Chapel Hill, USA; 4 University of Colorado, Cancer Center and Department of Pediatrics, Aurora, USA Background: MerTK, a receptor tyrosine kinase normally expressed in macrophages, epithelium and reproductive tissues, is expressed at higher levels in metastatic melanoma than in primary melanoma or nevi. We have previously shown that treatment of melanoma cell lines with UNC developed MerTK small molecule kinase inhibitors reduces growth and invasiveness (J Clin Invest. 2013; 123: 2257−67). MerTK in macrophages serves to detect and ingest apoptotic material with externalized phosphatidyl serine. In doing so, the MerTK kinase physiologic signal dampens the innate immune inflammatory cytokine response, polarizing macrophages to an M2-wound healing phenotype. Thus, inhibition of MerTK in cancer might have a dual anti-neoplastic action, first inhibiting a tumor cell survival signal produced by aberrantly expressed

Poster Session – Molecular Targeted Agents II MerTK and second reversing the immune suppressive tumor-associated macrophage phenotype in the tumor’s microenvironment. Methods: We treated genetically-engineered melanoma mouse models (GEMMs) using UNC2025, a small molecule MerTK inhibitor with sub nanomolar potency in vitro, high oral bioavailability, and pharmacokinetic properties that allowed the drug to be delivered solubilized in mouse chow. We used this mode of delivery to treat the Tria mouse (Tyrosinase Hras transgene in an INK4a/Arf null background) either as sporadic tumors as they arose or by using a Tria tumor-derived cell line implanted orthotopically in NSG mice. We also treated mouse melanomas when they arose in a mutant BRAF/PTEN null melanoma GEMM. Results: Previously, we reported the Tria GEMM melanomas exhibited growth inhibition of the initial tumor when treated with UNC2025. Here we update those results with expanded numbers (N = 12) and show that the median survival is extended from 21.5 days in vehicle treated animals to 70 days in UNC2025 treated animals (p = 0.005). There were three initial complete responses not seen with in over 10 types of therapy in our experience using this model. In addition, UNC2025 also significantly (n = 5 vehicle vs. treated each group p < 0.01) slowed the growth of the Tria tumors implanted in NSG mice. There were no complete responses and the result was not as substantial as that seen in the immune-competent GEMM potentially suggesting that MerTK inhibition had both anti-tumor cell and immuno-mediated anti-tumor effects. In addition, UNC2025 treatment of spontaneously arising established melanoma in Braf/PTEN deleted mice significantly suppressed the growth of the initial tumors versus vehicle treated mice (n = 4 p < 0.006). Conclusions: UNC2025 has desirable PD/PK properties allowing its efficacious use in melanoma pre-clinical models. Further experiments will be needed to determine anti-tumor mechanism and determine if innate immune stimulatory mechanisms plays a role. 504 POSTER (Board P076) Frequent loss-of-function mutations in MLK4 suppresses signaling in the JNK-cJUN-p21/p15 pathway to promote growth of colon cancer cells A. Marusiak1 , N. Stephenson1 , H. Baik1 , E. Trotter1 , Y. Li2 , E. Testoni1 , K. Blyth3 , S. Mason3 , L. Puto4 , C. Miller2 , T. Hunter4 , O. Sansom3 , J. Brognard5 . 1 Cancer Research UK Manchester Institute, Signaling in Cancer Group, Manchester, United Kingdom; 2 Cancer Research UK Manchester Institute, Computational Biology Group, Manchester, United Kingdom; 3 Beatson Cancer Research UK, Colorectal Cancer and Wnt Signalling Group, Glasgow, United Kingdom; 4 Salk Institute for Biological Studies, San Diego, USA; 5 Cancer Research UK Manchester Institute, SNC, Manchester, United Kingdom MLK4 is a member of the mixed-lineage kinase (MLK) family that regulates the activity of specific MAPK pathways. MLKs are activated by environmental stress, cytokines and growth factors and phosphorylate JNK, p38 and ERK signaling pathways. Mutations have been identified in MLK4 in various human cancers, especially in colon cancer at a frequency of approximately 7%. The aim of this study is to assess the contribution of these mutations in the process of tumor development and assess the signaling pathways influenced downstream of mutant MLK4. Biochemical and structural analysis of MLK4 mutations imply that the majority are lossof-function (LOF). Furthermore, reconstitution of MLK4 WT in colon cancer cells harboring these inactivating mutations reduced viability, proliferation and the ability to form colonies. Reduced tumor growth was observed following reconstitution of MLK4 WT in mouse xenograft models. Finally, restoring the function of MLK4 WT in these colon cancer cell lines induced selective activation of the JNK pathway and its downstream targets, cJUN, ATF3 and p21/p15. In summary, these results indicate that the presence of LOF mutations in MLK4 will suppress signaling in the JNK pathway to promote proliferation. 505 POSTER (Board P077) The anti-ErbB3 antibody, EV20, counteracts vemurafenib resistance in BRAF-mutated colorectal cancer stem cells G. Sala1 , P.R. Prasetyanti2 , D. Barcaroli3 , S. Volpe3 , E. Capone3 , C. Rossi3 , R. Carollo4 , M. Todaro4 , G. Stassi4 , J.P. Medema2 , S. Iacobelli1 , V. De Laurenzi3 . 1 “G. d’Annunzio University”, Mediapharma s.r.l., Chieti, Italy; 2 Academic Medical Center University of Amsterdam, Center for Experimental Molecular Medicine, Amsterdam, Netherlands; 3 “G. d’Annunzio University”, Department of Experimental and Clinical Sciences, Chieti, Italy; 4 University of Palermo, Central Laboratory of Advanced Diagnosis and Biomedical Research (CLADIBIOR), Palermo, Italy Background: Approximately 10% of metastatic colorectal cancers harbor V600E BRAF mutation and this has been correlated with resistance to

Poster Session – Molecular Targeted Agents II EGFR-targeted therapies and worse clinical outcome. Moreover, feedback re-activation of ErbB receptors, severely limited response to V600E BRAF selective inhibitor PLX4032 (vemurafenib) in these tumors. A growing body of evidence indicates that upregulation of ErbB-3 may occur in response to several targeted therapeutics, including vemurafenib, and NRG-1b-dependent re-activation of the PI3K/Akt survival pathway has been associated to therapy resistance. Here, we analyzed the antitumor activity of the ErbB3 blocking antibody, EV20, in combination with vemurafenib. Materials and Methods: Three V600E BRAF patient-derived colorectal cancer stem cells (CCSCs) were used in this study. Activation of the ErbB3/PI3K/Akt axis, cell proliferation, clonogenic cell growth and in vivo tumor growth, were evaluated by standard procedures. Results: All CCSCs were found to express EGFR, ErbB-2 and ErbB-3 on the membrane. The ErbB-3 ligand, NRG-1b, strongly induced Akt and Erk phosphorylation, cell proliferation, stemness and promoted resistance to vemurafenib. Inhibition of ErbB-3 by stable RNAi or EV20 treatment, impaired NRG-1b-driven Akt and Erk activation, colony formation and tumor growth in xenograft models. Moreover, EV20 was able to restore vemurafenib sensitivity in CCSCs exposed to exogenous NRG-1b. Conclusions: Targeting ErbB-3 by EV20 in combination with vemurafenib could represent a promising therapeutic approach in V600 BRAF mutated colon-rectal cancer. 506 POSTER (Board P078) Phase (Ph) 1/2a study of TSR-011, a potent inhibitor of ALK and TRK, in advanced solid tumors including crizotinib-resistant ALK positive non-small cell lung cancer J. Sachdev1 , H.T. Arkenau2 , J.R. Infante3 , M.M. Mita4 , S.P. Anthony5 , R.B. Natale6 , S. Ejadi7 , K. Wilcoxen8 , V. Kansra8 , H. Laken8 , L. Hughes8 , R. Martell8 , G.J. Weiss9 . 1 Scottsdale Healthcare/TGen, Virginia G. Piper Cancer Center Clinical Trials, Scottsdale AZ, USA; 2 Sarah Cannon Research Institute UK, London, United Kingdom; 3 Sarah Cannon Research Institute, Tennessee Oncology, Nashville TN, USA; 4 Samuel Oschin Comprehensive Cancer Institute/Cedars-Sinai Medical Center, Experimental Therapeutics, Los Angeles CA, USA; 5 Evergreen Hematology and Oncology, US Oncology Research Affiliate, Spokane WA, USA; 6 Samuel-Oschin Comprehensive Cancer Institute/Cedars-Sinai Medical Center, Los Angeles CA, USA; 7 Scottsdale Health Care, VGPCC Clinical trials, Scottsdale AZ, USA; 8 TESARO Inc., Waltham MA, USA; 9 Cancer Treatment Centers of America Western Regional Medical Center, Goodyear AZ, USA Background: Both intrinsic and acquired resistance to crizotinib (crz) has been observed in anaplastic lymphoma kinase (ALK) rearranged (+) non-small cell lung cancer (NSCLC). Additionally, gene rearrangements in NTRK1 have been identified as a potentially actionable oncogenic aberration. TSR-011 inhibits ALK and tropomyosin-related kinase (TRK) A, B, and C receptor activity with IC50 values <3 nM and exhibits inhibition of oncogenic echinoderm microtubule associated protein like 4 (EML4)-ALK and tropomyosin (TPM)-TRKA dependent tumor growth in mice. Materials and Methods: A Ph 1/2a dose escalation and cohort expansion study is underway to evaluate safety, tolerability, pharmacokinetics (PK), and preliminary efficacy of TSR-011. The ongoing Ph 1 portion is evaluating patients with ALK+ or TRK ligand/receptor + tumors (defined by immunohistochemistry (IHC) or fluorescent in-situ hybridization) including those with ALK+NSCLC progressing on, or na¨ıve to, ALK inhibitor therapy. This study was approved by the institutional review board at each institution and informed consent was obtained on all patients. Results: Thirty patients with advanced cancer have been enrolled at total daily doses between 30 and 480 mg, including 11 ALK+ (8 evaluable ALK+NSCLC) and 2 TRK+. Dose-limiting toxicities included dysesthesia and QTc prolongation. Bi-exponential PK was observed, with dose proportional Cmax, AUC and t1/2 of 12−24 h. PK modeling revealed that a fractionated dose of 60 mg daily and above achieves sustained trough concentrations above the IC50 for ALK inhibition and minimizes peak exposure. Four of six (67%) of evaluable ALK+ patients previously treated with crz achieved a response across dose levels evaluated. Two patients with crz na¨ıve ALK+ disease have treatment ongoing with stable disease after 4 cycles. Duration on TSR-011 exceeded that of crz treatment in a majority of responders, up to 10 months longer than treatment with crz for a patient still continuing on study. Twenty six patients with solid tumors have been screened by IHC for expression of TRKA or TRKB and the respective ligand. Approximately 40% of patients across tumor types screened express either the TRKA or TRKB receptor and respective ligand, however no patients have been identified with TRKA gene rearrangement. To date no responses have been observed in patients with TRK+ IHC overexpression. Conclusions: TSR-011 is a well-tolerated, promising second-generation agent for ALK-dependent and crz resistant NSCLC, and is being explored

Friday 21 November 2014 165 in ALK+ and TRK+ tumors. The ideal assay for TRK driven tumors is under active investigation. 507 POSTER (Board P079) Amplification of D-type cyclin genes CCND2 and CCND3 confers an oncogene addiction phenotype in tumor cells and hypersensitivity to the CDK4/6 inhibitor LY2835219 S. Buchanan1 , X. Gong1 , M. Lallena2 , L. Chio1 , F. Merzoug1 , J. Dempsey1 , R. Beckman1 , C. Reinhard1 . 1 Eli Lilly, Oncology Research, Indianapolis, USA; 2 Eli Lilly, Quantitative Biology, Alcobendas, Spain Background: It is well established that phosphorylation of Rb-family pocket proteins by CDK4 and CDK6 is important for the commitment of cancer cells to a new cell cycle and the initiation of the G1-S phase transition. LY2835219 is a potent inhibitor of the kinase activity of both CDK4 and CDK6 and, like other drugs in its class, very effectively arrests cancer cells at G1. Consequently, LY2835219 is currently undergoing clinical testing in various cancers. Methods: To identify the molecular determinants of its anti-proliferative activity, we tested LY2835219 mesylate across a panel of genomically characterized cancer cell lines. Statistical approaches were employed to uncover genomic features associated with the response. Candidate markers of sensitivity and resistance were further tested by genetic manipulations in vitro. In vivo models representing the candidate molecular marker of sensitivity were identified and drug efficacy examined. Results: Three broad classes of response were identified. The class of tumors cells most resistant to LY2835219 showed enrichment for RB1 mutations. Drug-sensitive tumor cells could be divided into a larger subset of cell lines that showed sensitivity consistent with G1 arrest, and a smaller group of solid tumor cell lines that showed a hypersensitive response with indications of cell death. Cell lines with amplification of CCND2 and CCND3 were among the very most sensitive tumor cells. Further investigation showed that, distinct from the more common G1 arrest phenotype, tumor cells with these markers showed rapid apoptosis after either depletion of the cognate D-cyclin or treatment with LY2835219. In vivo models of tumors harboring CCND2 and CCND3 gene amplification were very sensitive to LY2835219 treatment and showed evidence of tumor regression. Conclusions: Tumors with high copy, focal amplification of D-cyclin genes may be particularly sensitive to LY2835219 treatment. 508 POSTER (Board P080) A phase Ib trial of AMG386 and temsirolimus in patients with advanced sold tumors (PJC-008/NCI#9041) J.W. Chiu1 , S.J. Hotte2 , C.K. Kollmannsberger3 , D.J. Renouf3 , D.W. Cescon1 , D. Hedley1 , S. Chow1 , J. Moscow4 , M. Perry1 , I. Diaz-Padilla1 , D. Tan1 , H. Hirte2 , E. McWhirter2 , H. Chen4 , L.L. Siu1 , P.L. Bedard1 . 1 Princess Margaret Cancer Centre, Division of Medical Oncology & Hematology, Toronto, Canada; 2 Juravinski Cancer Centre and Escarpment Cancer Research Institute, Hamilton, Canada; 3 BC Cancer Agency, Vancouver, Canada; 4 National Cancer Institute, Cancer Therapy Evaluation Program, Bethesda, USA Background: There is crosstalk between the Ang-Tie2 and the PI3K/Akt/mTOR pathways. Combined Ang1/2 and mTOR blockade may have additive anti-cancer activity. The combination of AMG386, an inhibitor of Ang1/2-Tie2 interaction, with the mTOR inhibitor temsirolimus was evaluated in patients (pts) with advanced solid tumors to determine tolerability, maximum tolerated dose (MTD), preliminary antitumor activity and explore pharmacodynamic effects. Methods: Pts were enrolled using 3+3 design, and were given intravenous AMG386 and temsirolimus on Days 1, 8, 15 and 22 of a 28-day cycle. Dose limiting toxicities (DLTs) were evaluated during cycle 1. Peripheral blood was collected for evaluation of Tie2-expressing monocytes (TEMs) and thymidine phosphorylase (TP) (an angiogenic enzyme increased in TEMs upon Tie2 stimulation) by flow cytometry. Tumor response was assessed by RECIST v1.1 every 2 cycles. Results: 21 pts were enrolled, 6 at dose level (DL) 1, 7 at DL −1, and 8 at DL −2. Median age was 58 years, 13 tumor types were included, median previous chemotherapy lines was 3 (range 1−11). Dosage and DLTs are summarized in table below. The most common AEs (all Grades (Gr)) possibly related to drugs were: fatigue (81%), edema (62%), anorexia (57%), nausea (52%), rash (43%) and mucositis (43%). Common Gr3 AEs included lymphopenia (28%) and fatigue (28%). Six pts had 1 of the 2 drugs off during treatment due to toxicities. MTD was still exceeded at DL −2. Of 18 response evaluable pts, 1 PR was observed (ER+/HER2−/ PIK3CA mutant breast cancer) for 12 cycles and 4 pts had prolonged SD  24 wks (renal cell carcinoma, thyroid cancer, uterine sarcoma, bone chordoma). Of 10 pts evaluated for TP or Tie2 change from D1 to 3, no correlation with clinical benefit was observed.

166 Friday 21 November 2014

Poster Session – Molecular Targeted Agents II

Conclusions: The MTD was exceeded at AMG386 10 mg/kg weekly and temsirolimus 20 mg weekly. No tolerable dose could be identified for the combination, with frequent overlapping toxicities including fatigue, edema, and anorexia. No further dose de-escalation is planned and the study has been terminated. Table: Summary of DLTs Dose Level

AMG386 (mg/kg)

No. of patients

No. of patients

Temsirolimus (mg)

treated

with DLT

6 (1 inevaluable) 7

1

Gr2 pneumonitis

2

8 (2 inevaluable)

2

Gr3 Gr2 Gr4 Gr5 Gr4

1

15

25

−1

15

20

−2

10

20

DLT

mucositis intolerable edema sepsis, Gr4 diarrhea, multi-organ failure hypertriglyceridemia

509 POSTER (Board P081) Detecting gene amplification in circulating tumor cells of patients with gastric cancer for clinical trial Y. Mishima1 , S. Matsusaka1 , Y. Terui1 , M. Mikuniya1 , T. Takayama1 , N. Mizunuma2 , K. Hatake1 . 1 Japanese Foundation for Cancer Research, Cancer Chemotherapy Center, Tokyo, Japan; 2 Japanese Foundation for Cancer Research, Division of Chemotherapy Cancer Institute Hospital, Tokyo, Japan Background: A key to successful treatment with molecular targeted medicines is the ability to preselect the patients who will have clinical benefit by molecular-basis analysis of tumor tissue. Currently, the biomarker assessment is mostly measured using tumor biopsy samples. However, the suspicion always persists that biopsy samples may not truly represent the patient tumors. In addition, we should also consider that primary tumor samples may not be representative of a patient’s metastatic disease. One potential strategy that can complement primary tumor biopsy is the utilization of circulating tumor cells (CTCs). Recent studies have suggested that discordance may exist between HER-2 status of a patient’s primary breast tumor and CTCs. This finding provokes a question of whether the patients with HER-2 negative primary tumor and HER-2 amplified CTCs can have clinical benefit with trastuzumab treatment. To answer this question, we developed a sensitive, accurate, and robust method to analyze HER-2 status on CTCs. Material and Methods: We employed the method based on 3D multicolor cell imaging where CTCs are fluorescently-labeled by both immunoflorescence and FISH simultaneously. Briefly, CTCs of the gastric cancer patients were enriched by negative magnetic selection for hematopoietic linage. The enriched CTCs were labeled with fluorescent mAbs against pan-cytokeratin and CD45. The specimens were then hybridized with FISH probes and screened for Z-axis optical sections with a confocal microscope and 3D images were generated. This study was approved by the IRB of JFCR. Results: CTCs (Cytokeratin+/CD45−/DAPI+) were easily discriminated from remaining hematopoietic cells. The immunofluorescent staining had no crossover on FISH signal because of 3D imaging. Most CTC enrichment techniques previously established use EpCAM positivity. However, accumulating evidence suggests CTCs may have low or no surface EpCAM expression. Hence, we used intra-cytoplasmic cytokeratin staining instead of surface EpCAM staining. Our pilot study of primary tumor biopsies and CTCs from the same patients demonstrated that surface EpCAM expression level is lower on most CTCs than on matched primary tumors. The number of the patients detected CTCs per 7mL blood was significantly higher in our method compared to a conventional method (CellSearch System) (82% vs 46%, p < 0.01 by chi-square test). CTCs were detected in 134 out of 163 gastric cancer patients. Of these, 21 cases were determined to have HER-2 amplifications. Conclusions: Because our method does not depend on expression of EpCAM, even CTCs that express little or no EpCAM can be identified, providing a more accurate and sensitive test for CTCs. In addition, 3D imaging with high spatial resolution made it possible to analyze HER-2 gene status easily and accurately. Our on-going clinical trial utilized the technologies described herein, and compares clinical outcomes of patients with HER-2 FISH positivity in either their primary tumor, their CTCs, or both, in a trastuzumab containing chemotherapy regime. In summary, our work describes an important, novel method for personalized medicine.

510 POSTER (Board P082) Inhibition of STAT3 enhances the radiosensitising effect of temozolomide in malignant glioma cells in vitro and in vivo I. Kim1 , E. Choi1 , B. Cho1 , T. Han1 , S. Song1 , J. Kim1 , S. Paek2 . 1 Seoul National University, Radiation Oncology, Seongnam-si Gyeonggi-do, South Korea; 2 Seoul National University, Neurosurgery, Seoul, South Korea Background: Despite aggressive treatment with radiation therapy concurrently with the adjuvant temozolomide (TMZ), the prognosis of glioblastoma multiforme (GBM) remains poor. We investigated the potential of targeting signal transducer andactivator of transcription-3 (STAT3) to improve the therapeutic outcome of combined radiotherapy and TMZ in malignant glioma. Methods: We evaluated the preclinical potential of a STAT3 inhibitor, Cpd188 combined with temozolomide and radiation by in vitro clonogenic assays using two established glioblastoma cell lines (U251, U87) and two patient-derived glioblastoma cell lines (GBL12, GBL28) and in vivo studies using nude mice bearing intracranial U251 xenografts. Results: Cpd188 potentiated the radiosensitising effect of TMZ in the U251 glioma cell line which has high levels of p-STAT3 expression in vitro. Increased radiosensitising effects of TMZ were associated with the induction of apoptosis and the reversion of epithelial–mesenchymal transition (EMT). Cpd188 delayed in vivo tumor growth both alone and in combination with fractionated radiation and TMZ with a trend toward improved survival rates. Immunohistochemical staining of tumor sections showed that Cpd188 decreased the expression of CD31 (a marker of endothelial proliferation), vascular endothelial growth factor, and hypoxiainducible factor-1a, suggesting that Cpd188 also has anti-angiogenic effects. We also confirmed the radiosensitising effect of Cpd188 of GBL28 cell line which was originated from a patient who had a glioblastoma and in which a high level of STAT3 expression was also confirmed. Conclusion: These data indicate that Cpd188 has the potential to improve the therapeutic outcome of combined radiotherapy and TMZ in human glioblastoma, especially in patients whose tumor has a high level of STAT3 expression. 511 POSTER (Board P083) ComboPredictor: identification of synergy biomarkers and enrichment within tumor sample populations M. Tomilo1 , A. Bankhead III1 , M.E. Urick1 , S. Eddy1 , R. Rickles2 , J. Ledell2 , E.T. Bowden1 . 1 Thermo Fisher Scientific, Life Sciences Solutions Group, Ann Arbor MI, USA; 2 Horizon Discovery Group, Discovery Services, Cambridge MA, USA Background: Oncology drug development has suffered due to the incremental impacts that many targeted agents have exhibited in the clinic. Systematic approaches to support appropriate combination therapy clinical trial design are critical to understanding tumor heterogeneity and inherent or emerging resistance. The purpose of this study was to identify synergistic drug combinations, biomarkers of synergy and potentially responsive populations using a novel systematic combination therapy bioinformatics analysis. Materials and Methods: Thirty-nine cell lines were screened with a PI3K inhibitor (GDC-0941) as a single agent and in combination other compounds including a MEK inhibitor, PD0325901. Synergy scores were calculated from a growth inhibition endpoint and normalized using control compound combination treatments. Cell lines were assigned a categorical synergy status and integrated with genomic data for association analysis. Synergistic response biomarkers were applied to data from retrospective banked clinical samples using the Oncomine® Knowledgebase. Results: Significant growth inhibition-based synergy was identified when GDC-0941 was combined with PD0325901. Associations between each of 2,300+ biomarkers and the synergy response for the GDC0941/PD0325901 combination revealed significant (Q-value  0.01) biomarkers of synergy including RAS/RAF pathway mutation and a custom gene expression signature which was biologically enriched for MAPK and PI3K pathway signaling. Gene expression signature analysis in the Oncomine® Knowledgebase revealed that 18 of 20 major cancer types have activated MAPK pathway signaling and more than 50% of those samples are predicted to be MEK inhibitor resistant. Expression of the GDC-0941/PD0325901 synergy expression signature was found in cancer types predicted to be resistant to MEK single agent therapy including glioblastoma and 35% of breast cancer (specifically ~20% of triple negative breast cancers). Conclusions: We provide proof of concept that ComboPredictor analyses can reveal novel combination treatment strategies for specific cancer histologies and potential biomarker strategies, a major advancement to aid clinical trial design. Subsets of both glioblastoma and triple negative breast cancer were identified as potentially responsive to combined MEK and PI3K inhibition through a novel systematic bioinformatics analysis.

Poster Session – Molecular Targeted Agents II 512 POSTER (Board P084) Somatically mutated ABL1 represents an actionable and essential lung cancer survival gene E. Testoni1 , N.L. Stephenson1 , A.A. Marusiak1 , E.W. Trotter1 , A. Hudson1 , J. Brognard1 . 1 CRUK Manchester Institute, SNC, Manchester, United Kingdom Background: Non-small cell lung cancer (NSCLC) accounts for 89% of lung cancers and worldwide this affects 1.37 million patients each year. Lack of actionable mutations in lung cancer presents a significant hurdle in the administration of targeted therapies for this disease. Here we identify somatically mutated ABL1 as a genetic dependency that is required to maintain lung cancer cell survival. Materials and Methods: Phenotypic consequences of ABL mutations in NSCLC cell were sassed by viability assays (MTT, CFA) in response to ABL inhibition. Molecular dynamics simulations were used to predict the conformational changes of the mutant ABL kinases and changes in catalytic activities were assessed biochemically. Results: NSCLC cell lines carrying ABL1 mutations were sensitive to pharmacological ABL inhibition with imatinib and dasatinib. The specificity of the cytotoxic effects of these compounds was verified by overexpression of the drug resistant ABL1 kinase, which fully rescued the drug induced cell death. Furthermore, the ABL1 mutant cells, but not the control lung cell lines, were sensitive to ABL1 knockdown, indicating the importance of the mutant ABL1 kinases in maintaining lung tumorigenic phenotypes. Molecular dynamics simulations indicated that ABL1 mutations induce conformational changes in the kinase domain leading to increased activity of the mutant ABL1 proteins. Consistent with these data, transient overexpression of the ABL1 mutants identified in a lung cancer cell line and in primary lung cancer samples resulted in increased levels of CRKL phosphorylation relative to WT ABL. Conclusion: The current study demonstrates that mutationally active ABL1 represents a novel actionable mutation in non-small cell lung cancer and patients with ABL1 mutations (approximately 1.5% of NSCLC patients) may respond to already approved therapies targeting these kinases. 513 POSTER (Board P085) Synergistic inhibition of ovarian and endometrial cancer cell lines using combined treatment of ARQ 092 and ARQ 087 in vitro and in vivo Y. Yu1 , E. Nakuci1 , E. Chiesa1 , C.R. Chen1 , E. Marchlik1 , D.T. Dransfield1 . 1 ArQule Inc, Woburn, USA Background: Ovarian and endometrial cancers are the most common female gynecological malignancies. Although standard therapies have been used to inhibit cancer progression, the majority of patients have developed resistance and experienced recurrence. Dysregulation of the PI3K/AKT pathway or/and activating mutations of fibroblast growth factor receptor 2 (FGFR2) have been implicated in tumor initiation and development, and as such are considered as molecular targets. ARQ 092 is a potent and selective pan-AKT inhibitor with IC50 in the low nanomolar range for AKT1, 2, and 3 respectively. ARQ 087 is a pan-FGFR inhibitor against FGFR1, 2, and 3 with single digit nanomolar IC50 . Both ARQ 092 and ARQ 087 are in early stage clinical development as single agents. Methods: In vitro ARQ 092 and ARQ 087 combination studies were performed based on the Chou–Talalay method and combinatorial indices were calculated. The effects of this combination on downstream proteins of PI3K/AKT and FGFR pathways were assessed using western blot analysis, Reverse Phase Protein Microarray and/or IHC. Cell cycle analysis and apoptotic response were determined by flow cytometric analysis. Efficacy studies of this combination of ARQ 092 and ARQ 087 were performed in mouse xenograft models harboring AN3CA endometrial-derived and IGROV1 ovarian-derived tumors. Results: Thirty-eight percent (3/8) of ovarian cancer cell lines exhibited synergism upon combined treatment of ARQ 092 and ARQ 087 in antiproliferation assays. All 3 were adenocarcinomas harboring PIK3CA/R1 mutations. In endometrial cancer cell lines, 44% (4/9) of endometrial cancer cell lines exhibited synergism and three of them bear both PIK3CA/R1 and FGFR2 mutations. Pathway analysis showed that combined treatment with ARQ 092 and ARQ 087 suppressed downstream target proteins of PI3K/AKT and FGFR2 more potently than either single agent alone. Cell cycle analysis revealed enhancement of G1 phase arrest upon treatment with ARQ 092 and ARQ 087 as compared with either single agent. Furthermore, combined treatment with both agents induced a slight apoptotic response not observed by either single agent. In in vivo mouse xenograft models, combined treatment with ARQ 092 and ARQ 087 resulted in superior tumor growth inhibition in comparison to the single agent arms. Conclusions: ARQ 092 and ARQ 087 show synergistic anti-proliferative effects in in vitro and can be dosed in combination to yield synergistic

Friday 21 November 2014 167 anti tumor effects in vivo in ovarian and endometrial cancer cell models. The synergism is associated with the suppression of signaling pathways of PI3K/AKT and FGFR2, cell cycle arrest, and an initiation of an apoptotic response. Mutational analysis indicates that adenocarcinomas with PIK3CA/R1 mutations in ovarian cancer or endometrial cancers with both PIK3CA/R1 and FGFR2 mutations represent potential clinical indications for the combination therapy of ARQ 092 and ARQ 087. 514 POSTER (Board P086) Hypoxia inducible factor (HIF)-1a expression levels and p53 mutations are prognostic factors for survival in breast cancer patients treated with neoadjuvant chemotherapy M. Molina-Vila1 , S. Baulies2 , M. Gonzalez-Cao3 , N. Karachaliou3 , A. Rodriguez Capitan3 , S. Viteri3 , M. Cusido3 , R. Fabregas3 , C. Teixido3 , R. Rosell3 . 1 Institut Universitari Dexeus Quiron, Barcelona, Spain; 2 Institut Universitari Dexeus Quiron, Gynecology, Barcelona, Spain; 3 Institut Universitari Dexeus Quiron, Oncology, Barcelona, Spain Background: Hypoxia inducible factor (HIF1a) is a transcription factor that drives cellular adaptation to hypoxia, promoting chemoresistance and metastases. p53 mutations attenuate TAP63 activity, that regulates HIF1a proteosomal degradation. The role of HIF1a and p53 mutations has been described previously cellular lines of breast cancer. We have analysed HIF1a expression levels and p53 mutational status as a possible prognostic or predictive markers in a group of breast cancer patients treated with neoadjuvant chemotherapy (NAC). Patients and Methods: Gene expression analysis was performed in pretreatment samples of 92 breast cancer patients treated with NAC. Expression levels of HIF1 alpha were analysed by RT-PCR, in addition to mutational status of p53 and PI3K genes. We explored the correlation of molecular markers with immunohistochemical classification, pathological complete response (pCR) and survival. Results: High levels of HIF1a expression were more frequent in erb2 breast cancer subgroup (HER2+): high levels were found in 24% (8/33) of hormone receptor positive cases (HR+), 50% of HER2+ (9/18) and in 22% of triple negative patients (TN) (2/9) (p = 0.05). PI3k mutations were more frequent in HR+ tumors (24%) and p53 mutations in TN (28%). Although no correlation was found with the chance of obtaining pCR by p53 status or HIF1a levels, disease free survival at 10 years was 30% for p53 mutated patients versus 60% in wild type (p = 0.028) and patients with low levels of HIF1 alpha had an overall survival (OS) at 10 years of 100% versus 59% in patients with high levels (p = 0.071). Conclusions: Low expression levels of HIF1a predict a good outcome in patients with p53 wild type breast cancer tumors, in spite of not having pCR to NAC. 515 POSTER (Board P087) TAS-115, a potent MET/VEGFR-targeted kinase inhibitor, is a new therapeutic approach for the treatment of bone metastasis of lung cancer A. Gomori1 , M. Sakuragi1 , A. Hashimoto1 , K. Ito1 , T. Haruma1 , T. Suzuki1 , H. Fujita1 , Y. Fujioka1 , K. Yonekura1 , T. Utsugi1 . 1 Taiho Pharmaceutical Co. Ltd., Tsukuba Research Center, Tsukuba, Japan Background: Bone metastasis is observed in the late stage of cancer patients including prostate, breast, and lung cancer etc. Bisphosphonates, anti-RANKL antibody and 223 RaCl2 are approved as therapies for skeletal related events, but there still remain issues about efficacy for prolonged survival and toxicities. It was reported that a MET/VEGFR kinase inhibitor, cabozantinib, resolved bone scans in patients with prostate cancer bone metastasis, but the detailed mechanism of improved bone metastasis via MET/VEGFR inhibition remains unclear. TAS-115 is an orally active and highly potent MET/VEGFR inhibitor. Herein we investigated the efficacy of TAS-115 in bone metastasis model of lung cancer. Material and Methods: We established luciferase-transfected human lung adenocarcinoma cell line with bone metastatic potential (A549-Luc BM1) by in vivo selection. One week after inoculation of A549 Luc-BM1 into right tibiae of mice, TAS-115, a VEGFR inhibitor (sunitinib), and a MET inhibitor (crizotinib) were administered orally for 4 weeks. Tumor growth was monitored by bioluminescence imaging. Right tibiae were used for bone morphological analysis by micro-computed tomography (micro CT) and for histomorphometric analysis. The basis for the effects on bone disruption was investigated by immunoblot analysis in murine osteoclasts in vitro. Results: Bioluminescence imaging showed that TAS-115 and sunitinib suppressed tumor growth of A549 Luc-BM1 in tibiae. In contrast, crizotinib did not affect tumor progression. Thus, the antitumor efficacy of TAS-115 would be mainly due to VEGFR inhibition. Micro CT analysis showed that aberrant bone formation and resorption by A549 Luc-BM1 was suppressed by TAS-115 and by concomitant dosing of sunitinib with crizotinib, but

168 Friday 21 November 2014 not by each inhibitor alone. Histomorphometric analysis showed that number of TRAP positive osteoclasts around tumor in bone was markedly decreased by TAS-115. In vitro study showed that HGF or VEGF produced phosphorylation of each receptors (MET or VEGFR2) -related signaling factors (AKT, ERK1/2) in murine osteoclasts, and TAS-115 suppressed them. These results suggested that although VEGFR inhibition is important for antitumor efficacy, the amelioration of tumor-associated bone disruption requires dual inhibition of MET and VEGFR. Conclusions: VEGFR and MET signaling cooperatively play pivotal roles for tumor-associated bone disruption, and TAS-115 is an effective therapeutic treatment for bone metastases. 516 POSTER (Board P088) Different genetic profiles of resistant and sensitive patients with EGFR wild type NSCLC undergoing tyrosine kinase inhibitor (TKI) treatment P.U. Ulivi1 , E.C. Chiadini1 , A.D. Dubini2 , D.C. Calistri1 , M.P. Puccetti3 , M.A.B. Burgio4 , A.D. Delmonte4 , A.V. Verlicchi5 , A.G. Gamboni6 , M.P. Papi7 , W.Z. Zoli1 , L.C. Crino` 8 , C.D. Dazzi5 . 1 IRCCS Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (I.R.S.T.), Biosciences Laboratory, Meldola, Italy; 2 Morgagni-Pierantoni Hospital, Pathology Unit, Forl`ı, Italy; 3 S. Maria delle Croci Hospital, Pathology Unit, Ravenna, Italy; 4 IRCCS Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (I.R.S.T.), Medical Oncology, Meldola, Italy; 5 S. Maria delle Croci Hospital, Medical Oncology, Ravenna, Italy; 6 Degli Infermi Hospital, Medical Oncology, Faenza, Italy; 7 Infermi Hospital, Medical Oncology, Rimini, Italy; 8 Perugia Hospital, Medical Oncology, Perugia, Italy Background: Tyrosine kinase inhibitors (TKIs) are the first choice of treatment in a subset of patients with non-small cell lung cancer (NSCLC) harboring specific epidermal growth factor receptor (EGFR) mutations. However, about 15% of patients carrying wild type (wt) EGFR also respond to TKIs, and the reason for this sensitivity is unclear. Material and Methods: We considered a total of 60 patients with advanced NSCLC and EGFR wt who received erlotinib after one or more chemotherapy lines. Thirty patients were defined as responsive (stable disease or partial response for at least 6 months of treatment), while 30 were non-responsive. All patients had progressive disease at the start of treatment with erlotinib. Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue or cytological tumor samples. A 26gene panel was analyzed by MassArray (Sequenom) using the LungCarta panel kit (Sequenom). Results: Sequenom analysis revealed that, among responsive patients previously identified as EGFR wt by pyrosequencing analysis, 2 (7%) had an EGFR mutation that was probably present in cell subclones and only detectable with more sensitive methods, while 7 (23%) showed a mutation in genes involved in the control of proliferation: 2 in PIK3CA, 1 in AKT , 1 in NTRK2 and 2 in STK11 genes. Moreover, 5 (17%) of the responding patients had a KRAS mutation (2 G12V,1 G12D, 1 G13D, 1 G12C). Of the 30 non responsive patients, 2 (7%) showed a resistance mutation in EGFR (P753S and L747S), 3 (10%) a KRAS mutation (G12D, G12C, G12V), 4 (13%) a p53 mutation (R273L, G245C, R158C, R273C) and 3 (10%) a NOTCH1 mutation (all R2328W). Conclusions: EGFR wt NSCLC patients showed different gene mutation profiles on the basis of their responsiveness or resistance to erlotinib. Responsive patients carried underrepresented EGFR mutations that were only detectable using highly sensitive methods and also harbored mutations in genes involved in different proliferation pathways. Conversely, EGFR resistance mutations, p53 and NOTCH 1 alterations were more frequently found in non-responsive patients. KRAS mutations were equally present in both groups of patients and would not seem to play a role in predicting response to TKIs. 517 POSTER (Board P089) Identification and characterization of selective MELK kinase inhibitors P. Carpinelli1 , N. Amboldi1 , D. Ballinari1 , S. Re Depaolini2 , U. Cucchi2 , G. Canevari3 , M. Caruso4 , R. Galli5 , D. Donati4 , A. Isacchi2 , E.R. Felder3 , A. Montagnoli1 , A. Galvani1 . 1 Nerviano Medical Sciences Srl, Cell Biology, Nerviano (Milano), Italy; 2 Nerviano Medical Sciences Srl, Biotechnology, Nerviano (Milano), Italy; 3 Nerviano Medical Sciences Srl, Chemical Core Technologies, Nerviano (Milano), Italy; 4 Nerviano Medical Sciences Srl, Medicinal Chemistry, Nerviano (Milano), Italy; 5 San Raffaele Scientific Institute, Neural Stem Cell Biology Unit, Milano, Italy MELK is a serine-threonine kinase that has been implicated in various cellular processes, including stem cell renewal, cell cycle progression, pre-mRNA splicing and apoptosis. MELK is overexpressed in various

Poster Session – Molecular Targeted Agents II undifferentiated tumors and its knockdown results in decreased cell-cycle progression, proliferation and tumor growth. Its expression has further been found to be enriched in embryonic and adult neural stem/progenitor cells and to be required for their self-renewal capacity. In tumor tissue, increased MELK expression correlates with the pathological grade of brain tumors, and is significantly correlated with poor prognosis of glioblastoma, breast and prostate cancer patients. Here we report the identification and biological evaluation of novel, selective inhibitors of MELK kinase. We identified a series of MELK inhibitors via high-throughput screening of our proprietary compound collection. Some of these small ATP-competitive molecules exhibit potent inhibitory activity against MELK enzyme with IC50 concentrations in the nanomolar range and are highly selective for MELK compared to a panel of protein kinases. Treatment of different tumoral cell lines with such compounds resulted in reduced cell proliferation with dose-dependent induction of apoptosis in a particular setting. Furthermore the compounds reduced neurosphere formation of glioblastoma stem cells, suggesting that they can have an effect on the growth of cancer stem cells. The threedimensional structure of a MELK fragment, comprising the kinase and UBA domains in complex with one of these compounds, NMS-P664, resolved at 2.5 A˚ resolution, reveals compound binding mode in the active site of MELK, thus complementing compound kinetic characterization and providing useful information for further optimization. Overall, our data demonstrated that the novel MELK inhibitors described above represent a valuable tool to further validate this kinase as a promising biological target and for the development of potential new anticancer therapies. 518 POSTER (Board P090) The HSP90 inhibitor, AT13387, overcomes resistance to crizotinib and second generation ALK inhibitors A. Courtin1 , A. Smyth1 , K. Hearn1 , V. Martins1 , J. Lewis1 , N. Thompson1 , J. Lyons1 , N. Wallis1 . 1 Astex Pharmaceuticals, Cambridge, United Kingdom Background: ALK-positive non-small cell lung cancer (NSCLC) can be successfully treated with the first generation ALK inhibitor, crizotinib, but responses are transient and tumors often relapse through a variety of resistance mechanisms. The second-generation ALK inhibitors can overcome some, but not all of these resistance mechanisms leaving a need for novel therapies to deal with resistance. The chaperone, HSP90, has many client proteins, including EML4-ALK, and HSP90 inhibition results in the disruption of multiple signalling pathways. Here we tested AT13387, an HSP90 inhibitor currently in Phase 2 clinical trials, in a number of preclinical models of crizotinib resistance to investigate its potential in ALK inhibitorresistant NSCLC. Materials and Methods: Crizotinib-resistant cell lines were generated by ex-vivo culture of H2228 xenograft tumors which had been treated continuously with crizotinib until relapse. Cell lines were characterized by proliferation assay and analysis of signalling by western blot. Results: Analysis of crizotinib-resistant cell lines suggested that different mechanisms were probably responsible for resistance. Two cell lines had increased levels of ALK whilst one cell line appeared to have lost ALK. One cell line had an activation of the STAT3 pathway. Signalling was investigated in these cell lines after treatment with the second generation ALK inhibitors, ceritinib and alectinib, and the HSP90 inhibitor, AT13387. As expected, signalling through the AKT and ERK pathways were poorly affected by crizotinib, but response varied to the 2nd generation ALK inhibitors with 2 cell lines being resistant to both alectinib and ceritinib as well as crizotinib. In contrast, treatment of all cell lines with the HSP90 inhibitor, AT13387, led to decreases in the levels of pERK, pAKT and pS6 suggesting that HSP90 inhibition can overcome resistance to these ALK inhibitors. All four compounds inhibited tumor growth of the ALK-positive H2228 xenograft model. AT13387 also inhibited tumor growth of an H2228-derived crizotinibresistant xenograft model. Conclusions: The HSP90 inhibitor, AT13387, is active in crizotinibresistant models and can overcome mechanisms of crizotinib resistance which are not susceptible to the second generation ALK inhibitors. This suggests AT13387 has therapeutic potential in ALK inhibitor-resistant NSCLC. AT13387 is currently being tested in the clinic in combination with crizotinib in ALK-positive NSCLC.

Poster Session – Molecular Targeted Agents II 519 POSTER (Board P091) AD-O53.2 − novel biological anticancer therapeutic agent with a dual mechanism of action K. Poleszak1 , J. Pieczykolan1 , S. Pawlak1 , B. Zerek1 , P. Rozga1 , M. Teska-Kaminska1 , M. Galazka1 , M. Szymanik1 , A. Jaworski1 , A. Pieczykolan1 , K. Bukato1 , W. Strozek1 . 1 Adamed Sp. z.o.o., Drug Discovery, Warsaw, Poland Background: Cancer is the second leading cause of death in developed countries therefore, there is an urgent need to discover novel therapies and approaches. Promising therapeutics are naturally occurring molecules, which induce apoptosis in cancer cells omitting normal cells. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) initiates apoptosis through interactions with receptors on tumor cells. Due to its selective ability for killing tumor cells the TRAIL pathway has emerged as an attractive target for cancer therapy. However, many cancer cell lines have developed a number of resistance mechanisms to TRAIL. Overexpression of protein XIAP (X-linked inhibitor of apoptosis protein) is considered as one of the main mechanisms of resistance. To overcome the resistance and to increase the efficacy of TRAIL, we have developed a novel, fusion molecule (AD-O53.2) consisting of a soluble variant of TRAIL (carrier and in some cases an effector) and a peptide derived from Smac/DIABLO protein (effector). This peptide binds to XIAP and inhibits its antiapoptotic activity. AD-O53.2 contains a linker joining carrier and effector, that consist of a membrane transduction motif followed by sequences recognized by tumor-specific proteases (MMPs, uPa), that release effector peptide in tumor proximity. Materials and Methods: AD-O53.2 was expressed in E. coli, using pET expression system, and purified by IEX chromatography. The biophysical properties were verified by circular dichroism (CD) and size-exclusion chromatography (SEC). Interactions of AD-O53.2 with TRAIL receptors and AD-O53.2 with XIAP were analyzed using surface plasmon resonance (SPR). The proapoptotic and antiproliferative activity was tested with MTT cell cytotoxicity assays and flow cytometry method. The efficacy of ADO53.2 was examined on mice xenograft models of human cancers. Results: AD-O53.2 showed in vitro cytotoxic effect on various cancer cell lines at the level of IC50 below 0.1 ng/ml and no or very low activity on noncancer cells. We demonstrated also that AD-O53.2 is a potent apoptosis inducer. This protein efficiently interacted with TRAIL receptors and XIAP. Comparing with TRAIL and standard chemotherapeutic agents, AD-O53.2 displayed significant tumor volume regression on mice xenograft models of human cancers. Conclusions: We have developed a molecule with high pro-apoptotic activity, that can be considered as a novel therapeutic agent. 520 POSTER (Board P092) Validation of Nanostring for FGFR1 gene expression analysis in squamous non-small cell lung cancer (sqNSCLC) tissue C. Rooney1 , C. Geh1 , V. Williams2 , C. Cresswell2 , K. Al-Kadhimi1 , M. Dymond3 , T. French2 , P.D. Smith1 , C. Barrett1 , E.A. Harrington1 , E. Kilgour1 . 1 AstraZeneca, Oncology iMed, Macclesfield − Cheshire, United Kingdom; 2 AstraZeneca, Personalised Healthcare and Biomarkers, Macclesfield − Cheshire, United Kingdom; 3 AstraZeneca, Discovery Sciences, Macclesfield − Cheshire, United Kingdom FGFR family members are genetically deregulated in diverse cancers through gene amplification, fusion and mutation. These genetic aberrations are the focus of several clinical trials with FGFR inhibitors. FGFR1 amplifications occur in ~20% of sqNSCLC cases and trials with FGFR inhibitors are selecting patients with FGFR1 amplifications, as determined by FISH. A panel of lung cancer cell lines was profiled for sensitivity to AZD4547, a potent, selective inhibitor of FGFR1, 2 and 3. Sensitivity to FGFR inhibition was associated with, but not wholly predicted by increased FGFR1 gene copy number (GCN). DMS114, NCI-H520 and NCI-H1703 cell lines harbour an FGFR1 amplification and were sensitive to FGFR inhibition. Calu-3 cells were insensitive however, despite increased FGFR1 GCN. Further, NCI-H226 cells do not have increased FGFR1 GCN but were sensitive to AZD4547 treatment. Interestingly, gene expression analysis revealed that Calu-3 cells did not express FGFR1 to high levels whereas NCI-H226 cells expressed relatively high levels of FGFR1. The preclinical data presented here indicate that evaluation of the expression of FGFRs and the correlation between amplification and expression in clinical tissues is warranted. We validated the Nanostring platform for mRNA expression analysis of 194 genes, including FGFRs, from a 5 mm section of FFPE clinical tumour tissue. Intra- and inter-assay variability was low and validation of a subset of these genes by RT-PCR revealed a good correlation between the platforms. A panel of 90 sqNSCLC tumours was analysed for FGFR1 gene amplification by FISH and 13 samples (14.4%) were confirmed as

Friday 21 November 2014 169 FGFR1 amplified. FGFR1 gene expression was evaluated in a subset of these samples by Nanostring. Although mean FGFR1 expression was significantly higher in the amplified samples (q = 0.012), several nonamplified samples expressed FGFR1 to levels equivalent to amplified samples. Statistical analysis revealed expression of many genes on the 8p12 amplicon, for example BAG4, LSM1 and in particular WHSC1L1, was increased in the FGFR1 amplified tumours. FGFR1 expression was also significantly associated with increasing GCN as assessed by qPCR and consistent with the FISH analysis, the 8p12 amplicon genes WHSC1L1, BAG4, and LSM1 were strongly associated with increasing FGFR1 GCN (p < 0.0001). These data reveal a broad range of FGFR1 expression within amplified and non-amplified sqNSCLC samples. FGFR1 expression was significantly higher in the amplified cohort and amplification enriched for the highest expressing samples. However there was significant overlap in the levels of FGFR1 expression between the amplified and non-amplified cohorts. Nanostring analysis will be incorporated as part of a wider biomarker toolkit to determine the role of FGFR1 expression levels in predicting response to AZD4547 in the clinic. 521 POSTER (Board P093) Tisular expression of the EGFR and N-Glycolyl GM3 Ganglioside as recognized by ior egf/r3 and 14F7 Mabs in triple negative breast cancer 1 A. Calvo Perez ´ , R.I. Alvarez Goyanes1 , E. Rengifo Calzado2 , S. Franco Odio3 , R. Camacho Rodr´ıguez4 . 1 Instituto Nacional de Oncolog´ıa y Radiobiolog´ıa, Cell Biology and Banking of Biological Samples, Havana, Cuba; 2 Center of Molecular Immunology, Quality Control, Havana, Cuba; 3 ´ Instituto Nacional de Oncolog´ıa y Radiobiolog´ıa, Anatom´ıa Patologica, Havana, Cuba; 4 Instituto Nacional de Oncolog´ıa y Radiobiolog´ıa, Mastology, Havana, Cuba

Introduction: Triple negative breast cancers (TNBC, estrogen receptornegative, progesterone receptor-negative, and HER2-negative) are a high risk breast cancer lacking the benefit of specific therapy that targets these proteins. Therefore, there is a need to develop alternative treatment strategies in those tumors that fail to express known target molecules. The epidermal growth factor receptor (EGFR) as well as the N-glycolyl GM3 ganglioside (NeuGcGM3) have been previously reported to be expressed in breast cancer. These molecules have been considered as potential targets for both passive and active immunotherapy in breast tumors. However, an extended study of their expression in TNBC using ior egf/r3 and 14F7 Mabs has not been documented up to now. Purpose: To evaluate the recognition of two Cuban monoclonal antibodies raised against the N-glycolyl-GM3 ganglioside (14F7) and EGFR (ior egf/r3) in TNBC. Method: A total of 1509 formalin-fixed and paraffin-embedded tissues from Cuban women with diagnosis of breast cancer were taken. The expression of hormone receptors and HER2 were determined by immunohistochemistry and the TNBC group was obtained. The immunorecognition of both, 14F7 and ior egf/r3 Mabs in TNBC were studied. The intensity of reaction of each sample was judged qualitatively and the percentage of positive cells was estimated. The staining with ior egf/r3 was compared with those obtained using the EGFR pharmDx™ Kit. Results: ER, PR and HER2 expression were detected in 53%, 49% and 23.7% of breast tumors respectively, while 19.9% of these malignancies were TNBC. 14F7 Mab showed a moderate to intense reaction in 100% of TNBC. The staining with 14F7 showed a homogeneous and finely granular pattern and was localized mainly in the plasmatic membrane but also in the cytoplasm of more than 95% of malignant cells. EGFR over-expression was observed in 10.5% of these tumors. The classification of EGFR expression using EGFR pharmDx™ Kit was 3+ (17.3%), 2+ (5.9%), 1+ (20.3%) and 0 (76.5%). Similar patterns of recognition were obtained with ior egf/r3. Conclusions: The recognition of ior egfr/r3 and 14F7 Mabs in TNBC opens up the possibility of using passive and active immunotherapy against EGFR and NeuGcGM3 ganglioside, along or combined with other treatment modalities in this orphan of effective therapies malignancy. 522 POSTER (Board P094) Evaluation of the efficacy of melatonin in breast cancer metastasis mediated by ROCK-1 T. Borin1 , A.S. Arbab2 , L.C. Ferreira3 , G.B. Botaro3 , L.B. Maschio3 , G.M. Moschetta1 , N.N. Gon¸calves1 , G.R. Martins1 , D.A.P.C. Zuccari1 . 1 ˜ Jose´ do Rio Preto SP, Brazil; 2 Henry FAMERP, Molecular Biology, Sao ˜ Jose´ Ford Hospital, Radiology, Detroit, USA; 3 UNESP, Genetics, Sao do Rio Preto SP, Brazil The metastasis occurrence, an important prognostic factor, depends on peculiarities such as cellular invasiveness and cell migration, mechanisms

170 Friday 21 November 2014 controlled by regulatory and effector molecules such as Rho-associated kinase protein (ROCK-1). An increased expression of this protein promotes tumor growth and metastasis, a mechanism which can be restricted by the use of the effector’s inhibitors. Melatonin, a hormone secreted by the pineal gland, has shown oncostatic action and anti-metastatic effects and can modulate the ROCK-1 expression. The objective of this study was to investigate the anti-metastatic response mediated by ROCK-1 and through the action of melatonin and its specific inhibitor (Y27632) in vitro and in vivo breast cancer models. Cells from metastatic (MDA-MB-231) and non-metastatic (MCF-7) breast cancer lines were treated with melatonin and Y27632. Cell viability was verified by MTT assay, cell migration/invasion assays in Boyden chamber, ROCK-1 protein and gene expression by western blot and quantitative real time PCR, respectively. In addition, the in vivo lung metastasis study was performed using female athymic nude mice induced by injection of 2×105 MDA-MB-231 viable cells by tail vein for 3 and 6 weeks. The animals were treated with melatonin for 2 and 5 weeks. The metastasis developments were evaluated by single photon emission computed tomography (SPECT). Treatment with melatonin reduced cell viability and migration of both cell lines (p < 0.05). The use of melatonin and Y27632, in association or not, decreased the ROCK-1 protein expression in metastatic cells, not significantly altering its expression in the nonmetastatic line (p > 0.05). An statistically significant reduction of ROCK-1 gene expression was observed in all treatment groups (p < 0.05). ROCK-1 downexpression was more efficient in the group with associated treatments for both lines (p < 0.05). In vivo SPECT images showed multiple metastasis foci in the lungs (on Tc-99-tetrofomin images). The numbers of ‘hot’ spots were significantly higher in both periods of lung metastasis of control animals compared to treated groups. Semi quantitative analysis showed significantly lower activity in animal lungs that received melatonin treatment (p < 0.05). Besides that, melatonin was more effective on early treatment (2 weeks) when compared on delayed treatment (5 weeks), along time metastasis seem to develop resistance to treatment. Melatonin and Y27632 are effective drugs of metastatic breast cancer in vitro treatment, confirming their effects in decreasing cell viability, invasion, migration and protein expression of ROCK-1 in these cells. In vivo, melatonin treatments seem to be effective reducing early lung metastasis. Melatonin, in particular, appears to be more effective when combined to ROCK-1 inhibitor. 523 POSTER (Board P095) Antitumor activity of mTOR kinase and DNA-PK inhibitor CC-115 in a mouse model of glioblastoma H. Raymon1 , S. Peng1 , J. Katz2 , C. Zhao2 , C. Phan2 , M.F. Moghaddam2 , K.E. Fultz3 , S. Sankar3 , D.S. Mortensen4 , R.K. Narla1 . 1 Celgene, Pharmacology, San Diego, USA; 2 Celgene, DMPK, San Diego, USA; 3 Celgene, Oncology Research, San Diego, USA; 4 Celgene, Medicinal Chemistry, San Diego, USA CC-115 is an orally active dual small molecule inhibitor of DNA-PK (DNAdependent protein kinase) and mTOR (mammalian target of rapamycin) kinase (TORK) which is currently in clinical development. In cellular assays CC-115 inhibited pS6RP(Ser 235/236) and pAkt(Ser 473) and pDNA-PK(Ser 2056). The current study was aimed to determine the pharmacokinetic/pharmacodynamic (PK/PD) relationship, antitumor activity and mechanism of action of CC-115 in mice with U87MG human glioblastoma tumors. In PK/PD studies with U87MG tumor-bearing mice, CC-115 exhibited a dose and time dependent inhibition of tumor mTOR pathway biomarkers, pS6RP (mTORC1) and pAkt (mTORC2) which correlated with plasma exposure. Antitumor activity of CC-115 was tested in mice with subcutaneously implanted U87MG tumors of approximately 100– 150 mm3 in size. CC-115 consistently exhibited dose-dependent tumor growth inhibition. The minimum dose required to obtain approximately 65% tumor volume reduction compared with vehicle control (without causing significant body weight loss) was 0.5 mg/kg QD. The antitumor activity of CC-115 was further tested in mice with intracranially implanted U87MGluc glioblastoma cells. Daily treatment of CC-115 significantly inhibited tumor growth (as indicated by reduced luciferase signal) and prolonged the survival of mice with intracranial glioblastoma. In immunofluorescence assays, intracranial glioblastoma tumors sections from CC-115 treated animals showed a marked decrease in pDNA-PK and mTOR pathway markers (pS6RP and pAkt) when compared to vehicle controls. In conclusion, treatment with CC-115, a dual inhibitor of TORK and DNAPK, significantly inhibited U87MG glioblastoma tumor growth. Inhibition of mTOR pathway markers (pS6RP and pAkt) and pDNA-PK in CC115-treated tumors correlated with the antitumor activity of CC-115. The antitumor activity and biomarker inhibition observed with CC-115 treatment in an intracranial glioblastoma xenograft model suggests that CC-115 is brain permeable.

Poster Session – Molecular Targeted Agents II 524 POSTER (Board P096) Targeting MET for a differentiation therapy of rhabdomyosarcoma K. Skrzypek1 , A. Jagiello1 , B. Szewczyk1 , T. Adamus1 , K. Miekus1 , M. Majka1 . 1 Jagiellonian University Medical College, Department of Transplantation Polish-American Institute of Pediatrics, Krakow, Poland Background: Rhabdomyosarcoma (RMS) is a soft tissue sarcoma originating from a defect in myogenic differentiation, but precise pathomechanism of its development is not known. The aim of our studies was to investigate whether excessive activation of MET, the tyrosine kinase receptor for hepatocyte growth factor, might be responsible for RMS development and if MET can be a potential target for novel differentiation therapies of RMS. Materials and Methods: In vitro, MET signaling pathway was activated independent of HGF in mesenchymal stem cells (MSC) and SMS-CTR RMS cells by transduction with lentiviral vectors encoding TPR-MET. RH30, RH28, CW9019 RMS cell lines were differentiated in DMEM with 2% horse serum and 100 nM TPA. MET level was verified with flow cytometry. Expression of miRNA and genes was evaluated with quantitative realtime PCR both in RMS cell lines and paraffin embedded RMS samples from patients and tumors from mice. For in vivo experiments, SMS-CTR cells were subcutaneously implanted to NOD-SCID mice. Hematoxylineosin and Ki67 staining showed tumor morphology, vascularization and proliferation. Results: Mesenchymal stem cells expressing TPR-MET survive in culture for several months, whereas control cells stopped growing after the 5th passage, suggesting oncogenic capabilities of the excessive MET activation. In RMS tumors from patients MET level positively correlates with MyoD expression and it is lower in embryonal (ERMS) tumors than in alveolar (ARMS) type, which is associated with worse prognosis. To investigate it further, we introduced TPR-MET to benign SMS-CTR cells of ERMS type. Those cells poorly differentiate in vitro, whereas in vivo, after subcutaneous implantation to immunodeficient NOD-SCID, they form structures resembling muscle fibers. In contrast, tumors formed by TPRMET cells are composed of pleomorphic cells. Impaired differentiation in those tumors is associated with downregulation of myogenic factors, such as myogenin, myostatin and MEF2A. Moreover, TPR-MET tumors proliferate faster and grow bigger, they are better vascularized and they metastasize to lungs. Accordingly, in vitro their chemotactic capabilities are enhanced and they express more proangiogenic mediators, such as VEGF, MMP9 and miR-378. Growth of ERMS tumors with active MET signaling resembles the growth of ARMS tumors, which display higher basal MET level and are associated with worse prognosis. Accordingly, differentiation of RH30, RH28 and CW9019 ARMS cell lines results in downregulation of MET. Finally, inhibition of MET phosphorylation with SU11274 in RH30 ARMS cells reduces proliferation in vitro. Conclusions: Excessive MET activation is associated with myogenic differentiation defect responsible for RMS development. Therefore targeting MET receptor may be an innovative solution for novel therapies in future. 525 POSTER (Board P097) Analysing Src and phosphoinositide 3-kinase pathway inhibition in a radiotherapy context − pathway interplay and therapeutic response E. Rowling1 , N. Burrows2 , P. Elvin3 , K. Williams1 . 1 University of Manchester, Manchester Pharmacy School, Manchester, United Kingdom; 2 University of Cambridge, Cambridge Institute for Medical Research, Cambridge, United Kingdom; 3 AstraZeneca, Oncology Innovative Medicines, Macclesfield, United Kingdom Introduction: Radiation treatment can enhance cellular characteristics associated with metastatic progression. We have previously observed that both Src and phosphoinositide 3-kinase (PI3K) pathways are activated by radiotherapy in vitro and contribute to an aggressive cellular phenotype. Here, using thyroid tumour cells established as xenografts in nude mice, we have evaluated radiation-induced modification of molecular targets within these pathways and how these are influenced by pharmacological inhibitors targeting Src (AZD0530) and/or PI3K (GDC-0941) and related this to tumour response. Materials and Methods: Radiotherapy alone (5×2 Gy fractions; days 1−5), and combined with Src (AZD0530 25 mg/kg once daily) and/or PI3K (GDC-0941 50 mg/kg twice daily) inhibition (days 1−14) was evaluated in vivo using FTC133 xenografts. Molecular targets of PI3K (pAKT [pS473]) and src activity (pFAK [pY397 and pY861]; active src [clone 28]) were analysed in primary tumours by IHC and western blotting at day 5 or at the experimental endpoint, when tumours had quadrupled in size from first treatment (RTV4). For comparison, xenografts were treated with AZD0530 and/or GDC-0941 only and excised at RTV4. Results: At day 5, radiation increased the expression of active src and pAKT and caused pronounced localisation of staining at the tumour periphery. pFAK was also markedly enhanced, showing strong

Poster Session – Molecular Targeted Agents II membranous expression. Western blots confirmed the inhibitory effects of GDC-0941 on pAKT and AZD0530 on pFAK in irradiated tumours. Inhibition of both pathways resulted in more marked suppression of pAKT and pFAK compared with either treatment alone and countered the peripheral staining pattern observed in irradiated tumours. Surprisingly, in tumours at RTV4, AZD0530 treatment alone was associated with elevated levels of active src and pFAK that was not observed when combined with GDC-0941 and/or with radiation. AZD0530 also resulted in enhanced pAKT in RTV4 tumours. In this case elevated pAKT was maintained in tumours that had regrown following AZD0530 plus GDC-0941 and/or radiation treatment. Neither drug alone was able to enhance primary tumour response to radiation, but radiation response was significantly improved when combined with both agents. Further, AZD0530 alone significantly reduced the advent of lung metastasis, an effect that was much more pronounced with dual src and PI3K inhibition. Conclusions: Radiation treatment was shown to enhance PI3K and Src pathways, and by using rational drug combinations we saw improved local control and reduced metastatic spread in preclinical studies. Analysis of phosphoprotein targets suggests that there are clearly complex inter-relationships between these two pathways and their response to radiation which could impact both overall treatment response and acquired resistance. 526 POSTER (Board P098) A Phase I study of the dual, intravenous (IV) phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibitor PF-05212384 in combination with irinotecan in patients (pts) with colorectal cancer (CRC) and other advanced solid tumors Z. Wainberg1 , J. Tabernero2 , M.A. Maqueda2 , S. Leong3 , G. Del Conte4 , C. Britten5 , N. Brega6 , C. Davis7 , B. Houk8 , K. Pierce7 , J. Vermette9 , L. Siu10 , I. Brana10 . 1 University of California Los Angeles Center for the Health Sciences, Hematology/Oncology, Los Angeles CA, USA; 2 Vail d’Hebron Institute of Oncology Barcelona Spain, Oncology, Barcelona, Spain; 3 University of Colorado, Oncology, Denver, USA; 4 Ospedale San Raffaele, Oncology, Milan, Italy; 5 Medical University of South Carolina, Oncology, Charleston, USA; 6 Pfizer, Oncology, Milan, Italy; 7 Pfizer, Oncology, Groton, USA; 8 Pfizer, Oncology, La Jolla, USA; 9 Pfizer, Oncology, Cambridge, USA; 10 Princess Margaret Hospital, Oncology, Toronto, Canada Background: Signaling through the PI3K-AKT-mTOR pathway plays an important role in cell survival, proliferation, and metabolism. In preclinical studies, combining the PI3K/mTOR inhibitor PF-05212384 (PF-384) with irinotecan demonstrated enhanced anti-tumor activity, suggesting a potential benefit from this combination in cancer pts. Patients and Methods: Adult pts with advanced solid tumors were enrolled in the dose-escalation part (stage 1 [S1], based on a standard 3+3 design) in arm C of a multi-arm Phase I study or in an expansion cohort (stage 2 [S2]). In arm C, pts received biweekly irinotecan 180 mg/m2 in combination with IV PF-384 administered at 95, 110, or 130 mg on days 2, 9, 16, and 23 of each 28-day cycle. Study endpoints included safety, antitumor activity, pharmacokinetics (PK), and pharmacodynamic (PD) effects on circulating biomarkers. Results: In arm C, 13 pts were enrolled in S1 and 27 pts in S2. Median age was 64 yrs (range 43−77) and 57 yrs (range 33−78), respectively. CRC was the most frequent diagnosis. S2 included CRC pts with disease progression on irinotecan-based regimens (n = 17) and pancreatic cancer (PC) pts who progressed on front-line therapy (n = 10). Two patients, receiving PF-384 130 mg plus irinotecan, experienced DLTs: grade 4 (G4) febrile neutropenia and G3 fatigue (n = 1 each). Median duration of treatment was 7.3 (PF-384 95 mg), 9.9 (110 mg), and 19.3 (130 mg) weeks in S1, and 7.4 weeks in S2 (110 mg). Treatment-related adverse events (AEs) were mostly G1 or G2 in severity; the most frequent were nausea and diarrhea. G3/G4 AEs noted in S2 included G3 asthenia, hyperglycemia, and leukopenia (3.7% each); G3 (14.8%) and G4 (11.1%) neutropenia. No treatment-related deaths were reported in S1 or S2. In S1 (n = 13), 1/10 pts with CRC had a partial response, 6 pts (4/10 with CRC, 2/2 with PC) had stable disease (SD). In S2 (n = 27), 13 pts had SD (9/17 with CRC, 4/10 with PC). In S1, 2/6 pts with mt-KRAS CRC and 1/2 pts with wt-KRAS CRC had SD. In S2, 3/4 pts with mt-KRAS CRC and 4/11 pts with wt-KRAS CRC had SD. Conclusions: Treatment with weekly IV PF-384 in combination with irinotecan waswell tolerated and demonstrated antitumor activity in patients with advanced, irinotecan-refractory, mt- and wt-KRAS CRC or with advanced, pretreated PC. The MTD for this combination was determined to be PF-384 110 mg. The most relevant AEs were fatigue and gastrointestinal AEs. Updated safety, antitumor activity, PK, and PD results will be presented. Randomized trials with irinotecan and PF-384 are ongoing in pts with mCRC.

Friday 21 November 2014 171 527 POSTER (Board P099) Is CRAF required for the maintenance of KRAS mutant non-small cell lung cancer? P. Russell1 , J. Wickenden1 , K. Cadwallader1 , S. Maguire1 , J. Joel1 , M. Stockdale1 , A. Chicas2 , D. Banka2 , R. Darman2 , S. Perino2 , P. Fekkes2 , P. Smith2 , P. Zhu2 , S. Buonamici2 , J. Moore1 . 1 Horizon Discovery Ltd, Cambridge, United Kingdom; 2 H3 Biomedicine Inc, Cambridge, USA KRAS is one of the most frequently mutated genes in cancer, but has proved intractable to targeting with potent small molecules. Considerable attention has been applied to blocking KRAS function by interfering with its localization or inhibiting downstream signalling. Inhibitors of BRAF and MEK have proved beneficial, but only to melanoma patients with BRAF mutations. An effective therapy for KRAS-driven tumours remains a pressing unmet medical need. Knockout mouse experiments have indicated that simultaneous deletion of MEK1/MEK2 or ERK1/ERK2 leads to organ failure. In contrast, simultaneous deletion of BRAF and CRAF is tolerated in adult animals (Blasco, 2011). Strikingly, two studies employing lung-specific Crerecombinase expression have demonstrated that CRAF is required for the initiation of lung cancer by activated KRAS alleles (Blasco, 2011 & Karreth, 2011). Together these results imply optimised CRAF inhibitors might provide a well-tolerated therapy for lung cancer. One key question is: do existing tumours retain dependence on CRAF for viability? Here we report the results of a large shRNA validation study, where multiple shRNAs were used to assess dependence of a panel of human non-small cell lung cancer cells on KRAS and CRAF. 13 KRAS mutant and 7 KRAS wild-type cell lines were infected with lentiviruses directing doxycycline-inducible expression of shRNAs vs. KRAS (×3), CRAF (×3) and controls. Proliferation assays were then performed in the presence and absence of doxycycline. A subset of the KRAS mutant lung cancer lines proved notably sensitive to depletion of KRAS by RNA interference. Furthermore, a correlation (r = 0.5925, p = 0.0059) was observed between cell lines that exhibited sensitivity to KRAS depletion and those that showed sensitivity to CRAF depletion. Partial rescue of the antiproliferative effects of two shRNAs vs CRAF with cDNAs bearing silent mutations in Calu-1 cells were unsatisfactory, due in part, to heterogenous levels of rescue protein expression throughout the cell population. An engineered A549 cell line with reduced basal levels of CRAF expression, demonstrated increased sensitivity to further shRNA-mediated depletion. This suggested a greater reduction of CRAF that can be achived with shRNA, might lead to increased sensitivity. However, a confounding piece of data is that one of the five shRNAs vs CRAF suppressed CRAF expression without inhibiting proliferation in the KRAS dependent cell lines. These data provide some support for the hypothesis that CRAF is specifically required for the maintenance of KRAS-driven lung cancers but also illustrate some of the data interpretation issues raised by the use of shRNAs for validation. Further studies utilising new, proprietary Cas9/sgRNA (CRISPR) technologies are being undertaken that will provide a definitive answer regarding CRAF dependence. 528 POSTER (Board P100) A procaspase activator shows preclinical promise for glioblastoma therapy G. Riggins1 , A. Joshi1 , P.J. Hergenrother2 . 1 Johns Hopkins University, Neurosurgery, Baltimore MD, USA; 2 University of Illinois, Chemistry, Urbana IL, USA The treatment of glioblastoma (GBM), an aggressive brain tumor, is challenging as most systemically administered drugs do not reach intracranial tumors and fail to activate widespread cell death specifically in the malignant cells. We have performed extensive preclinical testing on an oral procaspase-3 activating drug, PAC-1. PAC-1 showed significant survival benefit in two aggressive orthotopic animal models of glioblastoma. Use of PAC-1 in an aqueous suspension with rat intracranial 9L glioblastoma improved survival compared to untreated controls. Moreover, when PAC-1 was administered as an oral capsule to rats with 9L glioblastoma, median survival increased over three fold. Oral PAC-1 improved survival significantly also in a human xenograft model compared to controls. PAC-1 demonstrates synergy in vivo in both models with the glioblastoma approved therapy temozolomide. In glioma patients high expression of procaspase-3 mRNA is correlated with a worse prognosis, indicating that procaspase-3 activation may be involved in the pathology of the tumor progression. PAC-1 has the ability to penetrate sufficiently within intracranial tumors and its synergy with temozolomide makes it a promising small molecule for further development.

172 Friday 21 November 2014

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Table (abstract 529). Tumour type

Total number of samples

Failed all tests

Wild type for all genes analysed

Mutation rate by gene

Gene rearrangement by fluorescent in situ hybridisation

Aberration in more than one gene

Breast cancer

1874

5.3%

45%

−

7%

Colorectal cancer

1634

0.9%

19%

−

33.1%

Lung cancer

1889

2.8%

65%

1.9%, ALK

0.65%

Malignant melanoma

535

3.6%

31%

−

2.4%

Ovarian cancer

559

3.2%

40.2%

−

4%

Prostate cancer

1359

4.0%

52.2%

BRAF 0.07%, PIK3CA 29%, PTEN 4.8%, TP53 23% BRAF 12%, KRAS 44%, NRAS 4%, PIK3CA 11%, TP53 67% BRAF 1%, EGFR 8.3%, KRAS 23% BRAF 43%, KIT 1.3%, NRAS 23%, PIK3CA 1.5% BRAF 2.3%, PIK3CA 6.3%, PTEN 4.2%, TP53 48% BRAF 1.2%, PTEN 5.2%

40%, TMPRSS2-ERG

2.1%

529 POSTER (Board P101) The Cancer Research UK Stratified Medicine Programme: From national screening to national trial I. Mirabile1 , E. Shaw1 , C. Lindsay1 , I. Walker1 , P.W.M. Johnson1 , Stratified Medicine Programme 1 Consortium. 1 Cancer Research UK, Stratified Medicine, London, United Kingdom Background: The increasing demand for analysis of multiple predictive genetic markers in tumour samples to guide therapeutic decision-making requires the rapid validation and uptake of new knowledge and technology, as well as collaboration between clinical teams, cellular pathology and molecular genetics laboratories. Material and Methods: Between 2011 and 2013, formalin-fixed paraffinembedded tumour samples from patients consented through a network of hospitals underwent targeted mutation analysis. This was initially performed using Sanger sequencing and similar methods but following cross-validation an amplicon-based targeted next generation sequencing panel was implemented. Clinical information based on the requirements of the national cancer registration dataset was also centrally collated for each participant. The current second phase of the initiative has a sole focus on patients with advanced lung cancer, and aims to expand the geographical reach to perform molecular pre-screening of samples to help determine patient eligibility for the National Lung Matrix Trial, a multi-arm stratified phase II clinical trial of novel therapeutics. Results: 10,754 patients (98% of those approached) consented to analysis of material during phase one, with 9010 samples submitted for analysis. The table shows the overall results of analysis for the first 7850 samples. Analysis by histological subtype enriches for certain mutations, with 925 pulmonary adenocarcinomas showing 36.5% KRAS mutations, 11.6% EGFR mutations and 2.5% ALK gene rearrangements and 56 mucinous colorectal adenocarcinomas showing 43.5% BRAF mutations and 50% KRAS mutations. Conclusions: A centralised national testing network for genetic analysis of formalin-fixed paraffin-embedded tumour samples is feasible and acceptable to patients and clinicians, with applications beyond genotyping for currently licensed oncologic therapies. Acknowledgements: Funding for the Stratified Medicine Programme is acknowledged from Cancer Research UK and programme founding partners AstraZeneca and Pfizer. 530 POSTER (Board P102) Anti-angiogenic therapy induces T-lymphocyte infiltration associated with poor survival in metastatic renal cell carcinoma patients X.D. Liu1 , A. Hoang1 , L. Zhou1 , S. Kalra1 , A. Yetil1 , M. Sun1 , Z. Ding1 , S. Bai1 , P. German1 , X. Zhang1 , P. Tamboli1 , P. Rao1 , J. Karam1 , C. Wood1 , S. Matin1 , A. Zurita1 , N. Tannir1 , K. Sircar1 , E. Jonasch1 . 1 The University of Texas M.D. Anderson Cancer Center, Houston TX, USA Rationale: Renal cell carcinoma (RCC) is an immunogenic and proangiogenic cancer. Recently, anti-angiogenic therapy has been widely used for RCC management. The interaction between immune cell infiltration and anti-angiogenic treatment in RCC, and its association with patient survival, is unknown. We assessed the interaction between anti-angiogenic therapy and the tumor microenvironment, with a specific focus on immune cell subtypes, and determined their impact on clinical outcome. Methods: Tissue microarrays (TMA) with triplicate cores for each case were generated from formalin-fixed, paraffin embedded tissues from 33 unaffected kidneys, 41 untreated primary RCC, 42 bevacizumab and 39 sunitinib pretreated primary RCC from patients with metastatic RCC. Immunohistochemistry was used to evaluate the infiltration of immune cells.

Staining quantitation was performed using a Vectra multispectral system (Perkin Elmer, Waltham, MA). Statistical analysis was performed using unpaired t test with Welch’s correction. Results: Compared with normal kidney tissues, RCC tissues harbored more infiltrating immune cells, including CD3+ T cells, CD4+ T cells, CD8+ T cells, granzyme B+ T cells and CD68+ macrophages (p<0.001). Both bevacizumab and sunitinib treated cases showed increased CD4+ T cell infiltration relative to untreated RCC controls: 15.3% and 16.3% vs 7.6% (p<0.01), respectively, and higher CD8+ T cell infiltration: 7.0% and 5.1% vs 2.3% (p < 0.05), respectively. Importantly, the infiltration of CD4+ and CD8+ T cells was inversely correlated with overall survival (OS) and progression free survival (PFS) (p < 0.05). To investigate the underlying mechanism, we found that anti-angiogenic therapy treated specimens showed higher infiltration of CD4+FoxP3+ regulatory T cells (Tregs) and expression of checkpoint ligand PD-L1 (p < 0.05). Both features were correlated with T cell infiltration and were negatively related to patient OS and/or PFS. In addition, sunitinib treated specimens showed elevated expression of adhesion molecule ICAM1, indicating a potential role in enhancing T cell infiltration. The infiltration of CD68+ macrophages was higher in sunitinib treated versus control RCC specimens (25.2% vs 20.3%, p < 0.05) but not in bevacizumab treated samples (21.3 vs 20.3%, p > 0.05), and macrophage infiltration was not associated with patient survival. Conclusion: Our study shows that anti-angiogenic treated RCC show enhanced T cell infiltration. This T cell infiltration is associated with worse clinical outcome in patients with metastatic RCC, and is associated with the upregulation of immune suppressive Tregs and PD-L1. These findings will guide further studies on the mechanistic drivers of T cell infiltration, the impact of these cell populations on therapy response and their influence on patient survival. 531 POSTER (Board P103) In vitro and in vivo anti-tumor activity of ARQ 092, a potent and selective pan-AKT inhibitor Y. Yu1 , S. Cornell-Kennon1 , C.R. Chen1 , E. Marchlik1 , T. Isoyama2 , K. Tazaki2 , K. Fujiwara2 , D.T. Dransfield1 . 1 ArQule Inc, Woburn, USA; 2 Daiichi Sankyo Co. Ltd, Tokyo, Japan Background: Dysregulation of the PI3K-AKT signaling pathway has been implicated as a key driver in cancer initiation and progression. AKT is a serine/threonine kinase and a critical component mediating the PI3KAKT signaling axis. It plays an important role in cell cycle progression, survival, proliferation and growth. AKT can be constitutively activated through activated receptor tyrosine kinases, gain-of-function mutations of PIK3CA, or inactivation of PTEN, and its amplification, activating mutations, or over-expression. ARQ 092 is a potent and selective pan-AKT inhibitor currently in early clinical development. Methods: Biochemical IC50 for AKT1, 2, and 3 and the selectivity profile of ARQ 092 against 303 kinases were determined. Broad antiproliferative effects of ARQ 092 were assessed using the Oncopanel profiling analysis on the growth of 240 human cancer cell lines. Threedimensional clonogenic assays were performed using patient-derived tumor. Somatic mutations for each cell line were obtained from the COSMIC database and correlations between somatic mutations and ARQ 092 sensitivity were assessed. Efficacy studies of ARQ 092 were performed in mouse xenograft models using the ZR-75−1 and KPL-4 breast cancer cell lines. Results: ARQ 092 is a potent AKT inhibitor with IC50 in the low nanomolar range for AKT1, 2, and 3 respectively. Kinase profiling showed 5 kinases had IC50 values less than 1 mM against ARQ 092 but with far lower potency (26 to161 fold) than AKT1. Oncopanel profile analysis revealed the cancer types most sensitive to ARQ 092 are: breast with 65% (11/17)

Poster Session – Molecular Targeted Agents II

Friday 21 November 2014 173

sensitivity rate (GI50 <1 mM), leukemia with 47% (8/17) and CRC with 41% (11/27). Furthermore, 56% (23 out of 41) of cell lines with PIK3CA/R1 mutations from Oncopanel analysis are sensitive to ARQ 092 (GI50 <1mM). In comparison, only 24% (46 out of 190) of the cell lines with wildtype PIK3CA/R1 are sensitive. Interestingly, 21% of mutant PTEN cell lines (4 out of 19 cell lines) and 25% of wild-type PTEN cell lines (42 out of 171 cell lines) among cell lines with wild-type PIK3CA/R1 exhibited a similar sensitivity to ARQ 092. In breast cancer cell lines with PIK3CA mutations, 88% (7 out of 8) are sensitive to ARQ 092. Three dimensional clonogenic assays using patient-derived tumor also showed 33% (3/9) sensitivity rate for breast cancer models, 100% (4/4) for female gynecologic cancers and 100% (3/3) for head and neck cancer, but only 9% (1/11) for CRC. In vivo efficacy studies with two breast cancer cell lines showed that ARQ 092 inhibited tumor growth in a dose-dependent manner. Conclusions: ARQ 092 has exhibited potent anti-proliferative effects on a subset of a large panel of cancer cell lines with particular potency inbreast cancer cells. This enriched activity in breast cancers was also demonstrated in the clonogenic assay using patient-derived tumors. Mutational analysis indicates that gain-of-function mutations of PIK3CA but not PTEN deficiency predict greater ARQ 092 sensitivity and represent potential predictive biomarkers for patient stratification. Our analysis suggests that leukemia, female gynecologic cancer and particularly breast cancer patients may represent a targeted indication for ARQ 092. 532 POSTER (Board P104) Evaluation of a BRCAness signature as a predictive biomarker of response to veliparib/carboplatin plus standard neoadjuvant therapy in high-risk breast cancer: results from the I-SPY 2 trial A. Glas1 , J. Peeters1 , C. Yau2 , D.M. Wolf2 , A. Sanil3 , Y. Li4 , T. Severson5 , S. Linn5 , I-SPY 2 TRIAL Investigators6 , M. Buxton2 , A. DeMichele7 , N. Hylton2 , F. Symmans8 , D. Yee9 , M. Paoloni6 , L. Esserman2 , D. Berry3 , H. Rugo2 , O. Olopade10 , L.J. van ’t Veer2 . 1 Agendia, Amsterdam, Netherlands; 2 University of California, San Francisco, USA; 3 Berry Consultants LLC, San Francisco, USA; 4 Agendia, Irvine, USA; 5 NKI, Amsterdam, Netherlands; 6 QuantumLeap Healthcare, San Francisco, USA; 7 University of Pennsylvania, Philadelphia, USA; 8 The University of Texas MD Anderson, Houston, USA; 9 University of Minessota, Minneapolis, USA; 10 University of Chicago, Chicago, USA Background: We developed a 77-gene BRCAness gene expression signature that predicts ‘BRCA1-like’ (vs. ‘Sporadic-like’) breast cancers with a validated high sensitivity and specificity rate. The BRCAness signature was developed as part of the RATHER project (EU#258967). We hypothesized that BRCA1-like tumors would have a higher sensitivity to PARP inhibitors, including veliparib. In the I-SPY II TRIAL, HER2− patients were randomized to receive standard chemotherapy or the oral PARP inhibitor veliparib in combination with carboplatin and chemotherapy (V/C). V/C graduated in the triple-negative (TN) signature. Here we assess the BRCAness signature as a specific biomarker of V/C response. Material and Methods: 113 HER2− patients (V/C: 71 and concurrent controls: 42) were considered in this analysis. The BRCAness classification is computed from Agilent full genome array data of the 77 signature genes using our validated Diagonal Linear Discriminant Analysis (DLDA) model. We assess association between BRCAness classification and response in the V/C and control arms alone (Fisher Exact test), and relative performance between arms (biomarker × treatment interaction, likelihood ratio test) using a logistic model. To assess the BRCAness signature in the context of the graduating signature, we added the BRCAlike patients to the graduating TN subset and evaluated the treatment effect in this new ‘biomarker-positive’ group. Our study is exploratory with no claims for generalizability of the data. Statistical calculations are descriptive (e.g. p-values are measures of distance with no inferential content). Our analyses do not adjust for multiplicities of other biomarkers in the trial but outside this study. Results: Of the 113 patients assessed, 55 are classified as BRCA-like. 16% of BRCA-like patients are HR+HER2-. The distribution of pCR rates among BRCAness signature dichotomized groups stratified by HR status are in Table 1.

Table 1.

TN (n = 58) HR+HER2− (n = 55)

V/C (n = 71) Sporadic-like (32)

BRCA1-like (39)

Control (n = 42) Sporadic-like BRCA1-like (26) (16)

4/6 1/26

18/32 4/7

2/6 4/20

3/14 0/2

The BRCAness signature classification associates with patient response in the V/C arm (OR = 6.8, p = 0.0005) but not in the control arm (OR = 0.77, p = 1). There is a significant biomarker × treatment interaction (OR in V/C arm relative to control arm = 9.1, p = 0.021), which remains significant upon adjusting for HR status (p = 0.019). When the BRCA1-like patients are added to the graduating TN subset, the OR associated with V/C is 4.7, which is comparable to that of the TN signature (OR: 4.1), while increasing the prevalence of biomarker-positive patients by ~8%. Evaluation of the BRCAness signature in the context of the graduating signature under the I-SPY 2 Bayesian model is pending. Conclusion: Our sample size is small. Our pre-specified analysis suggests the BRCAness signature is associated with response to veliparib/ carboplatin combination therapy relative to control. We will compare with results of other biomarker signatures developed to predict PARP inhibitor response. All I-SPY 2 qualifying biomarker signatures require validation in larger trials prior to consideration to assist in patient selection of future PARP inhibitor trials. 533 POSTER (Board P105) Detection of recurrent novel fusion transcripts from whole transcriptome sequencing of 120 primary breast cancer J. Kim1 , S.Y. Go2 , S.W. Kim3 , S. Lee4 , H.S. Lee3 , J. Park5 , M.J. Lee6 , H.G. Moon6 , D.Y. Noh6 , S. Kim2 , S. Kim7 , W. Han6 . 1 ASAN Medical Center College of Medicine Ulsan University, Surgery, Seoul, Korea; 2 Seoul National University, Computer Science and Engineering, Seoul, Korea; 3 Macrogen, Bioinformatics, Seoul, Korea; 4 Magrogen, Bioinformatics, Seoul, Korea; 5 Geference, Bioinformatics, Seoul, Korea; 6 Seoul National University Hospital College of Medicine Seoul National University, Surgery, Seoul, Korea; 7 Seoul National University, Graduate School of Convergence Science and Technology, Seoul, Korea Background: Although fusion genes serve as an effective target in hematologic malignancies, recurrent gene fusion events are relatively rare in solid tumors. To identify novel fusion transcripts we performed paired-end whole transcriptome sequencing in 120 primary breast cancer samples. Material and Methods: Whole transcriptome sequencing was done from 120 fresh frozen breast cancer samples and 5 adjacent normal breast tissues using Illumina HiSeq2000 platform. Both deFUSE and Chimerascan was applied for fusion detection. Among 120 samples, 27 (22.2%) were cases with distant metastasis during follow up. 49 (40.8%) were hormone receptor positive, 33 (27.5%) were triple negative subtypes and 29 (24.2%) had HER2 amplification. Highly nominated fusions were chosen for validation by reverse transcription PCR and Sanger sequencing. We then chose 10 validated fusion transcripts for screening in a 90 independent breast cancer cohort. MAGI3-AKT3, previously reported fusion gene found in 7% of triple negative breast cancer were included for screening likewise. Results: Total 1310 fusion transcripts were detected by deFUSE with an average of 24.4 per sample while adjacent normal tissue had 0−2 fusions. Fusion transcripts detected in multiple samples, genes associated with cancer and with high probability provided by deFUSE algorithm were prioritized. Among the 71 fusion candidates, 32 were validated by Sanger sequencing of the PCR product. Validation rate was higher when Chimerascan was applied (57.7%, 15/26) compared to deFUSE (45.1%, 32/71). None of the fusions were identified among the 90 independent cohort including the MAGI3-AKT3. To lower the false positivity, we also applied Chimerascan and the total number of fusion transcripts narrowed down to 399. The number of fusion transcripts were higher in HER2 amplified cases (22.4 vs 31.4, p = 0.014) and lower in triple negatives (28.0 vs 19.3, p = 0.012). The number of fusions were independently associated with distant metastasis-free survival (<25 versus 25, odds ratio 3.61, p = 0.009). We identified a recurrent fusion PPP1R1B–STARD3 − detected in 16.7% (20/120) which has been reported previously in breast and gastric cancer. Overexpression of this off-frame fusion is likely to activate AKT pathway with retained function of PPP1R1B and early truncation of STARD3. As the two genes are located within the 17q12 PPP1R1B–STARD3–ERBB2–GRB7 amplicon, we assessed copy number and expression of ERBB2, GRB7 genes. 7/20 had ERBB2 amplification and both genes showed higher expression in samples harboring PPP1R1B–STARD3 transcript (p = 0.021, <0.001 respectively). Conclusions: From sequencing the whole transcriptome of 120 brest cancer samples, 399 fusions were idenfied and 32/71 were validated. Although we failed to detect fusions among the 90 independent cohort, we identified highly recurrent fusion transcript with oncogenic potential. Applying multiple algorithms may contribute greatly in overcoming high false positivity of next generation sequencing.

174 Friday 21 November 2014 534 POSTER (Board P106) Expression of c-MET in invasive meningioma S. Yun1 , J. Koh1 , K. Lee1 , A. Seo2 , K. Nam3 , G. Choe4 . 1 Seoul National University Hospital, Pathology, Seoul, Korea; 2 Kyungpook National University School of Medicine, Pathology, Daegu, Korea; 3 Haeundae Paik Hospital, Pathology, Busan, Korea; 4 Seoul National University Bundang Hospital, Pathology, Seongnam, Korea Background: Meningiomas show high recurrence rate even after curative tumor removal and invasiveness of tumor may contribute to the high recurrence rate. Recently, c-MET and HGF have been reported to be involved in cancer invasion. We investigated whether expressions of c-MET and HGF were associated with clinicopathologic variables as well as brain and bone/scalp invasion in large scale of meningiomas. Materials and Methods: We examined immunohistochemical expression of c-MET and HGF in consecutive 100 cases of meningiomas treated with complete tumor removal (Simpson grade I). We harvested one core with a diameter of 3 mm from the most representative tumor areas of the archival donor block and we made TMA blocks. We graded the expression of c-MET and HGF into c-MET−high /HGF−high (>25% positive cells) and c-MET−low /HGF−low (0−25% tumor cells) according to previous reports. Results: c-MET−High was observed in 60% meningiomas with brain invasion and c-MET−High significantly correlated with brain invasion (P = 0.033). c-MET−High was also observed in 33.3% of meningiomas with bone/scalp invasion. There was a tendency for increased c-MET−High in meningiomas with bone/scalp invasion compared with meningiomas without bone/scalp invasion, although statistical significance was not reached. (P = 0.119) HGF−High did not show statistical association with brain invasion or bone/scalp invasion. (P = 0.375, P = 0.653, respectively) c-MET−high showed shorter recurrence free survival (93.467±8.211 months vs. 96.131±1.911 months); however, it did not reach statistical significance. (P = 0.139) There was no association of HGF−high expression with recurrence free survival. Conclusions: We demonstrated that c-MET−High was associated with brain invasion of meningiomas and that c-MET expression could be a useful predictive marker for meningioma recurrence. Invasive meningiomas with high expression of c-Met might be good indications for targeted therapy using selective c-Met inhibitors. 535 POSTER (Board P107) Tumor targeting and tissue distribution of solitomab (AMG 110; ® anti-EpCAM BiTE ) in human EpCAM-positive tumor bearing mice F.J. Warnders1 , S. Waaijer2 , M.N. Lub-de Hooge1 , M. Friedrich3 , A.G.T. Terwisscha van Scheltinga1 , P. Deegen3 , S.K. Stienen3 , P.C. Pieslor4 , H.K. Cheung4 , J.G.W. Kosterink1 , E.G.E. de Vries2 . 1 University Medical Center Groningen, Clinical Pharmacy and Pharmacology, Groningen, Netherlands; 2 University Medical Center Groningen, Medical Oncology, Groningen, Netherlands; 3 Amgen Research Munich GmbH, Munich, Germany; 4 Amgen, Thousand Oaks CA, USA Background: Bispecific T-cell Engagers (BiTE® ) belong to a class of single-chain bispecific antibodies with dual target binding specificities. AMG 110 combines in one polypeptide chain the single-chain variable regions directed against the epithelial cell adhesion molecule (EpCAM) and the epsilon chain of the T cell receptor/CD3 complex. EpCAM is abundantly expressed by epithelial tumors and cancer stem cells. By labeling AMG 110 with Zirconium-89 (89 Zr) we aimed to study the tumor targeting and tissue distribution of AMG 110 in human tumor bearing mice. Material and Methods: AMG 110 was conjugated with desferal for 89 Zr labeling and for biological activity evaluation in vitro prior to in vivo use. 89 Zr-AMG 110 was injected into nude mice bearing subcutaneously implanted xenografts with high EpCAM expressing HT-29 human colorectal adenocarcinoma. MicroPET imaging was performed at 0.5, 3, 6, 24, 48 and 72 h after injection. Tissue was collected at 6, 24 and 72 h. To examine the impact of dose on 89 Zr-AMG 110 tumor uptake and biodistribution, 0, 20 and 480 mg of unlabeled AMG 110 were tested in different cohorts of mice in combination with 20 mg of 89 Zr-AMG 110. A non-EpCAM binding BiTE® , Mec14, was evaluated as negative control in HT-29 xenograft imaging and biodistribution studies. Results: In vitro analysis of desferal conjugated-AMG 110 showed biological activities comparable to unmodified AMG 110. MicroPET imaging revealed specific tumor uptake of 20 mg 89 Zr-AMG 110 in HT-29 tumors, maximally at 6 h after tracer injection, and prolonged tumor retention up to 72 h. Biodistribution analyses showed a dose- and time-dependent 89 ZrAMG 110 tumor uptake in HT-29 tumors. The highest tumor uptake was observed in the 40 mg dose group with tumor % ID/g at 6 h, 24 h & 72 h

Poster Session – Molecular Targeted Agents II ranging from 5.4±0.2, 5.3±0.3 to 2.7±0.4, respectively, and tumor-toblood %ID/g ratio escalating from 2±0.2, 33±3 to 58±12, respectively. 89 Zr-Mec14 control BiTE® (40 mg) did not accumulate to appreciable level in HT-29 xenografts (0.7±0.1% ID/g), and unlabeled Mec14 had no impact on HT-29 xenograft accumulation of 89 Zr-AMG 110. Conclusions: This study shows for the first time that PET imaging can be used to investigate 89 Zr-AMG 110 BiTE® antibody tumor targeting and tissue distribution non-invasively in vivo. Our data support using this approach to assess the distribution of a 89 Zr-labeled BiTE® in clinical trials. % ID/gram tissue 89

Zr-AMG 110 (40 mg) iv 6h 24 h 72 h

Tumor 5.4±0.2 Blood 2.8±0.1 Tumor:Blood 2±0.2

89 Zr-Mec 14 (40 mg) iv 24h

5.3±0.3 2.7±0.4 0.7±0.1 0.16±0.01 0.05±0.01 0.10±0.01 33±3 58±12 7.3±1.2

536 POSTER (Board P108) Shifts in microRNA expression pattern can facilitate the cancer cell stemness V. Halytskiy1 . 1 Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine, Molecular Immunology Department, Kiev, Ukraine Background: Tumor growth is tightly associated with regular shifts in microRNA (miRNA) expression pattern. Usually, expression of miRNAs miR-15/16, miR-128, miR-145, miR-150, miR-148/152 and miR-299 is down-regulated in cancer cells whereas expression of miRNAs miR-19, miR-23, miR-27, miR-155 and miR-223 is up-regulated. This investigation aims to identify in what way these shifts in miRNA expression pattern contribute to the cancer cell stemness. Methods: miRNA targets within gene transcripts were predicted in silico using TargetScan software. Results: Nanog gene transcript carries miR-128 and miR-150 binding sites. MiRNA miR-145 can silence Sox2 gene. MiRNA miR-299 can target transcript of gene POU5F1 encoding transcription factor Oct4. Tcf3 gene transcript carries miR-15/16 binding site. MiRNAs miR-148/152, miR-128 and miR-145 can silence Klf4 gene expression. At the same time, transcripts of these genes do not contain any targets for miRNAs overexpressed in cancer cells. Moreover, miRNAs miR-155, miR-19, miR23 and miR-223 can silence IL6ST gene encoding glycoprotein gp130 − subunit of oncostatin M, IL-6 and LIF receptors. miR-155 binding site was found within transcript of OSMR gene encoding another subunit of oncostatin M receptor. MiRNAs miR-27, miR-155 and miR-223 can silence gene encoding STAT1 molecule, a key downstream element in the oncostatin M receptor pathway. TP53 gene transcript contains miR19, miR-27 and miR-223 binding sites. Also, up-regulated miRNAs miR-23 and miR-155 can target transcripts of CDH1 gene encoding E-cadherin and CLDN1 gene encoding claudin 1, respectively. MiRNAs miR-23, miR-155 and miR-181 suppress OCLN gene coding occludin. Conclusions: miRNAs, expression of which is usual for normal differentiated cells, can silence genes encoding the key stemness factors Nanog, Oct4, Sox2, Klf4. Down-regulation of these miRNAs allows reactivation of mentioned stemness genes in transformed cells. Also, hyperexpression of other miRNAs, which are essential for abnormal proliferation and surviving of cancer cells, cancels down-regulation effect of oncostatin M receptor signal as well as of p53 on the stemness factor genes. Under the circumstances, it is possible, that DNA demethylation and chromatin acetylation, which can be also caused by shifts in miRNA expression in cancer cells, can lead to spontaneous reactivation of genes encoding factors Nanog, Oct4, Sox2 and Klf4 as well as other genes responsible for stemness. In addition, up-regulated miRNAs silence genes encoding the epithelial differentiation markers − especially, intercellular junction components. This predetermines the epithelial–mesenchymal transition of cancer cells acquiring stemness.

Poster Session – Molecular Targeted Agents II

Friday 21 November 2014 175

537 POSTER (Board P109) Absolute quantitation of MET using mass spectrometry for clinical application: assay precision, stability, and correlation with MET gene amplification in FFPE tumor tissue

539 POSTER (Board P111) Reversing the epithelial to mesenchymal transition with N-myc downstream regulated gene-1 and novel iron chelators in pancreatic cancer

F. Cecchi1 , W.L. Liao1 , S. Thyparambil1 , K. Bengali1 , J. Uzzell1 , M. Darflar1 , D. Krizman1 , J. Burrows1 , T. Hembrough1 , T. Veenstra1 , D.P. Bottaro2 , T. Karrison3 , L. Henderson4 , P. Xu4 , B. Rambo4 , S.Y. Xiao5 , L. Zhao5 , J. Hart5 , D. Catenacci4 . 1 OncoPlex Diagnostics, OncoPlex Diagnostics, Rockville, USA; 2 NCI, UOB, Bethesda, USA; 3 The University of Chicago Medicine, Department of Health Studies, Chicago, USA; 4 The University of Chicago Medicine, Dept of Medicine, Chicago, USA; 5 The University of Chicago Medicine, Dept of Pathology, Chicago, USA

L. Fouani1 , Z. Kovacevic1 , D. Richardson1 . 1 University of Sydney, Pathology Sydney Medical School, Sydney Nsw, Australia

Background: Overexpression of Met tyrosine kinase receptor is associated with poor prognosis. Overexpression, and particularly MET amplification, are predictive of response to Met-specific therapy in preclinical models. Immunohistochemistry (IHC) of formalin-fixed paraffin-embedded (FFPE) tissues is currently used to select for ‘high Met’ expressing tumors for Met inhibitor trials. IHC suffers from antibody non-specificity, lack of quantitative resolution, and, when quantifying multiple proteins, inefficient use of scarce tissue. Methods: After describing the development of the Liquid-Tissue-Selected Reaction Monitoring-mass spectrometry (LT-SRM-MS) Met assay, we evaluated the expression level of Met in 130 FFPE gastroesophageal cancer (GEC) tissues. We assessed the correlation of SRM Met expression to IHC and mean MET gene copy number (GCN)/nucleus or MET/CEP7 ratio by fluorescence in situ hybridization (FISH). Results: Proteomic mapping of recombinant Met identified 418 TEFTTALQR426 as the optimal SRM peptide. Limits of detection (LOD) and quantitation (LOQ) for this peptide were 150 and 200 amol/mg tumor protein, respectively. The assay demonstrated excellent precision and temporal stability of measurements in serial sections analyzed one year apart. Expression levels of 130 GEC tissues ranged (<150 amol/mg to 4669.5 amol/mg. High correlation was observed between SRM Met expression and both MET GCN and MET/CEP7 ratio as determined by FISH (n = 30; R2 = 0.898). IHC did not correlate well with SRM (n = 44; R2 = 0.537) nor FISH GCN (n = 31; R2 = 0.509). A Met SRM level of 1500 amol/mg was 100% sensitive (95% CI 0.69−1) and 100% specific (95% CI 0.92−1) for MET amplification. Conclusions: The Met SRM assay measured the absolute Met levels in clinical tissues with high precision. Compared to IHC, SRM provided a quantitative and linear measurement of Met expression, reliably distinguishing between non-amplified and amplified MET tumors. These results demonstrate a novel clinical tool for efficient tumor expression profiling, potentially leading to better informed therapeutic decisions for patients with GEC. 538 POSTER (Board P110) Development of a novel anti-tumor antibody targeting CXADR M. Kawada1 , H. Inoue1 , M. Kajikawa2 , M. Sugiura2 , S. Sakamoto1 , S. Urano2 , T. Masuda1 , A. Nomoto1 . 1 Microbial Chemistry Research Foundation, Institute of Microbial Chemistry Numazu, Shizuoka, Japan; 2 Medical & Biological Laboratories Co. Ltd., Nagoya, Japan Cancer chemotherapy using antibodies against cancer-associated molecules is one of promising approaches. In order to develop effective anti-tumor antibodies we have tried to find new molecular targets to raise antibodies. Previously we established highly tumorigenic subline LNCaPCR cells from human prostate cancer LNCaP cells. Using both cell lines we assessed the signal sequence trap by retrovirus-meditated expression (SST-REX) method. SST-REX method could identify membranous and secreted proteins and revealed that LNCaP-CR cells highly expressed coxsackie virus and adenovirus receptor (CXADR) compared to the parental cells. We then constructed monoclonal antibodies against human CXADR and examined their anti-tumor effects. Among them, 6G10A clone antibody did not directly affect the growth of LNCaP-CR cells in vitro, but it significantly inhibited the growth of LNCaP-CR xenograft in vivo. Furthermore, 6G10A antibody also exerted strong anti-tumor activity against LNCaP-CR cells implanted orthotopically in murine prostate. Moreover, 6G10A antibody significantly inhibited the growth of various cancer xenograft models expressing CXADR in vivo such as human prostate DU-145, pancreatic BxPC3, and colorectal DLD-1 cancer cells. The study of its action demonstrated that 6G10A antibody exerted its antitumor activity through both ADCC and CDC activities. Thus, these results show that anti-CXADR 6G10A antibody is a promising candidate for cancer chemotherapy.

Pancreatic cancer is a devastating disease, as it remains one of the most challenging neoplasms to both diagnose and treat. Unfortunately, due to frequent late diagnosis, metastasis is likely to have occurred. By this stage, the treatment options for pancreatic cancer patients are limited and carry a poor success rate. Hence, new and more efficient therapies targeting metastasis are urgently needed. To this end, understanding the metastatic process and the molecules involved is crucial in order to develop more effective therapies for pancreatic cancer. Therefore, the focus of the current study was to examine the molecular mechanisms behind the epithelial to mesenchymal transition (EMT), a process known to facilitate metastasis in pancreatic cancer. The current study aimed to examine the capacity of the metastasis suppressor gene, N-myc downstream regulated gene 1 (NDRG1), to promote an epithelial phenotype by attenuating oncogenic signalling pathways (e.g., transforming growth factor beta (TGF-b) and nuclear factor kappa B (NF-úB)) known to facilitate the EMT. Here, we report that NDRG1 is able to attenuate canonical NF-úB signalling in PANC1 pancreatic cancer cells through i. inhibiting the IúB kinase (Iúú) complex and its activation; ii. preventing the phosphorylation and subsequent degradation of the inhibitory IúBa protein bound to the NF-úB complex, that would otherwise prevent its nuclear translocation and transcriptional activity; and iii. NDRG1 reduces levels and nuclear expression of down-stream molecules of this latter pathway, namely claudin-1 and ZEB1. In metastatic cancer cells, the tight junctional protein claudin-1 shifts from the plasma membrane and is mis-localised in the nucleus where it may facilitate the transcription of the E-cadherin gene repressor, ZEB1. Here we demonstrate, that NDRG1 i. reduces claudin-1 transcription and nuclear localisation, while possibly restoring claudin-1 membrane expression, and ii. promotes an epithelial phenotype via up-regulating E-cadherin and down-regulating vimentin. Importantly, NDRG1 can be up-regulated using a novel class of anti-cancer agents, namely the iron chelators, Dp44mT and DpC. To this end, these agents were shown to reduce activation of the Iúú complex, and thus, attenuate NF-úB signalling. Interestingly, these agents were also found to reduce ZEB1 levels, and can be expected to reduce the repression of E-cadherin and inhibit metastasis. In conclusion, the current study demonstrates the potential molecular mechanisms by which NDRG1 promotes an epithelial phenotype, and thus, inhibits the EMT. Additionally, this study demonstrates the promising application of novel iron chelators as anti-metastatic agents through their molecular targeting of the NF-úB signalling pathway. 540 POSTER (Board P112) Vemurafenib alters glucose utilization in BRAF-driven human melanoma cells M. Falck Miniotis1 , T. Delgado-Goni1 , S. Wantuch1 , P. Workman2 , R. Marais3 , M.O. Leach1 , M. Beloueche-Babari1 . 1 The Institute of Cancer Research, Cancer Research UK Centre for Cancer Imaging, London, United Kingdom; 2 The Institute of Cancer Research, Cancer Research UK Cancer Thrapeutics Unit, London, United Kingdom; 3 Cancer Research UK Manchester Institute, Manchester, United Kingdom Background: Several BRAF/MEK inhibitors have recently been approved for the treatment of BRAF-driven melanoma and many more are undergoing clinical testing. Inhibitors of BRAF and MEK attenuate the glycolic activity of BRAF mutant human melanoma cells but the downstream metabolic consequences of this action are not clear. Here we explore the alterations in glycolytic pathway fluxes induced by the BRAF inhibitor vemurafenib in BRAF mutant human melanoma cells using 13 C-labelled glucose and NMR spectroscopy. Materials and Methods: BRAFV600D WM266.4 human melanoma cells were treated with 0.2, 0.5 and 1mM vemurafenib (i.e. 1×, 2× and 5×GI50 , respectively) for 24 h in standard culture media and levels of extracellular lactate were determined by 1 H NMR spectroscopy. For pathway flux analysis, cells were treated with 2.5×GI50 vemurafenib in media containing 50% (12.5mM) [1-13 C] glucose. Following 24 h incubation, cell extracts were prepared and analysed by 13 C NMR spectroscopy to assess the levels of aqueous 13 C-labelled intermediates formed. 1 H and 13 C NMR data were acquired at 500MHz and metabolite levels normalized to cell number and an internal standard. Inhibitor action was verified by western blotting for P-MEK1/2 and P-ERK1/2 protein levels. Data represent mean±SD.

176 Friday 21 November 2014 Results: As expected, vemurafenib induced a concentration-dependent reduction in P-MEK1/2 and P-ERK1/2 levels, consistent with signaling pathway inhibition. There was a concomitant fall in extracellular lactate (down to 70±11% of controls, p = 0.01) that coincided with reduced hexokinase-II expression, indicative of decreased glycolytic activity. Analysis of 13 C-labelled intracellular intermediates formed from [1-13 C] glucose revealed a reduction in [3-13 C] lactate (to 47±14% of controls, p = 0.002) coupled with a build-up of intracellular [1-13 C] glucose (up to 240±42%, p = 0.004) and [2,3-13 C] glutamate up to 157±26% (p = 0.02-pyruvate carboxylase activity) while [4-13 C] glutamate (pyruvate dehydrogenase activity) remained unchanged (101±20% of controls). These effects are consistent with reduced utilization of glucose as well as its preferential routing to the TCA cycle via pyruvate carboxylase. Conclusions: Our data show that BRAF inhibition with vemurafenib alters glucose utilization in BRAF mutant human melanoma cells in favour of TCA cycle metabolism. Further work is required to establish the significance of this metabolic shift and the potential to exploit it for improving therapeutic activity. 541 POSTER (Board P113) The abnormal bone remodeling associated with prostate cancer bone metastasis is attenuated by TAS-115, the dual inhibitor for HGF/VEGF signaling H. Fujita1 , C. Matsumoto2 , K. Yonekura1 , K. Watanabe2 , M. Hirata2 , C. Miyaura2 , T. Utsugi1 , M. Inada2 . 1 Taiho Pharmaceutical Co. Ltd., Tsukuba Research Center, Ibaraki, Japan; 2 tokyo University of Agriculture and Technology, Department of Biotechnology and Life Science, Tokyo, Japan Background: Prostate cancer frequently metastasizes to the bone, leading to significant bone resorption and abnormal bone formation, resulting in bone pain and decreased bone strength. We recently focused on the roles of the hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) signal transduction pathways in the bone metastasis of prostate cancer cells (PCs). In this study, we investigated the roles of HGF/VEGF signaling using an in vivo model of bone metastasis of prostate cancer. Materials and Methods: PCs were analyzed for their expression profiles of HGF, VEGF, their receptors and other molecules involved in these signaling pathways using RT-PCR, phosphorylation assays and Western blot analysis. A bone metastasis model of prostate cancer was generated using the intratibial (IT) injection of PCs (PC3) into nude mice. To investigate the changes in bone mass, bone mineral density (BMD) was measured by DEXA. A three-dimensional bone imaging analysis, including histomorphometry, was performed by micro-CT. Results: We analyzed the expression profiles of HGF, MET (HGF receptor), VEGF, and VEGF receptor (VEGFR) in osteoblasts, osteoclasts and PC3. All three cell types expressed HGF, VEGF and their respective receptors. In the in vivo IT model implanted PC3, we compared the effects of MET-, VEGFR- and dual MET/VEGFR-inhibitors (crizotinib, sunitinib and TAS115, respectively). IT injection of PC3 caused dramatically increased BMD, with irregularly deformed cancerous bone. Oral administration of these inhibitors suppressed the irregular bone formation. Notably, the suppressive activity of TAS-115 was the most potent among them. Micro-CT analysis showed that PC3 produced severe abnormal bone formation in both cancerous and cortical bone, with resorptive lacunae on the surface of the cortical bone. In the bone marrow space and the cancerous bone, severe bone resorption and subsequent irregular bone formation were shown. This abnormal bone remodeling was attenuated in all treatment groups. Above all, TAS-115 potently inhibited the progression of abnormal bone remodeling. Conclusions: These results indicate that the dual MET/VEGFR inhibitor, TAS-115, suppressed the prostate cancer-induced bone destruction following abnormal bone formation. Dual inhibition of MET/VEGFR signaling is a potential therapeutic approach for metastatic prostate cancer and its skeletal-related clinical events. 542 POSTER (Board P114) Tivantinib in combination with gemcitabine shows strong antitumor activity on mesothelioma cell lines and cytoskeletal effects via inhibition of actin M. Simonelli1 , P. Zucali1 , M. Suter1 , L. Rubino1 , A. Santoro1 , C. Carlo-Stella1 . 1 Istituto Clinico Humanitas − Humanitas Cancer Center, Clinical Oncology, Rozzano, Italy Background: Thoracic Malignant Mesothelioma (MM), a rare malignancy, has a poor prognosis and lacks effective treatments, especially for advanced disease. The MET receptor has a prominent role in MM growth and progression and represents a promising target. Materials and Methods: MM cell lines representative of the main histotypes were used, including H28 (epithelioid), H2452 and MSTO-211H

Poster Session – Molecular Targeted Agents II (biphasic), and H2052 (sarcomatoid) cell lines. All lines express MET. Study drugs were the MET inhibitor tivantinib and the nucleoside analogue gemcitabine. Efficacy of single and combined drugs was assessed by cell viability assay WST-1 and scratch assay. We explored several combination schedules (Table 1). Analysis of tivantinib effects on the cytoskeleton was performed by confocal microscopy. Results: Tivantinib alone was effective on all cell lines (IC50, <1000 nM), while gemcitabine alone was active on 3 lines, with H2452 being resistant. Both drugs exerted their maximum activity after 48 hours. Tivantinib/Gemcitabine combination showed best results at the Sequential G schedule. The combination resulted in additive antitumor effects on H28 and MSTO-211H [viability decreased by 64%, versus 21% (tivantinib alone, p < 0.001), and 43% (gemcitabine alone, p < 0.01)]. In the scratch assay on the H28 line, the sequential G combination was the only treatment that inhibited motility to the point of widening the inter-cellular gap. Exposure to tivantinib resulted in phospho-MET membrane and cytoplasmic reduction, and in actin collapsing by confocal microscopy with clusters instead of single strands being observed. These changes were also mirrored by an altered distribution of phospho-MET on actin with a loss of co-localization after treatment. Conclusions: Tivantinib/Gemcitabine combination resulted in potent antitumor activity against MM cell lines. Moreover, a specific effect of tivantinib on actin with clustering of strands was observed. These data support clinical evaluation of Tivantinib/Gemcitabine combination in MM. Table 1. 24 h Pulsatile T1 Pulsatile G1 Pulsatile T2 Pulsatile G2 Concomitant Sequential T Sequential G

24 h

24 h

24 h

Tivantinib Medium Gemcitabine Medium Gemcitabine Medium Tivantinib Medium Tivantinib Medium Gemcitabine Gemcitabine Medium Tivantinib Gemcitabine+Tivantinib Tivantinib Tivantinib+Gemcitabine Gemcitabine Gemcitabine+Tivantinib

543 POSTER (Board P115) MEK1/2 specific inhibitor, SMK-17 selectively induces apoptosis in b-catenin mutated tumors M. Kiga1 , A. Nakayama2 , Y. Sasazawa2 , Y. Shikata2 , H. Ikeda2 , E. Tashiro2 , M. Imoto2 . 1 Daiichi Sankyo Co. Ltd, Shinagawa R&D Center, Tokyo, Japan; 2 Keio University, Yokohama, Japan Although several MEK1/2 inhibitors have been evaluated in clinical studies, there is probably little chance of clinical study of MEK1/2 inhibitors being succeeded. BRAF mutation has been proposed as a responder marker of MEK1/2 inhibitor in preclinical study. However, moderate sensitivity of MEK1/2 inhibitor by current clinical approaches focused on BRAF mutation has not sufficiently promoted clinical adoption of MEK1/2 inhibitors. Further characterization of MEK inhibitor-sensitive tumor holds great promise for optimizing therapy of these drugs. Here we report that b-catenin mutation accelerates MEK1/2 inhibitor-induced apoptosis. Our selective MEK1/2 inhibitor, SMK-17 induced apoptosis in b-catenin mutated cell lines at the effective concentration. Furthermore, cell lines harboring highly TCF4 transcription activity, which is result from b-catenin mutation, seemed to be sensitive to SMK-17-induced apoptosis. To confirm the possibility of mutation of b-catenin and highly TCF4 transcriptional activity as a prediction marker of MEK inhibitor, we evaluated the effects of dominant negative TCF4 and active mutated b-catenin on MEK inhibitor-induced apoptosis. As a result, dominant negative TCF4 reduced MEK inhibitor-induced apoptosis. In contrast, mutated b-catenin accelerated apoptosis. Our findings provide b-catenin mutation as an important predictive biomarker for treatment of MEK1/2 inhibitor. 544 POSTER (Board P116) Preclinical studies of a dual Bcl-2/Bcl-xL inhibitor APG-1252 with strong anti-tumor efficacy and significantly reduced platelet toxicity H. Wang1 , G. Wang1 , Z. Du1 , M. Wu1 , D. McEachern2 , A. Aguilar2 , Y. Lin3 , X. Lin3 , J. Wen3 , L. Gu3 , M.I.N.G. Guo3 , Y. Zhai1 , S. Wang2 , D. Yang1 . 1 Ascentage Pharma Group, Biological Sciences, Taizhou, China; 2 University of Michigan, Comprehensive Cancer Center and Departments of Internal Medicine Pharmacology and Medicinal Chemistry, Michigan, USA; 3 Ascentage Pharma Group, Pharmaceutical Sciences, Taizhou, China Background: Pro-survival Bcl-2 family proteins play a central role in evasion of apoptosis, tumorigenesis and resistance to chemotherapy,

Poster Session – Molecular Targeted Agents II and represent compelling therapeutic targets. Unfortunately, Bcl-2/Bcl-xL inhibitors cause prominent on-target platelet toxicity due to pharmacologic inhibition of Bcl-xL. We have developed a unique strategy to tactically reduce mechanism-based platelet toxicity with APG-1252, a novel dual Bcl-2/Bcl-xL inhibitor, while maintaining potent in vivo antitumor activity. Methods and Results: APG-1252 demonstrated better in vivo antitumor activity than ABT-263 and achieved complete and long-term tumor regression in both H146 and H1963 SCLC xenograft models with the majority of mice still remaining tumor-free after treatment stopped. APG-1252 at weekly dosing inhibited tumor growth ofH1963 xenografts with complete or partial tumor regression, suggesting the potential of APG-1252 for greater clinical efficacy without triggering dose-limiting thrombocytopenia, namely, broader therapeutic window in clinic. APG-1252 in combination with irinotecan synergistically suppressed tumor growth of SW620 xenografts. In PK/PD study, less than 1% of its active metabolite APG-1252-M1was detected in plasma, whereas high concentration of APG1252-M1 was found in tumor tissues and increased over time, indicating little or no conversion of APG-1252 into APG-1252-M1 in blood, but efficient conversion in tumor tissues. Preclinical studies revealed that APG-1252 was 30-fold less potent in platelet killing thanAPG-1252-M1 in dogs, and was >10 fold less toxic against platelets relative to ABT-263 in mice, rats, dogs and monkeys, suggesting a much superior therapeutic index over ABT-263 for platelet toxicity which limited the clinicalapplication of ABT263. APG-1252 exhibited excellent PK properties with low clearance (0.03– 0.18 L/h/kg) and moderate T1/2 (4.04–7.76 hours) in preclinical species. APG-1252 showed higher plasma exposure than ABT-263 did at respective dose on platelet killing, indicating that APG-1252 might have greater ability than ABT-263 to drive drug concentration into highly efficacious range in clinic. In14-day repeated dose exploratory toxicity studies, APG-1252 demonstrated favorable toxicity profile in rats and dogs with no significant abnormalitiesin clinical observation, gross necropsy, and clinical pathology except for the moderate platelet drop. Conclusions: APG-1252 demonstrates robust anti-tumor activity, excellent PK and PD properties and favorable toxicity profile, particularly the superior therapeutic window for platelet toxicity, which warrants further development of APG-1252 as a promising dual Bcl-2/Bcl-xLinhibitor for the treatment of Bcl-2/Bcl-xL-dependent hematologic cancers and solid tumors. Currently, APG-1252 is being evaluated in IND-enabling studies and phase I trial is expected to start in early 2015. 545 POSTER (Board P117) MiRNAs related to KRAS mutational status in resectable non-small cell lung cancer S. Gallach1 , S. Calabuig-Farinas ˜ 1 , E. Jantus-Lewintre1 , D. Montaner2 , E. Escorihuela1 , J. Castellano1 , S. Figueroa3 , A. Blasco4 , C. Hernando4 , ´ para la Investigacion ´ el Hospital R. Guijarro5 , C. Camps5 . 1 Fundacion General de Valencia, Oncolog´ıa Molecular, Valencia, Spain; 2 Centro de ´ Pr´ıncipe Felipe, Departamento de Bioinformatica, ´ Investigacion Valencia, Spain; 3 Consorcio Hospital General Universitario de Valencia, Servicio 4 ´ de Cirug´ıa Toracica, Valencia, Spain; Consorcio Hospital General ´ Universitario de Valencia, Servicio de Oncolog´ıa Medica, Valencia, Spain; 5 Universitat de Valencia, Departamento de Medicina, Valencia, Spain Background: Non-Small Cell Lung Cancer (NSCLC) harbours different genetic and epigenetic alterations among them, oncogene mutations such as EGFR and KRAS, being mutations of KRAS the most common molecular change. Furthermore, microRNAs regulate post-transcriptional gene expression and several studies have revealed their implication in tumorogenesis. The main goal of this study is to analyse the correlation between deregulated miRNAs identified in resectable NSCLC (obtained in a previous study performed by NGS) and the KRAS mutational status in this cohort. Material and Methods: Fresh tumor and normal adjacent lung tissue samples (n = 163) were obtained by surgical resection from NSCLC patients and storage at −80ºC with RNAlater® until being used. Nucleic acids were isolated from fresh tumor and normal lung samples by standard methods. KRAS mutations were carried out by qPCR (Therascreen). We assessed the miRNA expression levels in NSCLC of deregulated miRNAs (data obtained from a previous study using NGS technologies) in paired tumor/normal samples by RTqPCR using a TaqMan microRNA assay kit. The statistical analyses were performed using non-parametric methods. All statistical analyses were considered significant at p < 0.05. Results: Baseline characteristics of the patients: median age, 65 years [26−82]; 85.3.0% male; 66.3% ECOG-PS 0; 41.1% adenocarcinomas (ADC); 12.3% KRAS mutated (frequencies: 8 12ASP, 6 12CYS, 5 12VAL and 1 12SER). Survival analysis showed that KRAS-mutated patients have a trend to shorter PFS and OS (p = 0.06 and P = 0.07, respectively). Analysis of correlation between KRAS mutational status and miRNAs expression levels showed that miR-133a-3p, miR-135b-5p and miR-21 were significantly associated with KRAS mutations (p = 0.05, p = 0.001 and

Friday 21 November 2014 177 p = 0.049, respectively). Using miRTarBase, an experimentally validated microRNA-target interactions database, we found that miR-133a-3p and miR-21 target some genes downstream KRAS pathway such as PI3KR2, CDC42, RHO and PTEN. However, any target genes related to KRAS pathway were found to miR-135b-5p. Conclusions: We found miR-133a-3p downregulated and mir-21 upregulated in NSCLC, both implicated in KRAS pathway. This finding could explain that tumors with mutated KRAS and deregulation of these miRNAs have a largest cell survival and growth. This work was supported in part, by a grant [RD12/0036/0025] from RTICC, ISCIII, and Beca Roche-Oncohematolog´ıa 2009. 546 POSTER (Board P118) The PI3Kbeta/delta inhibitor AZD8186 combines with the dual mTORC1/2 inhibitor AZD2014 to give comprehensive PI3K pathway inhibition and drive tumour regression in vivo B. Simon1 , U. Hancox1 , U. Polanska1 , L. Hanson1 , P. Dudley1 , R. Ellston1 , J. Maynard1 , M. Kraus2 , J. Curwen1 , T. Klinowska1 , L. Ward1 , F. Cruzalegui1 , S. Symeonides1 , K. Cronin1 . 1 AstraZeneca, Oncology iMED, Macclesfield, United Kingdom; 2 AstraZeneca, Oncology iMED, Boston, USA AZD8186 inhibits the PI3K isoforms PI3Kb and d. In solid tumours when the tumour suppressor PTEN is deleted, mutated or downregulated, PI3Kb becomes a key driver of tumour cell growth. In haematological tumours such as DLBCL where PTEN can also down-regulated, PI3Kd is additionally important in signaling from the B-cell receptor, creating potential for targeted treatment of haematological malignancies. AZD8186 has single agent activity in a range of pre-clinical models representative of different tumour types. However efficacy of agents in the PI3K pathway are anticipated to be limited by compensatory PI3K isoform activation or relief of feedback loops. As a result it is likely that maximal benefit will be seen when combining different agents that target the PI3K pathway. AZD8186 is differentiated from many other agents that target PI3K family members as peripheral glucose control is not affected at efficacious doses. Supporting this in the PTEN null renal model, 786−0, AZD8186 specifically modulated of FDG uptake in tumours, but not normal tissue. This is associated with tumour specific modulation of the Pi3K pathway biomarkers. Therefore in PTEN null tumours AZD8186 can be combined with other PI3K pathway inhibitors to give increased pathway suppression without increasing normal tissue toxicity. To test this further AZD8186 was combined with the dual mTORC1/2 inhibitor AZD2014 in a number of different PTEN null tumour models. This significantly reduces tumour growth, and even achieves tumour regression, associated with increase depth or duration of pathway suppression. Interestingly tumour regressions can be achieved with intermittent of both AZD8186 and AZD2014, implying that acute complete pathway suppression is sufficient to drive the antitumour effect. This data establishes the potential for AZD8186 to be used in combination with mTOR inhibitors with the ability to customise dose and schedule to optimise therapeutic index. Further exploration of the combination opportunities for AZD8186 would inform on the potential for inhibitors of Pi3Kb and d to give benefit in broader tumour types. 547 POSTER (Board P119) Preclinical activity of Debio 1347, an oral selective FGFR1, 2, 3 inhibitor, in models harboring FGFR alterations 3 A. Vaslin Chessex1 , C. Moulon2 , V. Nicolas-Metral ´ 3 , A. Men ´ etrey ´ , H. Maby-El Hajjami1 , S. Rigotti4 , C. Zanna5 , G. Vuagniaux1 . 1 Debiopharm International S.A., Pharmacology & Screening, Lausanne, Switzerland; 2 Debiopharm International S.A., Research & Evaluation, Lausanne, Switzerland; 3 Debiopharm International S.A., Drug Metabolism & Pharmacokinetics, Lausanne, Switzerland; 4 Debiopharm International S.A., Research & Development, Lausanne, Switzerland; 5 Debiopharm International S.A., Medical Affairs, Lausanne, Switzerland

Background: Dysregulation of the fibroblast growth factor receptor (FGFR) signaling pathway due to receptor overexpression, gene amplification, point mutations or fusions/chromosomal translocations is associated with cancer development and progression. The oral selective FGFR1, 2, 3 inhibitor Debio 1347 (CH5183284), is currently investigated in a Phase I trial in selected patients harboring FGFR genetic alterations (NCT01948297). This study evaluated Debio 1347 activity in 2 preclinical models harboring FGFR genetic alterations. Material and Methods: Frequency of administration on the antitumoral activity of Debio 1347 was evaluated in RT112 bladder cancer model bearing the FGFR3-TACC3 fusion. Antitumoral activity was also assessed in a patient derived xenograft (PDX) FGFR amplified squamous lung cancer model.

178 Friday 21 November 2014 Results: Dose dependent antitumoral activity was observed in both animals models. FGFR inhibition resulted in dynamic modulation of circulating FGF23 levels, as well as inhibition of FGFR downstream signaling in tumor tissue. Frequency of administration impacted on efficacy and potential toxicity. The PKPD relationship was explored. Conclusions: Debio 1347 administration resulted in a dose-dependent reduction in tumor growth in both animal models, displaying either a FGFR fusion or amplification. These results suggest that Debio 1347 will provide therapeutic opportunities for patients who have FGFR genetic alterations. 548 POSTER (Board P120) Development of a small molecule activator of protein phosphatase 2A for the treatment of prostate cancer K. McClinch1 , D. Callejas2 , M. Cooper2 , A. Stachnik1 , D. Kastrinsky3 , M. Ohlmeyer3 , M. Galsky1 , G. Narla4 . 1 Mount Sinai School of Medicine, Medicine, New York, USA; 2 Case Western Reserve University, Medicine, New York, USA; 3 Mount Sinai School of Medicine, Structural Biology, New York, USA; 4 Case Western Reserve University, Medicine, Cleveland, USA Background: Several new therapies have recently been approved for patients with castration-resistant prostate cancer (CRPC), however, none are curative and tumors ultimately develop resistance. Successful combating of CRPC requires novel approaches and therapies. While protein kinases have been a major focus of drug development efforts in cancer treatment, protein phosphatases, the key negative regulators of oncogenic signaling pathways, have been largely unexplored. We have recently developed a first-in-class series of molecules, exemplified by TRC-794 and DT-1154, capable of binding, and activating, protein phosphatase 2A (PP2A), a serine/threonine phosphatase. PP2A acts as a tumor suppressor and dephosphorylates, several critical nodes in prostate cancer pathogenesis including the androgen receptor (AR). Decreased PP2A expression and/or activity have been correlated with castrationresistance in cell culture and human prostate cancer studies. Methods: The functional and biological effects of TRC-794 and DT-1154 were explored in a panel of prostate cancer cell lines. Cell viability and survival were measured using MTT and colony formation assays. Flow cytometry was utilized to evaluate the induction of apoptosis, and Western blotting was performed to analyze changes in target protein expression. Global phosphoproteomic profiling was performed using TiO2 enriched chromatography coupled with MS/MS analysis. In vivo activity was assessed in castration-resistant LNCaP/AR xenografts.

Poster Session – New Therapies with Pleiotropic Activity significant activity in vitro and in vivo in prostate cancer models through a unique mechanism of action − AR degradation. Combined these data highlight the potential for PP2A activation for both the treatment of castration-resistant prostate cancer and potentially for diverse PP2A inactivated tumor types and diseases. 549 POSTER (Board P121) A novel cancer marker and potential therapeutic target M.P. Parri1 , S.C. Campagnoli1 , A.G. Grandi1 , A.S. Santi2 , E.D.C. De Camilli3 , G.V. Viale3 , P.C. Chiarugi2 , L.T. Terracciano4 , P.P. Pileri1 , R. Grifantini5 . 1 Externautics, R&D, Siena, Italy; 2 University of Florence, Human Health Medical School, Florence, Italy; 3 European Institute of Oncology, Pathology, Milan, Italy; 4 Basel medical University Hospital, Pathology, Milan, Italy; 5 Externautics, Siena, Italy In our recent research activities, we identified 89 novel candidate markers forprevalent cancers by a systematic Tissue microarray analysis (TMA) of a large collection of polyclonal antibodies (approximately 1600) raised against membrane-associated and secreted human proteins currently marginallycharacterized. Among them, here we report a novel cancerassociated protein, referred as EXN32, so far only marginally characterized. EXN32 is a metallo-protease highly conserved across species, known to be involved in normal ovarian folliculogenesis. An anti-EXN32 monoclonal antibody (mAb) detects the protein in a high percentage of in breast, lung, ovary, and colo-rectal (CRC) cancers (high-to-medium intensity ranging from 30 to 70% in different cancer types). An analysis of high density tissue microarrays (300–700 highly characterized clinical specimens per each cancer) shows that EXN32 is over-expressed in all cancer stages and grades. In breast cancer, a EXN32 it is over-expressed in Her-2+, PR+ and ER+ patients and also in triple negative cases. In colon cancer, EXN32 is over-expressed both in B-RAF and K-RAS mutant and wild type cancers. Preliminary evidence indicates that EXN32 is associated with favorable prognosis in lung cancer. This evidence opens the way to further investigations on the EXN32 role in the marker-associated pathways. Currently, we found that EXN32 over-expression in colon cancer tissues is associated to a low activation of cadherin and beta-catenin pathways. A characterization of the protein biological role showed that alteration of EXN32 expression significantly hampers cell proliferation, migration and invasiveness. Initial studies aimed at identifying the molecular pathways in which the protein is involved show that it plays a role in the response to the reticular stress and hypoxia. Our findings reveal that EXN32 is strongly affected by reticular stress induced by thapsigargin. In addition, forced silencing of EXN32 during reticular stress appears to protect cells from expressing the stress marker Bip/Grp78. In addition, EXN32 is likely involved also in handling of oxidative stress. Indeed, the silencing of EXN32in cells exposed to 1% O2 hypoxia, causing a mitochondrial delivery of oxidants and hence sustaining oxidative stress, leads to inhibition of the Nrf2-mediated anti-oxidant response and to reduction of accumulation of HIF-1, the master transcription factor instructing cells to respond to hypoxic stress. We are investigating the possibility that EXN32 may participate as a molecular starter to the survival response induced by extracellular stresses. Results indicate EXN32 as a potential target for the design of novel drugs, such as aptamers, protease inhibitors and small molecules. Moreover, EXN32 and its specific mAb represent an interesting toolfor the molecular characterization of specific cancer subtypes. Investigations are ongoing to understand the predictive value of EXN32 for state-of art and innovative therapeutic strategies.

Figure: Structure of TRC-794. Results: Treatment of LNCaP, 22RV1, and VCAP cells with TRC-794 and DT-1154 led to a decrease in cell viability and clonogenicity through the induction of caspase-dependent apoptosis. Phosphoproteomic analysis of drug treated cells revealed AR signaling to be one of the top perturbed pathways. Western blot analysis of cells demonstrated dosedependent degradation of the AR resulting in PSA reduction and changes in additional canonical AR targets. Mechanistic studies demonstrated SMAP treatment resulted in increased PP2A recruitment to the AR resulting in decreased AR phosphorylation and increased CHIP E3 ligase binding. DT-1154 demonstrated significant activity in vivo in castrationresistant LNCaP/AR xenografts resulting in either significant tumor growth inhibition or tumor regression. In addition, this small molecule series demonstrated significant single agent activity in a MDV3100 resistant LUCAP in vivo tumor model. The effects on tumor volume correlated strongly with target engagement as evidenced by significant decreases in PSA and AR expression in vivo. Pharmaceutic characterization of this series demonstrates significant tolerability (in mice and rats), and good oral bioavailability and pharmacokinetic (PK) profile. Conclusion: Together, these data demonstrate that our newly discovered first-in-class small molecule activators of PP2A (SMAP) demonstrates

New Therapies with Pleiotropic Activity 550 POSTER (Board P122) Nupharidine inhibits NF-kB activity, induces apoptosis and has synergistic cytotoxic activity with cisplatin and etoposide J. Gopas1 , J. Ozer2 , N. Eisner2 , D. Benharroch3 , A. Golan-Goldhirsh4 . 1 Ben Gurion University, Dept. of Microbiology Immunology and Genetics and Dept. of Oncology Soroka Medical Center, Bear Sheva, Israel; 2 Ben-Gurion University of the Negev, Microbiology Immunology and Genetics, Bear Sheva, Israel; 3 Soroka University Medical Center, Dept. of Pathology, Bear Sheva, Israel; 4 Ben Gurion University, Jacob Blaustein Institute for Desert Research, Bear Sheva, Israel Background: The nuclear factor-kappa B (NF-úB) family of transcription factors plays a pivotal role in inflammation, proliferation, and prevention of apoptosis. Due to its central role in many physiological and pathological processes, including cancer, they constitute attractive targets for therapy.

Poster Session – New Therapies with Pleiotropic Activity Materials and Methods: The use of plant extracts to alleviate inflammatory diseases is centuries old and continues to this day, therefore we screened thirty-four methanolic plant extracts for inhibition of constitutive NF-úB activity by a NF-úB-luciferase reporter gene assay. There was strong inhibition of NF-úB activity by Nuphar lutea L. SM. (Nuphar), leaf and rhizome extracts. An active fraction containing a mixture of dimeric sesquiterpene thioalkaloids was purified by solvent extraction, pH adjustment silica gel chromatography and HPLC. One- and twodimensional NMR spectroscopy indicated the presence of nupharidine, 6-hydroxythionuphlutine as a major component. Results: Nupharidine showed a dose dependent inhibition of NF-úB activity by luciferase reporter gene assay as well as reduction of nuclear NF-úB subunits expression as tested by western blots and immunohistochemistry. Diminution of DNA binding was demonstrated by Electro Mobility Shift Assay (EMSA). Nupharidine inhibited both inducible and constitutive NF-úB activation and affected the canonical and alternative pathways. Suppression of NF-úB was not cell type specific. Conclusions: Induction of apoptosis by Nupharidine was demonstrated by time and dose dependent cleavage of procaspase-9 and PARP. Synergistic cytotoxicity of nupharidine with cisplatin and etoposide was demonstrated, therefore suggesting its possible use as a ‘sensitizer’ in anticancer treatment.

551 POSTER (Board P123) Phase 1B study of CC-486 (oral azacitidine) in tumors associated with a viral etiology D.D. Von Hoff1 , N. Isambert2 , J. Lopez-Martin3 , P.N. Munster4 , D.W. Rasco5 , J.C. Bendell6 , J.H.M. Schellens7 , J. Tomaro8 , R. Sarmiento9 , K. Liu10 , A. Nguyen8 , G.L. Bray8 , S.R. Hatty9 , J.F. DiMartino8 , C. Le Tourneau11 . 1 TGen/Scottsdale Healthcare Research Institute, Translational Research, Scottsdale, USA; 2 Centre Georges-Fran¸cois Leclerc, Medical Oncology, Dijon, France; 3 ‘12 de Octubre’ University Hospital/Research Institute, Medical Oncology/Translational Oncology, Madrid, Spain; 4 UCSF Helen Diller Family Comprehensive Cancer Center, Department of Medicine, San Francisco, USA; 5 South Texas Accelerated Research Therapeutics, Oncology, San Antonio, USA; 6 Sarah Cannon Research Institute, GI Oncology and Drug Development, Nashville, USA; 7 Nederlands Kanker Instituut/Antoni van Leeuwenhoek, Molecular Pathology, Amsterdam, Netherlands; 8 Celgene Corporation, Translational Development, San Francisco, USA; 9 Celgene Corporation, Celgene Institute Translational Research Europe, Seville, Spain; 10 Celgene Corporation, Biostatistics, Berkeley Heights, USA; 11 Institut Curie, Medical Oncology, Paris, France Background: Management of virally-associated tumors (e.g. EBVassociated nasopharyngeal carcinomas [NPC]; HPV-associated cervical carcinoma [CC] and head and neck squamous cell carcinomas [HNSCC]) that recur after treatment for locally-advanced or metastatic disease represents a significant unmet need. Low or undetectable expression of viral proteins by these tumors may allow them to evade recognition and killing by T cells. CC-486 induces DNA hypomethylation and may enhance the immunogenicity of these tumors by upregulating the expression of viral antigens, thereby allowing recognition and elimination by the adaptive immune system. Methods: Part 1 of this study assessed the maximally tolerated dose (MTD) and schedule of CC-486, either alone or in combination with carboplatin or nab-paclitaxel, in several patient cohorts with results reported previously (Mol Cancer Ther 2013; 12: abstracts A120 & B217). In Part 2, eligible subjects were stratified according to specific tumor types, including one cohort of subjects with virally-associated tumors, who were treated with CC-486 alone at 300 mg/day for 14 consecutive days in a 21-day cycle until progression or intolerable toxicity. All subjects were evaluated for safety, pharmacokinetics, and tumor response per RECIST 1.1. Results from subjects with virally-associated tumors across both study parts are reported here. Results: As of 19 May 2014, a total of 19 subjects with virally-associated tumors (7 NPC, 4 CC, 3 Merkel cell tumors, 4 HNSCC, and 1 anal carcinoma) were treated and evaluated. The median number of prior regimens for this cohort was 3 (range 1−6). The median number of CC486 cycles administered was 4 (range 1−16). Treatment was well tolerated. Grade 3−4 adverse events reported in >10% of study subjects included neutropenia (36.8%), anemia (10.5%), hyponatremia (10.5%), and asthenia (10.5%). Four subjects, 3 with NPC and 1 with CC, achieved an objective response. Duration of responses ranged from at least 36 days to 192 days. An additional 2 NPC subjects achieved a best response of stable disease lasting >180 days. The disease control rate among NPC patients was particularly noteworthy with 5/7 subjects achieving a response of SD or better and 3 subjects still receiving CC-486 treatment after 64, 203, and

Friday 21 November 2014 179 240 days. Analysis of blood and NPC tissue samples for methylation and expression status of EBV genes is ongoing. Conclusion: CC-486 merits further clinical evaluation in subjects with NPC who are unresponsive to or progress following conventional, multimodality therapy. 552 POSTER (Board P124) Discovery of an unexpected vulnerability of cancer stem-like cells via a functional dissection of EMT biology Y. Feng1 , E.S. Sokol2 , C.A. Del Vecchio1 , S. Sanduja1 , J.H. Claessen1 , T.A. Proia1 , D.X. Jin2 , F. Reinhardt1 , H.L. Ploegh1 , Q. Wang3 , P.B. Gupta1 . 1 Whitehead Institute for Biomedical Research, cambridge, USA; 2 Massachusetts Institute of Technology, Department of Biology, cambridge, USA; 3 Duke University, Department of Chemistry, Durham, USA Epithelial-to-mesenchymal transition (EMT) induces a number of malignant traits of cancer cells, including invasiveness, drug resistance, and stemlike properties, yet very few vulnerabilities of cells that undergo this cell state switch have been identified. Using selective small molecules as cellular probes, we discovered that induction of EMT profoundly sensitizes cells to chemicals that interfere the homeostasis of endoplasmic reticulum (ER). This sensitivity to ER perturbations is largely due to the substantial increase in synthesis and secretion of extracellular matrix (ECM) proteins. Consistent with their increased secretory output, EMT cells exhibit a branched ER morphology and constitutively activate the PERK-eIF2a axis of the unfolded protein response (UPR). Interestingly, constitutive PERK activation is also required for EMT cells to invade and metastasize. Taken together, our findings identify a novel vulnerability of EMT cells, and demonstrate that the PERK–eIF2a axis of the UPR plays a critical role in their malignancy. 553 POSTER (Board P125) PRPF6, a tri-snRNP spliceosome protein, regulates the alternative splicing of a distinct oncogenic ZAK variant and promotes colon tumor growth R. Firestein1 , A. Adler1 , M. McCleland1 , M. Yaylaoglu1 , Z. Zhang1 , J. Liu2 , Z. Jiang2 . 1 Genentech, Pathology, South San Francisco, USA; 2 Genentech, Bioinformatics, South San Francisco, USA The spliceosome machinery is composed of multi-meric protein complexes that generate a diverse repertoire of mRNA through coordinated splicing of heteronuclear RNAs. While somatic mutations in spliceosome components have been discovered in several cancer types, the molecular bases and consequences of spliceosome aberrations in cancer are poorly understood. Here we report for the first time that PRPF6, a member of the tri-snRNP spliceosome complex, drives cancer proliferation by preferential splicing of genes associated with growth regulation. Inhibition of PRPF6 and other tri-snRNP complex proteins, but not other snRNP spliceosome complexes, selectively abrogated growth in cancer cells with high tri-snRNP levels. High-resolution transcriptome analyses revealed that reduced PRPF6 alters the constitutive and alternative splicing of a discrete number of genes, including an oncogenic isoform of the ZAK kinase. These findings implicate an essential role for PRPF6 in cancer via splicing of distinct growth related gene products. 554 POSTER (Board P126) Dual EZH2 and EHMT2 histone methyltransferase inhibition increases biological efficacy in breast cancer cells R. Brown1 , M. Fuchter2 , E. Curry1 , I. Green1 , S. Kandil1 , F. Cherblanc2 , L. Payne1 , N. Chapman-Rothe1 , E. Shamsaei1 , N. Srimongkolpithak2 , J. Snyder3 , M. Vedadi4 . 1 Imperial College London, Surgery & Cancer, London, United Kingdom; 2 Imperial College London, Chemistry, London, United Kingdom; 3 Emory University, Chemistry, Atlanta, USA; 4 University of Toronto, Structural Genomics Consortium, Atlanta, USA Repressive chromatin histone marks, such as H3K27 and H3K9 methylation maintained by histone methyltransferases (HKMT) EZH2 and EHMT2 respectively, are associated with transcriptional silencing. Although selective HKMT inhibitors reduce levels of individual repressive marks, removal of H3K27me3 by specific EZH2 inhibitors, for instance, may not be sufficient for inducing expression of genes with multiple repressive marks. Using dual siRNA knockdown of EZH2 and EHMT2 or combination of EZH2 inhibitor, GSK343, and EHMT2 inhibitor, UNC0638, we show that induction of gene expression and inhibition of triple negative breast cancer cell growth (MDA-MB-231) are markedly increased when both inhibitors are given in combination, rather than independently. Indeed, the expression

180 Friday 21 November 2014 of certain genes is only induced upon dual inhibition. We therefore sought to identify compounds which showed evidence of dual EZH2 and EHMT2 inhibition. Using a cell-based assay and based on the substratecompetitive EHMT2 inhibitor BIX01294, we have identified proof-of-concept compounds that induce re-expression of a subset of genes consistent with dual HKMT inhibition. Chromatin immunoprecipitation verified a decrease in silencing marks (H3K27me3, H3K9me3) and an increase in permissive marks (H3K4me2/3, H3K27ac) at the promoter of re-expressed genes. Biochemically, the compounds are substrate competitive inhibitors against both EZH2 and EHMT1/2. In conclusion, we have demonstrated that dual inhibition of EZH2 and EHMT2 is more effective at eliciting biological responses of gene transcription and growth inhibition compared to inhibition of single HKMTs, and have shown the potential to identify dual EZH2/EHMT2 inhibitors that are functional in cells. 555 POSTER (Board P127) CB-5083, a first in class inhibitor of the AAA-ATPase p97/VCP, induces irresolvable ER stress that results in antitumor activity in solid and hematological tumor models F. Yakes1 , R. Le Moigne2 , S. Wong2 , D.J. Anderson3 , S. Djakovic3 , E. Valle1 , M.K. Menon1 , J. Rice3 , B. Yao4 , F. Soriano5 , J. Wang4 , S. Kiss von Soly3 , M. Chesi6 , P.L. Bergsagel7 , H.J. Zhou4 , M. Rolfe8 , D. Wustrow4 . 1 Cleave Biosciences, Translational Biology, Burlingame CA, USA; 2 Cleave Biosciences, Pharmcology, Burlingame CA, USA; 3 Cleave Biosciences, Biology, Burlingame CA, USA; 4 Cleave Biosciences, Chemistry, Burlingame CA, USA; 5 Cleave Biosciences, Pharmacology, Burlingame CA, USA; 6 Comprehensive Cancer Center Mayo Clinic, Molecular Biology, Scottsdale AZ, USA; 7 Comprehensive Cancer Center Mayo Clinic, Hematology, Scottsdale AZ, USA; 8 Cleave Biosciences, Discovery, Burlingame CA, USA Background: The AAA-ATPase p97/VCP utilizes the energy derived from ATP hydrolysis to structurally remodel or unfold client proteins. P97 plays a central role in protein homeostasis by extracting misfolded proteins from the ER and other organelles and chaperoning them through the ubiquitin– proteasome system (UPS) for degradation. In cancer cells, the UPS maintains protein homeostasis promoting cell survival. Pharmacological abrogation of p97 function that leads to irresolvable ER stress, disruption of protein homeostasis, and cancer cell death may be of clinical benefit. In this report, we demonstrate that CB-5083 is a potent inhibitor of p97 that elicits a durable and irresolvable ER stress response leading to tumor growth inhibition in solid tumor and hematological models. Methods: Cancer cell lines were treated with CB-5083 and tested in proliferation and apoptosis assays. Western blotting, immunofluorescence and ELISA assays were utilized to monitor pharmacodynamic responses. In vivo, animals were administered vehicle or CB-5083 (30–100 mg/kg) as a single dose or on a weekly qd4/3 off schedule. Results: CB-5083 is a potent and selective inhibitor of p97 (IC50 17 nM). In cancer cell lines, CB-5083-mediated inhibition of p97 resulted in polyubiquitin accumulation (PUA), blockade of ER-associated degradation, induction of an unfolded protein response, decreased cell viability, and apoptosis. In vivo, oral administration of CB-5083 to tumor-bearing mice resulted in PUA in both tumor and tissues including peripheral blood mononuclear cells. Concurrently, CB-5083 promoted an unfolded protein stress response and induction of apoptosis markers that was both time and concentration dependent. In vivo, solid and hematological tumors treated with CB-5083 showed significant tumor growth inhibition with body weight loss of 10%. Further, CB-5083 in combination with multiple myeloma standard of care agents resulted in tumor regression when compared to single agent treatment. Additionally, translational assays applicable to monitoring p97 target engagement and induction of irresolvable ER stress have been developed in support of CB-5083 clinical trials. Conclusions: These findings demonstrate that CB-5083 is a potent inhibitor of p97 eliciting an irresolvable ER stress response that translates to tumor growth inhibition. Further, these results validate targeting of p97 and protein homeostasis in the treatment of cancer, and support clinical evaluation of CB-5083. 556 POSTER (Board P128) Antagonizing microRNA mediated epigenetic reprogramming as therapeutic strategy for aggressive prostate cancer C. Dallavalle1 , D. Albino1 , G. Civenni1 , P. Ostano2 , M. Mello-Grand2 , R. Garcia-Escudero1 , G. Chiorino2 , C.V. Catapano1 , G.M. Carbone1 . 1 Institute of Oncology Research (IOR), Tumor Biology and Experimental Therapeutics, Bellinzona, Switzerland; 2 Fondazione Edo ed Elvo Tempia Valenta, Laboratory of Cancer Genomics, Biella, Italy MicroRNAs (miRNAs) play important roles in cell differentiation and transformation controlling post-transcriptional processing of mRNAs and

Poster Session – New Therapies with Pleiotropic Activity attenuating production of the encoded proteins. Oncogenic miRNAs can lead to epigenetic reprogramming of the cancer cell transcriptome and are attractive therapeutic targets. We have performed miRNA microarray profiling in a cohort of normal prostate (n = 21) and primary tumors (n = 56). We found that miR-424 was upregulated in tumors compared to normal prostate and was significantly higher in a subgroup of tumors characterized by low expression of the ETS gene ESE3/EHF and enriched of aggressive molecular and biological features, including expression of epithelial-to-mesenchymal transition (EMT) and cancer stem cell (CSC) genes. Consistently, miR-424 was elevated in prostate epithelial cells with stable knockdown of ESE3/EHF (ESE3-kd cells) that acquire similar EMT and CSC features. To understand the functional role of miR-424 in prostate tumors we performed functional studies in multiple prostate cell models. We found that miR-424 possessed oncogenic properties and miR-424 over-expression in immortalized RWPE-1 prostate epithelial cells and LNCaP prostate cancer cells promoted anchorage-independent growth, cell migration and CSC properties. The oncogenic effects of miR-424 were effectively counteracted by ablating the miRNA function with a locked nucleic acid (LNA) antagomiR. The miR-424 antagomir reduced anchorage-independent growth and cell migration and significantly affected the CSC compartment reducing the CD44+/CD24− cell fraction and prostatosphere formation in transformed ESE3-kd RWPE1 and LHS prostate epithelial cells and in metastatic DU145 cells with high level of miR-424. Furthermore, inhibition of miR-424 strongly reduced in vivo tumor formation of DU145 cells in nude mice. Growth of the limited ensuing tumor xenografts from antagomiR treated cells was significantly reduced compared to control tumors. Importantly, cells harvested from antagomiR treated tumors had reduced ability to form prostatospheres ex vivo, indicating that inhibition of miR-424 induced persistent depletion of the CSC compartment. Thus, our results demonstrate that miR-424 has an important role in sustaining transformed phenotype and stemness in prostate cancer. Targeting miR-424 may represent an effective therapeutic strategy to revert the malignant phenotype and self renewal in aggressive prostate cancer. 557 POSTER (Board P129) A rational approach for discovery of inhibitors of YAP−TEAD interaction L. Chene1 , A. Soude2 , C. Valaire3 , S. Delaporte3 , S. Jacquet2 , Y. Cambet2 , I. Braccini2 , M. Barth2 , C. Montalbetti4 , P. Broqua5 , C. Fromond6 . 1 Inventiva, Head of Target Validation, Dijon, France; 2 Inventiva, Scientist, Dijon, France; 3 Inventiva, Lab technician, Dijon, France; 4 Inventiva, Head of Chemistry Dept, Dijon, France; 5 Inventiva, CSO, Dijon, France; 6 Inventiva, Head of Biology & Pharmacology Dept, Dijon, France The Hippo signalling pathway plays a major role in organ growth regulation and tumorigenesis. By sensing contact inhibition, the central kinase network of the hippo pathway controls nuclear translocation and activity of the YAP transcription factor. Once translocated in the nucleus, YAP interacts with the TEAD family of transcription factors and drives expression of gene involved in cell survival and proliferation. Deregulation of the hippo pathway frequently occurs in cancers and high nuclear expression of YAP has been documented in many tumors including lung, colorectal, ovarian and skin cancers. These data suggest that targeting the hippo pathway is a new therapeutic option for the treatment of a large number of cancers. Several approaches are currently proposed to target the hippo pathway such as inhibition of kinases and GPCRs that are upstream regulators of YAP nuclear translocation. However, because YAP and TEAD are the downstream effectors of the Hippo pathway, we believe that targeting YAP–TEAD interaction is the most promising approach to selectively counteract abnormal activity of the Hippo pathway. We have started a drug discovery program aimed at identifying potent inhibitors of YAP–TEAD interaction, based on a combined FBLD/HTS strategy. Druggability of the TEAD protein was assessed by a fragment screen using NMR and SPR technologies. This approach, conforted by assigned HSQC protein NMR information, provided fragment hits which are used as starting points for the identification of more potent binders. We have also designed a dedicated AlphaScreen assay to validate the ability of our compounds to disrupt the YAP–TEAD interaction. Fifty thousand compounds of the Inventiva’s library were screened using the AlphaScreen. Several hits in the micromolar range were identified and further confirmed using SPR. Hits are currently tested for their ability to disrupt YAP–TEAD interaction in acellular context with a transactivation assay and for their ability to block tumoral cell proliferation.

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558 POSTER (Board P130) RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer

560 POSTER (Board P132) ZEN3365 is a novel BET bromodomain inhibitor for the treatment of hematologic malignancies and solid tumors

H. Wendel1 , K. Singh1 , A. Wolfe1 , Y. Zhong1 , P. Drewe1 , J. Porco2 , 1 1 J. Pelletier3 , G. Ratsch ¨ . Memorial Sloan-Kettering Cancer Center, Cancer Biology & Genetics, New York NY, USA; 2 Boston University, Pharmacology, Boston, USA; 3 McGill, Biochemistry, Montreal, Canada

K.G. McLure1 , R. Jahagirdar2 , O.A. Kharenko1 , E.M. Gesner1 , E. Campeau1 , D. Gilham1 , J. Wu1 , L. Tsujikawa1 , S. Attwell1 , C. Calosing1 , N. Sharma2 , J. Tobin2 , H.C. Hansen3 . 1 Zenith Epigenetics Corp, Translational Biology, Calgary Alberta, Canada; 2 Zenith Epigenetics Corp, Pharmacology, Calgary Alberta, Canada; 3 Zenith Epigenetics Corp, Medicinal Chemistry, Calgary Alberta, Canada

Background: The translational control of oncoprotein expression is implicated in many cancers. This may provide an opportunity to block their production. However, at this time the mechanisms and translation regulatory elements (TREs) that control the translation of subsets of oncogenic mRNAs are largely unknown. Material and Methods: We use transcriptome-scale ribsome footprinting to define eIF4A dependent transcripts and their hallmark features. We further use preclinical cancer models to explore the therapeutic activity and safety of eIF4A inhibitors (Silvestrol and the synthetic CR31B analogue). Results: We report an eIF4A/DDX2 RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of Silvestrol and related compounds. For example, eIF4A promotes leukemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with Silvestrol has powerful therapeutic effects in vitro and in vivo. We use transcriptomescale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5 UTR sequences such as 12-mer guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and Silvestrol-sensitive transcripts are a number of oncogenes, super-enhancer associated transcription factors, and epigenetic regulators. In fact, we find c-MYC amongst the most eIF4A dependent mRNAs whose expression is effectively blocked by eIF4A inhibitors. Accordingly, therapeutic eIF4A inhibition has striking single agent activity against xenograft models of leukemia and lymphoma at dose levels that can be safely administered in vivo. Conclusion: The 5 UTRs of selected cancer genes harbour a targetable requirement for the eIF4A RNA helicase. This defines a gene-selective mechanism of translational control encoded by a new translation regulatory element (TRE). EIF4A-driven oncogene translation is suitable for therapeutic intervention and in vivo studies demonstrate a therapeutic index. 559 POSTER (Board P131) Development of a unique biologic for treating cysteine-dependent malignancies E. Stone1 , S. Cramer2 , A. Saha3 , S. Tiziani4 , J. Digiovanni3 , G. Georgiou2 . 1 University of Texas at Austin, Molecular BioSciences, Austin TX, USA; 2 University of Texas at Austin, Chemical Engineering, Austin TX, USA; 3 University of Texas at Austin, College of Pharmacy, Austin TX, USA; 4 University of Texas at Austin, College of Natural Sciences, Austin TX, USA Major limitations in treating prostate cancer (PCa) are resistance to androgen ablation therapy and chemotherapy associated toxicities that severely compromise quality of life. The finding that PCa (and other cancer types) display an excessive requirement of the amino acid L-cysteine relative to non-malignant tissues has led to the development of a completely novel therapeutic approach to treat PCa (and other indications) that exploits this critical vulnerability by using an engineered human enzyme to deprive tumor cells of L-cysteine (L-Cys). Most if not all normal tissue can make L-Cys from the essential amino acid L-Methionine, thus only tumors are highly impacted when L-Cys is unavailable. Our team at the University of Texas capitalized on the idea that enzymemediated systemic depletion of the serum L-Cys/CSSC pool would constitute a powerful and completely novel therapeutic approach both as a cancer monotherapy and as a mechanistically synergistic agent with existing chemotherapeutic modalities. This concept was borne out by strong preliminary data demonstrating that bi-weekly administration of an engineered human enzyme that depletes L-Cys (and L-cystine (CSSC), the oxidized form of L-Cys) in allograft and xenograft mouse models of PCa: i. results in near complete depletion of the serum L-Cys/CSSC pool; ii. mediates sustained and complete cessation of tumor growth with cancer cells experiencing cell cycle arrest accompanied by increased oxidative stress and autophagy and iii. importantly treatment for over a month is well tolerated with no weight loss or gross toxicities. We discuss the results of numerous IND-enabling studies in bringing this therapeutic into the clinic.

ZEN3365 is an orally bioavailable small molecule discovered and developed from a BET bromodomain inhibitor platform. In vitro, ZEN3365 binds BRD4(BD1) vs non-BET bromodomains with >20-fold selectivity, and binds both the first and second bromodomains of BRD2, BRD3, BRD4 and BRDT pan-selectively in a biochemical AlphaScreenassay, competing for binding to acetylated histone peptides with IC50 values of 8−36 nM. ZEN3365 selectively displaces the BRD4 protein from MYC and BCL-2 promoters and from super-enhancers in cells, resulting in inhibition of MYC and BCL-2 expression in acute myeloid leukemia (AML) and B-cell lymphoma cell lines with sub-uM IC50 values. In hematologic tumor types, including most lymphomas and many leukemias, ZEN3365 inhibits proliferation (IC50: 0.1−0.5 uM) and induces cell cycle arrest and apoptosis, consistent with inhibiting MYC and BCL-2 expression. In AML xenograft tumor models, ZEN3365, administered orally, dose-dependently inhibits MYC and BCL-2 expression and can cause complete tumor regression with no regrowth for 6 months post cessation of dosing. ZEN3365 has also demonstrated strong activity against many solid tumor cell lines with sub-uM IC50 values, including breast, prostate, head and neck, and colorectal cell lines. Solid tumor xenograft studies conducted with ZEN3365 have demonstrated that it is efficacious at well-tolerated doses. Robust PK/PD relationships have been established across a number of in vitro, in vivo and ex vivo systems for ZEN3365 and will be explored further in the clinic. Promising target validation data, excellent pharmaceutical properties, clean off target selectivity profile, and robust activity of ZEN3365 across a variety of hematologic malignancy and solid tumor settings support the clinical development of ZEN3365 in both of these therapeutic indications. 561 POSTER (Board P133) Nonclinical characterization of the first in class investigational ubiquitin activating enzyme inhibitor MLN7243 in cellular and in vivo models of cancer in support of a phase I study M. Milhollen1 , M. Hyer1 , J. Ciavarri1 , T. Traore1 , D. Sappal1 , J. Huck1 , J. Shi1 , J. Duffy1 , J. Gavin1 , J. Brownell1 , Y. Yang1 , B. Stringer1 , Y. Ishii1 , E. Koenig1 , A. Lublinsky1 , R. Griffin1 , C. Xia1 , J. Powe1 , P. Fleming1 , N. Bence1 . 1 Takeda Pharmaceuticals International Co., Oncology, Cambridge Massachusetts, USA The Ubiquitin Proteasome System (UPS) is critical for maintaining cellular homeostasis and regulates an array of cancer relevant processes including cell cycle progression, protein homeostasis and the DNA damage response. Proteasome inhibitors (e.g. bortezomib) are approved for the treatment of human malignancies and additional targets within this pathway are being explored as novel cancer therapies. Here we report the identification of MLN7243, a small molecule inhibitor of the primary E1 activating enzyme for the ubiquitin conjugation cascade, the Ubiquitin Activating Enzyme (UBA1 or UAE). MLN7243 is a mechanism based inhibitor that in vitro achieves nanomolar UAE inhibition through the formation of a MLN7243-ubiquitin adduct. MLN7243 is selective against other Ubiquitin-like (Ubl) protein activating enzymes including the NEDD8, SUMO, ISG15 and autophagy Ubl activating enzymes. In vitro, MLN7243 induces a depletion of cellular mono and polyubiquitinated proteins resulting in a G2/M cell cycle arrest. In addition, MLN7243 induces endoplasmic reticulum stress and an unfolded protein response (e.g. BIP induction) and disrupts the DNA damage response by impairing 53BP1 foci formation and the monoubiquitination of PCNA and FANCD2. Consistent with these findings MLN7243 induces apoptosis and impairs viability in a large panel of cancer cell lines. To support further evaluation of MLN7243, we developed immunohistochemistry (IHC) based pharmacodynamic biomarker assays that measure UAE target engagement (MLN7243-ubiquitin adduct formation) and pathway inhibition via the loss of monoubiquitinated histone H2B (UbH2B) and cellular polyubiquitnated proteins (polyUb). In tumor xenografts in vivo, MLN7243 treatment induces a dose dependent accumulation of the MLN7243-ubiquitin adduct, loss of ubiquitinated proteins (polyUb and Ub-H2B) and an induction of apoptotic cell death as assessed by cleaved caspase-3 IHC. Consistent with these findings, MLN7243 administration on a twice weekly schedule leads to broad anti-tumor activity in a wide range

182 Friday 21 November 2014 of cell-line derived and primary human xenograft tumor models grown in mice. Supported by these studies, MLN7243 is currently being evaluated in a first in human phase I clinical trial in patients with advanced solid tumors. ClinicalTrials.gov identifier: NCT02045095. 562 POSTER (Board P134) Lysophosphatidic acid-induced breast cancer metastasis depends on LPA1/ZEB1/miR-21-activation pathway D. Sahay1 , R. Leblanc1 , J. Ribeiro1 , P. Clezardin1 , O. Peyruchaud1 . 1 INSERM UMR1033, Functional Genomics and Treatments of Bone Metastase, Lyon Cedex O8, France Lysophosphatidic acid (LPA) is a natural bioactive lipid that promotes metastasis of many types of cancer cells. We have shown that blocking LPA receptor type 1 (LPA1) activity inhibits early stage of bone metastasis by inhibiting motility and invasion of breast cancer cells. However, the signaling pathways and gene activation involved in this process have not been well characterized. Micro-RNAs (miR) are well known master regulators of gene expression. Based on the complete miR expression profile in human MDAMB-231 breast cells stimulated by LPA, we found that miR-21 was one of the highest up-regulated miR. Using the Taqman RT-QPCR system, we found that LPA induced miR-21 expression in MDA-MB-231 cells and in their highly osteotropic sub-clone MDA-B02 cells. MiR-21 is well known to act as an oncomiR promoting metastasis in multiple cancers. Also, it is known that miR-21 expression is controlled by several transcription factors. The full transcriptomic analysis of our breast cancer cell lines showed that among those transcription factors, ZEB1, STAT3 and cFos were up regulated by LPA. Interestingly, silencing ZEB1 expression in these cells using synthetic ZEB1-siRNAs abolished LPA-induced miR-21 expression whereas silencing STAT3 or cFos had no effect. Hence, silencing ZEB1 up-regulated the expression of miR-21 target genes PDCD4, PTEN and SPRY2 in MDA-MB-231 cells stimulated by LPA. RT-QPCR analyses also showed that LPA1 was the most abundant LPA receptor in MDA-MB-231 cells (LPA1>LPA2>>LPA6=LPA7>LPA5) whereas LPA3 and LPA4 were not detectable. We found that the treatment of our breast cancer cells with Ki16425, a LPA1/LPA3 antagonist, inhibited LPA-induced ZEB1 and miR-21 expression. Silencing LPA1 expression in these cells using synthetic LPA1siRNAs also abolished LPA-induced ZEB1 and miR-21 expression. MirVana miR-21 inhibitor, silencing LPA1 or ZEB1 totally blocked in vitro LPAinduced cell migration and invasion, and in vivo tumor bone colonization. In all cases the in vitro and in vivo breast cancer functions were rescued with miR-21 mimic. All together our results demonstrate that miR-21 controls the pro-metastatic activity of LPA involving LPA1-dependent activation of ZEB1 in breast cancer cells. 563 POSTER (Board P135) Hsp90 pharmacoproteomics: Harnessing pleiotropy for therapeutic synergy R. Goldstein1 , G. Chiosis2 , L.C. Cerchietti3 , A.M. Melnick3 . 1 Weill Cornell Medical College, New-York NY, USA; 2 Memorial Sloan Kettering Cancer Center, Molecular Pharmacology and Chemistry, New-York NY, USA; 3 Weill Cornell Medical College, Hematology and Oncology Division, New-York NY, USA Background: Tumor cells are enriched for Hsp90 complexes that preferentially chaperone oncoproteins. Because many Hsp90-substrate proteins are degraded upon exposure to Hsp90 inhibitors, these drugs are considered pleiotropic. We hypothesize that targeting Hsp90 in combination with its disease-relevant substrate proteins may create more precise and effective therapeutic regimens. PU-H71 (PU) is a non-toxic Hsp90 inhibitor in clinical trials for breast cancer, leukemia and lymphoma. As a chemical tool, PU immobilized on a solid support can be used to specifically identify active Hsp90 substrate oncoproteins. We demonstrate here that PU affinity capture can be used as a pharmacoproteomic method to discover lymphoma type-specific druggable molecular targets for combination therapies. Methods: PU chemical precipitation (CP): To identify the diffuse large B-cell lymphoma (DLBCL) HSP90-oncoproteome, we performed PU CP followed by mass spectrometry (MS) in 2 DLBCL cell lines. Candidate druggable oncoproteins were validated by immunoblotting. Anti-lymphoma effect: DLBCL cell lines were treated with 6 doses of each drug or combination. Cell viability was measured by a fluorometric resazurin reduction or ATP luminescent method and normalized to vehicle treated controls. Dose-effect curves and combination index values were calculated with the method of Chou–Talalay. In vivo: NOD-SCID mice were injected subcutaneously with ABC DLBCL cell lines. When tumors were palpable, mice were treated with vehicle, ibrutinib (12.5 mg/kg/d ad libitum), PU (75 mg/kg/d i.p.) or the combination for 10−12 days.

Poster Session – New Therapies with Pleiotropic Activity Results: PU CP found 844 Hsp90 substrate proteins with 52% overlap between the two cell lines. We performed pathway analysis to identify druggable signaling pathways critical for DLBCL survival and found B cell receptor (BCR) signaling highly enriched. Mutations in BCR pathway proteins in the aggressive, NFúB-driven ABC subtype of DLBCL result in its chronic activation. Here we show that BCR pathway stability and function requires Hsp90 activity. To test this pathway for therapeutic synergy, we combined PU with BCR pathway inhibitors − ibrutinib (BTK), R406 (SYK), BKM120 (PI3K), MI-2 (MALT1) and sotrastaurin (PKCb). Targeting Hsp90 in combination with BCR pathway inhibitors maximally suppressed BCR signaling, resulting in synergistic growth inhibition (GI) of ABC DLBCL cell lines. The PU-ibrutinib combination was most highly synergistic, and showed greater GI than either drug alone in 2 xenograft models (p < 0.01) and additive and synergistic GI in ex vivo ABC DLBCLs. Conclusions: We demonstrate that Hsp90 pharmacoproteomics can be used to discover molecular targets for combinatorial therapies and are expanding this method to additional cancer types. We have identified novel, clinically relevant combinatorial treatment regimens for DLBCL as well as a novel role of Hsp90 in regulating the BCR pathway. 564 POSTER (Board P136) OTX015, a novel BET-BRD inhibitor is active in non-small-cell lung cancer cell (NSCLC) lines harboring different oncogenic mutations M. Riveiro1 , L. Astorgues-Xerri1 , N. Ijaz1 , M. Bekradda1 , R. Vazquez2 , R. Frapolli2 , A. Rinaldi3 , I. Kwee3 , E. Cvitkovic4 , E. Raymond5 . 1 Oncology Therapeutic Development, Clichy, France; 2 Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; 3 IOR Institute of Oncology Research, Lymphoma and Genomics Research Program, Bellinzona, Switzerland; 4 Oncoethix SA, Lausanne, Switzerland; 5 CHUV, Medical Oncology Department, Lausanne, Switzerland Background: The human BET bromodomain protein family is a druggable target through inhibitors that show potent antiproliferative effects in several tumors including NSCLC. We report preclinical findings of the BETBRD inhibitor OTX015 in NSCLC cell lines harboring different oncogenic mutations (KRAS, MYC amplifications, fusion protein EML4−ALK) as a single agent or combined with targeted therapies. Material and Methods: OTX015 growth inhibition 50% (GI50) values were determined with the MTT assay after 72 h exposure in 5 established NSCLC cell lines (H2228, H3122, A549, HOP62, HOP92). Protein levels were analyzed by Western Blot with commercial antibodies; RT-PCR was done with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System at baseline, 4 and 24 h post-treatment. For cell cycle analysis cells were stained with propidium iodide and DNA content analyzed with a FACScan flow cytometer. OTX015 was combined with the targeted agents everolimus and crizotinib and the combination index (CI) determined by the Chou–Talalay method. Results: OTX015 had antiproliferative effects in 4 of 5 NSCLC cell lines (two harboring the fusion protein EML4−ALK+) with GI50 values from 110 to 940 nM. In A549 cells, concurrent mutations in KRAS and LKB1 genes abrogated OTX015 effects, as described for JQ1, an OTX015 analog, while HOP92 and HOP62 are LKB1 wild-type. Percent cells in the S phase were reduced by 40%, 42% and 52% in HOP92, H2228 and H3122, respectively after 24 h exposure at 500 nM. OTX015 combined with the ALK+-inhibitor crizotinib had additive effects (CI = 0.98) after 48 h concomitant exposure in H2228 cells and additive/synergistic effects with everolimus in HOP62, HOP92, A549 and H2228. OTX015-sensitive and -resistant lines had similar basal mRNA and protein expression of BRD2/3/4, HEXIM1, BCL-2 and P21. CMYC and NMYC were overexpressed in HOP92 and H3122, respectively. Following 24 h OTX015 (500 nM), CMYC protein and mRNA were downregulated in HOP92 cells and NMYC downregulated in H3122 and HOP62 cells, while HEXIM mRNA was upregulated in OTX015sensitive cell lines. Conclusion: NSCLC cell lines, including those harboring the EML4-ALK fusion gene or KRAS mutation, are sensitive to OTX015 BET inhibition, downregulating MYC family members and upregulating HEXIM, suggesting the therapeutic potential of OTX015 in NSCLC. In vivo studies of single agent OTX015 and combined with everolimus in HOP92 xenografts will be presented.

Poster Session – New Therapies with Pleiotropic Activity 565 POSTER (Board P137) Preclinical evaluation of OTX015, a novel BET-BRD inhibitor, on small cell lung cancer (SCLC) cell lines N. Ijaz1 , L. Astorgues-Xerri1 , E. Odore2 , M. Bekradda1 , E. Cvitkovic3 , K. Noel3 , E. Raymond4 , M. Riveiro1 . 1 Oncology Therapeutic Development, Clichy, France; 2 Institute Rene´ Huguenin Hospital, Pharmacology Department, Saint Cloud, France; 3 Oncoethix SA, Lausanne, Switzerland; 4 CHUV, Medical Oncology Department, Lausanne, Switzerland Background: Standard combination chemotherapy for advanced SCLC has not changed substantially in the last two decades. The challenge when identifying novel SCLC therapies is the prevalence of multiple inactivating mutations in tumor suppressor genes (TP53, PTEN, RB1) and gain-of-function oncogenic mutations (PI3K3CA, MYC family). BET bromodomain inhibitors such as OTX015 (OncoEthix SA) which binds to human bromodomain proteins BRD2/3/4, modulate the transcription of target genes via epigenetic mechanisms. OTX015 shows preclinical activity in hematologic and solid tumors and is currently in clinical development. Here we characterized the effects of OTX015 on a series of SCLC cell lines. Material and Methods: OTX015 growth inhibition 50% (GI50) values were determined with the MTT assay after 72 h exposure in four established SCLC cell lines (H82, H69, DMS79, DMS114). Protein levels were analyzed by Western Blot using commercial antibodies and RT-PCR was performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System at baseline, then 4 and 24 h post-treatment. For cell cycle analysis, cells were stained with propidium iodide and analyzed for DNA content with a FACScan flow cytometer. Results: DMS114 cells showed dose-dependent sensitivity to OTX015 [GI50 = 120 (84–172) nM], while H82, H69, DMS79 cells were resistant [GI50 >6 mM], although they overexpressed CMYC and/or NMYC proteins. While all four SCLC cell lines are KRAS wild-type and TP53 mutated, OTX015-resistant SCLC cell lines harbor a homozygous mutation in RB1, while DMS144 cells express RB1 wild-type. All cell lines exhibited similar basal mRNA levels of BRD2/3/4, CMYC, NMYC, HEXIM, BCL-2 and p21. Following OTX015 treatment at 500 nM, CMYC and NMYC mRNA levels were unchanged, while mRNA levels of HEXIM and genes coding for histones (HIST1H2BK, HIST2H2BJ) were upregulated in all four cell lines. In DMS114 cells, OTX015 caused cell cycle arrest in G1 in a timedependent manner, which may be explained by an upregulation at the protein level of the cell cycle inhibitor p27. Conclusion: Our findings suggest that the MYC family does not mediate OTX015 antitumor effects in SCLC cell lines. The presence of functional RB1 protein, controlling cell progression at G1, may explain the cytostatic effects of OTX015. Further studies investigating the therapeutic potential of OTX015 in SCLC are ongoing. 566 POSTER (Board P138) GNS396 and analogues are potent new small molecules to target and kill chemotherapy-resistant subpopulation cells in acute myeloid leukemia F. Bassissi1 , R. Castellano2 , E. Josselin2 , C. Motersino2 , L. Pouyet2 , A. Goubard2 , A. Rostouin2 , G. Nicolas1 , S. Brun1 , J. Courcambeck1 , 3 ´ , N. Vey3 , A. Beret ´ 1 , P. Halfon1 , Y. Collette2 . C. Dubray1 , T. Prebet 1 Genoscience Pharma, Preclinical Development, Marseille Cedex 06, France; 2 Cancer Research Center of Marseille (CRCM), Platform TrGET preclinal assay, Marseille Cedex 09, France; 3 Paoli-Calmettes, Hematology, Marseille Cedex 09, France Despite efforts to understanding and treat acute myeloid leukemia (AML), there remains a need to more comprehensive therapies to prevent AML relapses and to reach sustainable clinical responses. To explain this phenomenon, the cancer stem cells/Leukemia initiating cells (CSCs/LICs) hypothesis suggests that tumors contain a small number of tumor-initiating cells, self-renewing cancer stem cells. Unlike most cells within the tumor, CSCs/LICs are resistant to chemotherapy, and after treatment, they can regenerate all the cell type in the tumor through their stem cell-like behavior. For this reason, innovative drugs which could tackle CSCs/LICs improve cancer treatment of patients. We screened a library of lysosomtropic compounds and identified new drugs GNS396 and analogues as compound that kills AML cells and LSCs in a panel of leukemia cell lines and primary tumor. The effect of GNS396 and analogues on LICs population was measured using ALDH activity marker, as evaluated by their AldefluorTM dehydrogenase activity by flow cytometry. A synergy study combining the cytarabine with the GNS 396 was performed on MOLM-14 14 cell Line. In vivo tumor growth was evaluated in an orthotopic NSG-mice AML model of transplanted human MOLM-14 cell line displaying Cytarabine resistance (unpublished data).

Friday 21 November 2014 183 GNS396 demonstrates autophagy inhibition and apoptosis induction activities which are probably related to lysosome disruption. GNS 396 shows potent anti-proliferation activity when assayed against the NCI60 panel of human tumor cell lines, notably against AML cell lines (Mean GI50 6mM), as well as an original dose–response cytotoxic activity against AML cell lines subpopulation displaying high ALDH activity. Furthermore GNS396 shows a synergic effect in combination with Cytarabine on MOLM-14 cell viability. In vivo GNS396 that is well tolerated by mice induces significant reduction of leukemia growth by about 60% in MOLM14 xenografted mice. Our results provide a rationale for testing lysosome disruption as a novel therapeutic strategy for AML. GNS396 and analogues offer great promise for AML treatment and prevention of relapse in AML patients in particular in combination with chemotherapy. By simultaneously targeting the tumorigenic (by GNS396) and non-tumorigenic populations (by both GNS396 and Cytarabine), both leukemia cell heterogeneity and plasticity could be overcome. This compound is selected as a drug candidate for future investigation in AML clinical trial. 567 POSTER (Board P139) OTX015, a BET-bromodomain (BET-BRD) inhibitor, potentiates the in vitro effects of chemotherapy drugs and targeted agents in human leukemic cell lines L. Astorgues-Xerri1 , C. Canet-Jourdan1 , M. Bekradda1 , E. Cvitkovic2 , P. Herait2 , E. Raymond3 , M. Riveiro1 . 1 Oncology Therapeutic Development, Clichy, France; 2 Oncoethix SA, Lausanne, Switzerland; 3 CHUV, Medical Oncology Department, Lausanne, Switzerland Background: The human BET-bromodomains family is a target for gene transcription inhibitors. OTX015 (OncoEthix SA, Switzerland) is a new selective oral inhibitor of BET proteins in clinical development in hematological malignancies. We report the results obtained with OTX015 in combination with a broad spectrum of standard anti-leukemic drugs using concomitant and sequential schemes. Material and Methods: Eight human cell lines from acute (HL-60, U-937) and chronic (K-562, NALM-1) acute myeloid leukemia, and acute lymphoblastic leukemia (Jurkat, CCRF-CEM, MOLT-3/-4) were treated with increasing OTX015 doses and MTT assays were performed after 72 hours. Protein was analyzed by Western blot and qRT-PCR was performed with Fast SYBR Green on a StepOnePlus Real-Time PCR System. Simultaneous and sequential schedules of OTX015 combined with demethylating agents (azacitidine, decitabine), a broad-spectrum HDAC inhibitor (panobinostat), mTOR inhibitor (everolimus), glucocorticoids (dexamethasone), and conventional cytotoxic drugs (daunorubicin, cytarabine and methotrexate) were evaluated. Combination Index (CI) was determined using Chou & Talalay analysis; CI < 1 reflects synergy, CI = 1 additivity and CI > 1 reflects antagonism. Results: Antiproliferative effects of single-agent OTX015 gave GI50s between 230 and 384nM in HL60, U937 and Jurkat, and 6,000nM for other cell lines. Combination studies were performed in 3 OTX015-sensitive cell lines (HL60, U937, Jurkat) and one resistant (K-562). 48 h-concomitant combination of OTX015 with azacytine (CI = 0.6; 0.6−0.7), decitabine (CI = 0.6; 0.3−0.9), cytarabine (CI = 0.8; 0.4−0.9), daunorubicin (CI = 0.8; 0.4−0.9) and methotrexate (CI = 0.2; 0.1−0.3) was synergistic in all 4 cell lines. Dexamethasone, everolimus and panobinostat were synergytic with OTX015 in HL60, K562 and Jurkat cell lines. Sequential combination of azacytidine or panobinostat followed by OTX015 for 48−48 h, 72−24 h or 72−48 h displayed stronger synergy than concomitant treatment, with lowest CI values after 72−24 h combination in the 4 cell lines. In HL60 cells, 24 h-combination of OTX015 (500nM) with azacytidine (3mM) or panobinostat (20nM) increased caspase-3 and PARP cleavage, whereas in K562 cells the combination increased P21 expression and enhanced C-MYC downregulation. Conclusion: Our findings support that OTX015 in combination with demethylating or HDACi agents potentiates cell cycle arrest or apoptosis induction in leukemic cell lines.

184 Friday 21 November 2014 568 POSTER (Board P140) The BET bromodomain inhibitor OTX015 shows synergy with several anticancer agents in preclinical models of mantle cell lymphoma (MCL) and multiple myeloma (MM) E. Bernasconi1 , C. Tarantelli1 , E. Gaudio1 , I. Kwee1 , A. Stathis2 , E. Riveiro3 , P. Herait4 , E. Cvitkovic4 , E. Zucca2 , F. Bertoni1 . 1 Institute of Oncology Research (IOR), Lymphoma & Genomics Research Program, Bellinzona, Switzerland; 2 Oncology Institute of Southern Switzerland (IOSI), Bellinzona, Switzerland; 3 Oncology Therapeutic Development, Clichy, France; 4 Oncoethix, Lausanne, Switzerland Background: Single agent OTX015 has shown activity in several preclinical models of lymphoid tumors, including MCL and MM (AACR 2014), and notably in the ongoing phase I study (AACR 2014). We evaluated the synergy of OTX015 administered in combination with other anticancer compounds in several MCL and MM cell lines. Material and Methods: Two mantle cell lymphoma (REC1, MAVER1) and three multiple myeloma (RPMI8226, U266B1, KMS11) cell lines were exposed to increasing doses of OTX015 alone or in combination with increasing doses of other anticancer drugs. MTT assays were performed after 72 hours exposure. Synergy was assessed by Chou– Talalay combination index (CI) with the Synergy R package: CI < 0.3, strong synergy; 0.3−0.9, synergy; 0.9−1.1, additive effects. Changes in CCND1 protein and mRNA expression after OTX015 exposure were assessed in 4 MCL cell lines (REC1, MAVER1, Granta519, JeKo1). Results: Additive effects were seen in 2/2 MCL cell lines when OTX015 was combined with the demethylating agent 5-AZA (CI = 0.8 and 1), the HDAC-inhibitor vorinostat (CI = 0.8 and 1), and the proteasome inhibitor carfilzomib (CI = 0.8 and 0.9). Whereas, synergy was observed with the BTK-inhibitor ibrutinib in 2/2 MCL cells (CI = 0.7 and 0.4), however in ibrutinib-resistant MAVER1 cells, synergy was only seen with high ibrutinib doses (5−10 mM). Synergy was observed in 1/2 MCL cell lines with OTX015 combined with the dual PI3K/mTOR inhibitor BEZ235 (CI = 0.1), the mTOR inhibitor everolimus (CI = 0.5), the glucocorticoid dexamethasone (CI = 0.1), and the immunomodulator pomalidomide (CI = 0.2). CCND1 mRNA and protein levels were not altered in 4/4 MCL cell lines after OTX015 (500 nM) treatment for 4 h and 24 h, suggesting that the antiproliferative activity of OTX015 is not CCND1mediated. Synergy was observed in 3/3 MM cell lines following OTX015 combined with carfilzomib (CI = 0.5, 0.7 and 0.9), or vorinostat (CI = 0.7, 0.6 and 0.7), in 2/3 cell lines with pomalidomide (KMS11, CI = 0.3; RPMI8226, CI = 0.4;) or 5-AZA (KMS11, CI = 0.4; RPMI8226, CI = 0.4) and in 1/3 with dexamethasone (U266B1, CI = 0.4). Conclusions: Preclinical experiments support the exploration of OTX015 combined with other anticancer agents in the clinical setting. Additional studies should be performed to clarify the mechanistic or molecular expression profile associated with sensitivity/resistance to individual combinations. 569 POSTER (Board P141) Biological consequences of selective inhibition of the first BET bromodomain J.G. Seitzberg1 , M.H. Hansen1 , T.T.A. Kronborg1 , C.R. Underwood1 , V. Polyak1 , G. Friberg1 , B. Tonnesen1 , M. Nørregaard-Madsen1 , L. Teuber1 , T. Franch1 , S. Nielsen1 . 1 Nuevolution A/S, Copenhagen, Denmark Introduction: The bromodomain (BD) acetyl-lysine ‘readers’ is an emerging target class with particular interest centered around the BET family proteins BRD2, 3, and 4. Target validation of BET proteins using genetic tools and/or small molecule BD inhibitors have established preclinical proof-of-concept in several cancers, and a number of BET inhibitors are currently in early clinical trials within oncology. BET proteins use tandem BDs (BD1 and BD2), to mediate binding to acetylated lysines within partner proteins. However, the contribution of individual BDs to biological activity is currently unclear, and domain-specific inhibition (intra- and/or inter-BET) of BET proteins remains largely unexplored. Here, using our recently developed BD1-selective small molecules, we characterize the biological consequences of BD1-selective inhibition. Methods: Chemetics DNA-encoding small molecule libraries were used to identify small molecule BET inhibitors. Binding of hit compounds to BDs was investigated using AlphaScreen and FRET assays. Selectivity for individual BDs was determined using DiscoveRx Bromoscan technology. Compounds were then assayed for AML and lymphoma cell growth inhibition using MTS and CellTiter-Glo assays, and for effect on release of a panel of 12 cytokines and chemokines from human PBMCs following LPS stimulation. Results: To discover novel inhibitors of BET BDs, we screened 300 million small molecule compounds for binding towards BD1 of human BRD4.

Poster Session – New Therapies with Pleiotropic Activity Representative hits were resynthesized and confirmed binding to BRD4 in the high nM range. Chemical optimization of one series yielded compounds with low to sub-nM affinity against BRD4 BD1. Interestingly, the compounds display marked intra-BET BD selectivity, with a strong preference (20−>100 fold) for binding to BD1 over BD2. In cellular growth inhibition assays, the compounds show activity against AML, BL and DLBCL-derived cell lines. LPS-induced release of cytokines and chemokines from human PBMCs is strongly inhibited by both the pan-BET inhibitors I-BET762 and our BD1-selective compounds, with qualitatively similar profiles of inhibition. In contrast, the BD2-selective compound RVX-208 show limited activity in the cell models tested here. Conclusions: Using DNA-encoding technology, we discovered potent small molecule compounds that selectively inhibit the first BD of BET proteins. The compounds strongly inhibit BET-dependent cellular processes. Furthermore, our data suggest that a number of BET biological activities are mediated primarily by BD1, and that BD1 blockade is sufficient to elicit BET inhibition. Comparative gene expression profiling using compounds with different BD selectivity profile is on-going and will be presented. 570 POSTER (Board P142) Modulation of chromatin-related processes in DNA damage response as a potential strategy to treat acute myeloid leukemia K. Chomej1 , M. Lagergren Lindberg1 , D. Zong2 , T. Juntti1 , L. Kanter1 , L. Stenke1 , R. Lewensohn1 , K. Viktorsson1 , P. Ha˚ ag ˚ 1 . 1 Karolinska Institutet, Department of Oncology/Pathology, Stockholm, Sweden; 2 National Cancer Institute National Institute of Health, Laboratory of Genome Integrity, Bethesda MD, USA Background: Despite treatment with high-dose chemotherapy, most acute myeloid leukemia (AML) patients fail to reach long-term complete remission (CR). Thus there is urgent need for new treatment solutions. Based on our previous observations that phenothiazines (phenos) interfere with DNA repair signaling and chemosensitize solid tumor cells, we analyzed if phenos also can sensitize AML cells to conventional and targeted therapies. Methods: Gene expression analysis study was performed in diagnostic samples from 42 AML patients with CR longer or shorter than 6 months. In silico analyses were performed with Connectivity Map (cmap) on public domain gene expression data from different tumor cells treated with phenos. For combination treatments, phenos were added 2 h prior to chemotherapy. Cell viability was measured with MTT after 72 h treatment and apoptosis with FACS using antibodies recognizing active Bax and caspase-3. DNA damage signaling was analysed using western blotting. Results: Gene expression analysis of AML patient cohorts revealed altered expression of histone deacetylase (HDAC), histone acetyltransferase 1 (HAT1) and chromodomain helicase DNA binding protein 3 (CHD3). Cmap analyses showed similarities of pheno-responsive signature with histone deacetylase inhibitors (HDACi) trichostatin A and vorinostat suggesting phenos may regulate chromatin-related processes involved in DNA damage response. Accordingly phenos were found to inhibit AML cell growth either alone or in combination with daunorubicin or gemtuzumab ozogamicin (GO). Interestingly, AML cells that were refractory to GO were re-sensitized when treated in combination with phenos, showing reduced viability and increased Bax- and caspase-3 activation. Moreover, phenos were found to cause hyperphosphorylation of DNA-PK, ATM and their downstream chromatin-associated substrates when used in combination with daunorubicin or GO. Further analyses of chromatin remodeling compounds, alone and in combination with daunorubicin and GO demonstrated that the class III HDACi Sirtinol, the broadspectrum HDACi Panobinostat and Trichostatin A (TSA), and the DNA methyltransferse inhibitor 5-aza-2 -deoxycytidine all reduced viability in AML cells. Conclusions: As epigenetic control of chromatin relaxation is altered in AML, combination treatment with phenos may offer new therapeutic strategies enabling a novel personalized therapy approach of this hematological malignancy.

Poster Session – New Therapies with Pleiotropic Activity 571 POSTER (Board P143) Novel potent inhibitors of the Histone Demethylase KDM1A: Synthesis, pharmacological evaluation and in vivo activity M. Varasi1 , O. Botrugno1 , A. Cappa1 , R. Dal Zuffo1 , P. Dessanti1 , A. Mai2 , A. Mattevi3 , C. Mercurio1 , G. Meroni1 , S. Minucci1 , F. Thaler1 , P. Trifiro’1 , S. Valente2 , P. Vianello1 , M. Villa1 . 1 European Institute of Oncology, Dpt of Experimental Oncology, Milano, Italy; 2 University “La Sapienza” Rome, Drug Chemistry and Technologies, Roma, Italy; 3 University of Pavia, Biology and Biotechnology, Pavia, Italy KDM1A, a FAD dependent histone demethylase with high homology to amino-oxidases, is responsible of the demethylation of mono and dimethyl lysine 4 on histone H3. KDM1A is part of various transcriptional corepressor complexes and interacts with the co-repressor complex CoREST and histone deacetylases (HDAC) 1 and 2. KDM1A is an essential gene with important roles in different biological relevant processes, including hematopoietic maturation. Its activity has also been demonstrated on non histone substrates such as p53, DNMT1 and MYPT1. High expression and correlation with poor prognosis has been reported for KDM1A in several cancer types, such as neuroblastoma, prostate cancer and non small cell lung cancer. Moreover the high expression of KDM1A and its inverse correlation in hematological malignancies, mostly in acute myeloid leukemia, was observed. Furthermore the enzyme has been demonstrated to sustain the in vivo leukemiogenic potential of MLLAF9 expressing leukemia stem cells. In the light of these findings, KDM1A has been increasingly recognized as an attractive therapeutic target in oncology. We have developed a novel series of potent and irreversible KDM1A inhibitors based on tranylcypromine. The compounds were obtained by a versatile and scalable synthetic process which allows a stereoselective synthesis of the trans enantiomers of the cyclopropane ring. Biochemical and biological characterization of these inhibitors, including their transcriptional effect on KDM1A targets and efficacy in reducing colony forming ability in leukemia cells, will be reported, together with ADME and pharmacokinetic properties of selected compounds. Finally, the best inhibitors of the series induced after oral administration a significant survival increase and provided evidences of target modulation in an in vivo murine promyelocytic leukemia model. 572 POSTER (Board P144) Oral panobinostat in patients with advanced tumors and impaired renal function: Relationship between pharmacokinetics and key safety parameters M. Porro1 , S. Sharma2 , P.O. Witteveen3 , M.P. Lolkema3 , D. Hess4 , H. Gelderblom5 , S.A. Hussain6 , E. Waldron1 , S. Valera7 , S. Mu7 . 1 Novartis, Basel, Switzerland; 2 University of Utah, Huntsman Cancer Institute, Salt Lake City, USA; 3 University Medical Center, Department of Medical Oncology, Utrecht, Netherlands; 4 Kantonsspital St. Gallen, ¨ Klinik fur ¨ Onkologie und Hamatologie, St. Gallen, Switzerland; 5 Leiden University Medical Center, Department of Clinical Oncology, Leiden, Netherlands; 6 University of Liverpool, The Clatterbridge Cancer Centre NHS, Liverpool, United Kingdom; 7 Novartis, East Hanover, USA Background: Panobinostat is a potent pan-deacetylase inhibitor (HDACi) that is active in hematologic and non-hematologic malignancies. Panobinostat has been associated with certain toxicities typical of HDACi’s, namely thrombocytopenia and cardiac QT interval prolongation. This study assessed the effect of renal dysfunction on key pharmacokinetic parameters and these two safety parameters. Methods: Patients with advanced cancer, Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0 to 2, and varying degrees of renal function (estimated creatinine clearance as per FDA guidance) were given a single oral dose of 30 mg panobinostat for safety and pharmacokinetic (PK) measurements. Serial plasma samples and urine samples were obtained to evaluate PK parameters using non-compartmental analysis. The maximum QT change and platelet count change from baseline for each patient in all renal function groups was recorded. Analysis of the relationship between peak plasma concentration (Cmax) and area under the curve (AUC) versus thrombocytopenia or QT prolongation was carried out for each renal function group. Results: Thirty seven patients were enrolled median age was 64 (range, 40−81) years. Eleven patients had normal renal function, 10, 10, and 6 patients had mild, moderate, and severe renal impairment, respectively. Geometric means of Cmax (%CV) were 31.0 (117), 18.2 (68.6), 29.6 (92.5) and 14.0 (82.2) ng/mL, for the normal, mild, moderate and severe renal function groups, respectively. Geometric means of AUC in the normal, mild, moderate, and severe groups were 224.5, 144.3, 223.1, and 131.7 ng*h/mL, respectively. No patients recorded a maximum QTcF value

Friday 21 November 2014 185 of >480 msec or a maximum QTcF increase from baseline of >60 msec in any renal function status group. The number of patients with increases in QT interval of >30 msec were 3 (50%), 1 (16.7%), 1 (16.7%) and 0 in the normal, mild, moderate and severe renal impairment groups, respectively. The number of patients with grade 3/4 thrombocytopenia were 0, 3 (30%), 3 (30%) and 1 (16.7%) in the normal, mild, moderate and severe renal function group. Conclusions: No significant QT prolongation was observed in the presence of any renal function status. No apparent relationship was seen between grade >3 thrombocytopenia and panobinostat exposures across patients with various degree of renal impairment. In this study it appeared that panobinostat toxicity was an intrinsic effect and not related to exposure levels, in patients with normal or impaired renal function, within the clinically applicable dose range. 573 POSTER (Board P145) Suppression of c-Myc oncogene and hematological tumor growth by a novel benzazepine BET inhibitor ´ an ´ 1 , R. Neuhaus1 , K.A. Gelato1 , P. Lejeune1 , A.E. Fernandez-Montalv N. Schmees1 , S. Siegel1 , H. Weinmann1 , V. Gekeler1 , B. Haendler1 . 1 Bayer HealthCare, Global Drug Discovery, Berlin, Germany Background: BRD4 is a protein that belongs to the bromodomain and extra-terminal domain (BET) protein family and binds to acetylated histone tails via its two bromodomains. It forms a complex with the positive transcription elongation factor b (P-TEFb) which controls phosphorylation of RNA polymerase II, ultimately leading to stimulation of transcription elongation in a subset of target genes involved in cell proliferation and playing an essential role in different pathologies including cancer. Material: Several small molecule antagonists of BET bromodomains have been identified, including the benzodiazepines JQ1 and I-BET762 which inhibit the binding of BRD4 to acetylated histone tails, thus suppressing transcription of target genes. Several chemical variations were introduced into the benzodiazepine core to generate novel structures with improved properties. Here we present a novel benzazepine, BAY6356, with an overall favorable pharmacological profile and good DMPK properties. Methods and Results: BAY6356 was first characterized in biochemical (TR-FRET) and cellular (target gene expression, ChIP, and proliferation) assays. We observed that BRD4 inhibition by BAY6356 resulted in a strong reduction of c-Myc expression in acute myeloid leukemia (AML) and multiple myeloma (MM) cell lines, which correlated with a loss of BRD4 binding to promoter and enhancer regions of the c-Myc gene. BRD4 binding was also reduced at additional target genes such as cyclin D1 and c-Fos. Correspondingly, BAY6356 strongly inhibited the proliferation of AML and MM cell lines with IC50 values of approximately 100 nM. The efficacy of BAY6356 was further evaluated in in vivo xenograft tumor models. MOLM13 (AML) and MOLP-8 (MM) tumor cells were implanted into SCID mice. Daily oral treatment at the maximal tolerated dose (MTD) of 30 mg/kg led to a strong tumor reduction in MOLM-13 (17% T/C on day 13 post tumor implantation) and in MOLP-8 (4% T/C on day 19 post tumor implantation) xenografts (T/C 40% = active, T/C 10% = highly active, according to NCI criteria). Further, in the MOLP-8 model, intermittent dosing every 2 or 3 days while keeping the same weekly total dose proved to be as active as daily dosing, when tested in dose response up to 250 mg/kg/week (MTD), demonstrating exposure-driven efficacy. Conclusion: Altogether, the promising pharmacological profile of BAY6356 supports its evaluation as a candidate for preclinical development. 574 POSTER (Board P146) M-COPA, a Golgi disruptor, inhibits cell surface expression of MET protein and exhibits antitumor activity against MET-addicted cancers Y. Ohashi1 , M. Okamura1 , A. Hirosawa1 , A. Akatsuka1 , I. Shiina2 , K. Yoshimatsu3 , T. Yamori1 , S. Dan1 . 1 Japanese Foundation for Cancer Research, Division of Molecular Pharmacology, Tokyo, Japan; 2 Tokyo University of Science, Graduate School of Science, Tokyo, Japan; 3 Eisai Co. Ltd, Eisai Product Creation Systems, Tokyo, Japan Background and Purpose: The Golgi apparatus plays an essential role in the transport, processing and sorting of numerous proteins including receptor tyrosine kinases (RTKs) expressed on the cell surface. We previously demonstrated that M-COPA (2-methylcoprophilinamide, also called ‘AMF-26’), disrupted the structure of Golgi apparatus via inhibiting the activation of Arf-1, which plays an essential role in the formation of COPI-mediated transport vesicle between endoplasmic reticulum (ER) and Golgi, and finally suppressed tumor growth. In this study, to evaluate the antitumor activity of M-COPA against RTK-addicted cancers, we exploited human gastric cancer cell lines with or without MET amplification. Material and Methods: Drug sensitivities in vitro were measured by sulforhodamine B assays after 48 h exposure of M-COPA. Cell surface

186 Friday 21 November 2014

Poster Session – New Therapies with Pleiotropic Activity

expression of MET was analyzed by flowcytometry. Processing of MET protein and phosphorylation status of signaling molecules including Akt and S6 ribosomal protein were examined by immunoblot analysis. The in vivo efficacy of M-COPA (50 mg/kg, p.o., qd×5) was examined using nude mice bearing xenografted tumors derived from human gastric cancer MKN-45 cells. Results: As expected, MKN-45, a MET-addicted cell line, exhibited a better drug response than those without MET amplification did. Upon treatment with M-COPA, MET expression on the cell surface was dramatically downregulated in a dose response manner. Concurrently, the amount of c-MET precursor form was accumulated; meanwhile cleaved mature form was reduced. Moreover, M-COPA reduced phosphorylated forms of MET and its downstream molecules such as Akt and S6. Finally, we confirmed in vivo antitumor activity of M-COPA against MKN-45 xenografts. Conclusions: M-COPA could be a promising therapy for treating METaddicted tumors by inhibiting the processing and the transport of MET protein onto the cell surface. 575 POSTER (Board P147) SF3B1 mutations are associated with alternative splicing in ER-positive breast cancer S. Maguire1 , A. Leonidou1 , P. Wai1 , C. Marchio2 , C.K.Y. Ng3 , B. Weigelt3 , A. Sapino2 , A. Vincent-Salomon4 , J.S. Reis-Filho3 , R. Natrajan1 . 1 The Institute of Cancer Research, Breast Cancer, London, United Kingdom; 2 University of Turin, Department of Medical Sciences, Turin, Italy; 3 Memorial Sloan-Kettering Cancer Center, Department of Pathology, New York, USA; 4 Institut Curie, Department of Tumour Biology, Paris, France Background: Mutations in genes encoding proteins involved in RNAsplicing such as SF3B1 have been found to occur at relatively high frequencies in a number of haematological malignancies and solid tumours. We investigated whether dysfunction in RNA splicing would be implicated in the pathogenesis of breast cancer. Materials and Methods: We performed a re-analysis of published DNA and RNA massively parallel sequencing data. In addition, we performed massively parallel targeted sequencing of genes involved in RNA splicing in a cohort of special histological subtypes of breast cancer (n = 19), and RNA sequencing in a subset of these (n = 14) of tumours in order to identify differential splicing events. Cell lines with and without mutations in SF3B1 were screened using siRNA against a panel of identified differentially spliced genes, and dose–response assays to the small molecule inhibitor Spliceostatin A. Results: Mutations in spliceosomal component genes occurred in 5.6% of unselected breast cancers, including SF3B1 hotspot mutations in 1.8% of unselected breast cancers. SF3B1 mutations were significantly associated with oestrogen receptor-positive (ER) disease, AKT1 mutations and distinct patterns of copy number alterations. Additional targeted profiling identified 16% (3/19) and 6% (1/17) of papillary and mucinous carcinomas of the breast respectively to harbour the K700E mutation. RNA sequencing identified differentially spliced events expressed in SF3B1 mutant tumours including TMEM14C, RPL31, DYNL1, ICA1, OBSL1 and RPL24 and CRNDE. Furthermore, SF3B1 mutant cell lines were selectively sensitive to Spliceostatin A, a selective inhibitor of the SF3b complex, which upon treatment significantly inhibited the alternative splicing signature. Conclusions: Our findings indicate that SF3B1 mutations result in alternative splicing events, and may constitute drivers of a subset of ERpositive breast cancers. Treatment with Spliceostatin A may offer new therapeutic opportunities for tumours with SF3B1 mutations. 576 Novel inhibitors of peritoneal seeding

POSTER (Board P148)

Y. Shen1 , X.L. Li1 , X. Lu1 , S. Kuwada1 . 1 University of Hawaii, Medicine, Honolulu, USA Many cancer cells are resistant to TRAIL or FASL yet paradoxically express their cognate death receptors (FAS, DR4, DR5). Resistance to death receptor signaling is often mediated by over expression of proteins that inhibit death receptor-induced apoptosis. Aims: To identify signaling pathways that inhibit death receptor-mediated apoptosis in cancer cells. Methods: TRAIL- and FASL-insensitive HT-29 colon and SU.86.86 pancreatic cancer cells were were stably transfected with Firefly luciferase to allow their detection. The cancer cells were suspended and allowed to adhere to adherent mesothelial cells that express FASL and TRAIL in the presence of absence of 80 compounds, and, FASL +/− TRAIL blocking antibodies. The percentages of surviving cancer cells were detected by luciferase activity. Athymic female adult mice were pre-treated by intraperitoneal (IP) injection of 2.5 mg/kg of lead active compounds

identified in the co-culture assay or vehicle 3 hours before receiving 106 cancer cells IP. The peritoneal implants were quantified by bioluminescence 2−3 weeks later. Results: 2−5mM BAY 11–7085 showed the greatest activity of the compounds and reduced HT-29 and SU.86.86 cell survival by >90% at 4 hrs in co-cultures with mesothelial cells. This decrease in cell survival was due to increased caspase activity and was almost completely reversed by preincubation of mesothelial cells with TRAIL or FASL blocking antibodies, indicating that BAY 11–7085 restored death receptor-mediated apoptosis. While HT-29 cells were resistant to TRAIL concentrations up to 1 mg/ml, pre incubation of HT-29 cells with 2.5mM BAY 11–7085 for 15 minutes lowered the EC50 for TRAIL-induced apoptosis to 50 ng/ml. Kinome analysis of HT-29 cells treated for 15 minutes with 1−2.5mM BAY 11–7085 revealed strong and concentration dependent activation of MEKK4 and JNK, and, inhibition of c-FLIP protein expression. Pretreatment of HT-29 and SU.86.86 cells with soluble JNK inhibitors (CC-401, SP600125) or transfection with a DN-JNK1 construct significantly inhibited BAY 11–7085-mediated c-FLIP protein degradation and apoptosis in the cancer cell lines. DN-JNK1 expression in HT-29 cells significantly increased while DA-JNK1 expression significantly decreased peritoneal implants in mice compared with control cells. Overexpression or RNAi of c-FLIP in HT-29 cells increased or decreased peritoneal implants, respectively. Cancer cells with higher c-FLIP protein expression were more resistant to BAY 11–7085 than cells with low c-FLIP expression in the co-culture assay as well. Conclusions: cFLIP expression inhibits death receptor-mediated apoptosis in cancer cells when they adhere to FASL- and TRAIL-expressing mesothelial cells. BAY 11–7085 activates JNK1 signaling which inhibits c-FLIP and restores death receptor-mediated apoptosis in cancer cells. These studies reveal the novel role for BAY 11–7085 in preventing peritoneal seeding and carcinomatosis, but add caution to the proposed use of JNK inhibitors in cancer. 577 POSTER (Board P149) Characterization of the cellular mechanism of action of the first in class investigational inhibitor of the Ubiquitin Activating Enzyme, MLN7243 M. Milhollen1 , D. Sappal1 , J. Duffy1 , K. Hoar1 , J. Huck1 , P. Sha1 , E. Koenig1 , M. Hyer1 , J. Ciavarri1 , N. Bence1 . 1 Millennium: The Takeda Oncology Company, Molecular and Cellular Oncology, Cambridge MA, USA MLN7243 is a first in class investigational small molecule inhibitor of the Ubiquitin Activating Enzyme (UAE or UBA1) currently in a Phase I clinical trial in patients with advanced solid tumors. MLN7243 is a mechanism based inhibitor that achieves nanomolar UAE inhibition through the formation of an MLN7243-ubiquitin adduct. MLN7243 is highly selective against the closely related E1 enzymes; NAE, SAE and ATG7 and demonstrates anti-proliferative effects in both in vitro cell line and in vivo xenonograft studies. MLN7243 demonstrates inhibition of global protein ubiquitination preventing subsequent proteasomal regulation of numerous cellular proteins within key biological pathways relevant to cancer cell survival. A series of laboratory studies was conducted to evaluate the mechanism of action of MLN7243 on cancer cells in vitro. Cells administered with MLN7243 revealed numerous cellular affects consistent with known UAE biology including induction of ER stress, inhibition of DNA damage repair and modulation of pro and anti-apoptotic proteins leading to cancer cell death. Here we demonstrate that MLN7243 activates an unfolded protein response (UPR) as a result of dysregulation of global protein turnover which initiates ER stress and lipid biosynthetic pathways that manifest in significant ER expansion and cellular vesiculation. The persistence of this ER stress ultimately leads to the activation of apoptosis. MLN7243 increases the accumulation of the BH3-only protein NOXA which associates with the anti-apoptotic protein MCL-1 to release pro-apoptotic proteins Bim, Bak and Bax leading to cell death. In addition to the effects on global ubiquitination and protein turnover, we also demonstrate that MLN7243 can impair the mono-ubiquitination and regulation of several key proteins within DNA damage repair pathways including PCNA and the Fanconia Anemia protein FANCD2. Comet assay analysis shows MLN7243 inhibits repair of UV insult. The inhibition of UAE impacts numerous biological pathways relevant to cancer cell survival and therefore warrants the investigation of MLN7243 for the treatment of human malignancies.

Poster Session – New Therapies with Pleiotropic Activity 578 POSTER (Board P150) Invadopodia are required for cancer cell extravasation and are a therapeutic target for metastasis H.S. Leong1 , A.E. Robertson2 , K. Stoletov3 , S.J. Leith2 , C.A. Chin2 , A.E. Chien2 , M.N. Hague2 , A.L. Ablack2 , K. Carmine Simmen3 , V.A. McPherson2 , C.O. Postenka2 , E.A. Turley2 , S.A. Courtneidge4 , A.F. Chambers2 , J.D. Lewis3 . 1 London Regional Cancer Program, Urology, London Ontario, Canada; 2 London Regional Cancer Program, Oncology, London Ontario, Canada; 3 University of Alberta, Oncology, Edmonton Alberta, Canada; 4 The Sanford-Burnham Medical Research Institute, Oncology, La Jolla CA, USA Tumor cell extravasation is a key step during cancer metastasis, yet the precise mechanisms that regulate this dynamic process are unclear. To examine the dynamics of extravasation in vivo, we utilized an intravital microscopy platform that we have developed to directly visualize human tumor growth, invasion, cell migration, and extravasation in real time (Nature Medicine 2006; Cancer Cell 2008; Nature Protocols 2010; Oncogene 2012 among others). This platform has allowed us to visualize at unprecedented resolution the protrusions that cancer cells form as they migrate and extravasate in vivo. Indeed, we observe that cancer cells dynamically form invadopodia during intravascular migration, some of which project between endothelial cells into the underlying stroma. We determined that invadopodia formed by multiple types of cancer cells are required for transendothelial migration in vivo and that inhibition of invadopodia function blocks tumor cell extravasation and the subsequent formation of metastases. Inhibition of key components of invadopodia, including cortactin, Tks4, and most specifically Tks5, resulted in an abrogation of both cancer cell extravasation and metastatic colony formation in an experimental metastasis mouse lung model. This study establishes tumor cell extravasation as a critical step of metastasis and reveals a novel opportunity for therapeutic intervention. This is particularly exciting in light of growing evidence that cancer cells are shed into the circulation subsequent to core needle biopsy or surgery, where an antiextravasation approach could mitigate potential risk. 579 POSTER (Board P151) Eph/ephrin-B interactions modulate a BAFF-R/TACI dependent survival of chronic lymphocytic leukemia (CLL) cells mediated in vitro by bone marrow stromal cells L.M. Alonso-Colmenar1 , A.G. Zapata1 , P. Fortea1 , M.A. Flores1 , F. Ortuno ˜ 2 , G. Soler2 , M.D. Garc´ıa2 , J. Garc´ıa-Cantalejo3 . 1 Universidad Complutense de Madrid, Faculty of Biology Cell Biology, Madrid, Spain; 2 HGU Morales Meseguer, Hematology and Medical Oncology Department, Murcia, Spain; 3 Universidad Complutense de Madrid, Genomic Unit Moncloa Campus, Madrid, Spain Background: To identify the possible role of Eph−ephrin interactions in the survival of CLL cells mediated by bone marrow stromal cells (BMSC). Material and Methods: CLL cells were purified from peripheral blood (PB) and bone marrow (BM) aspirates from 20 CLL patients, who gave informed consent to be involved in this study. Primary human bone marrow stromal cells (BMSC) were obtained after 2−3 weeks culture of cell suspensions obtained from the BM aspirates. Eph/ephrin expression was analysed by real-time polymerase chain reaction (Q-PCR) and flow cytometry. The role of Eph/ephrin, BAFF-R/TACI in the survival of CLL cells was determined in vitro, in cultures of CLL cells alone or in co-culture with BMSCs, by employing recombinant human proteins to block or induce signaling through them. Viability was determined by flow cytometry analysis of antiCD19FITC, Annexin-V-PE and 7AAD triple triple stained cell suspensions. Immunofluorescently stained sections from BM biopsies were examined by laser confocal microscopy. Results: We found a differential expression of several Eph/ephrin members between the CLL cells of BM and PB compartments and that of ephrinB2 was significantly associated with patients expressing worse prognosis markers, including unmutated IGVH genes and ZAP70 expression. BMSCs obtained from those CLL patients contained a subset of mesenchymal stromal cells (MSC) expressing CD90, CD105 and CD73 which highly co-expressed EphB2 and EphB4. Blocking Eph/ephrin class B interactions in CLL-BMSC co-cultures, through addition of soluble recombinant EphB4, resulted in a marked drop of CLL survival not observed in cultures of CLL cells alone. EphrinB reverse signaling in the CLL cells, achieved through culturing them alone onto plate bound recombinant EphB4, improved their viability and led to upregulation of TACI followed by downregulation of BAFF-R. In these conditions, blocking soluble BAFF produced by the CLL cells, through addition of soluble recombinant TACI, largely inhibited the ephrinB mediated CLL survival. Immunofluorescence microscopy analysis of BM biopsies confirmed the presence of EphB4 expressing stromal cells within the CLL infiltrates.

Friday 21 November 2014 187 Conclusions: Collectively, these results suggest that ephrinB2 plays an important role in the survival of CLL cells within the BM microenvironment through interacting with EphB4 expressed by a subset of stromal cells. These survival effects are largely mediated by BAFF-R/TACI signaling. The EphB4-ephrinB2 pair could represent a novel target for therapy in CLL. 580 POSTER (Board P152) Evaluation of the pan-BET-bromodomain inhibitor OTX015 as a single agent and in combination with everolimus (RAD001) in triple-negative breast cancer models 1 R. Vazquez ´ , L. Astorgues-Xerri2 , M.E. Riveiro2 , M. Di Marino1 , L. Beltrame1 , M. Bekradda2 , E. Cvitkovic3 , E. Erba1 , R. Frapolli1 , M. D’Incalci1 . 1 IRCCS − Istituto di Ricerche Farmacologiche Mario Negri, Oncology, Milano, Italy; 2 Oncology Therapeutic Development, Research, Clichy, France; 3 Oncoethix SA, Research, Lausanne, Switzerland

Background: Recent evidence shows that triple-negative breast cancer (TNBC) is driven by C-MYC. BET-bromodomain inhibitors, including OTX015 (OncoEthix SA), display antiproliferative activity accompanied by C-MYC downregulation in several tumor cell lines. Efficacy data in TNBC models, which have elevated MYC levels and altered expression of MYC regulatory genes, are lacking. Herein, we evaluated the antitumor activity of OTX015 in TNBC models in vitro and in vivo. Materials and Methods: OTX015 growth inhibition concentrations 50% (GI50 ) value was determined in HCC1937, MDA-MB-231 and MDA-MB-468 human-derived TNBC cell lines after 72 h, using the MTT assay. Protein levels were analyzed by Western Blot with commercial antibodies; RT-PCR were performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System at baseline and after treatments. For cell cycle analysis, cells were stained with propidium iodide and DNA content was analyzed with a FACScan flow cytometer. OTX015 was combined with everolimus, and the combination index (CI) was determined by the Chou– Talalay method (CI < 1, synergy; CI ≈ 1, additivity; CI > 1, antagonism). In vivo assays were performed in 8-week-old female MDA-MB-231 nude mice-derived xenografts. Mice were injected with 107 cells. Once tumor volume had reached 100 mg, mice were treated during 4 weeks with 50 mg/kg OTX-015 alone (daily, twice a day, po) or in combination with 2 mg/kg of RAD001 (thrice a week, ip). In all cases, n = 9. Results: OTX015 showed antiproliferative activity in the 3 cell lines after 72 h, with GI50 values ranging from 81.7 to 448.3 nM. Everolimus had an additive effect when combined with OTX015 in HCC1937 and MDA-MB231 cells (CI = 1.02 and 0.94, respectively), but was antagonistic in MDAMB-468 cells (CI = 1.60). Baseline protein and mRNA expression levels of BRD2/3/4 and C-MYC did not correlate with sensitivity to OTX015, nor did the combination effect of both drugs. Interestingly, C-MYC protein and mRNA levels were unchanged by OTX015 in the 3 cell lines. In vivo, OTX015-treated xenografts showed a significant (p < 0.05) reduction in tumor mass with respect to vehicle-treated mice (best T/C% = 40.7). Although everolimus alone was not active, combination with OTX015 was the most effective treatment strategy (best T/C% = 20.7). Conclusion: Our findings provide the first evidence that OTX015 is a potential clinical alternative for the treatment of TNBC, either alone or in combination with mTOR inhibitors. 581 POSTER (Board P153) Identification of genomic and chromatin features that predict transcriptional response to BET bromodomain inhibition J. Mertz1 , H.R. Huang1 , N. Follmer1 , A. Reddy1 , R. Centore1 , B. Bryant2 , C. Hatton2 , H. Franco3 , W.L. Krause3 , R. Sims III1 . 1 Constellation Pharmaceuticals, Biology, Cambridge, USA; 2 Constellation Pharmaceuticals, Computational Biology, Cambridge, USA; 3 UT Southwestern Medical Center, Green Center for Reproductive Biology Sciences, Dallas, USA Pharmacologic inhibition of BET family bromodomains results in the reversible transcriptional silencing of key cancer genes, such as MYC, MYB, and BCL-2. This transcriptional modulation is accompanied by robust anti-proliferative effects in cell lines and anti-tumor activity in animal models. However, the molecular and phenotypic response to BET inhibition (BETi) is not uniform, as some cancer subtypes and select genes display disproportionate sensitivity. It has been suggested that BRD4 loading at superenhancers explains the selective response to BETi, perhaps identifying molecular features that could aid in patient selection. Here, we performed comprehensive genomic analysis using ChIP-seq, RNA-seq, and GRO-seq to catalogue the dynamic transcriptional response following BETi. We find that BRD2 and/or BRD4 loading at transcriptional start sites or superenhancers are poor predictive indicators of transcriptional response to BETi, in part due to a transcriptional rebound that occurs as early as 30

188 Friday 21 November 2014 minutes after treatment. We identify a chromatin architecture that defines a durable response to BETi, specifically in the context of immunoglobulin heavy chain (IgH) re-arrangements, classifying patient populations suitable for BET treatment. Our results suggest that the molecular response to BETi is highly dynamic and adapts over time, while high-occupancy BRD4 loading is generally a poor predictor of BET transcriptional control. 582 POSTER (Board P154) Rational for targeting chromatin-modifying genes in clear-cell renal cell carcinomas ˆ G. Malouf1 , J. Zhang2 , D. Khayat1 , X. Su3 , J.P. Spano1 . 1 Hopital de la ´ ˆ ere, ` Pitie-Salp etri Medical Oncology, Paris, France; 2 MD Anderson Cancer Center, Department of Bioinformatics, Houston, USA; 3 MD Anderson Cancer Center, Bioinformatics, Houston, USA Background: Currently, standard of care of metastatic clear-cell renal cell carcinoma (ccRCC) is VEGF- or m-TOR targeted agents. The overall survival with these agents reached a plateau and the identifying novel targets is urgently needed. Recently, several chromatin-modifying genes (CMG) have been identified as frequently mutated in ccRCC, however, this often leads to gene inactivation. Of note, it is not known if there are any of those which expression is correlated with poor patient’s outcome. Methods: We analyzed RNA-seq data of 475 primary ccRCC which we extracted from The Cancer Genome Atlas (TCGA) project. Firstly, we performed unsupervised clustering for gene expression and correlated it with patients’ outcomes. Furthermore, using a cox model, we analyzed prognostic values of gene expression of 162 chromatin-modifying genes (CMG). Results: Unsupervised clustering for gene expression identified four clusters bearing different overall survival. Cluster 2 was associated in multivariate analysis with poor outcome and was enriched for mutations in BAP1. Out of the 162 selected CMG, there were 33 and 37 CMGs which gene expression was associated with poor and good patients overall survival, respectively. Biocarta pathway analysis using DAVID software showed that overexpressed CMGs were enriched for the polycomb repressive complex 2 (PRC2) sets of long-term silencing genes and which acts through modification of histone tails; those includes EZH2, EED, CBX4 and HDAC1 (p = 2×10−5 ). There was no enrichment found for under-expressed CMGs using the same criteria. Ingenuity pathway analysis revealed that overexpressed genes in cluster 2 were related to EZH2 (p = 5.3×10−7 ) and BRD4 (p = 3.7×10−4 ) targets. Furthermore, EZH2 expression was overexpressed in cluster 2 as compared to other clusters (FC = 1.5; p = 0.006). Conclusion: This is the first study to assess the comprehensive expression of CMGs in predicting patients’ outcome in ccRCC. It provides rational for targeting the PRC2 complex in patients with aggressive ccRCC. 583 POSTER (Board P155) The 8p11 amplicon in luminal breast cancers harbors multiple interacting epigenome modifying oncogenes: implications for epigenome-targeted therapy S. Ethier1 , J. Irish2 , R. Wilson1 , B. Turner1 . 1 Medical Univ of South Carolina, Pathology and Laboratory Medicine, Charleston, USA; 2 Wayne State University School of Medicine, Oncology, Detroit, USA Luminal breast cancers exhibit a focal amplification in the chromosome 8p11−p12 region in approximately 40% of cases. This chromosomal region harbors a number of candidate oncogenes that influence the malignant potential of the cells as well as their response to therapy. We have identified three oncogenes from this region that are coordinately amplified and over-expressed at high frequency when the amplicon is present and all three of these genes modify the epigenome. The genes include WHSC1L1, an H3K36 methylase, KAT6A a histone acetyl transferase, and ASH2L, an H3K4 methylase. As these genes are coordinately amplified and over expressed in amplicon-bearing cells, their over expression has profound effects on the epigenome by influencing the self-renewal potential, the differentiation state, and the response of the cells to hormonally based drugs. Indeed, WHSC1L1 and KAT6A cooperate to regulate the expression of the ESR1 gene (estrogen receptor), as well as HER4, C-MYC, and others. Recent data demonstrates that the short isoform of WHSC1L1, which contains histone recognition and binding domains, but not the SET domain, is the transforming variant of this oncogene and acts to reduce H3K36 methylation globally by dominant negative effects on the full lenght isoform of WHSC1L1. ChIP-Seq experiments demonstrate that WHSC1L1-short influences the binding of ER-a at specific genomic regions, particularly those enriched for FOXA1 binding motifs and the ERE. Knock-down of WHSC1L1-short or KAT6A in breast cancer cells bearing the amplicon results in changes in expression of many of the genes previously associated with ER-positive breast cancers that have a poor

Poster Session – New Therapies with Pleiotropic Activity clinical outcome, and dramatically reduces their proliferative and clonogenic potential. Thus, the presence of the 8p11−p12 amplicon influences the biology of the estrogen receptor in luminal breast cancers and influences their response to anti-estrogen therapies. Since patients with ER-positive breast cancers that fail hormonal therapy have few therapeutic options, these results indicate that patients with breast cancer cells bearing the amplicon are candidates for treatment with epigenome-targeting drugs such as BET-bromo domain inhibitors, histone methylase inhibitors, and histone acetyl transferase inhibitors. Since the biology of the amplicon is similar in squamous lung cancer, where it occurs in approximately 25% of cases, these results have implications for treatment strategies in this sub-set of lung cancer as well as in breast cancer. 584 POSTER (Board P156) The correlation between EMT and cancer stemness in lung adenocarcinoma affects on its prognosis T. Sowa1 , T. Menju1 , T. Nakanishi1 , K. Shikuma1 , N. Imamura1 , T. Yamada1 , A. Aoyama1 , M. Sato1 , T. Sato1 , F. Chen1 , M. Sonobe1 , M. Omasa1 , H. Date1 , T. Sozu2 . 1 Kyoto University Hospital, General Thoracic Surgery, Kyoto, Japan; 2 Kyoto University School of Public Health, Biostatistics, Kyoto, Japan Background: Lung cancer is the leading cause of cancer death worldwide, and adenocarcinoma is the most common histologic subtype of primary lung cancer. Currently the drug therapeutics for lung cancer have been developing and shifting from cytotoxic to molecular targeted reagents. But novel mechanisms by which tumor cells are resistant to these drugs have been appeared as one of the most challenging cancer properties to overcome, epithelial–mesenchymal transition (EMT) and cancer stemness. EMT and cancer stemness are also known to be the pivotal phenomena engaged in metastasis, recurrence and resistance for treatment in lung cancer, but the approaches for these factors are not sufficiently achieved. Previously EMT and cancer stemness have been separately reported in terms of its unfavorable effects on prognosis. In in vitro experiments, the correlations of each property have been reported, whereas the playing role of the mutual correlations in clinical settings remains elusive. If these correlations and effects on tumor progression are revealed, the approach for novel targeted therapy is expected to be promoted. The purposes of our present study are to investigate the correlation of EMT with cancer stemness and its effect on the prognosis in lung adenocarcinoma. In this study we presented and discussed the key factors which linked EMT and cancer stemness, and how these factors affected on tumor malignancy. Materials and Methods: Two hundred fifty-seven specimens of lung adenocarcinomas in tissue microarrays were collected from the patients with lung cancers who were operated in Kyoto University Hospital from January 2001 to December 2007. Both EMT (E-cadherin/vimentin) and cancer stemness (CD133/CD44/aldehyde dehydrogenase-A) markers were examined in these specimens by using immunohistochemistry staining. Expression levels of each markers were integrated with the patients’ data including the prognosis, and then, the correlations of EMT with cancer stemness were statistically analyzed. Results: E-cadherin, vimentin, and CD133 were significantly related to the prognosis independently (Log-rank test, P < 0.01). E-cadherin and vimentin had a negative correlation, whereas vimentin and CD133 had a positive one (Pearson’s chi-square test, P = 0.02). Only CD133 was significantly correlated with EMT conversion instead of other stemness markers. Indeed, more active EMT conversion leads to worse prognosis, but additional CD133 expression further impaired it. CD133 was considered to be the signature of which cancer with EMT conversion gains nature of cancer stemness. Conclusions: EMT and cancer stemness were mutually correlated in lung adenocarcinoma. Especially, CD133 is suggested to be the key factor which links EMT and cancer stemness directing further tumor progression. 585 POSTER (Board P157) The investigational HSP90 inhibitor ganetespib displays robust single agent activity in gastric cancer models both as monotherapy and in combination with standard of care therapeutics S. He1 , C. Zhang1 , J.P. Jimenez1 , J. Sang1 , M. Sequeira1 , D. Smith1 , J. Acquaviva1 , M. Nagai1 , R. Bates1 , D.A. Proia1 . 1 Synta Pharmaceuticals, Biology, Lexington MA, USA Background: Gastric cancer is the second leading cause of cancer mortality worldwide and treatments for this disease have evolved only minimally in recent years. HER2 overexpression and/or amplification has been observed in 10−38% gastric cancer patients and this phenotype has

Poster Session – New Therapies with Pleiotropic Activity been linked to more aggressive disease and poor clinical outcomes. HER2 is particularly reliant on the chaperoning activity of heat shock protein 90 (HSP90) for its stability and function. Pharmacological blockade of HSP90 results in cellular depletion and loss of its client proteins, in turn providing a means to simultaneously disrupt multiple oncogenic pathways. Ganetespib is a clinically advanced small molecule inhibitor of HSP90. Here, we have evaluated the antitumor activity and efficacy of ganetespib, both as a single agent and in combination with clinically approved drugs, in models of human gastric cancer. Material and Methods: Cell viability was determined by CellTiterGlo. The anticancer effect of ganetespib as single agent or coupled with other chemotherapeutic agents was examined via immunoblot, immunofluorescence, flow cytometry and xenograft models in SCID mice. Results: Ganetespib demonstrated potent in vitro cytotoxic activity in a panel of 22 gastric cancer cell lines in vitro, with an average IC50 value 20 nM. The panel included 3 HER2-overexpressing (HER2+) lines and antiproliferative activity was independent of p53 status. At the molecular level, ganetespib treatment promoted destabilization of multiple HSP90 client proteins and effectors, including HER2, EGFR, IGF-IR, cMET, cKit, P-AKT, P-ERK1/2 and a number of cell cycle modulators, which ultimately resulted in apoptosis. These effects of ganetespib were durable, with kinetic analysis showing client suppression was maintained for over 48 hours following a 1 hour drug exposure. Combinations of ganetespib with chemotherapeutic agents (doxorubicin, docetaxel, cisplatin) as well as the dual HER2/EGFR kinase inhibitor lapatinib promoted significant increases in total cell death in comparison to monotherapy. Synergistic effects were also observed when ganetespib was combined with the PI3K/mTOR inhibitor BEZ235, due in part to ganetespibs’ ability to overcome the rebound in AKT activity that arises from PI3K/mTOR inhibition. The combinatorial benefit observed in vitro translated to improved efficacy in vivo, where combinations of ganetespib with capecitabine or docetaxel induced tumor regressions in NCI-N87 xenografts. Conclusions: Taken together, these data highlight the potent antitumor activity of ganetespib for gastric cancer cells, including HER2+ lines, both in vitro and in animal models. The findings support the potential therapeutic value of ganetespib, particularly in combination with standardof-care agents, for the treatment of patients with gastric cancer. 586 POSTER (Board P158) Phase I study of pan-histone deacetylase inhibitor abexinostat in combination with cisplatin in patients with advanced solid tumors M. Campone1 , N. Isambert2 , R. Sabatier3 , H. Castanie1 , S. Zanetta2 , I. Sudey4 , F. Cantero4 , J. Pauly4 , E. Leroux4 , S. Malasse5 , A. Goncalves3 . 1 ´ ´ Institut de Cancerologie de l’Ouest, Cancerologie, Saint Herblain-Nantes ´ Cedex, France; 2 Centre Georges-Fran¸cois Leclerc, Oncologie Medicale, 3 ´ Dijon, France; Institut Paoli-Calmettes, Oncologie Medicale, Marseille, France; 4 IRIS, Oncologie Innovation Therapeutic Pole, Suresnes, France; 5 IRIS, Biostatistic, Suresnes, France Background: Abexinostat (Abx) is an oral pan-histone deacetylase inhibitor (HDACi) currently in phase I/II. Due to their important role in epigenetic regulation HDACi may promote accessibility of DNA to chemotherapeutic agents like cisplatin (C) and help enhance cytotoxicity. Based on preclinical studies showing synergistic effects of Abx and C, the aim of this phase I dose-escalation study was to assess the safety of the combination in patients (pts) with advanced solid tumors (STs). Methods: Primary objective was to determine the maximum tolerated dose (MTD) based on the dose-limiting toxicities (DLTs) using a 3+3 design. Secondary objectives included the pharmacokinetics (PK) and the evaluation of efficacy. Pts received Abx po bid, on days 1−4 and 8−11 of a 21-day cycle. C (75 mg/m2 , iv) was given on day 3 for a maximum of 6 cycles due to the risk of cumulative toxicity. Abx was given at a starting dose 80 mg bid (dose level 1 [DL1]) and then de-escalated. Results: 19 heavily pretreated pts (18 F/1 M) were enrolled having a median age of 55 years (range 25−70), ECOG 1, and a variety of advanced STs including ovary (7), breast (7), cervix (2), colon (1), pancreas (1), neuro-endocrine (1) cancers. Twelve pts had prior exposure to platinum compounds (2 received C). A total of 18 pts were treated (DL1 = 80 mg bid, n = 7; DL −1 = 60 mg bid, n = 5; DL −2 = 40 mg bid, n = 6) with a median number of 2 cycles (range, 1−8). Five pts experienced DLTs during cycle 1: thrombocytopenia grade (G) 4 (n = 2), hepatic enzymes increased G3 (n = 2), ECG repolarisation abnormalities G3 (n = 1). MTD of Abx in combination with C 75 mg/m2 was 60 mg bid. The most frequent adverse events (AEs) related to Abx and/or C included nausea (18%), asthenia (11%), vomiting (11%), thrombocytopenia (10%), decreased appetite (7%), anemia (7%) and were mostly G1/2 (85%). A majority of G3/4 AEs were considered as related to both study drugs and led to drug withdrawal for 6 pts. Among the 15 evaluable pts, one partial response at cycle 4 (cervix) and 5 cases of stable disease lasting

Friday 21 November 2014 189 4 cycles were observed. The exposure of Abx in combination with C was in accordance with monotherapy results, suggesting no PK interaction between Abx and C. Conclusions: These preliminary results showed that in spite of some signs of activity, the safety profile of concomitant administration of Abx and C is limiting. Even though AEs were generally manageable, the tolerability issues might affect treatment duration and completion. 587 POSTER (Board P159) Cellular pharmacokinetics and molecular pharmacodynamics studies of the BRD-BET inhibitor OTX015 in sensitive and resistant leukemic cell lines E. Odore1 , L. Astorgues-Xerri2 , M. Bekradda2 , E. Cvitkovic3 , P. Herait3 , F. Lokiec1 , K. Rezai1 , M. Riveiro2 . 1 Curie Institute, Pharmacology Department, Saint Cloud, France; 2 Oncology Therapeutic Development, Clichy, France; 3 Oncoethix SA, Lausanne, Switzerland Background: OTX015 (OncoEthix SA, Switzerland) is a novel oral bromodomain and extraterminal (BET) protein family inhibitor, which is a potential cancer target particularly in hematologic malignancies. The relation between the cellular pharmacokinetics (PK) and targeted-gene regulation governing the biological effects of BRD inhibitors is still largely not understood. We analyzed cellular PK properties and CMYC and related genes modulation in OTX015-sensitive and resistant leukemic cell lines. Materials and Methods: Established human cell lines from acute and chronic myeloid leukemia (HL-60, U937, K562) and acute lymphoblastic leukemia (Jurkat, MOLT-3) were used. Anti-proliferative effects of OTX015 were assessed by MTT after 72 h-exposure. For cellular PK studies, cells were seeded at 2×106 cells/ml and exposed to 240 ng/ml OTX015 for 0, 5, 15, 30, 60, 120 and 360 min. At each time point, OTX015 extracellular and intracellular concentrations were analyzed in cell supernatants and pellets respectively, using Ultra Performance Liquid Chromatography with tandem Mass Spectrometry (concentration range 1–250 ng/mL). In cell pellets, protein modulation was analyzed by Western blot using commercial antibodies and qRT-PCR was performed using Fast SYBR Green on a StepOnePlus RT-PCR System. Results: GI50 values were between 230 and 384 nM in HL60, U937 and Jurkat, and were 6,000nM for other cell lines. Cellular uptake of OTX015 was rapid (<5 min) in both sensitive and resistant leukemic cell lines, with a mean concentration of ~15 ng·mL−1 /106 cells (3−23 ng·mL−1 /106 cells), whereas extracellular levels of OTX015 were stable with up to 6 h exposure (~200 ng·mL−1 ). In the resistant cell line, K562, we observed CMYC mRNA downregulation after 2 h exposure, even though OTX015 intracellular levels were detected after 5 min exposure. In the sensitive cell lines, HL60, U937 and Jurkat, a rapid downregulation of CMYC protein and mRNA levels was observed, whereas other OTX015-targeted genes, such as HEXIM, P21 and BCL2 mRNA levels were modulated after longer exposure time points in sensitive cell lines. Conclusions: OTX015 intracellular accumulation occurred to a similar extent and timing in OTX015 sensitive and resistant leukemic cell lines. Rapid modulation of OTX015-target genes was observed at mRNA and protein levels only in sensitive cell lines. 588 POSTER (Board P160) Neddylation as a therapeutic target in refractory pediatric malignancies: Evaluation of the activating enzyme inhibitor MLN4924 Y. Ruan1 , A. Jayanthan1 , T. Cooper2 , A. Narendran1 . 1 Alberta Children’s Hospital, Calgary Alberta, Canada; 2 Emory University, Department of Pediatrics, Atlanta Georgia, USA Recently, the neddylation pathway has been identified as a therapeutic target in cancer. The activity of cullin-RING dependent ubiquitin E3 ligases (CRLs) requires neddylation (i.e. conjugation of NEDD8) to their cullin subunit. The CRLs control the turnover of many key proteins in regulating cell-cycle progression, DNA damage, stress responses, and signal transduction. Therefore, suppression of neddylation, and subsequently CRLs activity, can decrease cancer cell proliferation. MLN4924 is an investigational first-in-class small molecule inhibitor of NEDD8-activating enzyme (NAE). It has shown excellent efficacy in preclinical studies of adult cancers as well as neuroblastomas and osteosarcoma. Therefore, we tested the efficacy of this compound in cell lines established from refractory pediatric tumors, with a focus on refractory leukemia and atypical teratoid/rhabdoid tumors (ATRT). Methods: A panel of cell lines (5 ALL lines; 3 AML lines; and 3 ATRT lines), were treated with MLN4924 and cell growth inhibition was quantified by Alamar blue assay. Cell cycle analysis was performed by PI staining and FACS. Target modulation was evaluated by western blotting. Drug combination studies were interpreted using the Chou–Talalay method.

190 Friday 21 November 2014 Results: There was variable efficacy of MLN4924 in pediatric cancer cell lines with IC50 values ranging from 35 nM in leukemia, to 1500 nM in an ATRT. MLN4924 resulted in decreased neddylation of CRLs and the stabilization of CRL substrates p21 and p-IúBa and led to DNA rereplication and delay in S phase and G2/M phase progression. MLN4924induced cell death was partially mediated by the production of reactive oxygen species (ROS), as ROS scavenger N-acetylcysteine protected MLN4924-treated leukemia cells. In drug combination assays MLN4924 did not synergy with many conventional chemotherapeutics including etoposide, methotrexate, and doxorubicin. Interestingly, additive effect to weak synergy was observed with the bromodomain inhibitor JQ1 (CI: 0.7−1.0). Conclusion: We demonstrated that MLN4924 has variable but excellent in vitro efficacy towards pediatric cancer cell lines. It induced cell death and cycle arrest by the inhibition of cullin activity and the production of ROS. MLN4924 displayed combinatorial effects with BET inhibition. These preliminary observations provide the rationale for additional studies and to explore the use of MLN4924 in early phase clinical trials in children with refractory malignancies. 589 POSTER (Board P161) N-myc downstream regulated gene 1 (NDRG1) as a novel anti-angiogenic and therapeutic target for VEGF/VEGF receptor signaling by vascular endothelial cells K. Watari1 , A. Shinoda1 , T. Shibata1 , A. Kawahara2 , T. Nakama3 , S. Yoshida3 , M. Kage2 , M. Kuwano4 , M. Ono1 . 1 Kyushu University, Dept. of Pharmaceutical Oncology, Fukuoka, Japan; 2 Kurume University Hospital, Dept. of Diagnostic Pathology, Kurume, Japan; 3 Kyushu University, Dept. of Ophthalmology, Fukuoka, Japan; 4 Kyushu University, Lab. of Molecular Cancer Biology, Fukuoka, Japan Background: Angiogenesis is essential for the malignant progression of cancer. Therefore, development of a novel type of angiogenesis inhibitor is essential for further improvement of therapeutics against cancer patients. N-myc downstream regulated gene 1 (NDRG1) has been known to play pleiotropic roles in cell proliferation, development, differentiation, and tumorigenesis. We have previously reported that NDRG1 expression levels in cancer cells are closely correlated with tumor angiogenesis and tumor growth in both experimental models and human tumors (Hosoi et al., Cancer Res., 2009; Murakami et al., J Biol Chem., 2013). However, whether tumor angiogenesis could be affected by NDRG1 in host remains unclear. In our present study, we asked whether NDRG1 could specifically modulate tumor angiogenesis, and impaired tumor angiogenesis could be observed by NDRG1 deficiency. Material and Methods: NDRG1 deficient mice: The NDRG1 deficient mice on C57BL6 background have been kindly donated to us by Prof. Toshiyuki Miyata (National Cerebral and Cardiovascular Center) (Okuda et al., Mol Cell Biol., 2004). Isolation of mouse endothelial cells: CD31+ endothelial cells were isolated from mouse lung by magnetic sorting using CD31 MicroBeads. Aorta ring assay: 1 mm mouse aortic rings were embedded in 3-dimensional growth factor reduced Matrigel, treated with or without FGF-2 (50 ng/mL) or VEGF (25 ng/ml), and incubated at 37ºC. Vascular length and branching point were measured at day 7. Results: In NDRG1 deficient mice as compared to their wild type counterparts, (1) tumor growth and angiogenesis by subcutaneous transplantation of syngeneic cancer cells were markedly suppressed; (2) tumor angiogenesis by cancer cells was suppressed in dorsal air sac assay; (3) exogenous administration of VEGF could not induce angiogenesis in corneas whereas FGF-2 could induce angiogenesis; (4) VEGF-induced angiogenesis was markedly impaired in aorta ring assay; (5) VEGF could not induce cell growth and phosphorylation of Akt and Erk in vascular endothelial cells in vitro; (6) in addition to VEGF, an inflammatory cytokine IL-1b also induced impaired angiogenesis in corneas. Conclusion: NDRG1 deficiency in host induced functional impairment of endothelial cells in response to VEGF, affecting impaired tumor angiogenesis, and also inflammatory angiogenesis. Based on our present study, we will discuss whether targeting NDRG1 and its downstream signaling molecules could be useful for further development of novel antiangiogenic drug.

Poster Session – New Therapies with Pleiotropic Activity 590 POSTER (Board P162) A dose dense schedule improves antitumor activity of trabectedin in myxoid liposarcoma with type III FUS-CHOP chimera S. Uboldi1 , R. Frapolli1 , E. Bello1 , S. Brich2 , F. Bozzi2 , R. Sanfilippo3 , P.G. Casali3 , A. Gronchi4 , C.M. Galmarini5 , J.M. Fernandez Sousa-Faro5 , S. Pilotti2 , M. D’Incalci1 . 1 IRCCS-Istituto di Ricerche Farmacologiche Mario Negri, Oncology, Milano, Italy; 2 Fondazione IRCCS Istituto Nazionale Tumori, Pathology, Milano, Italy; 3 Fondazione IRCCS Istituto Nazionale Tumori, Cancer Medicine, Milano, Italy; 4 Fondazione IRCCS Istituto Nazionale Tumori, Surgery, Milano, Italy; 5 PharmaMar S.A., R&D, Colmenar Viejo, Spain Background: The exquisite sensitivity of myxoid liposarcomas (MRCLS) to trabectedin seems to be related to a selective effect of the drug on the oncogenic mechanism of this tumor. MRCLS is characterized by the chromosomal translocation t(12;16)(q13;p11), with consequent expression of the fusion gene FUS-CHOP that is the main pathogenetic lesion, responsible for the arrest of adipocytic differentiation. Trabectedin is able to specifically displace FUS-CHOP chimera from its target promoters, thus inducing cEBP a and b and consequently adipocytic differentiation. The sensitivity to trabectedin appears to be dependent on the type of transcript, being the type III transcript less sensitive than the more frequent type I and type II. The lower susceptibility of type III variants than of type I and II was clearly demonstrated in MRCLS xenografts. Using xenografts representative of type I, II and III it was found that in all three subtypes trabectedin treatment causes the displacement of FUS-CHOP from DNA but the kinetic of the reattachment was different being much faster in type III than in the other types. The aim of the study was to assess whether the low sensitivity of type III MRCLS could be improved by using a different schedule. Materials and Methods: MRCLSs were xenografted in nude mice. When tumor load achieves about 200–400 mg mice were divided into the experimental groups. Trabectedin was administered i.v. at the cumulative dose of 0.45 mg/kg: 0.15 mg/kg every 7 days for three times (q7d×3) or with a dose-dense schedule 0.075 mg/kg every three days for six times (q3d×6). Tumor growth was monitored by caliper. The binding of FUSCHOP to some of its target promoters was monitored by Chromatin Immunoprecipitation. H&E staining was performed to evaluate tumor response. Results: We found that trabectedin was more effective on type III MRCLS xenografts when administered q3d×6. Only with this schedule, the response to trabectedin was associated with focal pathological response (adipocytic maturation). Molecular analysis revealed that trabectedin at the dose of 0.075 mg/kg was able to remove FUS-CHOP type III from its own target genes 24 hours after treatment. 72 hours after the third dose (thus immediately before the forth dose), FUS-CHOP type III remained unbound. Conclusions: These results indicate that shortening the interval between doses is necessary to overcome the low sensitivity to trabectedin of type III MRCLS by maintaining FUS-CHOP detached from its target genes. 591 POSTER (Board P163) Mechanistic analysis of reversible FASN inhibition in preclinical tumor models identifies highly susceptible tumor types and enriches biomarker discovery for clinical applications T.S. Heuer1 , R. Ventura1 , J. Waszczuk1 , K. Mordec1 , J. Lai1 , M. Fridlib1 , R. Johnson1 , L. Hu1 , H. Cai1 , A. Wagman1 , M. O’Farrell1 , D. Buckley1 , G. Kemble1 . 1 3-V Biosciences, Biology, Menlo Park CA, USA Reduction of de novo lipid synthesis via fatty acid synthase (FASN) inhibition provides a novel approach to cancer therapy with strong biological rational. FASN expression increases with tumor progression and associates with chemoresistance, tumor metastasis, and diminished patient survival in numerous solid and hematopoietic tumor types. FASNsynthesized palmitate and palmitate-derived lipids comprise diverse cellular components and function in processes required for tumor cell proliferation and survival; examples include energy metabolism and storage, membrane biosynthesis and architecture and protein localization and activity. TVB-3166, which belongs to a series of orally available, reversible, potent, and selective FASN inhibitors discovered and developed by 3-V Biosciences, inhibits FASN and exhibits tumoricidal activity against a diverse array of tumor types. Anti-tumor activities of TVB-3166 include induction of tumor cell apoptosis, inhibition of anchorage-independent tumor cell growth under lipid-rich conditions, and in vivo xenograft tumor growth inhibition. Ongoing mechanism-of-action studies demonstrate inhibition of multiple signal transduction pathways, modulation of gene expression, disruption of lipid raft architecture and altered localization of raft-associated signaling proteins. Studies to understand the fundamental biological consequences of FASN inhibition are guiding identification of tumors most dependent on FASN

Poster Session – New Therapies with Pleiotropic Activity activity for their growth, proliferation, and survival. Using both genomics and directed analysis of mRNA, protein and lipids, we find FASN inhibition modulates lipid synthesis, signal transduction, cell cycle and apoptosis pathways in both in vitro cell culture and in vivo tumor xenograft studies. Results in patient-derived tumor xenografts strengthen findings from tumor cell line studies, and comparison of TVB-3166-induced changes in different tumor types is identifying changes that are both common and specific to defined tumor types. The results from these studies are advancing the discovery of multiple mechanisms of action that can operate in specific tumor types and the discovery of biomarkers with potential utility for selecting responsive patients and measuring tumor response. Taken together, these preclinical data support 3-V Biosciences’ recently-initiated clinical study of a first-in-class FASN inhibitor, and are informing potential applications that include drug-combination therapy. 592 POSTER (Board P164) TAS-116, a highly selective inhibitor of heat shock protein 90a/b, inhibits tumor growth in biliary tract cancer mouse models S. Ohkubo1 , H. Muraoka1 , Y. Kodama1 , K. Ito1 , S. Ito1 , A. Hashimoto1 , C. Yoshimura1 , T. Utsugi1 . 1 Taiho Pharmaceutical Co. Ltd., Tsukuba Research Center, Tsukuba, Japan Background: Patients with unresectable or metastatic biliary tract cancer (BTC) have poor prognosis. Despite the use of gemcitabine-based chemotherapy in the treatment of BTC, more effective therapy is urgently needed for treatment of unresectable or metastatic cancers. Since aberrations of several heat shock protein (HSP)90 clients have been reported in BTCs, we evaluated the therapeutic potential of TAS-116, which is an orally available, highly selective inhibitor of HSP90a/b that is currently undergoing clinical trial, against tumor growth in preclinical BTC mouse models. Material and Methods: The effect of TAS-116 and select protein kinase inhibitors on cell growth was evaluated in eight BTC cell lines. The effects of TAS-116 on protein expression and phosphorylation status were evaluated by using western blotting and a phospho-receptor tyrosine kinase (RTK) antibody array. The antitumor activity of TAS-116 was evaluated in BTC xenograft mouse models. Results: Though specific RTKs play important roles in certain human cancers, we did not detect a distinctive RTK phosphorylation profile in the BTC cell lines examined except that aberrant MET activation was observed in OCUG-1 cells. Indeed, erlotinib and lapatinib had little effect on the growth of these cell lines. In addition, although the phosphorylated forms of AKT and ERK were detected in each of the BCT cell lines, treatment with MK2206 or selumetinib only slightly reduced cell growth. In contrast, TAS-116 markedly inhibited cell growth in all of the BTC cell lines. TAS-116 simultaneously suppressed the phosphorylation of multiple RTKs through downregulation of those proteins, leading to inhibition of both PI3K/AKT and MAPK/ERK signaling. Daily administration of TAS-116 at a dose of 14 mg/kg for 2 weeks resulted in tumor growth inhibition (TGI) of 63% in a human cholangiocarcinoma (TFK-1) xenograft model. TAS-116 also resulted in a TGI of 68% in a human gallbladder cancer (TGBC2TKB) xenograft model, compared with weekly administration of gemcitabine at a dose of 300 mg/kg that resulted in a TGI of only 10%. Conclusions: TAS-116 was broadly active against BTC cell lines through pleiotropic downregulation of HSP90 clients. TAS-116 also inhibited tumor growth in BTC xenograft models. Therefore, treatment with an HSP90 inhibitor such as TAS-116 is a potential approach for treating BTC. 593 POSTER (Board P165) Strategies to overcome resistance to BET bromodomain inhibitor in KRAS/LKB1 mutant NSCLC M. Soucheray1 , E. Kikuchi2 , I. Pulido3 , E. Akbay4 , J.H. Becker1 , C.L. Christensen4 , N. Johnson5 , T.B. Patel6 , J. Carretero3 , K.K. Wong4 , T. Shimamura7 . 1 Loyola University Chicago, Oncology Research Institute, Maywood, USA; 2 Dana-Farber Cancer Institute, Medical Oncology, Boston, USA; 3 University of Valencia, Physiology, Valencia, USA; 4 Dana-Farber Cancer Institute, Medical Oncology, Boston, USA; 5 Fox Chase Cancer Center, Developmental Therapeutics, Philadelphia, USA; 6 Loyola University Chicago, Pharmacology, Maywood, USA; 7 Loyola University Medical Center, Oncology Research Institute, Maywood, USA Approximately 15−30% of human non-small cell lung cancer (NSCLC) patients harbor a somatic KRAS mutation resulting in aberrant activation of downstream signaling pathways that control cell proliferation, cell growth, and cell survival. Thus developing effective therapies for the significant subpopulation of NSCLC patients with KRAS mutation significantly impacts public health. However, it has been proven difficult to develop small molecule inhibitors that directly target mutant KRAS activities.

Friday 21 November 2014 191 Consequently, efforts have focused on developing inhibitors of downstream effectors of mutant KRAS that are essential for cell survival. Based on the finding that transcription factor MYC is important for the survival and proliferation of mutant KRAS NSCLC tumors in vivo, we have recently demonstrated significant tumor regression in transgenic murine lung cancers driven by mutant kras when treated with JQ1, a small molecule that predominantly inhibits MYC transcription. We also demonstrated that the loss of the tumor suppressor lkb1 rendered the mutant kras murine NSCLC resistant to JQ1. However, how LKB1 loss attenuates the efficacy of JQ1 remains elusive. Using LKB1 isogenic NSCLC cell lines and genetically engineered mouse models, we investigated the mechanisms of the JQ1 resistance and formulated rationale-based combination therapies to overcome JQ1 resistance in LKB1 deficient NSCLC. LKB1 depletion in mutant KRAS NSCLC cells promotes reduced MYC expression and phospho-ERK1/2. We also discovered that LKB1 depletion results in aberrant activation of SRC and PI3K pathways. The activation of PI3K and SRC pathways was sufficient to promote MYC-independent cell survival in LKB1-deficient mutant KRAS NSCLC. Importantly, the inhibition of these pathways sensitized LKB1-deficient mutant KRAS NSCLC to JQ1. We also discovered that JQ1 inhibits the initiation of DNA double-strand break (DSB) repair and mismatch repair mechanisms in LKB1-deficient mutant KRAS NSCLC through BRD4 and MYC inhibition. While JQ1 treatment was not efficacious in LKB1-deficient mutant KRAS NSCLC cells, JQ1 and gemcitabine synergistically killed the cells. We elucidated previously unknown roles of LKB1 in controlling MYC-dependent cell survival and discovered how the inactivation of LKB1 deregulates pathways to promote MYC-independent cell survival in mutant KRAS NSCLC. The identification of pathways that bypass MYC-dependent cell survival break new ground to test different inhibitors that target these pathways in order to restore the sensitivity to inhibitor of MYC-dependent transcription in hard-to-treat mutant KRAS NSCLC with LKB1 loss. The results provide a rationale for early stage human clinical trials. 594 POSTER (Board P166) Identification of novel EZH2 inhibitor scaffolds X. Espanel1 , L. Chene2 , A. Soude3 , S. Estevez4 , V. Adarbes4 , B. Loillier4 , B. Boubia5 , P. Masson6 , C. Montalbetti7 , P. Broqua8 , C. Fromond9 . 1 Inventiva, Head of HCS, Dijon, France; 2 Inventiva, Head of Target Validation, Dijon, France; 3 Inventiva, Scientist, Dijon, France; 4 Inventiva, Lab Technician, Dijon, France; 5 Inventiva, Expert scientist, Dijon, France; 6 Inventiva, Head of Biology & Screening, Dijon, France; 7 Inventiva, Head of Chemistry Dept, Dijon, France; 8 Inventiva, CSO, Dijon, France; 9 Inventiva, Head of Biology and Pharmacology Dept, Dijon, France The histone-lysine N-methyltransferase (HKMT) EZH2 is often over activated by gain-of-function mutations in lymphomas or over expressed in solid tumors. This over activation is associated with increase in H3K27 methylation in tumors, leading to decreased suppressor gene expression and poor prognosis. Regarding the therapeutic potential of EZH2 inhibitors, several groups have initiated drug discovery programs leading to the identification of potent drugs. Surprisingly, most of these independent efforts led to the discovery of molecules harbouring the same pyridone pharmacophore, with probable similar binding mode to EZH2 and pharmacokinetic properties. In this context identifying EZH2 inhibitors within new chemical classes is required to optimize chance of clinical success and patient access to this target class. We screened our 200K proprietary compound library using an alphascreen assay aiming at identifying new EZH2 inhibitors. Several potent inhibitors were identified and validated by other technologies such as chemiluminescence and radioactive methyl donor. False positive compounds were carefully rejected using counterscreen technology and assays identifying redox compounds. Thirteen inhibitors with different chemical scaffold were identified, some showing activity at sub micromolar range and importantly none of them harboured the pyridone pharmacophore. Interestingly, several compounds did not behave as SAM competitor. When tested against four EZH2 mutants, Y641F, Y641N, A677G and A687V, some compounds showed better inhibitory activity to the mutant isoforms than to the WT isoform, suggesting the possibility to have higher therapeutic index in lymphomas harbouring these mutations. This work demonstrates that the screening of our proprietary library for EZH2 inhibitors resulted in the discovery of new classes of potent EZH2 inhibitors. Other HKMTs of therapeutic relevance in oncology and immunology are currently being screened for hit identification using our proprietary library.

192 Friday 21 November 2014 595 POSTER (Board P167) Improving specificity of epigenetic therapy through combined targeting of DNA and histone methylation T. Sato1 , M. Cesaroni1 , J. Jelinek1 , J.P. Issa1 . 1 Temple University, Fels Institute for Cancer Research and Molecular Biology, Philadelphia PA, USA Background: A major obstacle facing the use of epigenetic therapies such as HDAC inhibitors in cancer is the lack of specificity in reactivating gene expression, which could lead to unwanted side effects or treatment ineffectiveness. Combining the targeting of DNA and histone methylation is an unexplored and promising alternative to current therapies due to the role of both modifications in silencing of various tumor suppressor genes in cancer. Materials and Methods: qRT-PCR and RNA-seq experiments were performed on SW48 colon cancer cells treated with a DNA methyltransferase inhibitor (DAC) in combination with a LSD1 inhibitor (S2101), G9a inhibitor (UNC0638), EZH2 inhibitor (GSK343) or a HDAC inhibitor (Depsipeptide). DNA methylation of differentially regulated genes was analyzed by Digital Restriction Enzyme Analysis of Methylation (DREAM). Results: Initial qRT-PCR experiments on a set of 10 silenced tumor suppressor genes demonstrated that DAC in combination with S2101, UNC0638, or GSK343 reactivate distinct sets of genes, contrary to the non-specific global gene reactivation observed with the combination of DAC and Depsipeptide. Furthermore, genome-wide gene expression analysis demonstrated that S2101 (103 genes), UNC0638 (152 genes), and GSK343 (244 genes) by themselves induced limited gene upregulation in comparison to DAC (668 genes) and Depsipeptide (4388 genes). Interestingly, UNC0638 had a preference to upregulate genes that contain high levels of DNA methylation in their promoters, similar to DAC. DAC in combination with S2101, UNC0638, or GSK343 was able to synergistically or additively upregulate many DAC target genes (44, 128, and 187 genes respectively) compared to levels achieved by DAC alone, demonstrated limited overlap between the upregulated genes (only 4.42% of genes commonly upregulated), and formed distinct clusters in a hierarchical cluster analysis. Each DAC+inhibitor combination also induced a significant number of genes that were only upregulated with the combination therapy and not with DAC or inhibitor alone (583, 641, and 1018 genes respectively), and there was again limited overlap between the upregulated genes (only 7.84% of genes commonly upregulated). Gene ontology analysis demonstrated that genes induced uniquely by the inhibitor combinations belong in critical cancer regulation pathways such as cell proliferation, cell motility, and cell adhesion. Conclusions: These results demonstrate the ability to target specific, non-overlapping sets of genes through different combinations of DAC and histone methyltransferase or demethylase inhibitors, which is contrary to the nonspecific effects observed by traditional epigenetic therapies. Combined targeting of DNA and histone methylation is a promising novel epigenetic therapy that may allow for the targeting of specific tumor types based on their gene expression profiles.

Poster Session – New Therapies with Pleiotropic Activity

th

26 EORTC–NCI–AACR Symposium on Molecular Targets and Cancer Therapeutics

Late Breaking Abstracts

Plenary Session 3

Wednesday 19 November 2014

Late Breaking Abstracts, Wednesday 19 November 2014 195

13:15–15:20

PLENARY SESSION 2

Proffered Paper Session 1LBA LATE BREAKING ABSTRACT Clinical safety and activity in a phase I trial of AG-120, a first in class, selective, potent inhibitor of the IDH1-mutant protein, in patients with IDH1 mutant positive advanced hematologic malignancies D.A. Pollyea1 , S. de Botton, A.T. Fathi3 , E.M. Stein4 , M.S. Tallman4 , S. Agresta5 , C. Bowden5 , B. Fan5 , M. Prah5 , H. Yang5 , K. Yen5 , R.M. Stone6 . 1 University of Colorado Cancer Center, Aurora CO, USA; 2 Institut Gustave Roussy, Villejuif, France; 3 Massachusetts General Hospital Cancer Center, Boston MA, USA; 4 Memorial Sloan-Kettering Cancer Center, New York NY, USA; 5 Agios Pharmaceuticals, Cambridge MA, USA; 6 Dana-Farber Cancer Institute, Boston MA, USA This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

Wednesday 19 November 2014

16:00–17:50

PLENARY SESSION 3

Oncolytic Viruses 3LBA LATE BREAKING ABSTRACT Initial report of a first-in-human study of the first-in-class fatty acid synthase (FASN) inhibitor, TVB-2640 J. Infante1 , M. Patel2 , D. Von Hoff3 , A. Brenner4 , C. Rubino5 , W. McCulloch6 , V. Zhukova-Harrill7 , M. Parsey6 . 1 Sarah Cannon Research Institute, Tennessee Oncology, Tennessee, USA; 2 Sarah Cannon Research Institute, Florida Cancer Specialists, Sarasota, USA; 3 Scottsdale Healthcare Research Institute, Oncology, Scottsdale, USA; 4 Cancer Therapy & Research Center, Oncology, San Antonio, USA; 5 Institute for Clinical Pharmacodynamics, University at Buffalo, Buffalo, USA; 6 3-V Biosciences, Clinical Development, Menlo Park, USA; 7 Ockham Oncology, Medical Affairs, North Carolina, USA This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

2LBA LATE BREAKING ABSTRACT The identification of potent and selective inhibitors of oncogenes in medullary thyroid carcinoma and lung adenocarcinoma disease models S. Fritzl1 , H. Small1 , B. Acton1 , S. Holt1 , G. Hopkins1 , S. Jones1 , A. Jordan1 , N. March1 , R. Newton1 , I. Waddell1 , B. Waszkowycz1 , M. Watson1 , D. Ogilvie1 . 1 CRUK Manchester Institute, Drug Discovery Unit, Manchester, United Kingdom This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

196 Late Breaking Abstracts, Thursday 20 November 2014

Plenary Session 5

Thursday 20 November 2014

10:30–12:30

PLENARY SESSION 5

Epigenetic Targets

5LBA LATE BREAKING ABSTRACT Results of a first-in-man phase I trial assessing OTX015, an orally available BET-bromodomain (BRD) inhibitor, in advanced hematologic malignancies

4LBA LATE BREAKING ABSTRACT A phase 1 study of first-in-class microRNA-34 mimic, MRX34, in patients with hepatocellular carcinoma or advanced cancer with liver metastasis M. Beg1 , A. Brenner2 , J. Sachdev3 , M. Borad4 , J. Cortes5 , R. Tibes4 , Y. Kang6 , A. Bader7 , J. Stoudemire7 , S. Smith7 , S. Kim7 , D. Hong8 . 1 UT Southwestern Medical Center, Dallas, USA; 2 University of Texas Health Science Center San Antonio, Hematology Oncology, San Antonio, USA; 3 Scottsdale Healthcare Research Institute, Hematology/Oncology, Scottsdale, USA; 4 Mayo Clinic, Hematology/Oncology, Phoenix, USA; 5 University of Texas MD Anderson Cancer Center, Department of Leukemia, Houston, USA; 6 Asan Medical Center Korea, Oncology, Seoul, Korea; 7 Mirna Therapeutics Inc., Austin, USA; 8 University of Texas MD Anderson Cancer Center, Investigational Cancer Therapeutics, Houston, USA This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

A. Stathis1 , B. Quesnel2 , S. Amorim3 , C. Thieblemont3 , E. Zucca1 , E. Raffoux3 , H. Dombret3 , Y. Peng4 , A. Palumbo5 , N. Vey6 , X. Thomas7 , M. Michallet7 , C. Gomez-Roca8 , C. Recher8 , L. Karlin7 , K. Yee9 , K. Rezai10 , C. Preudhomme2 , T. Facon2 , P. Herait11 . 1 Istituto Oncologico della Svizzera Italiana, Lymphoma Unit, Bellinzona, Switzerland; 2 CHRU Lille, ˆ Dept Hematology, Lille, France; 3 Hopital Saint Louis, Dept Hematology, Paris, France; 4 Royal Marsden Hospital, Dept Hematology, Sutton, United Kingdom; 5 Ospedale San Giovanni Battista, Dept Hematology, Torino, ˆ Italy; 6 Institut Paoli Calmettes, Leukemia Unit, Marseille, France; 7 Hopital Lyon Sud, Dept Hematology, Pierre-Benite, France; 8 Oncopole, Dept Medical Oncology, Toulouse, France; 9 Princess Margaret Cancer Centre, Leukemia Unit, Toronto, Canada; 10 Institut Curie, Leukemia Unit, Paris, France; 11 Oncoethix, Medical Oncology, Lausanne, Switzerland

This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

Plenary Session 6

Late Breaking Abstracts, Thursday 20 November 2014 197

6LBA LATE BREAKING ABSTRACT Phase 1 first-in-human study of the enhancer of zeste-homolog 2 (EZH2) histone methyl transferase inhibitor E7438 as a single agent in patients with advanced solid tumors or B cell lymphoma 1

2

3

4

5

V. Ribrag , J.C. Soria , L. Reyderman , R. Chen , P. Salazar , N. Kumar6 , G. Kuznetsov6 , H. Keilhack7 , L.H. Ottesen8 , A. Italiano9 . 1 Institut Gustave Roussy, Laboratoire de recherche translationnelle, Villejuif, France; 2 Institut Gustave Roussy, Drug Development Department, Villejuif, France; 3 Eisai Inc, Clinical Pharmacology and Translational Medicine Oncology PCU, Woodcliff Lake, USA; 4 Eisai Inc, Biostatistics and Programming Oncology PCU, Woodcliff Lake, USA; 5 Eisai Ltd, Clinical Operations Oncology PCU, Hatfield, United Kingdom; 6 Eisai Inc, Biomarkers and Personalized Medicine, Andover, USA; 7 Epizyme, Biological Sciences, Cambridge, USA; 8 Eisai Ltd, Early Clinical Development Oncology PCU, Hatfield, United Kingdom; 9 Institute ´ Early Phase Trials and Sarcoma Unit, Bordeaux, France Bergonie, This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

Thursday 20 November 2014

13:30–15:35

PLENARY SESSION 6

Proffered Paper Session 7LBA LATE BREAKING ABSTRACT Phase 1 dose-expansion study of AMG 900, a pan-Aurora kinase inhibitor, in adult patients with advanced taxane-resistant solid tumors B. Markman1 , D. Mahadevan2 , S. Hurvitz3 , D. Kotasek4 , M. Shaheen5 , M. Carducci6 , O. Goodman7 , X. Jiang8 , V. Chow8 , G. Juan8 , G. Friberg8 , E. Gamelin8 , J. Desai9 . 1 Monash Health, Melbourne, Australia; 2 West Cancer Center/UTHSC, Memphis, USA; 3 University of California Los Angeles, Los Angeles, USA; 4 Ashford Cancer Centre Research, South Australia, Australia; 5 University of New Mexico Cancer Center, Albuquerque, USA; 6 Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Baltimore, USA; 7 Comprehensive Cancer Centers of Nevada, Las Vegas, USA; 8 Amgen Inc., Thousand Oaks, USA; 9 Royal Melbourne Hospital, Melbourne, Australia This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

198 Late Breaking Abstracts, Friday 20 November 2014

Plenary Session 8

Friday 20 November 2014

11:00–13:00

PLENARY SESSION 8

Targeting RAS and Other Driver Oncogenes

Thursday 20 November 2014

16:00–17:50

PLENARY SESSION 7

Novel Mechanisms for Drug Resistance 8LBA LATE BREAKING ABSTRACT Activity of the PARP inhibitor olaparib in ATM-deficient gastric cancer: from preclinical models to the clinic D. Hodgson1 , H. Mason1 , L. Oplustilova1 , C. Harbron1 , X. Yin2 , S.A. Im3 , H. Jones1 , L. Zhongwu1 , B. Dougherty1 , M. McLoughlin1 , A. Dickinson1 , A. Fielding1 , J. Robertson1 , W.H. Kim3 , C. Womack1 , Y. Gu2 , Y.J. Bang3 , A. Lau1 , J.C. Barrett1 , M.J. O’Connor1 . 1 AstraZeneca, Macclesfield Cheshire, United Kingdom; 2 AstraZeneca, Shanghai, China; 3 Cancer Research Institute, Seoul, Korea This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

9LBA LATE BREAKING ABSTRACT Antitumor activity of ASP8273, an irreversible mutant selective EGFR-TKI, in NSCLC patients with tumors harboring EGFR activating mutations and T790M resistance mutation H. Murakami1 , H. Nokihara2 , T. Shimizu3 , T. Seto4 , A. Keating5 , A. Krivoshik5 , K. Uegaki6 , S. Morita7 , K. Nakagawa3 , M. Fukuoka8 . 1 Shizuoka Cancer Center, Thoracic Oncology, Sunto-gun, Japan; 2 National Cancer Center Hospital, Thoracic Oncology, Chuo-ku, Japan; 3 Kinki University Hospital, Medical Oncology, Osakasayama, Japan; 4 National Hospital Organization Kyushu Cancer Center, Thoracic Oncology, Minami-ku, Japan; 5 Astellas Pharma Global Development Inc., Northbrook, USA; 6 Astellas Pharma Inc., Clinical Development 3, Chuo-ku, Japan; 7 Kyoto University Graduate School of Medicine, Department of Biomedical Statistics and Bioinformatics, Kyoto, Japan; 8 Izumi Municipal Hospital, Izumi-city, Japan This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

Plenary Session 8 10LBA LATE BREAKING ABSTRACT Interim phase 2 results of study CO-1686-008: A phase 1/2 study of the irreversible, mutant selective, EGFR inhibitor rociletinib (CO-1686) in patients with advanced non small cell lung cancer J. Soria1 , L.V. Sequist2 , J.W. Goldman3 , H.A. Wakelee4 , S.M. Gadgeel5 , A. Varga1 , H.A. Yu6 , B.J. Solomon7 , S.H. Ou8 , V. Papadimitrakopoulou9 , G.R. Oxnard10 , L. Horn11 , R. Dziadziuszko12 , B. Chao13 , A.I. Spira14 , S. Liu15 , T. Mekhail16 , S. Matheny17 , J. Litten17 , R.D. Camidge18 . 1 Institut Gustave Roussy, Villejuif, France; 2 Massachusetts General Hospital, Boston, USA; 3 UCLA Santa Monica Hematology-Oncology, Santa Monica, USA; 4 Stanford Cancer Institute, Stanford, USA; 5 Karmanos Cancer Institute Wayne State University, Detroit, USA; 6 Memorial Sloan-Kettering Cancer Center, New York, USA; 7 Peter MacCallum Cancer Centre, Melbourne, Australia; 8 University of California, Irvine, USA; 9 The University of Texas MD Anderson Cancer Center, Houston, USA; 10 Dana Farber Cancer Institute, Boston, USA; 11 Vanderbilt-Ingram Cancer Center, Nashville, USA; 12 Medical University of Gdansk, Department of Oncology and Radiotherapy, Gdansk, Poland; 13 The Ohio State University, Division of Medical Oncology, Columbus, USA; 14 Virginia Cancer Specialists, Fairfax, USA; 15 Georgetown University Hospital, Washington, USA; 16 Florida Hospital, Orlando, USA; 17 Clovis Oncology, Boulder, USA; 18 University of Colorado Cancer Center, Aurora, USA This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

Late Breaking Abstracts, Friday 20 November 2014 199 11LBA LATE BREAKING ABSTRACT Phase I study of the selective BRAFV600 inhibitor encorafenib (LGX818) combined with cetuximab and with or without the a-specific PI3K inhibitor alpelisib (BYL719) in patients with advanced BRAF mutant colorectal cancer J. Tabernero1 , R. van Geel2 , J.C. Bendell3 , A. Spreafico4 , M. Schuler5 , T. Yoshino6 , J.P. Delord7 , Y. Yamada8 , M.P. Lolkema9 , J.E. Faris10 , F.A.L.M. Eskens11 , S. Sharma12 , R. Yaeger13 , H.J. Lenz14 , Z. Wainberg15 , E. Avsar16 , A. Chatterjee16 , S. Jaeger17 , T. Demuth18 , J.H.M. Schellens2 . 1 ´ University Hospital, Medical Oncology Department, Vall D’Hebron Barcelona, Spain; 2 The Netherlands Cancer Institute, Department of Medical Oncology, Amsterdam, Netherlands; 3 Sarah Cannon Research Institute, Department of Oncology, Nashville TN, USA; 4 Princess Margaret Cancer Centre, Department of Oncology, Toronto, Canada; 5 West German Cancer Center University Hospital Essen University Duisburg-Essen, Department of Medical Oncology, Essen, Germany; 6 National Cancer Center Hospital East, Department of Gastroenterology and Gastrointestinal Oncology, Chiba, Japan; 7 Institut Claudius Regaud, ´ ´ Departement Medecine, Toulouse, France; 8 National Cancer Center Hospital, Division of Gastrointestinal Oncology, Tokyo, Japan; 9 University Medical Center Utrecht, Department of Medical Oncology, Utrecht, Netherlands; 10 Massachusetts General Hospital, Department of Medicine, Boston MA, USA; 11 Erasmus MC Cancer Institute, Department of Medical Oncology, Rotterdam, Netherlands; 12 Huntsman Cancer Institute University of Utah, Department of Internal Medicine, Salt Lake City UT, USA; 13 Memorial Sloan-Kettering Cancer Center, Department of Medicine, New York NY, USA; 14 Keck School of Medicine at the University of Southern California, Department of Medicine, Los Angeles CA, USA; 15 UCLA Medical Center, Department of Medicine, Santa Monica CA, USA; 16 Novartis Pharmaceutical Corporation, Oncology Translational Medicine, East Hanover NJ, USA; 17 Novartis Institutes for Biomedical Research, Oncology Translational Medicine, Cambridge MA, USA; 18 Novartis Pharma AG, Oncology Translational Medicine, Basel, Switzerland This abstract is part of the media programme and is embargoed until the day of presentation, when it will be published online at 08:00.

201

Presenting Authors List A Abe, T., 120 (373) Abi-Habib, R., 126 (394) Addou Klouche, L., 99 (303) Aguirre, E., 64 (198) Agullo-Ortu ´ no, ˜ M.T., 111 (344) Al-Awar, R., 95 (287) Al-Zaubai, N., 58 (176) Ali, M., 75 (221) Ali, S., 132 (414) Alonso-Colmenar, L.M., 187 (579) Alvarado, D., 130 (407) Amit, M., 100 (305) An, C., 49 (143) An, E., 159 (489) Anderson, D.J., 180 (555) Andrews, D., 91 (275) Ang, A.L., 161 (496) Angibaud, P., 115 (356) Aracil, M., 17 (35) Astorgues-Xerri, L., 183 (567) Augustin, E., 26 (65) Austin, L., 37 (103) Avadisian, M., 93 (282) Aviles, P., 23 (55) Aviles, P.M., 164 (502) Azaro, A., 124 (386)

B Bacus, S., 74 (218) Bai, L., 109 (338) Bailly, C., 40 (113) Bajrami, I., 50 (147) Balani, S.K., 54 (162) Ballesteros, J., 77 (230), 136 (427) Baloglu, E., 156 (480) Bando, H., 36 (101) Barault, L., 23 (54) Bardia, A., 163 (500) Barlaam, B., 90 (272) Barradas, M., 128 (402) Bassissi, F., 183 (566) Batey, M., 53 (158) Baugher, M., 9 (8) Baulies, S., 85 (254) Baxter, M., 18 (40) Beeram, M., 107 (329) Beg, M., 196 (4LBA) Beloueche-Babari, M., 175 (540) Bergamaschi, A., 158 (485) Bernard, B., 90 (271)

Bertoni, F., 184 (568) Bhattacharya, B., 29 (74) Bignall, G., 56 (167) Bignan, G., 141 (430) Bissery, M., 45 (130) Blagden, S., 7 (3) Borin, T., 169 (522) Bouhana, K., 125 (391) Boulos, N., 63 (193) Boyko, A., 14 (25) Bradley, B., 125 (390) Brameld, K., 157 (483) Bray, G.L., 179 (551) Briest, F., 149 (455) Broudy, T., 59 (178) Brown, A.C.N., 41 (117) Brown, B.D., 115 (358) Brown, R., 179 (554) Brunton, V.G., 160 (491) Brynzak, E., 53 (156) Buchanan, S., 165 (507) Bugano, D., 79 (237) Bunz, F., 157 (482) Bupathi, M., 136 (425) Burdelya, L., 40 (114)

C Caffo, N., 38 (105) Cairo, S., 25 (61), 58 (174) Calvo Perez, ´ A., 169 (521) Campagne, C., 81 (241) Campiglio, M., 56 (168) Cano, C., 141 (429) Cardnell, R.J., 81 (243), 124 (388) Carmona Sanz, F., 27 (69) Carneiro-Lobo, T., 103 (317) Carpinelli, P., 16 (31), 168 (517) Castanon, E., 65 (199) Castel, P., 29 (75) Castro, C., 54 (161) Cautain, B., 24 (59) Cavazzoni, A., 129 (404) Cecchi, F., 175 (537) Cerezo, A., 110 (339) Chalugun, P., 36 (98) Chang, N., 46 (131) Charych, D., 16 (33) Chen, H., 22 (53) Chen, J., 98 (297) Chen, Z.X., 135 (423) Chenchik, A., 60 (184)

Cheng, H., 108 (335) Chimmanamada, D., 87 (260) Chiu, J.W., 165 (508) Chmielecki, J., 84 (250) Choi, M., 76 (224) Chomej, K., 184 (570) Chowbay, B., 32 (86) Chu, P., 118 (366) Civenni, G., 108 (333), 159 (487) Claret, F., 155 (477) Clarke, N., 98 (300) Clarkson, R., 122 (379) Clausen, M., 145 (441) Coimbra de Sousa, S., 59 (179) Corcoran, R., 141 (428) Courtin, A., 168 (518) Covre, A., 47 (134) Crochiere, M., 160 (492) Croset, A., 48 (139) Cruz, C., 82 (244) Cubillo, A., 55 (166) Cumaraswamy, A.A., 94 (285) Cuppens, T., 11 (15)

D da Silva, S.R., 94 (283) Dallavalle, C., 180 (556) Damia, G., 110 (340) D’Arcangelo, M., 114 (353) Dasgupta, T., 57 (172) Daud, A., 48 (140) Davies, B.R., 150 (457) De Angulo, A., 49 (142) de Jonge, M., 121 (378) de Lint, K., 31 (81) Denmeade, S.R., 143 (434) Depil, S., 118 (368) Deshane, J., 43 (123) Deuker, M., 100 (304) Deutsch, E., 74 (219) Devi, G.R., 31 (80) Dey, N., 128 (399) Dib, C., 96 (291) Dilruba, S., 34 (92) Dittamore, R., 45 (129), 127 (397) Doi, T., 72 (213) Dornetshuber-Fleiss, R., 16 (30) Dovat, S., 134 (419) Dreyling, M., 162 (499) Dubbink, E., 53 (157) Dunn, B., 75 (222)

202 Duval, D.L., 11 (13)

E Earp, S., 164 (503) Edwards, Z.C., 29 (76) Efstathiou, A., 106 (328) Ekmekcioglu, S., 150 (460) Elmquist, W., 86 (259) Elvin, J.A., 104 (319) Enderle, D., 102 (313), 149 (454) Engeland, C.E., 48 (137) Ethier, S., 188 (583) Eun, Y., 50 (144) Evans, D., 30 (78)

F Fang, C., 88 (265) Farras, R., 115 (357) Feng, Y., 81 (242), 179 (552) Ferguson, P., 21 (49) Fernandez de Farias, C., 26 (64) Figg, W., 13 (21) Figueroa, A., 17 (37) Finetti, M.A., 132 (413) Firestein, R., 179 (553) Fouani, L., 175 (539) Frattini, M.G., 96 (293) Freres, P., 53 (159) Friedman, J., 154 (471) Fromond, C., 180 (557), 191 (594) Fujita, H., 176 (541)

G Gabrielli, B., 97 (294) Gallach, S., 177 (545) Gameiro, S., 101 (308) Garcia-Fernandez, L.F., 20 (47), 24 (57, 58) Garon, E.G., 44 (127) Gelato, K.A., 185 (573) Geles, K.G., 145 (443) Gerami-Moayed, N., 52 (152) Gerber, D., 84 (253) Glas, A., 173 (532) Goldman, J.W., 162 (498) Goldstein, R., 182 (563) Gomori, A., 167 (515) Goncalves, A., 189 (586) Gonzalez-Cao, M., 114 (354) Gopas, J., 178 (550) Goschl, ¨ S., 22 (52) Graham, D., 114 (352) Grifantini, R., 41 (116), 178 (549) Guichard, S.M., 123 (383) Gupta, I., 119 (370)

H Ha, J.M., 79 (236) Haber, M., 135 (422) Halonen, P., 59 (177) Haluska, P., 72 (214) Halytskiy, V., 174 (536) Hardcastle, I.R., 91 (276) Harmon, T., 89 (267) Harms, B.D., 144 (438)

Presenting Authors List Hawe, D., 64 (197) Hawtin, R.E., 39 (109) He, S., 188 (585) Henke, E., 89 (266) Hernandez, F., 79 (234) Hernandez-Bernal, ´ F., 38 (107) Heuer, T.S., 190 (591) Higgins, B., 106 (327) Hoffman, R.M., 96 (292), 111 (343), 121 (376) Hoffman-Luca, G., 32 (87) Holbeck, S., 36 (99) Holmstrom, ¨ T., 142 (432) Hong, J.H., 116 (360) Hsieh, H.P., 92 (279) Hung, L., 19 (43) Hunter, F.W., 66 (204)

I Ikram, F., 130 (409) Imoto, M., 176 (543) Inazu, M., 58 (175) Infante, J., 195 (3LBA) Innocenti, F., 26 (66), 55 (163) Inoue, S., 101 (309) Ishida, K., 13 (22) Ishii, Y., 78 (231), 181 (561)

J Jacobsen, K., 30 (79) Janku, F., 56 (169), 57 (170) Jastrzebski, K., 34 (91) Jiang, J., 52 (154) Jimenez, A.M., 60 (183) Jimeno, J., 156 (478) Jivrajani, M., 85 (256) Johnson, S., 80 (238) Jonasch, E., 172 (530) Jones, R., 125 (392) Jordan, A., 94 (284), 195 (2LBA) Josse, ´ R., 83 (248) Juan, G., 40 (112) Jung, K.H., 42 (119) Jung, S., 107 (331)

K Kang, W.Y., 23 (56) Kapoun, A., 152 (465) Kashyap, T., 83 (247) Kato, M., 128 (401) Kawada, M., 175 (538) Kawamura, T., 14 (26) Kelley, M.C., 33 (90) Kim, C., 98 (298) Kim, I., 166 (510) Kim, J., 45 (128), 173 (533) Kim, M., 117 (363) Kim, S., 65 (201) Kirkpatrick, L., 160 (490) Klinghammer, K., 10 (10) Klinguer-Hamour, C., 113 (351) Koh, S.B., 16 (32) Kolev, V., 144 (439) Komai, K., 51 (150) Korn, R., 61 (186) Korn, W., 148 (451)

Koromilas, A., 148 (452) Kounnis, V., 86 (258) Krishnan, B., 98 (299) Kumari, R., 60 (185) Kuntz, K., 92 (277) Kurmasheva, R., 131 (410, 412) Kuroda, M., 105 (323) Kuwada, S., 186 (576) Kwon, M., 119 (371)

L Larsen, A.K., 130 (406) LeBeau, A., 145 (442) Lee, C.K., 20 (44) Lee, K., 153 (468) Lee, T.C., 31 (82) Lewis, J.D., 187 (578) Li, G., 101 (310) Li, H., 112 (348) Li, W., 95 (288) Lih, C., 78 (233) Lin, C., 17 (34) Littlefield, B.A., 20 (46) Liu, G., 60 (181) Lockley, M., 38 (106) Lokiec, F., 142 (431) Lorens, J., 117 (362) LoRusso, P., 71 (210) Lu, J., 89 (268)

M Maag, D., 82 (246) Maddalena, F., 60 (182) Malouf, G., 188 (582) Mansfield, J.R., 8 (6) Mantaj, J., 19 (42) Marchlik, E., 155 (475) Markman, B., 197 (7LBA) Martin, M., 106 (325) Martinez, J., 111 (345) Martinez, J.F., 108 (334) Martini, J.F., 116 (359) Marusiak, A., 164 (504) Massaoka, M.H., 43 (124) Maxuitenko, Y.Y., 107 (330) Mazerska, Z., 27 (67) Mazzocca, A., 161 (494) McCoon, P., 73 (217), 163 (501) McDaniel, A.S., 61 (187) McLure, K.G., 181 (560) Meetze, K., 156 (479) Merchant, M., 124 (387) Meric-Bernstam, F., 3 (1) Mertz, J., 187 (581) Middleton, M., 117 (364) Milhollen, M., 186 (577) Miller, D., 120 (372) Miller, R.E., 103 (318) Minamiguchi, K., 103 (315) Mirabile, I., 172 (529) Mirjolet, C., 67 (207) Mishima, Y., 166 (509) Mittal, D., 40 (115) Mittempergher, L., 54 (160) Mohammad, H., 72 (212) Molina-Vila, M., 167 (514)

Presenting Authors List Monks, A., 57 (171) Moore, P., 48 (138) Moreno-Acosta, P.M.A., 119 (369) Morgan, S.R., 126 (395) Morosini, D., 132 (415) Morris, J., 89 (269) Mortensen, D.S., 150 (459) Muders, M., 64 (196) Mulet Margalef, N., 110 (341) Munoz ˜ Hidalgo, L., 51 (151) Murakami, H., 198 (9LBA) Murphy, D., 143 (436) Murphy, S., 79 (235)

Poole, J.C., 51 (149) Poon, D.C.S., 93 (281) Porro, M., 185 (572) Pors, K., 95 (289) Prahallad, A., 151 (463) Prell, R., 136 (424) Pruschy, M.N., 65 (202)

N

Rainard, J., 123 (384) Ramachandra, M., 104 (321) Ramanathan, R.K., 87 (261) Rapisarda, A., 147 (450) Ratain, M.J., 13 (23) Raymon, H., 170 (523) Raynaud, F.I., 148 (453) Reed, B., 121 (377) Renner, O., 118 (365) Ribrag, V., 197 (6LBA) Richardson, K., 76 (227) Richter, G.H.S., 134 (420) Riggins, G., 171 (528) Riveiro, M., 182 (564), 183 (565) Robillard, L., 83 (249) Rodilla Mart´ın, A., 15 (28) Rolfo, C., 65 (200), 96 (290), 97 (295), 112 (347) Romano, M., 21 (48) Ronca, R., 100 (307) Rooney, C., 169 (520) Rose, A., 22 (51) Rosen, L., 122 (382) Rosen, O., 115 (355) Rozga, P., 118 (367) Ruan, Y., 42 (121) Russell, P., 171 (527)

Naito, M., 52 (155) Nakaigawa, N., 154 (473) Nakatsuru, Y., 127 (398) Narendran, A., 131 (411), 189 (588) Narla, G., 158 (486), 178 (548) Natrajan, R., 186 (575) Ner-Kluza, J., 12 (17) Ng, K.W., 35 (95) Nielsen, S., 184 (569)

O O’Connor, M.J., 198 (8LBA) Odore, E., 189 (587) Ogo, N., 91 (274) Oguchi, K., 15 (27) Ohashi, Y., 185 (574) Ohkubo, S., 191 (592) Ono, M., 32 (84) Osman, A., 109 (337) Ouafik, L., 153 (469) Ozawa, Y., 17 (36) Ozorio dos Santos, E.O., 129 (405)

P Pachter, J., 99 (302) Packer, L., 35 (94) Paiva, S., 95 (286) Pan, J., 28 (70) Pandha, H., 41 (118) Papadopoulos, K., 125 (389) Park, C.H., 126 (393) Park, E.K., 153 (470) Park, Y.M., 24 (60), 25 (62) Patierno, S., 105 (322) Patnaik, A., 7 (2) Pawlak, S.D., 152 (467) Paz, K., 63 (195) Pecqueur, J., 133 (417) Pesonen, S., 47 (136) Pessoa, C., 87 (262) Pettitt, S.J., 33 (88) Pfister, S.X., 71 (211) Phillips, D.C., 130 (408) Pieczykolan, J.S., 47 (135) Pisano, C., 62 (189) Pitt, C., 113 (350) Poleszak, K., 169 (519) Pollock, P., 122 (381) Pollyea, D.A., 195 (1LBA) Pommier, Y., 84 (252) Ponz-Sarvise, M., 63 (194)

Q Quan, Y., 92 (278) Quetglas, I.M., 152 (466)

R

S Sabzichi, M., 28 (72) Sachdev, J., 165 (506) Saha, D., 68 (208) Sahay, D., 182 (562) Sakoff, J., 36 (100) Sala, G., 164 (505) Saleh, M., 104 (320) Saletta, F., 135 (421) Sandy, P., 31 (83) Sanjiv, K., 81 (240) Sarkaria, J., 10 (12) Sato, M., 30 (77) Sato, T., 192 (595) Savage, R., 50 (145) Sawada, T., 129 (403) Schaider, H., 34 (93) Schalck, A., 102 (314) Schuessler-Lenz, M., 44 (125) Schuler, ¨ J., 10 (11), 11 (16) Shah, M.H., 151 (461) Shao, J., 39 (110) Sharma, A., 67 (206) Shaw, K., 159 (488) Sheng, X., 161 (495)

203 Shi, Q., 11 (14), 12 (19) Shi, W., 49 (141), 73 (216) Shiah, S.G., 147 (449) Shimamura, T., 191 (593) Shimizu, T., 97 (296) Shingler, W., 44 (126) Shoemaker, R.H., 76 (225) Simon, B., 177 (546) Simonelli, M., 176 (542) Siziopikou, K., 14 (24) Skrzypek, K., 170 (524) Sloane, R., 144 (437) Smith, P., 149 (456) Sobrevals Amieva, L., 87 (263) Sootome, H., 142 (433) Soria, J., 199 (10LBA) Sos, M., 28 (73) Sowa, T., 188 (584) Sridhar, S., 116 (361) Stathis, A., 196 (5LBA) Steele, V.E., 76 (226) Steinberg, G., 67 (205) Stone, E., 181 (559) Strauss, S.J., 82 (245) Su, D., 8 (5) Su, T.L., 20 (45) Sukumaran, S., 71 (209) Sullivan, D., 74 (220) Summa, J., 19 (41) Sun, L., 18 (38) Suominen, M.I., 25 (63) Swisher, E., 73 (215)

T Tabernero, J., 199 (11LBA) Takahashi, T., 127 (396) Talasaz, A., 151 (462) Tang, K., 33 (89) Taplin, M.E., 8 (4) Tedesco, D., 52 (153) Tellez Gabriel, M., 62 (191) Terui, Y., 136 (426) Tervonen, T., 57 (173) Testoni, E., 167 (512) Thamm, D., 9 (9) Thatcher, G., 103 (316) Thatte, J., 50 (146) Thibault, A., 39 (108) Thomas, A., 102 (312) Tognolini, M., 90 (270) Tolcher, A., 111 (342) Tomek, P., 91 (273) Tomilo, M., 166 (511) Toska, E., 28 (71) Toth, R., 55 (165) Tovar, V., 75 (223) Traore, T., 85 (255) Trapani, F., 88 (264) Tsimafeyeu, I., 155 (476) Tsuji, T., 100 (306) Tu, Y., 144 (440)

U Uboldi, S., 190 (590) Uitdehaag, J.C.M., 113 (349) Ulivi, P.U., 168 (516)

204 Unger, F.T., 151 (464) Uno, Y., 112 (346) Uso, ´ M., 43 (122), 46 (133)

V Van den Bossche, J., 27 (68) van der Meel, R., 147 (448) van ’t Veer, L., 154 (472) van Wageningen, S., 51 (148) Varasi, M., 185 (571) Varda, J., 42 (120) Vasilevskaya, I., 154 (474) Vaslin Chessex, A., 177 (547) Vazquez, ´ R., 187 (580) Vecchione, L., 61 (188) Vena, F., 146 (445) Vialard, J., 128 (400) Viswanadha, S., 108 (332) Voloshanenko, O., 32 (85) Vuaroqueaux, V., 9 (7), 62 (192)

W Wainberg, Z., 171 (526) Wan, X., 101 (311)

Presenting Authors List Wang, G., 162 (497) Wang, H., 176 (544) Wang, J., 143 (435) Wang, X., 106 (326) Ward, G., 122 (380) Warnders, F.J., 174 (535) Watari, K., 190 (589) Wei, Y.M., 93 (280) Weitsman, G., 77 (228) Wendel, H., 181 (558) Weng, Z., 120 (374) Wick, M.J., 12 (18), 35 (96, 97), 37 (102), 156 (481) Wilcoxen, K.M., 80 (239) Willetts, L., 147 (447) Williams, K., 170 (525) Williams, M., 78 (232) Wisell, J., 59 (180) Wong, K., 120 (375) Wood, K., 158 (484) Wright, K.D., 133 (416, 418) Wu, H.C., 86 (257) Wu, Y., 46 (132)

X Xu, Q., 146 (446)

Y Yamada, T., 150 (458) Yang, S., 55 (164) Yang, S.X., 77 (229) Yano, W., 15 (29) Yates, J., 84 (251), 99 (301) Ye, X., 123 (385) Yu, Y., 167 (513), 172 (531) Yun, S., 174 (534)

Z Zaman, G., 37 (104) Zanna, C., 146 (444) Zapata, J.M., 18 (39) Zhao, Z., 66 (203) Zhou, X., 21 (50), 105 (324), 109 (336) Zhu, Y., 39 (111) Zimon, D., 62 (190) Zoni, E., 12 (20) Zopf, C.J., 161 (493)

205

Author Index A Abbadessa, G., 104 (320) Abbasian, M., 150 (459) Abe, T., 120 (373) Abes, R., 118 (368) Abi-Habib, R., 126 (394) Ablack, A.L., 187 (578) Abrams, M., 115 (358) Abu-Yousif, A., 144 (438) Ackermann, S., 130 (409) Acosta, G., 156 (478) Acquaviva, J., 188 (585) Acton, B., 94 (284), 195 (2LBA) Adachi, Y., 17 (36) Adamo, B., 124 (386) Adamus, T., 170 (524) Adarbes, V., 191 (594) Addou Klouche, L., 99 (303) Adjei, A., 107 (329) Adler, A., 179 (553) Admunson, K., 80 (239) Adurthi, S., 40 (113) Aftimos, P., 39 (108) Agarwal, S., 72 (214), 80 (239) Agasti, S., 54 (161) Agelopoulos, K., 134 (420) Aggarwal, C., 44 (127) Agresta, S., 195 (1LBA) Agresti, R., 56 (168) Agudo-Lopez, ´ A., 111 (344) Aguilar, A., 109 (338), 176 (544) Aguilera, C., 110 (339) Aguirre, E., 64 (198) Agullo-Ortu ´ no, ˜ M.T., 111 (344) Ahmed, L., 117 (362) Ahmed, S., 104 (321) Ahmet, J., 123 (384) Ahn, M.J., 44 (127) Ahronian, L.G., 141 (428) Aida, H., 97 (296) Ainscough, B., 29 (75) Aird, D., 149 (456) Ajaz, M., 41 (118) Akatsuka, A., 185 (574) Akbay, E., 191 (593) Akinseye, C., 98 (300) Akiyama, Y., 129 (403) Akla, B., 113 (351) Al-Awar, R., 95 (287) Al-Awar, R.S., 95 (287) Al-Kadhimi, K., 169 (520)

Al-Zaubai, N., 58 (176) Albarran, M.I., 64 (198), 118 (365) Albericio, F., 156 (478) Albers, A.E., 10 (10) Albino, D., 159 (487), 180 (556) Alderson, R., 48 (138) Alexander, R., 141 (430) Alfieri, R., 129 (404) Alfredo Romero-Rojas, A.R.R., 119 (369) AlHilli, M., 72 (214) Alhoniemi, E., 25 (63) Ali, M., 75 (221) Ali, S., 132 (414) Ali, S.M., 84 (250), 104 (319), 132 (415) Allali-Hassani, A., 95 (287) Allard, B., 40 (115) Allensworth, J.L., 31 (80), 155 (477) Alonso-Colmenar, L.M., 187 (579) Alsina, M., 124 (386) Alvarado, D., 130 (407) Alvarez, C., 114 (354) Alvarez, R., 3 (1) Alvarez Goyanes, R.I., 169 (521) Alzani, R., 16 (31) Amant, F., 11 (15) Amato, C.M., 59 (180) Amato, K., 98 (297) Amatu, A., 23 (54) Amboldi, N., 168 (517) Ames, W., 111 (342) Amick, J., 130 (407) Amit, M., 100 (305) Amorim, S., 196 (5LBA) An, C., 49 (143) An, E., 159 (489) An, J.Y., 20 (44) Anandra, S., 7 (3) Anderson, A., 40 (112) Anderson, A.A., 161 (496) Anderson, D., 101 (310) Anderson, D.J., 180 (555) Andre, ´ T., 130 (406) Andrews, D., 91 (275) Andrews, D.W., 49 (141), 73 (216) Ang, A.L., 161 (496) Ang, J.E., 148 (453) Angevin, E.A., 74 (219) Angibaud, P., 115 (356), 128 (400) Anil, B., 91 (276) Anil, J., 104 (321) Annels, N., 41 (118) Annis, M.G., 22 (51)

Annunziata, C., 117 (363) Anscombe, E., 106 (325) Ansell, P., 111 (342) Anthony, S.P., 165 (506) Anthony, T., 142 (432) Anton, P., 82 (244) Anton-Aparicio, ´ L.M., 17 (37) Antonarakis, E.S., 8 (4), 143 (434) Antonio Huertas Salgado, A.H.S., 119 (369) Antony, J., 57 (172) Aoki, M.N., 129 (405) Aoyagi, H., 103 (315) Aoyagi, Y., 128 (401) Aoyama, A., 188 (584) Apuy, J., 150 (459) Arabnia, A., 104 (320) Aracil, M., 17 (35) Arbab, A.S., 169 (522) Ardiani, A., 101 (308) Ardini, E., 101 (310) Ardito-Abraham, C., 63 (194) Argiles, G., 124 (386) Arkenau, H.T., 44 (127), 126 (395), 165 (506) Armengol, C., 25 (61) Armer, R., 123 (384) Armstrong, G.T., 133 (416) Armstrong, S., 72 (212) Arnieri, B., 122 (382) Arnoldussen, Y.J., 161 (495) Arriaga, Y., 84 (253) Arribas, J., 82 (244) Arroyo, E., 77 (230), 136 (427) Arvan, R., 115 (358) Asad, Y., 148 (453) Asai, A., 91 (274) Asano, M., 17 (36), 20 (46) Ashton, S., 99 (301) Ashworth, A., 33 (88), 50 (147), 103 (318) Astorgues-Xerri, L., 153 (469), 182 (564), 183 (565, 567), 187 (580), 189 (587) Atmodimedjo, P.N., 53 (157) Attwell, S., 181 (560) Au, G., 41 (118) Au-Yeung, S.C.F., 93 (280, 281) Auer-Grzesiak, I., 131 (411) Augustin, E., 26 (65), 27 (67) Augustin, I., 32 (85) Auman, J.T., 55 (163) Austin, L., 37 (103) Avadisian, M., 93 (282)

206 Avery, T., 37 (103) Avila, S., 24 (57) Aviles, P., 23 (55) Aviles, P.M., 164 (502) Aviles, ´ P.M., 82 (244) Avitahl-Curtis, N., 115 (358) Avsar, E., 199 (11LBA) Awada, A., 39 (108) Ayadi, M., 130 (406) ´ Ayala Avila, M., 38 (107) Ayoub, N., 35 (95) Azab, M., 47 (134) Azaro, A., 124 (386) Azevedo, R.A., 26 (64), 43 (124) Azuma, K., 32 (84)

B Baas, P., 99 (302) Babu, G., 108 (332) Bachmann, F., 65 (202) Back, J., 48 (139) Backman, C., 47 (136) Bacon-Trusk, P., 74 (218) Bacus, S., 74 (218) Bader, A., 196 (4LBA) Bae, J.S., 79 (236) Baer, B., 125 (391) Baggertman, G., 96 (290) Bagley, R.G., 96 (291) Bahamon, ´ B., 78 (231) Bai, L., 109 (338) Bai, R.Y., 157 (482) Bai, S., 172 (530) Baia, G., 63 (195) Baik, H., 164 (504) Bailey, A.M., 159 (488) Bailey, S., 132 (413) Bailey, S.T., 98 (299) Bailly, C., 40 (113), 113 (351) Bajrami, I., 33 (88), 50 (147), 103 (318) Baker, J., 158 (485) Baker, L., 63 (194) Bakken, K., 86 (259) Balani, S., 161 (493) Balani, S.K., 54 (162) Balasubramanian, S., 125 (390) Baldwin, A.S., 103 (317) Baldwin, P., 116 (361) Bales, N., 127 (397) Balheda, R., 74 (219) Ball, C.R., 32 (85) Ballard, P., 99 (301), 125 (392) Ballesteros, J., 77 (230), 136 (427) Ballinari, D., 16 (31), 168 (517) Balmana, ˜ J., 80 (238), 82 (244) Balmanoukian, A.S., 44 (127) Baloglu, E., 83 (247), 156 (480), 160 (492) Balsubramanian, S., 84 (250) Baltes, S., 9 (7) Bancerz, M., 94 (283), 95 (286) Bandla, S., 61 (187) Bando, H., 36 (101) Bandyopadhyay, S., 28 (73) Banerji, U., 148 (453) Banfi, P., 101 (310) Bang, S.Y., 153 (470)

Author Index Bang, Y.J., 198 (8LBA) Banka, D., 171 (527) Bankhead III, A., 166 (511) Bao, L., 123 (383) Bar Ad, V., 73 (216) Barault, L., 23 (54) Barbarino, A., 62 (189) Barber, P.R., 77 (228) Barcaroli, D., 164 (505) Bardelli, A., 23 (54) Bardia, A., 163 (500) Bargfrede, M., 21 (50) Bargfrede, M.J., 105 (324) Barker, D., 44 (126) Barlaam, B., 90 (272) Barnes, J., 84 (251) Barocelli, E., 90 (270) Barradas, M., 128 (402) Barrett, C., 169 (520) Barrett, J.C., 198 (8LBA) Barrett, L., 141 (429) Barrett, S., 158 (484) Barsyte-Lovejoy, D., 95 (287) Barth, M., 180 (557) Bartusik, E., 26 (65) Basavaraju, A., 104 (321) Baselga, J., 27 (69), 28 (71), 29 (75), 80 (238), 82 (244) Basseres, D.S., 103 (317) Basseres, ` D.S., 129 (405) Bassissi, F., 183 (566) Baston, E., 44 (126) Basturk, O., 84 (250) Bates, R., 188 (585) Batey, M., 53 (158) Bauer, T., 21 (50) Bauer, T.M., 109 (336) Baugher, M., 9 (8) Baulies, S., 85 (254), 167 (514) Baumhoer, D., 134 (420) Bawn, R., 141 (429) Baxter, M., 18 (40) Bayever, E., 87 (261) Beadle, J., 41 (117) Bean, S., 136 (425) Beasley, E., 158 (485) Beau-Larvor, C., 113 (351) Beck, H.C., 30 (79) Becker, J.H., 191 (593) Becker, M.A., 72 (214) Beckman, R., 165 (507) Bedard, P.L., 165 (508) Bednarz-Knoll, N., 106 (328) Beeram, M., 35 (96), 107 (329) Beg, M., 196 (4LBA) Beg, M.S., 84 (253) Behrens, C., 124 (388) Behrens, J., 156 (479) Beijersbergen, R., 59 (177), 61 (188) Beijersbergen, R.L., 31 (81), 34 (91) Bejrowska, A., 27 (67) Bekaii-Saab, T.B., 148 (451) Bekdash, A., 126 (394) Bekradda, M., 153 (469), 182 (564), 183 (565, 567), 187 (580), 189 (587) Belhadj, A., 99 (303) Belitskin, D., 57 (173)

Bell, K., 163 (501) Bellahcene, A., 55 (165) Belleri, M., 100 (307) Bello, E., 21 (48), 110 (340), 190 (590) Beloueche-Babari, M., 175 (540) Belsanova, B., 51 (149) Beltrame, L., 21 (48), 187 (580) Belvin, M., 124 (387) Bence, N., 78 (231), 181 (561), 186 (577) Bence, N.B., 85 (255) Bendaoud, A., 99 (303) Bendell, J.C., 152 (465), 179 (551), 199 (11LBA) Bender, S., 67 (206) Benesova, L., 51 (149) Bengali, K., 175 (537) Benharroch, D., 178 (550) Benoukraf, T., 29 (74) Bentink, S., 102 (313), 149 (454) Benyelles, M., 99 (303) Bequet-Romero, M., 38 (107) Berdel, W.E., 134 (420) Berdini, V., 115 (356), 128 (400) Berenguer, C., 153 (469) Berens, M.B., 10 (12) Beret, ´ A., 183 (566) Bergamaschi, A., 158 (485) Berger, M., 27 (69) Berger, M.F., 29 (75) Berger, S., 113 (351) Berger, W., 16 (30), 22 (52) Berglund, U.W., 81 (240) Bergqvist, M., 114 (353) Bergsagel, P.L., 180 (555) Berking, C., 102 (313) Bernard, B., 90 (271) Bernards, R., 51 (148), 54 (160), 59 (177), 61 (188), 154 (472) Bernasconi, E., 184 (568) Bernet, A., 118 (368) Bernstam, E.V., 159 (488) Berros, J.P., 114 (354) Berry, D., 173 (532) Berthold, F., 130 (409) Bertino, E., 151 (461) Bertoni, F., 184 (568) Bertran, J., 156 (478) Bertucci, F., 31 (80) Berzosa, M., 124 (386) Betz, R.C., 161 (494) Beurdeley, A., 58 (174) Bexon, A., 142 (431) Beyer, J., 136 (424) Bhansali, S., 163 (500) Bhattacharya, B., 29 (74) Bianchi, F., 56 (168) Bianchino, E., 62 (189) Bianski, B., 63 (193) Bignall, G., 56 (167) Bignan, G., 141 (430) Bilke, S.W., 84 (252) Bingham, M., 123 (384) Birnbaum, D., 99 (303) Birrer, M., 54 (161) Bisaha, J., 142 (431) Bischoff, J., 141 (430) Bissery, M., 45 (130)

Author Index Bjorkman, ¨ M., 142 (432) Blachier, J.G., 118 (368) Blackburn, E., 91 (276) Blackburn, T., 91 (276), 141 (429) Blackhall, F., 88 (264) Blagden, S., 7 (3) Blakey, D., 150 (457) Blanco, M., 17 (37) Blanco-Aparicio, C., 64 (198), 118 (365) Blasco, A., 43 (122), 46 (133), 177 (545) Bleeker, F.E., 23 (54) Blein, S., 48 (139) Block, N.L., 107 (330) Blyth, K., 164 (504) Bo, L., 67 (205) Boehme, K.A., 107 (331) Bohnert, T., 54 (162) Boidot, R., 67 (207) Bokhari, F., 97 (294) Bola, B., 144 (437) Bolluyt, J., 84 (253) Bolshan, Y., 95 (287) Bolton, G., 41 (118) Bomfim, I.S., 87 (262) Bonelli, M., 129 (404) Bonneau, K., 52 (153), 60 (184) Bonneterre, J., 142 (431) Bonvini, E., 48 (138) Bonyadi Rad, E., 34 (93) Boothman, D.A., 84 (253) Borad, M., 196 (4LBA) Bordoni, C., 122 (379) Borin, T., 169 (522) Boring, D.L., 76 (225) Borodovsky, A., 157 (482) Borowa-Mazgaj, B., 26 (65) Bosch, A., 28 (71) Bosma, A.J., 54 (160) Bossi, G., 44 (126) Bossow, S., 48 (137) Botaro, G.B., 169 (522) Botling, J., 114 (353) Botrugno, O., 185 (571) Bottaro, D.P., 175 (537) Bottega, S., 120 (374) Bouabdallah, K., 162 (499) Boubia, B., 191 (594) Boudon, J., 67 (207) Bouhana, K., 125 (391) Bouillette-Marussig, M., 45 (130) Boukerroucha, M., 53 (159) Boulos, N., 63 (193) Bours, V., 53 (159) Boute, N., 113 (351) Boutros, M., 32 (85) Bouvet, M., 121 (376) Bouwman, R., 38 (106) Bouygues, A., 130 (406) Bovy, N., 53 (159) Bowden, C., 195 (1LBA) Bowden, E., 53 (158) Bowden, E.T., 166 (511) Bowman, D., 78 (231) Boyce, R., 16 (32) Boyko, A., 14 (25) Boyle, B., 16 (32) Bozon, V., 54 (162)

Bozon, ´ V., 105 (324), 117 (364) Bozzi, F., 190 (590) Braccini, I., 180 (557) Brackett, C., 40 (114) Bradley, B., 125 (390) Bradley, J., 67 (205) Bradshaw, J.M., 157 (483) Brameld, K., 157 (483) Brana, I., 171 (526) Brancale, A., 122 (379) Branchereau, S., 25 (61) Branstetter, D., 114 (353) Brantley-Sieders, D., 98 (297) Bravo, N., 110 (339) Bray, G.L., 179 (551) Brega, N., 171 (526) Breitwieser, W., 29 (76) Brekken, R., 117 (362) Brenner, A., 195 (3LBA), 196 (4LBA) Brentjens, R., 96 (293) Brenton, J., 73 (215) Briasoulis, E., 86 (258) Brich, S., 190 (590) Briest, F., 149 (455) Brinkmann, K., 102 (313), 149 (454) Brion, R., 59 (179) Britten, C., 171 (526) Broaddus, R., 3 (1) Broggini-Tenzer, A., 65 (202), 67 (206) Brognard, J., 29 (76), 164 (504), 167 (512) Bron, D., 162 (499) Bronsert, P., 62 (192) Brooke, H., 123 (384) Brooks, N., 150 (457) Brooks, N.A., 128 (402) Broqua, P., 180 (557), 191 (594) Broudy, T., 50 (146), 59 (178), 80 (239) Brough, R., 33 (88), 50 (147), 103 (318) Broussas, M., 40 (113), 113 (351) Brown, A.C.N., 41 (117) Brown, B.D., 115 (358) Brown, C.E., 94 (285) Brown, J., 11 (13) Brown, M., 53 (158) Brown, R., 179 (554) Browne, A.K., 38 (106) Brownell, J., 181 (561) Bruckheimer, E., 154 (471) Brugieres, ` L., 25 (61) Brun, S., 183 (566) Brunen, D., 154 (472) Brunner, A., 152 (465) Brunton, V.G., 160 (491) Brusco, L., 3 (1) Bryant, B., 31 (83), 125 (390), 187 (581) Brynzak, E., 22 (52), 53 (156) Brzezicha, B., 10 (10) Buch, T., 134 (420) Buchanan, S., 165 (507) Buck, M., 7 (3) Buckley, D., 190 (591) Budde, P., 107 (331) Bugano, D., 79 (237) Buijsman, R.C., 37 (104), 113 (349) Bukato, K., 47 (135), 118 (367), 152 (467), 169 (519) Bukhalid, R., 52 (152)

207 Bull, C., 9 (8) Bulychev, A., 54 (162) Buno, ˜ I., 18 (39) Bunz, F., 157 (482) Buonamici, S., 149 (456), 171 (527) Bupathi, M., 136 (425) Burdach, S., 134 (420) Burdelya, L., 40 (114) Burenkova, O., 52 (152) Burgio, M.A.B., 168 (516) Burke, K., 78 (231) Burke, S., 48 (138) Burkett, D., 61 (186) Burkett, M., 147 (450) Burkhart, C., 135 (422) Burlingame, A.L., 28 (73) Burrows, J., 159 (489), 175 (537) Burrows, N., 170 (525) Buscaglia, R., 120 (372) Butts, J., 159 (488) Buxton, M., 173 (532) Byakhov, M., 155 (476) Byers, L.A., 81 (242, 243), 124 (388), 143 (435) Byers, R.J., 8 (6) Byrne, J., 135 (421) Byron, A., 160 (491) Byron, S., 35 (94), 122 (381) Byron, S.B., 10 (12)

C Cabal-Hierro, L., 154 (474) Cadwallader, K., 171 (527) Caetano, W.S., 87 (262) Caffarra, C., 129 (404) Caffo, N., 38 (105) Cai, H., 190 (591) Cai, J., 60 (181), 112 (348) Cai, X., 57 (170) Cai, Y., 94 (283) Cairo, S., 25 (61), 58 (174) Calabuig Farinas, ˜ S., 115 (357) Calabuig-Farinas, ˜ S., 43 (122), 46 (133), 177 (545) Caldas, C., 54 (160) Calistri, D.C., 168 (516) Callaghan, R., 51 (151) Callejas, D., 178 (548) Calosing, C., 181 (560) Calvo, A., 65 (199) Calvo Perez, ´ A., 169 (521) Camacho Rodr´ıguez, R., 169 (521) Cambet, Y., 180 (557) Cameron, B., 44 (126) Cameron, J., 144 (440) Camidge, R.D., 199 (10LBA) Campagne, C., 81 (241) Campagnoli, S.C., 41 (116), 178 (549) Campbell, H., 150 (457) Campbell, J., 33 (88), 50 (147), 103 (318) Campbell, M.A., 123 (384) Campbell, P., 56 (167) Campeau, E., 181 (560) Campiglio, M., 56 (168) Campone, M., 142 (431), 163 (500), 189 (586)

208 Campos-Olivas, R., 110 (339) Camps, C., 43 (122), 46 (133), 115 (357), 177 (545) Canamero, ˜ M., 110 (339) Canan, S., 150 (459) Cancilla, B., 152 (465) Canet-Jourdan, C., 183 (567) Canevari, G., 168 (517) Cani, A., 61 (187) Cano, C., 141 (429) Cantero, F., 189 (586) Cao, S., 80 (238) Capdevila, J., 122 (382) Capone, E., 164 (505) Cappa, A., 185 (571) Carbone, G.M., 159 (487), 180 (556) Carceny Costa, E., 44 (127) Cardnell, R., 81 (242) Cardnell, R.J., 81 (243), 124 (388), 143 (435) Carducci, M., 197 (7LBA) Carducci, M.A., 143 (434) Cargnelli, S.M., 67 (205) Carlile, J., 12 (18) Carlo-Stella, C., 176 (542) Carlson, R., 83 (247), 160 (492) Carmine Simmen, K., 187 (578) Carmona Sanz, F., 27 (69) Carnegie-Clark, A., 135 (422) Carneiro-Lobo, T., 103 (317) Carnero, A., 118 (365) Carol, H., 131 (410, 412) Carollo, R., 164 (505) Carpinelli, P., 16 (31), 168 (517) Carpten, J.C., 10 (12) Carrasco, L., 108 (334) Carreca, A.P., 65 (200), 96 (290), 112 (347) Carretero, J., 191 (593) Carrillo, D., 96 (293) Carter, D., 135 (422) Caruso, M., 168 (517) Casali, P.G., 190 (590) Casalini, P., 56 (168) Cascajares, S., 24 (57) Cassier, P., 146 (444) Cassingena, A., 23 (54) Castanie, H., 189 (586) Castanon, E., 65 (199) Castel, P., 29 (75) Castellano, J., 177 (545) Castellano, R., 183 (566) Castiglia, M., 65 (200), 96 (290), 112 (347) Castillo Correa, F.J., 24 (59) Castosa, R., 17 (37) Castro, C., 54 (161) Castronovo, V., 55 (165) Cataluna, ˜ O., 23 (55) Catapano, C.V., 108 (333), 159 (487), 180 (556) Catenacci, D., 175 (537) Cathers, B., 150 (459) Cattrini, C., 21 (48) Cautain, B., 24 (59) Cavazzoni, A., 129 (404) Cayol, M., 153 (469)

Author Index Cebria, A., 64 (198), 118 (365) Cebrian, D., 64 (198), 118 (365) Ceccarelli, M., 62 (189) Cecchi, F., 175 (537) Cecilia, Y., 118 (365) Centore, R., 187 (581) Cerchietti, L.C., 182 (563) Cerda Nicolas, M., 51 (151) Cerezo, A., 110 (339), 128 (402) Ceriani, L., 110 (340) Ceruti, R., 16 (31) Cesaroni, M., 192 (595) Cescon, D.W., 165 (508) Chachoua, A., 108 (335) Chaffanet, M., 99 (303) Chai, F., 104 (320) Chaires, J., 120 (372) Chakravarty, A., 161 (493) Chakravarty, A.C., 85 (255) Chalmers, Z.R., 84 (250), 104 (319) Chalugun, P., 36 (98) Chambers, A.F., 187 (578) Chambers, G., 125 (389) Champion, B., 41 (117) Chan, B., 149 (456) Chan, D., 114 (353) Chan, J., 124 (387) Chang, J., 19 (43) Chang, J.Y., 147 (449) Chang, N., 46 (131) Chang, T., 95 (288) Chao, B., 199 (10LBA) Chapman-Rothe, N., 179 (554) Charati, M., 145 (443) Chardot, C., 25 (61) Charlton, P.A., 81 (240) Charych, D., 16 (33) Chatterjee, A., 199 (11LBA) Chau, I., 95 (287) Chavez, R., 156 (481) Chavez-MacGregor, C., 163 (500) Checkley, S., 84 (251) Chelur, S., 142 (432) Chen, A.P., 78 (233) Chen, B., 66 (203), 68 (208) Chen, C., 95 (288) Chen, C.R., 167 (513), 172 (531) Chen, C.T., 92 (279) Chen, D., 115 (358) Chen, F., 48 (138), 188 (584) Chen, G., 123 (385) Chen, H., 22 (53), 165 (508) Chen, J., 94 (283), 98 (297), 109 (338) Chen, M., 22 (53) Chen, N., 19 (43) Chen, R., 197 (6LBA) Chen, S., 87 (260) Chen, T., 19 (43) Chen, Y., 19 (43), 102 (312) Chen, Z., 154 (472) Chen, Z.X., 135 (423) Chenchik, A., 52 (153), 60 (184) Chene, L., 180 (557), 191 (594) Cheng, A., 17 (34) Cheng, A.L., 72 (213) Cheng, H., 108 (335) Cheng, J., 124 (387)

Cheng, X.R., 94 (285) Cheong, K.H., 98 (298) Cherblanc, F., 179 (554) Cherubin, S., 76 (227) Chesi, M., 180 (555) Chessari, G., 122 (380) Chesworth, R., 92 (277) Cheung, A., 125 (392) Cheung, H.K., 174 (535) Chevrier, S., 67 (207) Chi, D., 80 (238) Chi, K.N., 8 (4) Chiadini, E.C., 168 (516) Chiarugi, P.C., 178 (549) Chicas, A., 171 (527) Chichili, G.R., 48 (138) Chiemlicki, J., 104 (319) Chien, A.E., 187 (578) Chiesa, E., 155 (475), 156 (481), 167 (513) Chila, ` R., 110 (340) Childs, B.H., 162 (499) Chimmanamada, D., 87 (260) Chin, C.A., 187 (578) Chin, S.F., 54 (160) Ching, L., 91 (273) Chio, C., 63 (194) Chio, L., 165 (507) Chiorino, G., 180 (556) Chiosis, G., 182 (563) Chiron, M., 96 (291), 130 (406) Chishima, T., 121 (376) Chiu, H., 45 (128), 71 (209), 136 (424) Chiu, J.J., 92 (279) Chiu, J.W., 165 (508) Chmielecki, J., 84 (250), 132 (414, 415) Cho, B., 166 (510) Cho, N., 52 (155) Choe, G., 153 (468), 174 (534) Choi, E., 166 (510) Choi, M., 76 (224) Choi, S.U., 126 (393) Choi, Y., 49 (143) Chomej, K., 184 (570) Chondrogiannis, G., 86 (258) Chong, M.L., 29 (74) Choo, Z., 135 (423) Choueiri, T.K., 126 (395) Chouitar, J., 161 (493) Chow, S., 165 (508) Chow, V., 197 (7LBA) Chowbay, B., 32 (86) Choyke, P., 8 (5) Christen, F., 149 (455) Christensen, C.L., 191 (593) Christiansen, J., 143 (436) Chu, C.H., 31 (82) Chu, F., 8 (4) Chu, J., 87 (260) Chu, P., 118 (366) Chung, H., 50 (144) Chung, H.C., 20 (44) Chung, J.H., 20 (44), 157 (482) Churchill, M., 96 (293) Ciavarri, J., 181 (561), 186 (577) Ciavarri, J.C., 85 (255) Ciccarone, V., 48 (138)

Author Index Ciomei, M., 16 (31), 101 (310) Civenni, G., 108 (333), 159 (487), 180 (556) Ciznadija, D., 63 (195) Claes, B., 56 (169) Claessen, J.H., 179 (552) Claret, F., 155 (477) Clark, E., 126 (395) Clark, K., 164 (503) Clarke, N., 98 (300) Clarkson, R., 122 (379) Clausen, M., 145 (441) Cleary, J.M., 158 (484) Cleasby, A., 115 (356), 128 (400) Clevers, H., 63 (194) Clezardin, P., 182 (562) Clifford, S., 132 (413) Coats, S., 106 (326) Cochran, J., 8 (4) Cochrane, D., 41 (117) Coenegrachts, L., 11 (15) Coffee, E.M., 141 (428) Cohen, L., 54 (162) Cohen-Barnhouse, A., 9 (8) Cohn, A., 152 (465) Coimbra, M., 147 (448) Coimbra de Sousa, S., 59 (179) Cole, A., 62 (189) Coleman, R.L., 73 (215) Coleman, T., 125 (392) Collantes, M., 65 (199) Collette, Y., 183 (566) Collins, J.M., 36 (99) Collins, M., 163 (501) Colombel, H., 115 (356), 128 (400) Coltrini, D., 100 (307) Comer, F., 106 (326) Condelli, V., 60 (182) Conery, A., 31 (83) Conklin, D., 162 (498) Conley, B., 78 (232) Conley, B.A., 78 (233) Connelly, J., 147 (450) Connolly, P., 141 (430) Cook, R., 98 (297) Coombes, K.R., 143 (435) Cooper, M., 178 (548) Cooper, T., 189 (588) Cooper, Z.A., 102 (314) Copeland, R., 92 (277) Coppe, J.P., 154 (472) Coral, S., 47 (134) Coram, M., 60 (184) Corbo, V., 63 (194) Corcoran, R., 141 (428) Cornell-Kennon, S., 172 (531) Cornella, H., 75 (223) Corrie, P., 117 (364) Cortes, J., 114 (354), 196 (4LBA) Cortes-Funes, ´ H., 111 (344) Cortis, C., 87 (260) Corvaia, N., 113 (351) Corva¨ıa, N., 40 (113) Costa, M.P., 87 (262) Costales, P., 159 (487) Cosulich, S., 90 (272), 91 (275) Cote, G., 158 (484)

Cottu, P., 142 (431) Courcambeck, J., 183 (566) Courtin, A., 16 (32), 168 (518) Courtin, R., 148 (451) Courtneidge, S.A., 187 (578) Couture, J.F., 95 (287) Covre, A., 47 (134) Cozar, P., 82 (244) Craig, D.C., 10 (12) Craig, S., 18 (40) Cramer, S., 181 (559) Crandall, H., 33 (90) Creedon, H., 160 (491) Crehange, G., 67 (207) Cresswell, C., 169 (520) Cretella, D., 129 (404) Cribioli, S., 16 (31), 101 (310) Criekinge, W., 145 (441) Crino, ` L.C., 168 (516) Cristofanilli, M., 37 (103), 74 (218) Crochiere, M., 83 (247), 160 (492) Crona, D.J., 26 (66) Cronin, K., 177 (546) Croset, A., 48 (139) Crosier, S., 132 (413) Cross, D., 99 (301) Cruz, C., 80 (238), 82 (244), 124 (386) Cruzalegui, F., 177 (546) Csoka, I., 115 (356), 128 (400) Cubillo, A., 55 (166) Cubitt, C., 74 (220) Cubrich, C., 76 (227) Cucchi, U., 168 (517) Cuevas, C., 20 (47), 23 (55), 24 (57, 58), 108 (334), 164 (502) Cully, S., 91 (276) Cumaraswamy, A.A., 94 (285) Cummings, M., 141 (430) Cunningham, C., 75 (222) Cunningham, D., 162 (499) Cuppens, T., 11 (15) Curry, E., 179 (554) Curwen, J., 177 (546) Cusack, J.C., 102 (314) Cusan, M., 72 (212) Cusido, M., 167 (514) Cusido, M.T., 85 (254) Cvitkovic, E., 153 (469), 182 (564), 183 (565, 567), 184 (568), 187 (580), 189 (587)

D da Silva, S.R., 94 (283), 95 (286) Daeyaert, F., 155 (476) Dai, Y., 74 (220) Dal Zuffo, R., 185 (571) Dallavalle, C., 180 (556) Dalpozzo, K., 45 (128) Damia, G., 110 (340) Damico-Beyer, L., 45 (128), 136 (424) Damore, M., 40 (112) Damore, M.A., 161 (496) Damrauer, J.S., 98 (299) Dan, S., 185 (574) Danaee, H., 78 (231) D’Andrea, A., 80 (238)

209 D’Angelo, F., 62 (189) D’Angiolella, V., 71 (211) Danielsen, H.E., 161 (495) Danielson, U.H., 106 (325) Dapper, J.D., 63 (193) D’Arcangelo, M., 114 (353) Darflar, M., 175 (537) d’Ario, G., 61 (188) Darman, R., 149 (456), 171 (527) Darr, D., 164 (503) Daryani, V.M., 133 (416, 418) Das, B., 36 (101) Das, S., 34 (93) Dasgupta, T., 57 (172) Date, H., 188 (584) Datta, A., 78 (232) Datta, V., 78 (233) Dattani, R., 99 (301) Dauch, D., 152 (466) Daud, A., 48 (140) David, K.A., 151 (464) Davidson, N.M., 54 (160) Davies, B.R., 150 (457) Davies, M., 3 (1) Davies, S., 148 (453) Davis, C., 171 (526) Davis, S.L., 7 (2) Dawson, J., 74 (220) Dazzi, C.D., 168 (516) D’Cruz, C., 125 (392), 126 (395), 150 (457) De, P., 128 (399) De Angulo, A., 49 (142) De Bono, J., 148 (453) de Botton, S., 195 (1LBA) De Breucker, S., 141 (430) De Camilli, E.D.C., 41 (116), 178 (549) de Castro, B., 24 (57) De Cecco, L., 56 (168) de Corte, P., 147 (448) de Graan, A.J., 26 (66) de Gramont, A., 130 (406) de Jonge, M., 121 (378) de la Torre, A.V., 38 (107) de la Torre, J., 38 (107) De Laurenzi, V., 164 (505) de Lint, K., 31 (81), 34 (91) De Luca, P., 62 (189) de Man, J., 37 (104), 113 (349) De Mattos-Arruda, L., 114 (354) De Meyer, T., 145 (441) De Pedro, N., 24 (59) de Rink, I., 54 (160) de Roos, J.A.D.M., 113 (349) De Santis, F., 161 (494) de Vries, E.G.E., 174 (535) de Weger, V., 121 (378) Dean, W., 120 (372) Deas, ´ O., 25 (61), 58 (174) Deben, C., 27 (68), 97 (295) Debussche, L., 32 (87) DeCuypere, M., 63 (193) Deegen, P., 174 (535) Degorce, S., 90 (272) deGraffenried, L., 49 (142) Degrassi, A., 16 (31) DeGroot, J., 3 (1)

210 Dehkhoda, F., 122 (381) del Alamo, D., 83 (247), 160 (492) del Carpio Pons, A., 132 (413) Del Conte, G., 171 (526) Del Vecchio, C.A., 179 (552) Del Vecchio, L., 60 (182) Delaporte, S., 180 (557) Delcros, J.G., 118 (368) Delgado-Goni, T., 175 (540) Della Porta, M., 21 (48) Delmonte, A.D., 168 (516) Delon, C., 48 (139) Delord, J.P., 146 (444), 199 (11LBA) Delorenzi, M., 61 (188) Delosh, R., 30 (78) Delpero, A., 120 (374) Demetri, G., 121 (378) DeMichele, A., 173 (532) Dempsey, J., 165 (507) Demuth, T., 199 (11LBA) Deng, D., 52 (153), 60 (184) Denil, S., 145 (441) Denmeade, S.R., 143 (434) Depil, S., 118 (368) Depreeuw, J., 11 (15) DeRyckere, D., 114 (352) Desai, J., 197 (7LBA) Deschoolmeester, V., 27 (68), 65 (200), 97 (295) Deshane, J., 43 (123) Dessanti, P., 185 (571) Deuker, M., 100 (304) Deutsch, E., 74 (219) Devi, G.R., 31 (80) Dey, N., 128 (399) Dhadunia, S., 104 (321) Dhodheri, S., 40 (113) Dhudashia, A.A., 40 (113) Di Cresce, C., 21 (49) Di Marino, M., 187 (580) Di Nicolantonio, F., 23 (54) Di Salle, E., 100 (307) Diamond, J., 13 (23) Diana Mayorga, D.M., 119 (369) Dianov, G., 71 (211) Diao, L., 81 (243), 124 (388), 143 (435) Dias, G.G., 87 (262) Diaz, A., 35 (96) Diaz, D.B., 94 (285) D´ıaz-Garc´ıa, C.V., 111 (344) Diaz-Padilla, I., 165 (508) Dib, C., 96 (291) Dicker, A.P., 49 (141), 73 (216) Dickinson, A., 198 (8LBA) Dickson, M., 121 (378) Dienstmann, R., 124 (386) Dieras, V., 142 (431) Diezma, L., 128 (402) DiGiammarino, E., 82 (246) Digiovanni, J., 181 (559) Diliberto, A., 143 (436) Dilruba, S., 34 (92) DiMartino, J.F., 179 (551) D’Incalci, M., 21 (48), 187 (580), 190 (590) Ding, K., 28 (70) Ding, Z., 172 (530) Dinjens, W.N.M., 53 (157)

Author Index Diosdado, B., 59 (177), 61 (188) Dirix, L., 163 (500) Dirksen, U., 134 (420) Dito, E., 148 (451) Dittamore, R., 45 (129), 127 (397) Dituri, F., 161 (494) Ditzel, H.J., 30 (79) Dive, C., 88 (264), 144 (437) Divecha, N., 91 (275) Diwanji, R., 115 (358) Djaballah, H., 96 (293) Djakovic, S., 180 (555) Dochy, E., 130 (406) Dodheri, S., 104 (321) Doi, T., 36 (101), 72 (213) Doki, Y., 127 (396) Dolganov, G., 60 (184) Dolled-Filhart, M., 44 (127), 48 (140) Dombret, H., 196 (5LBA) Dombrovski, L., 95 (287) Dominguez, J.M., 164 (502) Donahue, R.N., 101 (308) Donati, D., 168 (517) Donawho, C., 82 (246) Dong, A., 95 (287) Dong, C., 60 (181) Dong, H., 162 (497) Dornetshuber-Fleiss, R., 16 (30) Doroshow, J., 78 (232), 83 (248) Doroshow, J.D., 36 (99), 57 (171) Doroshow, J.H., 77 (229), 78 (233), 147 (450) Dorsey, B., 154 (471) Dougall, W., 114 (353) Dougherty, B., 198 (8LBA) Dovat, S., 134 (419) Dowell, J.E., 84 (253) Dowers, A., 76 (227) Doxie, D.B., 33 (90) Drabik, A., 12 (17) Drakos, E., 155 (477) Dransfield, D., 155 (475) Dransfield, D.T., 156 (481), 167 (513), 172 (531) Dreidax, D., 130 (409) Drewe, P., 181 (558) Dreyling, M., 162 (499) Dronca, R., 48 (140) Drummond, C.J., 91 (276) Ds, S., 142 (432) Du, C., 45 (128) Du, Q., 106 (326) Du, W., 52 (154) Du, Z., 162 (497), 176 (544) Dubash, T.D., 32 (85) Dubbink, E., 53 (157) Dubini, A.D., 168 (516) Dubray, C., 183 (566) Ducarouge, B., 118 (368) Dudek, H., 115 (358) Dudley, M., 111 (342) Dudley, P., 177 (546) Duffey, J.D., 85 (255) Duffy, J., 181 (561), 186 (577) Dugas, M., 134 (420) Dukes, J., 44 (126) Dunn, B., 75 (222)

Dunn, B.K., 76 (225) Dupont, J., 7 (2), 55 (166), 152 (465) DuPont, R., 73 (217), 163 (501) Dutta, C., 115 (358) Duval, D.L., 11 (13) Dymond, M., 169 (520) Dziadziuszko, R., 199 (10LBA)

E Earp, H.S., 114 (352) Earp, S., 120 (375), 164 (503) Eary, J., 64 (197), 79 (234, 235) Eastham-Anderson, J., 124 (387) Ebbesen, S., 29 (75) Ebbinghaus, S., 48 (140) Eberhard, D., 55 (163) Ecay, M., 65 (199) Eccles, M., 57 (172) Eccles, S., 148 (453) Eckhardt, S.G., 7 (2), 120 (375) Eddy, S., 166 (511) Edenfield, W.J., 8 (4) Eder, J.P., 44 (127) Edwards, Z.C., 29 (76) Efstathiou, A., 106 (328) Eisenberger, M.A., 143 (434) Eisner, N., 178 (550) Ejadi, S., 165 (506) Ekman, S., 114 (353) Ekmekcioglu, S., 150 (460) Elkabets, M., 28 (71) Ellis, H., 29 (75) Ellston, R., 150 (457), 177 (546) Elmquist, W., 86 (259) Elsner, J., 150 (459) Eltoukhy, H., 151 (462) Elvin, J., 84 (250) Elvin, J.A., 104 (319) Elvin, P., 170 (525) Ely, H., 143 (436) Elyada, E., 63 (194) Emancipator, K., 44 (127), 48 (140) Emmenegger, U., 8 (4) Enderle, D., 102 (313), 149 (454) Endicott, J.A., 91 (276), 106 (325) Endo, I., 121 (376) Engeland, C.E., 48 (137) Engelman, J.A., 141 (428) Engelsen, A., 117 (362) Engle, D., 63 (194) English, P.A., 116 (359) Englund, J., 57 (173) Erba, E., 21 (48), 187 (580) Erdmann, G., 32 (85) Erguen, S., 89 (266) Erlander, M.G., 51 (149) Erlich, R., 132 (414) Escorcia, F., 89 (266) Escorihuela, E., 43 (122), 177 (545) Escudier, B., 116 (359), 126 (395) Eskens, F.A.L.M., 199 (11LBA) Espanel, X., 191 (594) Espinosa, A.B., 77 (230), 136 (427) Esquerre, M., 45 (130) Esser, N., 128 (400), 141 (430) Esserman, L., 173 (532)

Author Index Esteller, M., 23 (54), 152 (466) Esteva, F.J., 155 (477) Estevez, S., 191 (594) Ethier, S., 188 (583) Eun, Y., 50 (144) Evangelista, F.C., 87 (262) Evans, D., 30 (78), 147 (450) Evans, J.J., 49 (141), 73 (216) Evans, M., 31 (80)

F Fabre, M., 25 (61) Fabregas, R., 85 (254), 167 (514) Facon, T., 196 (5LBA) Fadel, E., 25 (61) Fagerlund, K.M., 25 (63) Fagerstrom, R.M., 75 (222) Fairweather, E., 94 (284) Falchook, G.S., 56 (169) Falck Miniotis, M., 175 (540) Fan, B., 195 (1LBA) Fan, J.B., 57 (170) Fan, Y., 81 (243), 124 (388), 143 (435) Fang, B., 89 (269) Fang, C., 88 (265) Fang, J., 57 (171) Faoro, L., 7 (2) Faris, J.E., 141 (428), 199 (11LBA) Faris, R., 49 (142) Farley, M., 37 (102) Farras, R., 115 (357) Farrell, C., 49 (141), 73 (216) Fathi, A.T., 195 (1LBA) Fattah, F., 84 (253) Fatunde, O., 84 (253) Fawdar, S., 29 (76) Fawell, S., 123 (383) Fazio, C., 47 (134) Fazli, L., 161 (495) Feala, J., 149 (456) Fedejko, B., 27 (67) Feeney, K., 49 (141) Feigin, M., 63 (194) Feitosa, A.C.S., 87 (262) Fekkes, P., 149 (456), 171 (527) Felder, E.R., 168 (517) Felip-Font, E., 44 (127) Fell, S.M., 135 (423) Feng, Y., 81 (242, 243), 179 (552) Fer, N.D., 57 (171), 147 (450) Ferguson, D., 123 (383) Ferguson, P., 21 (49) Fernandez de Farias, C., 26 (64) Fernandez-Montalv ´ an, ´ A.E., 185 (573) Fernandez Sousa-Faro, J.M., 190 (590) Fernandez-Teruel, C., 13 (23) Ferraroto, R., 7 (2) Ferre, P., 40 (113) Ferreira, L.C., 169 (522) Ferreiro-Neira, I., 102 (314) Fett, J., 156 (479) Fetterly, G.J., 87 (261) Fichtner, I., 10 (10) Fiebig, H.H., 9 (7), 10 (11), 62 (192) Fiedler, U., 107 (331) Fielding, A., 198 (8LBA)

Fietz, E., 144 (440) Figg, W., 13 (21) Figueiredo, C.R., 26 (64), 43 (124) Figueredo, R., 21 (49) Figueroa, A., 17 (37) Figueroa, S., 115 (357), 177 (545) Filannino, A., 161 (494) Finetti, M.A., 132 (413) Finn, R.S., 162 (498) Firestein, R., 179 (553) Fischer, J., 111 (342) Fischer, M., 130 (409), 135 (422) Fisher, K., 41 (117) Fitzek, M., 90 (272) Fitzgerald, A., 109 (337) Fitzgerald, D., 91 (275) Fitzgerald, J.B., 87 (261) Fjaellskog, M.L., 163 (500) Flaherty, K.T., 102 (314), 149 (454), 158 (484) Flanagan, J.U., 91 (273) Flecha, A., 9 (8) Fleming, J.B., 121 (376) Fleming, P., 181 (561) Fleming, P.F., 85 (255) Fleming, R., 106 (326) Flemington, V., 91 (275) Fletcher, S., 93 (282) Flores, J., 35 (97) Flores, M.A., 187 (579) Flynn, D., 115 (355) Fokas, D., 86 (258) Follettie, M., 145 (443) Follmer, N., 187 (581) Fontebasso, Y., 33 (88), 50 (147) Forbes, T.D., 78 (233) Ford, L.G., 75 (222) Forte, L., 56 (168) Fortea, P., 187 (579) Forteza, J., 46 (133) Fouani, L., 175 (539) Foucault, F., 62 (192) Fraiponts, E., 141 (430) Fram, R.J., 80 (239) Frampton, G.M., 84 (250) Francesch, A., 164 (502) Franch, T., 184 (569) Franco, H., 187 (581) Franco Odio, S., 169 (521) Frankel, A.E., 84 (253), 126 (394) Franklin, M., 9 (8) Frankum, J., 33 (88), 50 (147) Frapolli, R., 182 (564), 187 (580), 190 (590) Frattini, M.G., 96 (293) Frayo, S.M., 88 (265) Freas, E., 122 (382) Frederick, D.T., 102 (314) Frederick, M., 109 (337) Freedman, J., 105 (322) Freeman III, B., 63 (193) Freire, V.N., 87 (262) Freitag, H., 149 (455) French, T., 169 (520) Freres, P., 53 (159) Friberg, G., 184 (569), 197 (7LBA) Fridlib, M., 190 (591)

211 Friedlander, S., 83 (247) Friedman, J., 154 (471) Friedman, L., 124 (387) Friedrich, M., 174 (535) Friend, J., 8 (5) Frigault, M., 125 (392), 126 (395) Frink, R., 117 (362) Frith, S., 123 (384) Fritzl, S., 195 (2LBA) Fromond, C., 180 (557), 191 (594) Frye, S., 120 (375), 164 (503) Frye, S.V., 114 (352) Fu, S., 56 (169) Fuchter, M., 179 (554) Fuentes-Guirado, M., 110 (341) Fujioka, A., 13 (22) Fujioka, Y., 167 (515) Fujita, H., 167 (515), 176 (541) Fujita, N., 142 (433) Fujita, R., 103 (315), 120 (373) Fujiwara, K., 172 (531) Fujiwara, T., 96 (292) Fuks, Z., 81 (241) Fukuoka, M., 15 (29), 198 (9LBA) Fukushima, H., 142 (433) Fulgham, A., 144 (438) Fuller, J.D., 81 (241) Fultz, K.E., 150 (459), 170 (523) Fumarola, C., 129 (404) Fumuleau, P., 146 (444) Funahashi, Y., 17 (36) Funk, J.O., 157 (483) Furman, R., 149 (456) Fusi, A., 88 (264) Futami, T., 129 (403) Futreal, P.A., 56 (167)

G Gabra, H., 7 (3) Gabrielli, B., 34 (93), 97 (294) Gadakh, A., 104 (321) Gadgeel, S.M., 199 (10LBA) Gadhikar, M., 109 (337) Gaetzner, S., 89 (266) Gago, F., 108 (334) Gajjar, A., 63 (193), 133 (416) Galanski, M., 22 (52) Galazka, M., 47 (135), 118 (367), 152 (467), 169 (519) Galetti, M., 129 (404) Gallach, S., 43 (122), 177 (545) Gall`ı, A., 21 (48) Galli, R., 168 (517) Galmarini, C., 82 (244) Galmarini, C.M., 17 (35), 20 (47), 21 (48), 24 (57, 58), 108 (334), 111 (345), 164 (502), 190 (590) Galmiche-Rolland, L., 25 (61) Galsky, M., 178 (548) Galvani, A., 16 (31), 101 (310), 168 (517) Galvin, K., 161 (493) Gambino, V., 61 (188) Gamble, L., 135 (422) Gamboni, A.G., 168 (516) Gameiro, S., 101 (308) Gamelin, E., 197 (7LBA)

212 Gamez, L., 35 (96), 37 (102) Gan, L., 54 (162) Gandhi, L., 44 (127), 111 (342) Ganesh, S., 115 (358) Gangadhar, T.C., 48 (140) Gangolli, E., 105 (324), 117 (364), 161 (493) Gao, F., 78 (231) Gao, W., 82 (246) Gao, Y., 145 (443) Garc´ıa, C., 111 (345) Garc´ıa, M.D., 187 (579) Garc´ıa-Cantalejo, J., 187 (579) Garcia del Muro, X., 110 (341) Garcia-Escudero, R., 180 (556) Garcia-Fernandez, L.F., 20 (47), 24 (57, 58), 164 (502) Garc´ıa-Fernandez, ´ L.F., 108 (334) Garc´ıa-Inclan, ´ C., 159 (487) Garcia-Monclus, ´ S., 110 (341) Garcia-Navas, V., 77 (230), 136 (427) Garcia-Serelde, B., 118 (365) Garon, E.B., 162 (498) Garon, E.G., 44 (127) Garranzo, M., 164 (502) Garraway, L.A., 141 (428) Garrido-Castro, A., 124 (386) Gartside, M.G., 29 (76) Gaspar, M., 77 (230), 136 (427) Gasparini, P., 56 (168) Gaudio, E., 184 (568) Gausdal, G., 117 (362) Gautier, T., 67 (207) Gavillet, B., 146 (444) Gavilondo, J.V., 38 (107) Gavin, J., 181 (561) Gazzah, A., 74 (219) Geary, D., 13 (23) Geddie, M., 144 (438) Gedrich, R., 71 (210) Geeganage, S., 110 (339), 128 (402) Geh, C., 169 (520) Geitmann, M., 106 (325) Gekeler, V., 185 (573) Gelato, K.A., 185 (573) Gelderblom, H., 185 (572) Geles, K.G., 145 (443) Geletu, M., 94 (283, 285) Gemei, M., 60 (182) Genilloud, O., 24 (59) George, D., 105 (322) Georgiou, G., 181 (559) Gerami-Moayed, N., 52 (152) Gerber, D., 84 (253) Gerber, H.P., 145 (443) Gerigich, K., 48 (140) German, P., 172 (530) Germaschewski, F., 98 (300) Germonpre, P., 65 (200) Gerritsen, M., 157 (483) Gershenhorn, B., 19 (41), 132 (415) Gesner, E.M., 181 (560) Getlik, M., 95 (287) Geyer, M., 80 (238) Geyer, S., 151 (461) Ghaderi, D., 156 (479) Ghigna, M.R., 25 (61)

Author Index Giacchetti, S., 142 (431) Giaccone, G., 102 (312) Giacomini, A., 100 (307) Giannakou, A., 145 (443) Giannelli, G., 161 (494) Gibbons, D.L., 143 (435) Gibert, B., 118 (368) Gil-Bazo, I., 65 (199), 97 (295) Gil Benso, R., 51 (151) Gilbert, J., 36 (100) Gilbertson, R.J., 63 (193) Gilham, D., 181 (560) Gilissen, R., 115 (356), 128 (400), 141 (430) Gilles, E., 142 (431) Gillett, C., 77 (228) Gillis, N., 55 (163) Gilmour, R., 110 (339), 128 (402) Gimenez-Capitan, A., 156 (478) Giordano, H., 73 (215) Giorgio, C., 90 (270) Giovannetti, E., 65 (200), 112 (347) Girola, N., 26 (64), 43 (124) Girotti, M.R., 29 (76) Giurescu, M., 162 (499) Gladkov, O., 19 (41) Glas, A., 173 (532) Glass, J., 73 (216) Glimm, H., 32 (85) Gluck, W., 152 (465) Gniffke, J., 76 (227) Go, S.Y., 173 (533) Goel, S., 109 (336) Goetsch, L., 113 (351) Goffredo, V., 161 (494) Golan-Goldhirsh, A., 178 (550) Golas, J., 145 (443) Golding, B., 141 (429) Golding, B.T., 91 (276), 106 (325) Goldman, J., 122 (382) Goldman, J.W., 162 (498), 199 (10LBA) Goldschneider, D., 118 (368) Goldstein, D.M., 157 (483) Goldstein, P., 107 (330) Goldstein, R., 182 (563) Gomez-Roca, C., 196 (5LBA) Gomez-Roca, C.A., 146 (444) Gomori, A., 167 (515) Goncalves, A., 189 (586) Gon¸calves, N.N., 169 (522) Gonda, T., 97 (294) Gong, X., 165 (507) Gonzalez, A., 114 (354) Gonzalez, R., 105 (324), 117 (364) Gonzalez, X., 85 (254) Gonzalez-Cao, M., 85 (254), 114 (354), 167 (514) Gonzalez Darder, J., 51 (151) Gonzalez-Perez, R.R., 89 (267) Goodman, O., 197 (7LBA) Goodstal, S., 146 (445) Gooptu, M., 37 (103) Gopakumar, I., 29 (75) Gopas, J., 178 (550) Gordan, J.D., 28 (73) Gordon, M., 111 (342) Gore, L., 13 (23)

Gorlatov, S., 48 (138) Gorlick, R., 131 (410, 412) Gorrochategui, J., 77 (230), 136 (427) Gorse, A., 25 (61) Gorynia, S., 9 (7) Goschl, ¨ S., 22 (52) Goss, G., 111 (342) Gotzhein, F., 106 (328) Goubard, A., 183 (566) Goubier, A., 45 (130) Gowda, C., 134 (419) Grabowski, P., 149 (455) Grace, M., 66 (203) Gradinaru, C., 93 (282) Graham, D., 114 (352), 164 (503) Graham, D.K., 120 (375) Grandi, A.G., 41 (116), 178 (549) Grandi, G.G., 41 (116) Granot, Z.V.I., 89 (266) Grant, S., 43 (123), 74 (220) Grasberger, B., 141 (430) Grass, I., 149 (455) Gray, J., 127 (397) Gray, N., 98 (297) Gredy, C., 9 (7) Green, I., 179 (554) Green, S., 90 (272) Greene, S., 127 (397) Greenlee, E., 130 (407) Greenplate, A.R., 33 (90) Gregori Romero, A., 51 (151) Greifenberg, A.K., 80 (238) Grernrum, W., 54 (160) Grifantini, R., 41 (116), 178 (549) Griffin, R., 141 (429), 181 (561) Griffin, R.G., 85 (255) Griffin, R.J., 91 (276), 106 (325) Griffith, M., 29 (75) Griffith, O.L., 29 (75) Grimes, H.L., 94 (285) Grimm, E.A., 150 (460) Grimmond, S., 35 (94) Grochot-Przeczek, A., 118 (367) Groenendijk, F.H., 54 (160) Gronchi, A., 190 (590) Grossardt, C., 48 (137) Grosskurth, S., 123 (383) Grubbs, C., 76 (226) Grueso, J., 82 (244) Grunert, J., 162 (499) Gu, J.L., 52 (154) Gu, L., 162 (497), 176 (544) Gu, Y., 123 (385), 125 (392), 198 (8LBA) Gu, Z., 52 (154) Gubens, M.A., 44 (127) Gudkov, A., 40 (114) Gudkov, A.V., 135 (422) Guffanti, F., 110 (340) Guglielmi, M., 62 (189) Guha, R., 83 (248) Guichard, S.M., 123 (383) Guijarro, R., 43 (122), 46 (133), 177 (545) Guillen, M.J., 23 (55), 164 (502) Guillen, ´ M.J., 82 (244) Guley, K., 9 (8) Gunning, P., 93 (282) Gunning, P.T., 94 (283, 285), 95 (286)

Author Index Guo, J., 62 (192) Guo, M., 89 (268), 109 (338), 162 (497) Guo, M.I.N.G., 176 (544) Gupta, I., 119 (370) Gupta, P.B., 179 (552) Gurova, K., 135 (422) Gutin, A., 72 (214) Guy, R.K., 63 (193) ´ M., 82 (244) GuzmAn, Gyuris, J., 120 (374)

H Ha, J.M., 79 (236) Ha˚ ag, ˚ P., 184 (570) Haaland, G., 117 (362) Haber, M., 135 (421, 422) Hadari, Y., 130 (407) Haddock, K.M., 133 (416, 418) Haendler, B., 185 (573) Haeuw, J.F., 113 (351) Hagemann, T., 146 (445) Hagenkort, A., 81 (240) Haggerty, K., 91 (276) Hague, M.N., 187 (578) Hahn, A., 98 (300) Haie-Meder, C., 74 (219) Haimovitz-Friedman, A., 81 (241) Hajjar, J., 136 (425) Hakonen, T., 47 (136) Halfon, P., 183 (566) Hall, J., 108 (333), 123 (384) Hall, M., 7 (3) Hall, T., 50 (145), 125 (389) Halleen, J.M., 25 (63) Halmos, B., 108 (335) Halonen, P., 59 (177) Halpern, W., 136 (424) Haluska, P., 72 (214) Halytskiy, V., 174 (536) Hamid, O., 48 (140) Hamilton, A., 7 (3) Hamilton, N., 94 (284) Hamilton, S., 78 (232) Hammerich, B., 10 (11) Hammond, M.R., 102 (314) Hammonds, T., 141 (429) Han, J.W., 153 (470) Han, T., 166 (510) Han, W., 173 (533) Hancock, S., 51 (149) Hancox, U., 90 (272), 177 (546) Handly-Santana, A., 63 (194) Hann, B., 28 (73) Hann, C., 72 (212) Hanna, S.C., 98 (299) Hansen, H.C., 181 (560) Hansen, M.H., 184 (569) Hanson, L., 177 (546) Haraldsdottir, S., 151 (461) Harb, W., 107 (329) Harbron, C., 198 (8LBA) Harbuzariu, A., 89 (267) Hardcastle, I., 141 (429) Hardcastle, I.R., 91 (276) Harding, T.C., 83 (249) Harmon, T., 89 (267)

Harms, B.D., 144 (438) Harper, J., 44 (126) Harrell, M., 73 (215) Harrington, E.A., 169 (520) Harrington, K.J., 41 (118) Harrington, R.D., 78 (233) Harris, E., 147 (450) Harrison, E., 121 (377) Harstead, K.E., 133 (416, 418) Hart, J., 175 (537) Hart, L., 19 (41) Hartley, D., 125 (391) Hartley, J.A., 146 (445) Hartman, A.M., 72 (214) Haruma, T., 167 (515) Harvey, M., 45 (129), 127 (397) Hase, T., 30 (77) Hasegawa, Y., 30 (77) Hashii, C., 97 (296) Hashimoto, A., 91 (274), 127 (398), 128 (401), 167 (515), 191 (592) Hassan, N., 44 (126) Hassan, R., 102 (312) Hassan-Mohamed, I., 90 (270) Hatake, K., 136 (426), 166 (509) Hatton, C., 31 (83), 125 (390), 187 (581) Hattori, T., 52 (155) Hatty, S.R., 179 (551) Hawe, D., 64 (197), 79 (234, 235) Hawks, G., 8 (5) Hawryluk, M.J., 132 (414) Hawtin, R.E., 39 (109) Hayashi, Y., 103 (315) Hayes, N., 55 (163) Hayward, N.K., 29 (76) Haz, M., 17 (37) Hazemi, P., 16 (30) He, H., 95 (287) He, L., 48 (138) He, S., 188 (585) Hearn, K., 168 (518) Heath, E.I., 8 (4) Hedley, D., 165 (508) Heffeter, P., 16 (30), 22 (52), 53 (156) Helleday, T., 81 (240) Hellesøy, M., 117 (362) Hellman, U., 135 (423) Helman, L., 84 (252) Hembrough, T., 159 (489), 175 (537) Hemmer, M.C., 135 (423) Hemminki, A., 47 (136) Henderson, L., 175 (537) Hendrickson, A.W., 73 (215) Hendrix, M.J., 88 (264) Heng, D., 126 (395) Henke, E., 89 (266) Henrich, K.O., 130 (409) Heo, S.J., 20 (44) Herait, P., 183 (567), 184 (568), 189 (587), 196 (5LBA) Hergenrother, P.J., 171 (528) Herlyn, M., 34 (93) Hermann, J., 156 (479) Hermans, E., 11 (15) Hernandez, B., 11 (13) Hernandez, F., 79 (234, 235) Hernandez, F.R., 64 (197)

213 Hernandez, P., 77 (230), 136 (427) Hernandez-Bernal, ´ F., 38 (107) Hernando, C., 177 (545) Hero, B., 130 (409) Hersey, P., 48 (140) Hertfelder, D., 107 (331) Hertwig, F., 130 (409) Heske, C., 101 (311) Hess, D., 185 (572) Hess, K., 79 (237), 136 (425) Heuer, T.S., 190 (591) Heymach, J., 7 (2) Heymach, J.V., 124 (388), 143 (435) Heymann, D., 59 (179), 62 (191) Heynen, G., 51 (148) Hickman, M., 150 (459) Hidalgo, M., 23 (55), 55 (166), 83 (249) Hierro, C., 124 (386) Higgins, B., 106 (327) Higuera, M., 75 (223), 152 (466) Hill, A.A., 114 (352) Hill, D., 152 (465) Hill, R.J., 157 (483) Hilton, J.F., 158 (484) Hinz, S., 117 (362) Hirai, H., 127 (398), 142 (433) Hirata, M., 176 (541) Hirosawa, A., 185 (574) Hiroshima, Y., 96 (292), 121 (376) Hirota, S., 127 (396) Hirsch, F., 114 (353) Hirte, H., 165 (508) Hitchin, J., 94 (284) Hlman, L.J., 101 (311) Ho, C.Y., 22 (53) Ho, H., 17 (34) Hoang, A., 172 (530) Hoar, K., 186 (577) Hoch, U., 16 (33) Hochhauser, D., 146 (445) Hodge, J.W., 101 (308) Hodgkinson, C., 88 (264), 144 (437) Hodgson, D., 198 (8LBA) Hodi, F.S., 48 (140) Hodivala-Dilke, K.M., 38 (106) Hoefler, G., 34 (93) Hoey, T., 55 (166) Hoffman, R.M., 96 (292), 111 (343), 121 (376) Hoffman-Luca, G., 32 (87) Hoffmann, H., 89 (266) Hoffmann, J., 10 (10) Holbeck, S., 36 (99) Holbeck, S.L., 36 (101) Holen, K., 111 (342) Holla, V., 159 (488) Hollingshead, M., 89 (269) Holme, H., 82 (245) Holmes, B., 115 (358) Holmstrom, ¨ T., 142 (432) Holt, S., 94 (284), 195 (2LBA) Holtzhausen, A., 164 (503) Hong, D., 3 (1), 163 (501), 196 (4LBA) Hong, D.S., 56 (169), 79 (237) Hong, J.H., 116 (360) Honscheid, ¨ P., 64 (196) Hood, M.M., 115 (355)

214 Hoon, D.S., 150 (460) Hoon, D.S.B., 151 (462) Hopkins, G., 195 (2LBA) Hopkins, T., 82 (246) Hopson, C., 98 (300) Horie-Inoue, K., 101 (309) Horlings, H.M., 54 (160) Horn, L., 44 (127), 199 (10LBA) Horn-Lee, M., 60 (183) Horobin, J., 97 (296) Horsfield, J., 57 (172) Hose, C., 147 (450) Hose, C.D., 57 (171) Hotamisligil, G.S., 161 (495) Hotson, D., 39 (109) Hotte, S.J., 165 (508) Hou, S., 48 (139) Houghton, P., 131 (410, 412) Houk, B., 171 (526) Hovelson, D., 61 (187) Hoyt, C.C., 8 (6) Hsieh, H., 19 (43) Hsieh, H.P., 92 (279) Hsu, C., 17 (34) Hsu, F., 68 (208) Hsu, T.A., 92 (279) Hsueh, S., 17 (34) Hu, L., 190 (591) Hua, L., 52 (154) Huang, C., 17 (34) Huang, H.J., 56 (169), 57 (170) Huang, H.R., 187 (581) Huang, J., 64 (197), 79 (234, 235) Huang, L., 48 (138) Huang, S., 151 (462) Huang, S.A., 124 (387) Huart, V., 123 (384) Hubbard, J.M., 122 (382) Huck, J., 78 (231), 181 (561), 186 (577) Huck, J.H., 85 (255) Hudson, A., 29 (76), 167 (512) Hughes, L., 165 (506) Hugo, W., 29 (76) Huho, A., 104 (319) Hui, R., 44 (127) Hummel, M., 149 (455) Humphrey, T.C., 71 (211) Hung, C., 95 (288) Hung, L., 19 (43) Hunsaker, T., 124 (387) Hunter, F.W., 66 (204) Hunter, L., 164 (503) Hunter, T., 164 (504) Hurvitz, S., 197 (7LBA) Huseni, M., 45 (128), 71 (209), 136 (424) Hussain, A., 8 (4) Hussain, S.A., 185 (572) Hutchinson, K.E., 84 (250) Hutton, C., 94 (284) Hwang, C., 63 (194) Hwu, W.J., 48 (140) Hyer, M., 78 (231), 181 (561), 186 (577) Hyer, M.H., 85 (255) Hylarides, M.H., 88 (265) Hylton, N., 173 (532) Hyung, W.J., 20 (44)

Author Index

I I-SPY 2 TRIAL Investigators, 173 (532) Iacobelli, S., 164 (505) Iafrate, A.J., 78 (232), 141 (428) Ibrahim, Y., 82 (244) Ichikawa, K., 120 (373) Iglesias, J., 118 (366) Iglesias, L., 111 (344) Iijima, K., 129 (403) Ijaz, N., 182 (564), 183 (565) Ikeda, H., 176 (543) Ikeda, K., 101 (309) Ikeda, M., 73 (217) Ikram, F., 130 (409) Illingworth, S., 41 (117) Im, S.A., 198 (8LBA) Imamura, N., 188 (584) Imoto, M., 176 (543) Inada, M., 176 (541) Inazu, M., 58 (175) Incerti, M., 90 (270) Indarte, M., 160 (490) Infante, J., 195 (3LBA) Infante, J.R., 165 (506) Ingelman-Sundberg, M., 95 (289) Ingemarsdotter, C.K., 38 (106) Innocenti, F., 26 (66), 55 (163) Inoue, H., 175 (538) Inoue, S., 101 (309) Inoue, T., 87 (260), 154 (473) Iorio, F., 56 (167) Irish, J., 188 (583) Irish, J.M., 33 (90) Isaacs, J.T., 143 (434) Isacchi, A., 168 (517) Isambert, N., 146 (444), 179 (551), 189 (586) Ishida, K., 13 (22), 15 (27) Ishii, Y., 78 (231), 181 (561) Ishii, Y.I., 85 (255) Ishikawa, Y., 91 (274) Isoyama, T., 172 (531) Issa, J.P., 192 (595) Italiano, A., 142 (431), 197 (6LBA) Ito, K., 127 (398), 128 (401), 167 (515), 191 (592) Ito, S., 191 (592) Iwasawa, Y., 128 (401) Iyer, P., 125 (390)

J Jackson, P.J., 19 (42) Jacobs, J., 39 (108) Jacobs, V., 163 (501) Jacobsen, K., 30 (79) Jacquet, S., 180 (557) Jacquet-Bescond, A., 110 (340) Jacus, M.O., 63 (193), 133 (416, 418) Jaderberg, M., 47 (136) Jaeger, S., 199 (11LBA) Jaehde, U., 34 (92) Jager, ¨ D., 48 (137) Jager, E., 47 (136) Jagiello, A., 170 (524) Jahagirdar, R., 181 (560) Jakob, C., 64 (196)

Jakobsen, B., 44 (126) Jakupec, M.A., 22 (52), 53 (156) James, D., 94 (284) Jameson, G., 87 (261) Janku, F., 3 (1), 56 (169), 57 (170), 151 (461) Janssens, B., 141 (430) Jantus-Lewintre, E., 43 (122), 46 (133), 115 (357), 177 (545) Jao, S., 95 (288) Jaslow, R., 37 (103) Jasser, S., 109 (337) Jastrzebski, K., 34 (91) Jaworski, A., 47 (135), 118 (367), 152 (467), 169 (519) Jayanthan, A., 131 (411), 189 (588) Jeffrey, S.S., 60 (184) Jelinek, J., 192 (595) Jendrisak, A., 45 (129), 127 (397) Jensen, S., 76 (227) Jeon, E.K., 116 (360) Jeong, J., 92 (278), 158 (485) Jernigan, F., 18 (38) Jerusalem, G., 53 (159) Jiang, J., 52 (154), 87 (260) Jiang, X., 197 (7LBA) Jiang, Y., 71 (211) Jiang, Z., 179 (553) Jimenez, A.M., 60 (183) Jimenez, J.P., 188 (585) Jimenez, M., 77 (230), 136 (427) Jimenez, ´ S., 110 (339), 128 (402) Jimeno, A., 13 (23) Jimeno, J., 156 (478) Jin, B.J., 41 (116) Jin, D.X., 179 (552) Jin, L., 48 (138) Jin, S.L.C., 147 (449) Jin, T.H., 43 (123) Jin, Y., 161 (495) Jinneth Acosta, J.A., 119 (369) Jivrajani, M., 85 (256) Jo, M., 150 (457) Jodrell, D.I., 16 (32) Joel, J., 171 (527) Joensuu, H., 59 (179) Joensuu, T., 47 (136) Johannes, J., 123 (383) Johansson, I., 95 (289) Johnsen, J.I., 135 (423) Johnson, A., 44 (126), 84 (250), 159 (488) Johnson, B., 144 (438) Johnson, C.N., 122 (380) Johnson, E., 82 (246) Johnson, F., 109 (337) Johnson, G.L., 98 (299) Johnson, M., 66 (203) Johnson, N., 72 (212), 80 (238), 191 (593) Johnson, P.W.M., 172 (529) Johnson, R., 190 (591) Johnson, S., 48 (138), 80 (238) Johnstone, C., 58 (176) Jokela, T., 117 (362) Jolly, C., 49 (142) Jonak, Z., 98 (300) Jonasch, E., 126 (395), 172 (530) Jones, C., 91 (275)

Author Index Jones, D., 91 (275) Jones, H., 198 (8LBA) Jones, J., 72 (214) Jones, J.C., 88 (265) Jones, K.L., 11 (13) Jones, R., 125 (392) Jones, S., 94 (284), 195 (2LBA) Jordan, A., 94 (284), 195 (2LBA) Jordana-Ariza, N., 114 (354) Joseph, R., 48 (140) Joshi, A., 171 (528) Joshua, A.M., 48 (140) Josse, C., 53 (159) Josse, ´ R., 83 (248) Josselin, E., 183 (566) Juan, G., 40 (112), 197 (7LBA) Judde, J.G., 25 (61), 58 (174) Judy, K., 49 (141), 73 (216) Juhl, H., 151 (464) Jung, H.J., 126 (393) Jung, I.K., 20 (44) Jung, J.K., 79 (236) Jung, K.H., 42 (119) Jung, M.K., 20 (44) Jung, S., 107 (331) Jungwirth, U., 22 (52) Juntti, T., 184 (570) Junttila, M., 124 (387) Jurgens, ¨ H., 134 (420) Juric, D., 29 (75), 158 (484)

K Kadakia, E.K., 85 (255) Kadariya, Y., 146 (446) Kaemmerer, D., 149 (455) Kage, M., 190 (589) Kairemo, K., 47 (136) Kaiser, R., 136 (424) Kajikawa, M., 175 (538) Kajiwara, D., 103 (315) Kaklamani, V., 14 (24) Kak ¨ onen, ¨ S.M., 25 (63) Kakumu, T., 30 (77) Kalayda, G.V., 34 (92) Kalid, O., 160 (492) Kallio, P., 142 (432) Kalra, S., 172 (530) Kamat, S., 72 (212) Kamindla, R., 148 (452) Kamranpour, N., 162 (498) Kandil, S., 179 (554) Kaneda, M., 127 (396) Kang, C.H., 126 (393) Kang, J.H., 116 (360) Kang, M., 131 (410, 412) Kang, P., 48 (140) Kang, S., 153 (468) Kang, W.Y., 23 (56), 25 (62) Kang, Y., 106 (328), 196 (4LBA) Kang, Y.K., 73 (217), 163 (501) Kansra, V., 165 (506) Kanter, L., 184 (570) Kapoun, A., 152 (465) Kapoun, A.M., 7 (2) Karachaliou, N., 85 (254), 114 (354), 156 (478), 167 (514)

Karam, J., 172 (530) Karanth, S., 136 (424) Karaseva, N., 19 (41) Karbach, J., 47 (136) Karger, B., 156 (480) Karlin, L., 196 (5LBA) Karlsson, S., 142 (432) Karp, D., 136 (425) Karp, D.D., 56 (169) Karr, D., 157 (483) Karrison, T., 175 (537) Kashimoto, S., 112 (346) Kashofer, K., 34 (93) Kashyap, T., 83 (247), 160 (492) Kastrev, D., 50 (147) Kastrinsky, D., 158 (486), 178 (548) Katayama, K., 129 (403) Katella, T., 66 (204) Kato, M., 128 (401) Katz, A., 63 (195) Katz, J., 170 (523) Katz, M.H.G., 121 (376) Kauffman, M., 74 (220), 83 (247), 100 (305), 160 (492) Kaufman, J., 35 (97) Kaufman, M.D., 115 (355) Kaufmann, S., 73 (215) Kaur, G., 30 (78), 89 (269) Kawada, M., 175 (538) Kawahara, A., 190 (589) Kawamura, T., 14 (26) Kawatani, M., 14 (26) Kaye, S., 148 (453) Kazakin, J., 125 (389) Kazim, H., 100 (305) Kazim, S., 100 (305) Kazimierczak, U., 148 (452) Kazmi, S., 19 (41) Kazuno, H., 15 (27, 29) Keaney, G., 149 (456) Kearns, J., 52 (152), 107 (329) Keating, A., 198 (9LBA) Keegan, M., 97 (296) Kees, T., 62 (192) Kefford, R., 48 (140) Keilhack, H., 92 (277), 197 (6LBA) Keilholz, U., 10 (10) Keir, S., 131 (410, 412) Keller, H., 45 (130) Kelley, M.C., 33 (90) Kelley, R.K., 148 (451) Kelly, M., 97 (294) Kelly, T.J., 96 (293) Kelly, V., 21 (50), 109 (336) Kemble, G., 190 (591) Kenchappa, R.S., 135 (423) Kendra, K., 151 (461) Keppler, B.K., 22 (52), 53 (156) Kerman, K., 94 (285) Kern, O., 91 (275) Khambatta, G., 150 (459) Kharenko, O.A., 181 (560) Khayat, D., 188 (582) Kholodenko, B., 77 (228) Khose, S., 3 (1) Kiefer, F., 62 (192) Kiefer, M., 158 (485)

215 Kiga, M., 176 (543) Kikuchi, E., 191 (593) Kilgour, E., 169 (520) Kim, B., 92 (278) Kim, C., 98 (298) Kim, D., 153 (468) Kim, E., 79 (236) Kim, E.H., 119 (371) Kim, H., 92 (278) Kim, H.C., 65 (201) Kim, H.I., 20 (44) Kim, H.R., 126 (393) Kim, H.S., 20 (44) Kim, I., 166 (510) Kim, J., 45 (128), 87 (261), 136 (424), 166 (510), 173 (533) Kim, J.M., 71 (209) Kim, J.W., 119 (371) Kim, K.A., 98 (298) Kim, L., 73 (216) Kim, M., 49 (143), 117 (363) Kim, S., 65 (201), 173 (533), 196 (4LBA) Kim, S.J., 23 (56), 24 (60), 25 (62) Kim, S.W., 173 (533) Kim, S.Y., 119 (371) Kim, T.Y., 73 (217), 163 (501) Kim, W., 153 (468) Kim, W.H., 198 (8LBA) Kim, W.Y., 98 (299) Kim, Y., 150 (457) Kimmel, L., 71 (210) King, A., 52 (152) King, J., 60 (185) King, P., 115 (356) Kirkpatrick, L., 160 (490) Kishimoto, H., 96 (292) Kiss von Soly, S., 180 (555) Kitamura, R., 15 (27) Kittaneh, M., 125 (389), 163 (500) Klauck, P., 120 (375) Klausner, R., 57 (170) Klebanov, B., 83 (247), 160 (492) Klefstrom, ¨ J., 57 (173) Klein, B.D., 76 (225) Klein, H.U., 134 (420) Klimstra, D.S., 84 (250) Klinghammer, K., 10 (10) Klinghoffer, R., 38 (105) Klingner, K., 10 (11), 11 (16) Klinguer-Hamour, C., 113 (351) Klinowska, T., 160 (491), 177 (546) Klinz, S.G., 87 (261) Klotzbucher, ¨ A., 107 (331) Kneissl, M., 105 (324), 117 (364) Knutson, S., 92 (277) Ko, A.H., 148 (451) Kocak, H., 130 (409) Kodama, Y., 191 (592) Kodialbail, P., 41 (117) Koenig, E., 181 (561), 186 (577) Koenig, S., 48 (138) Koeppen, H., 124 (387) Koestler, T., 102 (313), 149 (454) Kogner, P., 135 (423) Koh, J., 174 (534) Koh, K.X., 29 (74) Koh, S.B., 16 (32)

216 Koh, Y., 129 (403) Kohler, ¨ G., 134 (420) Kohli, N., 144 (438) Koizumi, F., 129 (403) Kojouharov, B., 40 (114) Kolb, A., 131 (410, 412) Kolesnick, R.N., 81 (241) Koletsky, A., 8 (4) Kolev, V., 144 (439) Kolev, V.N., 99 (302), 146 (446) Kolinsky, K., 106 (327) Kollmannsberger, C.K., 165 (508) Komai, K., 51 (150) Koncarevic, S., 107 (331) Konde, A., 103 (318) Kondo, M., 30 (77) Kondoh, Y., 14 (26) Konecny, G., 73 (215) Kong, X., 29 (76) Konkol, Y., 25 (63) Kontny, U., 134 (420) Koolmeister, T., 81 (240) Kopetz, E.S., 56 (169) Kopetz, S., 3 (1) Korangath, P., 36 (98) Koritzinsky, M., 66 (204) Korjamo, T., 142 (432) Korn, R., 61 (186) Korn, R.L., 87 (261) Korn, W., 148 (451) Koromilas, A., 148 (452) Koropatnick, J., 21 (49) Korrodi-Gregorio, ´ L., 15 (28) Korsching, E., 134 (420) Koser, M., 115 (358) Kosterink, J.G.W., 174 (535) Kotasek, D., 197 (7LBA) Kounnis, V., 86 (258) Kovacevic, Z., 175 (539) Kovanen, P., 57 (173) Kovulchuk, A., 131 (411) Kovulchuk, O., 131 (411) Kozarewa, I., 33 (88) Kramer, B.S., 75 (222) Krastev, D., 33 (88) Kraus, M., 177 (546) Krause, W.L., 187 (581) Krepler, C., 34 (93) Krishnamoorthy, J., 148 (452) Krishnan, B., 98 (299) Krivoshik, A., 198 (9LBA) Krizman, D., 175 (537) Krohn, K., 64 (197), 79 (234, 235) Kronborg, T.T.A., 184 (569) Kros, J.M., 53 (157) Kruempel, A., 76 (227) Kruger, R., 72 (212) Krupa, R., 45 (129), 127 (397) Kryeziu, K., 16 (30) Krzycki, P., 76 (227) Kubbutat, M., 12 (17) Kubbutat, M.H.G., 107 (331) Kuhn, K., 107 (331) Kuhn, P., 148 (451) Kuijpers, A., 59 (177) Kukuk, K., 11 (16) Kumar, N., 197 (6LBA)

Author Index Kumar, P., 149 (456) Kumar, V., 89 (269) Kumar Pal, S., 126 (395) Kumari, R., 60 (185) Kummar, S., 77 (229), 78 (233) Kunii, K., 149 (456) Kuniyoshi, R., 136 (426) Kunkle, L., 125 (391) Kuntz, K., 92 (277) Kuo, C.C., 92 (279), 147 (449) Kuo, Y.H., 86 (257) Kurmasheva, R., 131 (410, 412) Kuroda, M., 105 (323) Kurokawa, Y., 127 (396) Kurzrock, R., 56 (169) Kuwada, S., 186 (576) Kuwano, M., 32 (84), 190 (589) Kuznetsov, G., 197 (6LBA) Kuznetsova, E., 95 (287) Kwak, E.L., 141 (428), 158 (484) Kwak, Y., 153 (468) Kwee, I., 182 (564), 184 (568) Kwilas, A.R., 101 (308) Kwok, B., 82 (245) Kwon, K.B., 55 (164) Kwon, M., 119 (371)

L La Monica, S., 129 (404) La Porta, I., 62 (189) LaBarge, M., 117 (362) Lackner, M., 122 (382) Lagergren Lindberg, M., 184 (570) Lai, C., 115 (358) Lai, J., 190 (591) Laister, R., 94 (285) Laken, H., 165 (506) Lallena, M., 165 (507) Lam, L., 130 (408) Lamar, M., 104 (320) Lambert-van der Brempt, C., 90 (272) Lambrechts, D., 11 (15) Lan, Y., 68 (208) Lana, S.E., 11 (13) Lancashire, M., 144 (437) Landesman, Y., 83 (247), 156 (480), 160 (492) Landriscina, M., 60 (182) Lane, H.A., 65 (202) Lang, A., 35 (96) Lange, N., 151 (464) Lardelli, P., 17 (35) Lardon, F., 27 (68), 97 (295) Larkin, J., 117 (364) Larsen, A.K., 130 (406) Larsen, A.R., 157 (482) LaStant, J., 157 (483) Lau, A., 84 (251), 198 (8LBA) Laudeman, J., 30 (78) Launchbury, F., 41 (118) LaVallee, T., 71 (210) Lawler, K., 77 (228) Lawrence, D.P., 102 (314) Lawrence, Y.R., 73 (216) Lawson, D., 163 (501) Le Cesne, A., 121 (378)

Le Moigne, R., 180 (555) Le Morvan, V., 95 (289) Le Tourneau, C., 146 (444), 179 (551) Leach, M.O., 175 (540) Leary, A., 142 (431) Leary, C.B., 115 (355) LeBeau, A., 145 (442) Leblanc, R., 182 (562) LeClair, B., 54 (162) Ledell, J., 166 (511) Lee, B.S., 98 (298) Lee, C.K., 20 (44) Lee, C.O., 126 (393) Lee, H., 153 (468) Lee, H.K., 126 (393) Lee, H.S., 173 (533) Lee, J., 17 (34), 49 (143), 65 (201), 76 (224) Lee, J.H., 42 (119) Lee, J.K., 79 (236) Lee, J.S., 44 (127) Lee, K., 153 (468), 174 (534) Lee, K.H., 42 (119), 73 (217), 79 (236) Lee, K.S., 35 (95) Lee, K.T., 79 (236) Lee, M.J., 173 (533) Lee, N., 105 (322) Lee, P., 125 (391) Lee, P.R.E., 154 (472) Lee, S., 49 (143), 130 (407), 173 (533) Lee, S.C., 116 (360) Lee, S.J., 73 (217) Lee, S.Y., 55 (164) Lee, T.C., 20 (45), 31 (82) Lee, V., 66 (203) Lee, Y.C., 50 (144) Leff, R., 84 (253) Legendre, B., 76 (227) Leggatt, G., 97 (294) Lehnhard, D., 10 (11) Leighl, N.B., 44 (127) Leith, S.J., 187 (578) Lejeune, P., 185 (573) Lemmens-Gruber, R., 16 (30) Lemos, R., 160 (490) Lenz, H.J., 199 (11LBA) Leong, H.S., 187 (578) Leong, S., 122 (382), 171 (526) Leonidou, A., 186 (575) Leopold, W.R., 9 (8) Leppla, S.H., 126 (394) Leroux, E., 189 (586) LeSimple, T., 142 (431) Lettini, G., 60 (182) Leung, H., 141 (429) Leung, L., 39 (109) Levantini, E., 129 (405) Leverson, J.D., 130 (408) Levin, R.S., 28 (73) Levine, R.L., 98 (299) Levy, A., 74 (219) Levy, S., 158 (485) Lewens, F., 149 (455) Lewensohn, R., 184 (570) Lewicki, J.A., 7 (2) Lewis, A., 94 (285) Lewis, A.M., 94 (283)

Author Index Lewis, J., 122 (380), 168 (518) Lewis, J.D., 187 (578) Leyland-Jones, B., 128 (399) Lhomme, C., 74 (219) Li, C., 39 (111) Li, F., 95 (287) Li, G., 101 (310) Li, H., 48 (138), 94 (283), 112 (348) Li, P., 54 (162) Li, S., 116 (359) Li, W., 95 (288) Li, X., 48 (140), 145 (443) Li, X.L., 186 (576) Li, Y., 3 (1), 164 (504), 173 (532) Li, Y.Y., 161 (494) Li Causi, E., 146 (445) Liang, S., 39 (109) Liang, X., 103 (316), 117 (362) Liao, M., 54 (162) Liao, W., 159 (489) Liao, W.L., 175 (537) Libener, R., 62 (189) Licandro, S., 62 (189) Licandro, S.A., 21 (48) Liddy, N., 44 (126) Lieftink, C., 34 (91), 61 (188) Lieu, C., 107 (329) Ligon, G.F., 130 (407) Lih, C., 78 (233) Lih, J., 36 (101), 78 (232), 147 (450) Lillo, M.P., 111 (345) Lillquist, J.S., 130 (407) Lim, E., 80 (238) Lim, H.Y., 73 (217), 163 (501) Lim, K., 149 (456) Lim, W.T., 32 (86) Lin, C., 17 (34) Lin, C.C., 72 (213), 73 (217), 163 (501) Lin, H., 19 (43) Lin, K., 73 (215), 83 (249), 136 (424) Lin, W., 122 (382) Lin, X., 162 (497), 176 (544) Lin, Y., 68 (208), 162 (497), 176 (544) Lin, Y.W., 20 (45) Lindbom, L., 125 (392) Lindsay, C., 172 (529) Linehan, W.M., 8 (5) Linetskaya, R., 148 (451) Ling, M., 33 (89) Lingnau, A., 12 (17) Link, W., 24 (59) Linn, S., 173 (532) Linnanen, T., 142 (432) Linton, K., 162 (499) Lipatov, O., 19 (41) Lipsitz, D., 8 (4) Lipson, D., 84 (250), 132 (414, 415) Litinovic, E., 130 (408) Litten, J., 199 (10LBA) Littlefield, B.A., 20 (46) Litvin, O., 28 (71) Liu, B., 93 (282) Liu, C.J., 61 (187) Liu, G., 60 (181) Liu, H., 39 (110) Liu, J., 91 (276), 179 (553) Liu, J.O., 36 (98)

Liu, K., 179 (551) Liu, L., 89 (268), 109 (338), 162 (497) Liu, S., 199 (10LBA) Liu, S.H., 126 (394) Liu, X., 108 (335) Liu, X.D., 172 (530) Liu, Y., 72 (212) Liu, Y.W., 152 (465) Liu, Z.H., 52 (154) Liv, P., 114 (353) Llovet, J.M., 75 (223), 152 (466) Lo, R.S., 29 (76) Loadman, P.M., 95 (289) Loberg, R., 40 (112) Loberg, R.D., 161 (496) Loch, D., 35 (94), 122 (381) Lock, R., 131 (410, 412) Lockhart, A., 21 (50) Lockley, M., 38 (106) Lodola, A., 90 (270) Lohmann, J.J., 90 (272) Loillier, B., 191 (594) Loiseau, A., 67 (207) Lokiec, F., 142 (431), 189 (587) Lolkema, M.P., 121 (378), 185 (572), 199 (11LBA) Longoni, N., 159 (487) Lopane, C., 161 (494) Lopez, ´ I., 65 (199) Lopez-Casas, P.P., 23 (55), 83 (249) Lopez ´ Gines, C., 51 (151) Lopez-Martin, J., 179 (551) Lopez Picazo, J.M., 65 (199) Lord, C.J., 33 (88), 50 (147), 103 (318) Lorens, J., 117 (362) Lorigan, P., 117 (364) LoRusso, P., 7 (2), 71 (210), 125 (389) Losada, A., 108 (334), 111 (345) Lospitao, E., 110 (339), 128 (402) Loßner, ¨ C., 107 (331) Louw, J., 45 (129), 127 (397) Low, S.H.H., 29 (74) Lowe, S.W., 29 (75) Lu, D., 45 (129), 46 (132), 127 (397) Lu, G., 87 (260) Lu, J., 32 (87), 89 (268) Lu, T., 141 (430) Lu, T.L., 51 (149) Lu, W.P., 115 (355) Lu, X., 186 (576) Lub-de Hooge, M.N., 174 (535) Lubbert, ¨ M., 10 (11) Lubeski, C., 71 (210) Lubet, R., 75 (222) Lubet, R.A., 76 (225, 226) Lubiniecki, G.M., 44 (127) Lublinsky, A., 181 (561) Lucas, J., 145 (443) Lucas, R., 115 (357) Lucic, M., 108 (333) Ludes-Meyers, J., 155 (476) Ludovici, D., 141 (430) Lugovskoy, A., 144 (438) Luider, J., 131 (411) Luna, X., 46 (132) Lunceford, J., 48 (140) Lund, R.R., 30 (79)

217 Lunec, J., 91 (276) Luo, J., 102 (312), 123 (385) Luthra, R., 3 (1) Luthra, R.G., 56 (169) Lynchbury, J., 72 (214) Lyne, P., 73 (217), 163 (501) Lyons, J., 168 (518)

M Ma, B., 163 (500) Ma, D.M., 10 (12) Ma, L., 73 (215) Maag, D., 82 (246) Ma¨ att ¨ a, ¨ J., 59 (179) Maawy, A., 121 (376) Maby-El Hajjami, H., 177 (547) MacBeath, G., 144 (438) MacCallum, L., 84 (251) MacDonald, E., 91 (275) Mace, ´ S., 121 (378) Macoin, J., 48 (139) Maddalena, F., 60 (182) Maderna, A., 145 (443) Maertens, G., 56 (169) Maguire, S., 171 (527), 186 (575) Mahadevan, D., 197 (7LBA) Mahon, C., 35 (94) Mahon, T., 44 (126) Mai, A., 185 (571) Mainardi, S., 59 (177), 61 (188) Maio, M., 47 (134) Maitland, N., 12 (20) Majewski, I.J., 54 (160) Majka, M., 170 (524) Mak, M.P., 143 (435) Makhanov, M., 52 (153), 60 (184) Malafa, M.P., 100 (305) Malasse, S., 189 (586) Maleki Vareki, S., 21 (49) Maloney, L., 73 (215) Malouf, G., 188 (582) Maman, S., 91 (275) Mamlouk, K., 8 (4) Mangatt, B., 98 (300) Mankoo, P., 96 (291) Mann, E., 73 (215) Manova-Todorova, K., 81 (241) Mansfield, J.R., 8 (6) Mantaj, J., 19 (42) Mantzaris, M.D., 86 (258) Manuel-Manresa, P., 15 (28) Manzano, J.L., 114 (354) Mappa, S., 162 (499) Maqueda, M.A., 171 (526) Mar, A.C., 31 (82) Marais, R., 29 (76), 175 (540) Marappan, S., 104 (321) Marceau, K., 87 (261) Marcellus, R., 95 (287) March, N., 195 (2LBA) Marchini, S., 21 (48) Marchio, C., 186 (575) Marchlik, E., 155 (475), 156 (481), 167 (513), 172 (531) Mardis, E.R., 29 (75) Maric, G., 22 (51)

218 Marino, D., 124 (386) Maris, J., 131 (410, 412) Maris, J.M., 132 (414) Markkanen, E., 71 (211) Markman, B., 197 (7LBA) Marrinucci, D., 8 (4), 45 (129), 127 (397) Marsh, V., 87 (261) Marsh Durban, V., 100 (304) Marshall, G., 135 (421) Marshall, G.M., 135 (422) Martell, R., 165 (506) Martin, J., 24 (59) Martin, M., 106 (325) Martin, N., 141 (429) Martin, S.E., 83 (248) Martin, Y., 38 (107) Martin Algarra, S., 114 (354) Martinetto, H., 51 (151) Martinez, J., 111 (345) Martinez, J.F., 108 (334) Mart´ınez, P., 24 (57) Martinez, R.T., 84 (253) Martinez, V., 164 (502) Martinez-Laperche, C., 18 (39) Martinez-Leal, J.F., 24 (58) Mart´ınez-Leal, J.F., 20 (47) Martinez-Lopez, J., 77 (230), 136 (427) Martinez-Romero, A., 115 (357) Martini, J.F., 116 (359) Martins, G.R., 169 (522) Martins, V., 168 (518) Martinsson, T., 135 (423) Martomo, S., 46 (132) Marusiak, A., 164 (504) Marusiak, A.A., 29 (76), 167 (512) Maruyama, E., 30 (77) Maschio, L.B., 169 (522) Mason, H., 123 (384), 198 (8LBA) Mason, S., 164 (504) Masrorpour, F., 81 (243) Massaoka, M.H., 26 (64), 43 (124) Masson, K., 144 (438) Masson, P., 191 (594) Masuda, M., 112 (346) Masuda, T., 175 (538) Masuko, N., 103 (315) Matheny, C., 98 (300) Matheny, S., 199 (10LBA) Mathijssen, R.H.J., 26 (66) Matin, S., 172 (530) Matsui, J., 17 (36), 20 (46) Matsumoto, C., 176 (541) Matsuno, K., 91 (274) Matsuo, K., 13 (22), 15 (27, 29) Matsusaka, S., 166 (509) Mattevi, A., 185 (571) Maudet, M., 90 (272) Maugeri, I., 56 (168) Maxuitenko, Y.Y., 107 (330) Maynard, J., 177 (546) Maynes, J., 122 (382) Mayo de las Casas, C., 114 (354) Mazeron, R., 74 (219) Mazerska, Z., 26 (65), 27 (67) Mazhar, S., 158 (486) Mazsa, E., 120 (374) Mazzaferro, V., 152 (466)

Author Index Mazzocca, A., 161 (494) Mazzola, A., 123 (383) Mazzola, A.M., 150 (457) Mazzucco, L., 62 (189) McAleer, D., 7 (3) McCauley, D., 83 (247), 160 (492) McCleland, M., 179 (553) McClinch, K., 178 (548) McCluskey, A., 36 (100) McConville, P., 9 (8) McCoon, P., 73 (217), 163 (501) McCormick, F., 28 (73), 113 (350) McCowage, G., 135 (421) McCulloch, W., 195 (3LBA) McCutchen, E., 76 (227) McDaniel, A.S., 61 (187) McDermott, U., 56 (167) McDonnell, D., 31 (80) McEachern, D., 32 (87), 89 (268), 109 (338), 162 (497), 176 (544) McEwan, M., 57 (172) McGonagle, A., 94 (284) McGonigle, S., 20 (46) McGrath, Y., 44 (126) McInnes, C., 18 (40) McIntosh, D., 120 (374) McKee, S., 97 (294) McKelvie, J., 91 (275) McKenzie, A., 60 (185) McLeod, A.R., 150 (457) McLeod, K., 160 (491) McLeod, M., 84 (253) McLoughlin, M., 198 (8LBA) McLure, K.G., 181 (560) McMahon, M., 100 (304) McMillan, N., 97 (294) McNeish, I., 73 (215) McNeish, I.A., 38 (106) McPherson, V.A., 187 (578) McWhirter, E., 165 (508) Meade, J., 12 (18), 35 (96, 97), 37 (102), 156 (481) Medema, J.P., 164 (505) Meek, C., 84 (253) Meerpoel, L., 115 (356), 128 (400), 141 (430) Meetze, K., 120 (374), 156 (479) Mefo, T., 121 (377) Mehaffey, M.G., 78 (233) Mehlen, P., 118 (368) Mei, G., 151 (462) Mekhail, T., 199 (10LBA) Melcher, A.A., 41 (118) Melchers, L.J., 145 (441) Melito, C., 62 (189) Mello-Grand, M., 180 (556) Mellor, M., 99 (301) Melnick, A.M., 182 (563) Meltzer, P., 84 (252) Mendelsohn, J., 3 (1), 159 (488) Mendoza, A., 82 (245), 101 (311) Men ´ etrey, ´ A., 177 (547) Menges, C.W., 146 (446) Meniawy, T., 7 (3) Menju, T., 188 (584) Menon, D.R., 34 (93) Menon, M.K., 180 (555)

Mercadante, G., 62 (189) Merchant, M., 124 (387) Mercurio, C., 185 (571) Meric-Bernstam, F., 3 (1), 56 (169), 57 (170), 79 (237), 136 (425), 159 (488) Meroni, G., 185 (571) Merqui-Roelvink, M., 121 (378) Mertens, I., 96 (290) Mertz, J., 109 (336), 187 (581) Merzoug, F., 165 (507) Mesange, ´ P., 130 (406) Meschini, E., 106 (325) Messikommer, A., 65 (202) Metcalf, R.L., 88 (264) Mettetal, J., 161 (493) Metz, T., 9 (7) Metzig, M., 32 (85) Meyer, C., 141 (430) Meza, M., 50 (146) Miao, H., 121 (378) Michallet, M., 196 (5LBA) Mickelm, D., 117 (362) Middleton, M., 105 (324), 117 (364) Middleton, S.A., 106 (327) Miekus, K., 170 (524) Miele, G., 121 (377) Miettinen, M., 102 (312) Mikuniya, M., 166 (509) Mileshkin, L., 7 (3) Milhollen, M., 181 (561), 186 (577) Milhollen, M.M., 85 (255) Miller, C., 164 (504) Miller, D., 120 (372) Miller, M., 90 (271) Miller, M.S., 76 (225) Miller, N., 54 (161) Miller, R.E., 103 (318) Miller, V.A., 84 (250), 104 (319), 132 (414, 415) Millhollen, M., 78 (231) Millot, N., 67 (207) Mills, G.B., 3 (1), 124 (388), 159 (488) Mills, I.G., 161 (495) Min, D.J., 117 (363) Min, P., 162 (497) Minamiguchi, K., 103 (315) Minana, ˜ R., 87 (263) Minarik, M., 51 (149) Minden, M.D., 94 (285) Minna, J., 117 (362) Minna, J.D., 124 (388) Minter, R., 106 (326) Minucci, S., 185 (571) Mirabile, I., 172 (529) Mirjolet, C., 67 (207) Mirsaidi, C., 50 (146), 59 (178) Mishima, Y., 136 (426), 166 (509) Mistry, P., 82 (245) Mita, M.M., 165 (506) Mittal, D., 40 (115) Mittapalli, R., 86 (259) Mittempergher, L., 54 (160) Miura, A., 127 (398), 142 (433) Miwa, S., 96 (292), 111 (343), 121 (376) Miyadera, K., 128 (401) Miyata, H., 127 (396) Miyaura, C., 176 (541)

Author Index Miyazaki, Y., 127 (396) Mizuarai, S., 142 (433) Mizui, Y., 149 (456) Mizunuma, N., 166 (509) Mkrtchian, S., 95 (289) Modi, S., 163 (500) Moeini, A., 75 (223), 152 (466) Moffat, J., 66 (204), 124 (387) Mogami, T., 129 (403) Moghaddam, M.F., 150 (459), 170 (523) Mohammad, H., 72 (212) Mohankumar, K.M., 63 (193) Mohr, T., 16 (30) Moilanen, A., 142 (432) Moiseyenko, V., 19 (41) Moisse, M., 11 (15) Molina, M., 32 (84) Molina, M.A., 156 (478) Molina-Guijarro, J.M., 24 (58), 111 (345) Molina-Vila, M., 167 (514) Molina-Vila, M.A., 85 (254), 114 (354) Molloy, M.E., 103 (316) Molloy, P., 44 (126) Momiyama, M., 121 (376) Momot, M., 45 (130) Monasterio, J.C., 156 (478) Moneo, V., 24 (57, 58) Monica Molano, M.M., 119 (369) Monkk ¨ onen, ¨ J., 59 (179) Monks, A., 30 (78), 57 (171), 147 (450) Monleon, D., 51 (151) Montagna, C., 108 (335) Montagne, E., 76 (227) Montagnoli, A., 16 (31), 168 (517) Montalbetti, C., 180 (557), 191 (594) Montaner, D., 177 (545) Montemurro, F., 27 (69) Montgomery, B., 8 (4) Moon, H.G., 173 (533) Moon, S.R., 55 (164) Moore, J., 171 (527) Moore, P., 48 (138) Mor, M., 90 (270) Mora Vidal, R., 106 (325) Moral, P., 108 (334) Moran, D.M., 74 (218) Morancho, B., 82 (244) Mordec, K., 190 (591) Moreno-Acosta, P.M.A., 119 (369) Morera, Y., 38 (107) Morgan, S.R., 126 (395) Morgentin, R., 90 (272) Mori, M., 127 (396) Mori, T., 103 (315) Moriarty, A., 37 (102) Morimoto, A., 129 (403) Moris, F., 159 (487) Morita, S., 198 (9LBA) Moriyama, H., 112 (346) Moro, M., 154 (472) Morosini, D., 132 (414, 415) Morris, B., 59 (177) Morris, J., 30 (78), 83 (248), 89 (269) Morris, S.R., 7 (3) Morrison, A., 123 (384) Morrow, C., 144 (437) Morrrow, C., 88 (264)

Morschhauser, F., 162 (499) Mortazavi, A., 151 (461) Mortensen, D.S., 100 (306), 150 (459), 170 (523) Mortimer, S., 151 (462) Mortusewicz, O., 81 (240) Moschetta, G.M., 169 (522) Moscow, J., 165 (508) Moser, B., 134 (420) Moshel, Y., 73 (216) Moskalenko, M., 45 (128) Moskaluk, C.A., 35 (97) Mosse, Y., 132 (414) Motersino, C., 183 (566) Motta, M., 163 (500) Motzer, R.J., 116 (359) Moulon, C., 177 (547) Moutinho, C., 23 (54) Moyo, V., 107 (329) Mu, S., 185 (572) Muders, M., 64 (196) Mueller, V., 106 (328) Muench, D., 94 (285) Muir, M., 160 (491) Mukaida, N., 161 (494) Mukherjee, S., 81 (243), 96 (293), 104 (321) Mulet Margalef, N., 110 (341) Muller, ¨ P., 11 (16) Muller, W.J., 22 (51) Muller-Tidow, ¨ C., 134 (420) Mullins, C., 59 (178) Mumberg, D., 25 (63) Munasinghe, W., 111 (342) Muniz-Medina, V., 106 (326) Munoz, ˜ E., 114 (354) Munoz-Alonso, ˜ M.J., 164 (502) Munoz-Calleja, ˜ C., 18 (39) Munoz ˜ Hidalgo, L., 51 (151) Munster, P., 7 (2) Munster, P.N., 179 (551) Munt, S., 164 (502) Murai, J., 84 (252) Murakami, H., 198 (9LBA) Murakami, T., 121 (376) Muraoka, H., 191 (592) Murell, M., 97 (294) Muroi, M., 14 (26) Murphy, D., 143 (436) Murphy, S., 64 (197), 79 (234, 235) Murray, C.W., 115 (356), 128 (400) Murray, J., 135 (422) Mussini, C., 25 (61) Muzi, M., 64 (197), 79 (234, 235) Myers, J.N., 109 (337) Myers, S., 141 (429) Myint, K., 149 (456)

N Nadano, S., 73 (217), 163 (501) Nadella, P., 163 (501) Nagai, K., 52 (155) Nagai, M., 188 (585) Nagi, A.H., 42 (120) Naik, C., 104 (321) Naing, A., 56 (169), 136 (425)

219 Nair, P., 50 (146), 59 (178) Naito, M., 52 (155) Naka, T., 127 (396) Nakagawa, F., 13 (22) Nakagawa, K., 97 (296), 198 (9LBA) Nakaigawa, N., 154 (473) Nakajima, K., 127 (396) Nakama, T., 190 (589) Nakanishi, T., 188 (584) Nakatsuka, R., 127 (396) Nakatsuru, Y., 127 (398) Nakayama, A., 176 (543) Nakuci, E., 167 (513) Nam, K., 174 (534) Namura, K., 154 (473) Nanda, N., 125 (391) Nandabhiwat, P., 82 (245) Narayan, K., 142 (432) Narayanan, K., 104 (321) Narendran, A., 42 (121), 131 (411), 189 (588) Narhi, ¨ K., 57 (173) Narla, G., 158 (486), 178 (548) Narla, R.K., 150 (459), 170 (523) Naseem, N., 42 (120) Nash, A.F., 126 (395) Natale, R., 19 (41) Natale, R.B., 165 (506) Natali, P.G., 47 (134) Nathan, S., 104 (321) Natoli, E.J., 130 (407) Natrajan, R., 186 (575) Neff, C., 72 (214) Nehls, M., 12 (18), 35 (97) Nemunaitis, J., 21 (50), 163 (501) Nenseth, H.Z., 161 (495) Ner-Kluza, J., 12 (17) Nering, R., 107 (329) Nerle, S., 31 (83) Nettelbeck, D.M., 48 (137) Neuhaus, R., 185 (573) Neumann, F., 73 (217) Neves, D., 118 (368) Newbold, R., 61 (186) Newell, D., 141 (429) Newell, D.R., 91 (276), 106 (325) Newell, H., 133 (417) Newth, G., 125 (389) Newton, R., 195 (2LBA) Ng, C.K.Y., 186 (575) Ng, K.W., 35 (95) Ng, P.K.S., 143 (435) Ng, T., 77 (228) Ngu, H., 124 (387) Nguyen, A., 179 (551) Nguyen, D., 77 (229) Nguyen, L.K., 77 (228) Ni, J., 123 (385) Nichols, G., 106 (327) Nicolas, G., 183 (566) Nicolas Magne, N.M., 119 (369) Nicolas-Metral, ´ V., 177 (547) Nicolas Morales, N.M., 119 (369) Nicolay, H.J.M.G., 47 (134) Nicolle, D., 25 (61) Nielsen, S., 184 (569) Nielsen, U., 52 (152)

220 Nielsen, U.B., 144 (438) Nieto, A., 17 (35) Nijkamp, W., 31 (81) Ning, J., 60 (181) Nishida, T., 127 (396) Nivsarkar, M., 85 (256) Niwa, Y., 51 (150) Nix, J., 8 (5) Njar, V.C., 8 (4) Noble, M.E.M., 91 (276), 106 (325) Noble, R.B., 7 (3) Noel, K., 183 (565) Noerholm, M., 102 (313), 149 (454) Noguchi, K., 15 (29) Noh, D.Y., 173 (533) Noh, S.H., 20 (44) Nokihara, H., 198 (9LBA) Nomoto, A., 175 (538) Nomoto, K., 20 (46) Nonaka, D., 88 (264) Nones, K., 35 (94) Nordquist, L., 8 (4) Normant, E., 31 (83), 125 (390) Nørregaard-Madsen, M., 184 (569) Norris, M., 135 (421) Norris, M.D., 135 (422) Norton, J., 118 (366) Notarangelo, T., 60 (182) Nunez, ˜ L.E., 159 (487) Nunez, ˜ P., 23 (55) Nunn, P., 157 (483) Nurnberg, ¨ P., 130 (409) Nytko-Karouzakis, K., 65 (202)

O Oberthur, A., 135 (422) Obilisetti, K.M., 104 (321) O’Connor, K., 80 (238) O’Connor, M.J., 198 (8LBA) Oda, N., 127 (398) Odagami, T., 121 (377) Odedra, R., 84 (251) Odore, E., 153 (469), 183 (565), 189 (587) O’Dwyer, P., 154 (474) O’Farrell, M., 190 (591) Ogilvie, D., 94 (284), 195 (2LBA) Ogle, C., 30 (78) Ogo, N., 91 (274) Oguchi, K., 13 (22), 15 (27) Oh, G.S., 55 (164) Oh, Y., 92 (278) Ohashi, Y., 185 (574) Ohkubo, M., 120 (373) Ohkubo, S., 191 (592) Ohlmeyer, M., 158 (486), 178 (548) ¨ Ohlund, D., 63 (194) Ohno, S., 105 (323) Ohoka, N., 52 (155) Ohtsu, A., 36 (101), 72 (213) Okada, M., 120 (373) Okajima, S., 103 (315) Okamoto, K., 17 (36) Okamura, M., 185 (574) O’Keeffe, B., 122 (382) Oksala, R., 142 (432) Okuhira, K., 52 (155)

Author Index Okusaka, T., 73 (217), 163 (501) Olbiol, C., 156 (478) Oliveira, M., 163 (500) Ollier, R., 48 (139) Olopade, O., 173 (532) Olson, A., 82 (246) Olszanski, A.J., 105 (324), 117 (364) O’Malley, D., 73 (215) Omasa, M., 188 (584) Onar-Thomas, A., 133 (416) O’Neill, B.O., 10 (12) Ong, A., 148 (451) Oni, T., 63 (194) Ono, M., 32 (84), 190 (589) Oplustilova, L., 198 (8LBA) Orecchia, S., 62 (189) O’Reilly, E., 152 (465) O’Reilly, T., 65 (202) Ormanoglu, P., 83 (248) Orr, C., 124 (387) Ortuno, ˜ F., 187 (579) Osada, H., 14 (26) Osarogiagbon, R., 61 (186) Oscar Gamboa, O.G., 119 (369) Oses-Prieto, J.A., 28 (73) Oshlack, A., 54 (160) Osman, A., 109 (337) Osman, S., 87 (260) Ostano, P., 180 (556) Osterling, D., 82 (246) O’Sullivan, F., 64 (197), 79 (234, 235) O’Sullivan, J., 64 (197), 79 (234, 235) Oswald, E., 10 (11) Otterson, G., 19 (41) Ottesen, L.H., 197 (6LBA) Ou, S.H., 199 (10LBA) Ou, T.H., 20 (45) Ouafik, L., 153 (469) Ouhtit, A., 119 (370) Oves Costales, D., 24 (59) Oxnard, G.R., 199 (10LBA) Oza, A., 73 (215) Ozawa, Y., 17 (36) Ozer, J., 178 (550) Ozorio dos Santos, E.O., 129 (405)

P Pablo, L., 163 (501) Pacheco-Sanchez, P., 45 (128) Pachmann, K., 62 (190) Pachmann, U., 62 (190) Pachter, J., 99 (302), 144 (439) Pachter, J.A., 146 (446) Packer, L., 35 (94), 122 (381) Packman, K., 106 (327) Padval, M., 97 (296), 144 (439) Paek, S., 166 (510) Page, M., 60 (185) Page, R., 11 (13) Paiva, S., 95 (286) Paiva, S.L., 94 (283) Pala, D., 90 (270) Palazzo, J., 37 (103) Paller, C.J., 143 (434) Palma, N., 104 (319) Palma, N.A., 132 (414)

Palmer, B.D., 91 (273) Palmer, G.A., 132 (414) Palumbo, A., 196 (5LBA) Pan, B., 154 (472) Pan, J., 28 (70) Panchal, S., 82 (246) Pandha, H., 41 (118) Pandher, R., 148 (453) Pandit, C., 104 (321) Panini, N., 21 (48) Pant, S., 21 (50), 57 (173) Pantel, K., 106 (328) Pao, W., 84 (250) Paoloni, M., 173 (532) Papadimitrakopoulou, V., 199 (10LBA) Papadopoulos, K., 125 (389) Papadopoulos, K.P., 12 (18), 35 (96, 97), 37 (102), 111 (342), 156 (481) Papadopoulou, N., 60 (185) Papadpoulos, K., 104 (320) Papanikolaou, N.A., 86 (258) Papi, M.P., 168 (516) Paproski, R., 147 (447) Paracchini, L., 21 (48) Parchment, R., 147 (450) Pardo, J.M., 115 (357) Parini, V., 14 (24) Paris, J., 67 (207) Park, C.H., 126 (393) Park, D.H., 79 (236) Park, E., 149 (456) Park, E.K., 153 (470) Park, J., 92 (278), 173 (533) Park, J.I.H.O., 92 (278) Park, J.W., 42 (119) Park, J.Y., 119 (371) Park, R.K., 55 (164) Park, S.H., 65 (201) Park, W.Y., 79 (236) Park, Y., 63 (194) Park, Y.M., 23 (56), 24 (60), 25 (62) Park, Y.S., 65 (201) Parker, J., 55 (163) Parker, M., 141 (430) Parkkola, H., 87 (263) Parri, M.P., 41 (116), 178 (549) Parrish, K., 86 (259) Parsey, M., 195 (3LBA) Partanen, K., 47 (136) Pasquest, M.J., 90 (272) Pastan, I., 102 (312) Paston, S., 44 (126) Pastor, E., 46 (133) Pastor, J., 64 (198), 118 (365) Patel, A., 109 (337) Patel, H.K., 103 (316) Patel, K.P., 3 (1) Patel, M., 21 (50), 105 (324), 161 (493), 195 (3LBA) Patel, R., 143 (436) Patel, S.P., 56 (169) Patel, T.B., 191 (593) Patel, V., 35 (95) Patel, Y.T., 63 (193) Patierno, S., 105 (322) Patnaik, A., 7 (2), 12 (18), 35 (96, 97), 37 (102), 44 (127), 48 (140), 104 (320)

Author Index Patniak, A., 125 (389) Patterson, L.H., 95 (289) Paula Galvao da Silva, A., 156 (479) Paulussen, C., 115 (356), 128 (400) Pauly, J., 189 (586) Pauwels, P., 27 (68), 39 (108), 65 (200), 96 (290), 97 (295), 112 (347) Pavlicek, D., 108 (333) Pavlick, A., 117 (364) Paweletz, K., 40 (112) Pawlak, S., 118 (367), 169 (519) Pawlak, S.D., 47 (135), 152 (467) Pawlowska, M., 27 (67) Payne, K.J., 134 (419) Payne, L., 179 (554) Paz, K., 63 (195) Pearson, J., 35 (94) Pease, E.J.E., 123 (383) Pecqueur, J., 133 (417) Pedersen, M., 111 (342) Pedley, R.B., 82 (245) Peer, C.J., 13 (21) Peeters, D., 141 (430) Peeters, J., 173 (532) Peeters, M., 27 (68), 96 (290), 97 (295) Peille, A.L., 11 (16), 62 (192) Peix, J., 75 (223), 152 (466) Pelissier, F., 117 (362) Pelletier, J., 181 (558) Pemberton, H., 33 (88), 50 (147) Peng, H.Y., 147 (449) Peng, S., 150 (459), 170 (523) Peng, S.P., 10 (12) Peng, Y., 196 (5LBA) Peregrina, S., 118 (365), 128 (402) Perez, ´ C., 111 (344) Perez, J., 124 (386) Perez-Chacon, G., 18 (39) Perez-Ferreiro, ´ C.M., 128 (402) Perez-Soler, R., 108 (335) Perez-Tom ´ as, ` R., 15 (28) Perino, S., 171 (527) Perinpanayagam, M., 131 (411) Perrin-Ninkovic, S., 150 (459) Perry, M., 165 (508) Peru, A., 90 (272) Pesenti, E., 16 (31) Pesonen, S., 47 (136) Pessoa, C., 87 (262) Peters, M., 29 (75) Peterson, S., 76 (227) Peterson, V., 54 (161) Petersson, K., 125 (392) Petronini, P.G., 129 (404) Pettitt, S.J., 33 (88), 50 (147) Peyruchaud, O., 182 (562) Pfister, S.X., 71 (211) Pfister, T., 83 (248) Phan, C., 170 (523) Phan, V.T., 157 (483) Phelps, M., 151 (461) Phillips, D.C., 130 (408) Phillips, W., 100 (304) Pichler, V., 22 (52), 53 (156) Pieczykolan, A., 47 (135), 118 (367), 152 (467), 169 (519) Pieczykolan, J., 118 (367), 169 (519)

Pieczykolan, J.S., 47 (135), 152 (467) Pierce, K., 171 (526) Pierrat, M., 110 (340) Pieslor, P.C., 174 (535) Piha-Paul, S., 56 (169) Piha-Paul, S.A., 3 (1) Pike, I., 107 (331) Pike, K., 91 (275) Pilatte, I., 115 (356), 128 (400) Pileri, P.P., 41 (116), 178 (549) Pili, R., 8 (4) Pilotti, S., 190 (590) Pinyol, R., 152 (466) Piperdi, B., 108 (335) Pirie-Shepherd, S., 145 (443) Pirlo, K., 38 (106) Pisano, C., 62 (189) Pitt, C., 113 (350) Pitts, S., 162 (498) Pitts, T., 120 (375) Pizon, M., 62 (190) Plath, T., 10 (10) Ple, P., 90 (272) Ploegh, H.L., 179 (552) Pluess, M., 48 (139) Plummer, R., 117 (364) Poda, G., 95 (287) Poell, J.B., 31 (81) Poi, M., 151 (461) Poindessous, V., 130 (406) Pokorny, J., 86 (259) Polanska, U., 177 (546) Polanski, R., 88 (264) Poleszak, K., 47 (135), 118 (367), 152 (467), 169 (519) Poli, A., 97 (296) Pollard, J., 96 (291) Pollard, J.R., 81 (240) Polley, E., 30 (78) Polley, E.C., 36 (101), 57 (171), 78 (233) Pollock, P., 35 (94), 122 (381) Pollock, R., 92 (277) Pollyea, D.A., 195 (1LBA) Polonskaya, Z., 46 (132) Polyak, V., 184 (569) Pommier, Y., 83 (248), 84 (252) Ponce, S., 111 (344) Poncelet, V., 115 (356), 128 (400) Pond, G., 67 (205) Ponnazhagan, S., 43 (123) Ponz, M., 65 (199) Ponz-Sarvise, M., 63 (194) Poole, J.C., 51 (149) Poon, D.C.S., 93 (281) Poon, T.P., 73 (217) Poonawala, R., 123 (384) Poos, K., 134 (420) Pope, J., 76 (227) Porciello, A., 62 (189) Porco, J., 181 (558) Porro, M., 185 (572) Pors, K., 95 (289) Portela, A., 152 (466) Porter-Scott, M., 92 (277) Post, L.E., 81 (242) Postenka, C.O., 187 (578) Poupon, M.F., 58 (174)

221 Pouyet, L., 183 (566) Powe, J., 181 (561) Powell, S.N., 81 (241) Power, L., 107 (329) Powis, G., 160 (490) Powles, T., 126 (395) Pradere, U., 108 (333) Prah, M., 195 (1LBA) Prahallad, A., 51 (148), 151 (463) Prahalled, A., 154 (472) Prakash, S., 54 (162) Prasetyanti, P.R., 164 (505) Prebet, ´ T., 183 (566) Prell, R., 136 (424) Press, O.W., 88 (265) Presta, M., 100 (307) Preudhomme, C., 196 (5LBA) Prey, J., 87 (261) Prieto, V.G., 150 (460) Prieto-Garc´ıa, E., 111 (344) Primack, B., 80 (238) Primo, D., 77 (230), 136 (427) Prinsen, M.B.W., 113 (349) Prinz, T., 107 (331) Proia, D., 87 (260) Proia, D.A., 188 (585) Proia, T.A., 179 (552) Proniuk, S., 142 (431) Pronk, L.C., 72 (213) Pruschy, M., 67 (206) Pruschy, M.N., 65 (202) Pry, K., 74 (218) Przewloka, T., 87 (260) Puccetti, M.P., 168 (516) Pulido, I., 191 (593) Purmal, A., 135 (422) Purnajo, I., 156 (479) Pursell, N., 115 (358) Puto, L., 164 (504) Putta, S., 39 (109) Puyang, X., 149 (456)

Q Qadir, M., 82 (245) Qian, S., 60 (181) Qiao, M., 11 (14) Qiao, W., 26 (66) Qing, W., 125 (392) Qiu, J., 11 (14) Quach, C., 42 (119) Quadt, C., 29 (75) Quan, Y., 92 (278) Quattrochi, B., 156 (481) Quereshi, H., 95 (286) Querolle, O., 115 (356), 128 (400) Quesada, R., 15 (28) Quesnel, B., 196 (5LBA) Quetglas, I.M., 75 (223), 152 (466)

R Radu, C., 96 (293) Raffoux, E., 196 (5LBA) Rafiq, R., 33 (88), 103 (318) Raghunand, N., 87 (261) Raguse, J.D., 10 (10) Rahman, K.M., 19 (42)

222 Rainard, J., 123 (384) Rajagopalan, S., 142 (432) Rajapakse, V., 84 (252) Ramachandra, M., 40 (113), 104 (321), 142 (432) Ramachandra, R., 142 (432) Ramanathan, R.K., 87 (261) Rambo, B., 175 (537) Ramezani, F., 28 (72) Ramirez, J., 26 (66) Ramos-Lima, F., 64 (198), 118 (365) Ramskold, ¨ D., 135 (423) Ramzanali, N.M., 57 (170) Ranall, M., 97 (294) Rance, C., 158 (484) Rane, J., 12 (20) Ranki, T., 47 (136) Ransohoff, D., 75 (222) Rao, C.V., 76 (226) Rao, K., 74 (218) Rao, P., 60 (183), 87 (260), 172 (530) Rapisarda, A., 57 (171), 147 (450) Raponi, M., 73 (215) Rasco, D., 111 (342), 125 (389) Rasco, D.W., 12 (18), 35 (96, 97), 37 (102), 105 (324), 117 (364), 179 (551) Rashal, T., 83 (247) Rasiah, N., 41 (117) Rassidakis, G.Z., 155 (477) Ratain, M.J., 13 (23), 26 (66) Ratsch, ¨ G., 181 (558) Ravi, V., 3 (1) Ray-Coquard, I., 146 (444) Raymon, H., 170 (523) Raymon, H.K., 150 (459) Raymond, E., 182 (564), 183 (565, 567) Raynaud, F.I., 148 (453) Razak, A.R.A., 79 (237) Re, C., 16 (31) Re Depaolini, S., 168 (517) Read, J., 121 (377) Reaper, P.M., 81 (240) Rebolleda, N., 18 (39) Recher, C., 196 (5LBA) Reddy, A., 187 (581) Reddy, J., 104 (321) Reddy, K., 104 (321) Reed, B., 121 (377) Reedy, B.A., 7 (3) Rees, D.C., 115 (356), 128 (400) Reich, S.J., 107 (330) Reimer, C., 163 (501) Reimer, J., 131 (411) Reinhard, C., 165 (507) Reinhardt, F., 179 (552) Reinhart, R., 30 (78) Reis-Filho, J.S., 186 (575) Rello-Varona, S., 110 (341) Ren, Y., 125 (392) Rengifo Calzado, E., 169 (521) Renner, O., 64 (198), 118 (365) Rennie, P., 161 (495) Renouf, D.J., 165 (508) Rentsch, C., 10 (11) Rettig, M., 8 (4) Reuillon, T., 106 (325), 141 (429)

Author Index Revill, C.H., 91 (276) Reyderman, L., 197 (6LBA) Reyes, C., 118 (366) Reyes, F., 24 (59) Reynolds, P., 131 (410, 412) Rezai, K., 142 (431), 153 (469), 189 (587), 196 (5LBA) Rezzola, S., 100 (307) Rezzoug, F., 120 (372) Rha, S.Y., 20 (44) Rhodes, D., 61 (187) Rhodes, J., 57 (172) Rhodes, S., 125 (391) Riabowol, K., 14 (25) Ribas, A., 48 (140) Ribeiro, J., 182 (562) Ribrag, V., 74 (219), 197 (6LBA) Riccio, A., 62 (189) Rice, J., 180 (555) Rich, S., 122 (380) Richards, F.M., 16 (32) Richards, K., 55 (163) Richardson, D., 175 (539) Richardson, K., 76 (227) Richardson, S., 150 (459) Richon, V., 92 (277) Richter, G.H.S., 134 (420) Rickles, R., 166 (511) Ricono, J., 50 (146), 59 (178), 80 (239), 144 (439) Riddick, M., 91 (275) Riedinger, C., 91 (276) Riesterer, O., 67 (206) Riggins, G., 171 (528) Riggins, G.J., 157 (482) Rigoreau, L., 141 (429) Rigotti, S., 177 (547) Rinaldi, A., 182 (564) Rini, B.I., 116 (359) Rinner, B., 34 (93) Risberg, B., 161 (495) Risbood, P., 89 (269) Rissanen, J.P., 25 (63) Riveiro, E., 184 (568) Riveiro, M., 182 (564), 183 (565, 567), 189 (587) Riveiro, M.E., 153 (469), 187 (580) Rizvi, N.A., 44 (127) Rizzacasa, M., 58 (176) Robb, G., 91 (275) Robert, A., 113 (351) Robert, C., 48 (140) Robert, J., 95 (289) Roberts, K., 91 (275) Roberts-Rapp, L., 130 (408) Robertson, A.E., 187 (578) Robertson, J., 198 (8LBA) Robillard, L., 83 (249) Robinet, G., 44 (127) Robinson, G.W., 133 (416) Robinson, W.A., 59 (180) Robles, A., 77 (230), 136 (427) Robson, M., 82 (245) Rocaboy, C., 141 (430) Rodilla Mart´ın, A., 15 (28) Rodon, J., 124 (386) Rodriguez, J., 159 (489)

Rodriguez, L., 82 (246) Rodriguez, M., 17 (37) Rodriguez Capitan, A., 85 (254), 167 (514) Rodriguez-Carunchio, L., 75 (223) Rodriguez de Miguel, M.C., 64 (198), 118 (365) Rodr´ıguez-Garzotto, A., 111 (344) Rodriguez Quesada, L., 24 (59) Roehrig, F., 89 (266) Roels, F., 130 (409) Rogers, L.K., 75 (221) Roh, J.L., 119 (371) Roh, K.S., 116 (360) Rojas, J.L., 77 (230), 136 (427) Rolfe, L., 73 (215) Rolfe, M., 180 (555) Rolfo, C., 39 (108), 65 (199, 200), 96 (290), 97 (295), 112 (347) Roman, K., 8 (6) Romano, M., 21 (48) Ronca, R., 100 (307) Rong, S., 89 (268) Roodenburg, J.L.N., 145 (441) Rooney, C., 169 (520) Roos, M., 108 (333), 122 (382) Rose, A., 22 (51) Rose, B.J., 9 (9) Rosell, R., 32 (84), 85 (254), 114 (354), 156 (478), 167 (514) Rosen, D., 39 (109) Rosen, L., 122 (382) Rosen, O., 115 (355) Rosenwald, A., 89 (266) Rosner, G.L., 26 (66) Ross, J.S., 84 (250), 104 (319), 132 (414, 415) Rossi, C., 164 (505) Rossi, J., 40 (112) Rossi, V., 27 (69) Rossig, ¨ C., 134 (420) Rosti, V., 21 (48) Rostouin, A., 183 (566) Rothstein, R., 51 (148) Rottey, S., 39 (108) Routbort, M.J., 3 (1) Roux, B., 115 (356), 128 (400) Rovira, H., 90 (271) Rowling, E., 170 (525) Roy, M., 145 (443) Rozga, P., 118 (367), 169 (519) Rozga, P.K., 47 (135), 152 (467) Ruan, Y., 42 (121), 131 (411), 189 (588) Rubino, C., 195 (3LBA) Rubino, L., 176 (542) Rubinstein, L., 77 (229) Rudolph-Owen, L., 72 (214) Ruggeri, B., 154 (471) Rugo, H., 173 (532) Ruhle, M., 135 (422) Ruits, E., 146 (444) Rundle, M., 156 (481) Ruppel, J., 71 (209), 136 (424) Russell, P., 171 (527) Russo, M., 16 (31) Russo, S., 90 (270) Rust, S., 106 (326)

Author Index Rutkoski, T.J., 115 (355) Rutledge, R., 44 (127) Ryan, A.J., 71 (211) Ryoo, B.Y., 73 (217) Rytelewski, M., 21 (49)

S Saatcioglu, F., 161 (495) Saba, C., 110 (340) Sabatier, R., 189 (586) Sabba, ` C., 161 (494) Sablin, M.P., 146 (444) Sablon, E., 56 (169) Sabzichi, M., 28 (72) Saccani, F., 129 (404) Sachdev, J., 61 (186), 165 (506), 196 (4LBA) Sachdev, J.C., 87 (261) Sacristan, ´ M.A., 111 (345) Sadis, S., 61 (187), 78 (232) Saenger, J., 149 (455) Saha, A., 181 (559) Saha, D., 68 (208) Sahay, D., 182 (562) Sai, Y., 125 (392) Sainis, I., 86 (258) Sakamoto, K., 13 (22), 15 (27, 29) Sakamoto, S., 175 (538) Sakoff, J., 36 (100) Sakuragi, M., 127 (398), 167 (515) Sala, G., 164 (505) Salazar, P., 197 (6LBA) Saleh, M., 104 (320) Saleh, T., 90 (272) Sales, F.A.M., 87 (262) Saletta, F., 135 (421) Salgia, R., 159 (489) Salt, M., 28 (73) Salviatto, A.C.P., 103 (317) Samadi, N., 28 (72) Samajadar, S., 142 (432) Samarakoon, T., 115 (355) Sami, M., 44 (126) Sanchez-Fenoy, J., 77 (230), 136 (427) Sanchez-Ronco, M., 156 (478) Sandal, T., 117 (362) Sandberg, R., 135 (423) Sanduja, S., 179 (552) Sandy, P., 31 (83) Sanfilippo, R., 190 (590) Sanford, E.M., 132 (414) Sang, J., 188 (585) Sangale, Z., 72 (214) Sangodkar, J., 158 (486) Sanil, A., 173 (532) Sanjiv, K., 81 (240) Sankar, S., 100 (306), 150 (459), 170 (523) Sanmamed, M., 114 (354) Sansom, O., 164 (504) Santamaria, G., 20 (47) Santi, A.S., 178 (549) Santoro, A., 176 (542) Santos, R., 96 (293) Santra, T., 77 (228) Sapino, A., 186 (575)

Sapinoso, L., 100 (306) Sappal, D., 181 (561), 186 (577) Sappal, D.S., 85 (255) Sapra, P., 145 (443) Sarantopoulos, J., 109 (336) Sarashina, A., 72 (213) Sarkar, S., 71 (211) Sarkaria, J., 10 (12), 86 (259) Sarmiento, R., 179 (551) Sarmientos, P.S., 41 (116) Sarno, F., 23 (55) Sarode, V., 84 (253) Sartor, O., 8 (4) Sartore-Bianchi, A., 23 (54) Sasaki, E., 103 (315) Sasazawa, Y., 51 (150), 176 (543) Sasikumar, P.G., 40 (113) Sasso, M., 56 (168) Sato, M., 30 (77), 188 (584) Sato, S., 121 (376) Sato, T., 188 (584), 192 (595) Satyam, L.K., 40 (113) Savage, R., 50 (145), 104 (320), 125 (389) Sawa, M., 112 (346) Sawada, J., 91 (274) Sawada, T., 129 (403) Sawant, A., 43 (123) Saxena, U., 115 (358) Saxty, G., 115 (356), 128 (400) Scaltriti, M., 27 (69), 28 (71), 29 (75) Schaaf, L.J., 151 (461) Schafer, C., 43 (123) Schaider, H., 34 (93) Schalck, A., 102 (314) Schally, A.V., 107 (330) Schauer, S., 34 (93) Scheinberg, D.A., 89 (266) Schellens, J.H.M., 121 (378), 179 (551), 199 (11LBA) Schenone, M., 156 (480) Schiffelers, R.M., 147 (448) Schiller, B.J., 151 (462) Schiller, J.H., 84 (253) Schimmer, A.D., 95 (286) Schlessinger, J., 130 (407) Schlicker, A., 61 (188) Schlisio, S., 135 (423) Schmees, N., 185 (573) Schmidt, E., 134 (420) Schmidt, J., 150 (457) Schneider, H., 64 (196) Schoeberl, B., 144 (438) Scholch, ¨ S., 64 (196) Scholz, A., 25 (63) Schrump, D., 13 (21) Schubert, C., 141 (430) Schuessler-Lenz, M., 44 (125) Schuler, ¨ J., 10 (11), 11 (16), 62 (192) Schuler, M., 199 (11LBA) Schuller, A., 123 (383), 125 (392) Schultz, N., 81 (240) Schuuring, E., 145 (441) Schwartz, B., 50 (145), 84 (253), 125 (389) Schwartz, E., 108 (335) Schwartz, G., 121 (378) Schweizer, M.T., 143 (434)

223 Scobie, M., 81 (240) Scott, A., 158 (485) Scott, K., 76 (227) Scott, M., 160 (490) Searcey, M., 95 (289) Seegers, N., 37 (104) Seger, C., 16 (30) Seiser, E., 55 (163) Seitova, A., 95 (287) Seitzberg, J.G., 184 (569) Seki, M., 103 (315) Sekido, Y., 30 (77) Selby, M., 30 (78), 132 (413) Selman-Housein, K.H., 38 (107) Selvakumaran, M., 154 (474) Selzer, S., 107 (331) Senapedis, W., 83 (247), 156 (480), 160 (492) Senba, T., 121 (377) Sendra, J., 87 (263) Senisterra, G., 95 (287) Senthil, K., 106 (326) Seo, A., 174 (534) Seong, Y.K., 79 (236) Sequeira, M., 188 (585) Sequist, L.V., 199 (10LBA) Serada, S., 127 (396) Serna, E., 51 (151) Serra, V., 80 (238), 82 (244) Seto, T., 198 (9LBA) Sevecka, M., 52 (152) Severson, T., 173 (532) Sexton, J., 151 (461) Sha, P., 186 (577) Shacham, S., 74 (220), 83 (247), 100 (305), 156 (480), 160 (492) Shackleford, T.J., 155 (477) Shafren, D., 41 (118) Shah, A., 115 (358) Shah, M.H., 151 (461) Shah, R., 131 (411) Shaheen, M., 197 (7LBA) Shain, K., 74 (220) Shalev, Z., 66 (204) Shamsaei, E., 179 (554) Shanmuganathan, S., 119 (370) Shannon, M., 48 (138) Shao, J., 39 (110) Shapiro, G.I., 80 (238), 158 (484) Sharma, A., 67 (206) Sharma, M., 13 (23) Sharma, N., 181 (560) Sharma, S., 185 (572), 199 (11LBA) Sharpless, N., 164 (503) Shaw, E., 172 (529) Shaw, K., 3 (1), 159 (488) Sheehan, C.E., 104 (319) Shelat, A.A., 63 (193) Sheldrake, H.M., 95 (289) Shen, J., 81 (243) Shen, Y., 28 (70), 81 (242), 186 (576) Sheng, X., 161 (495) Shevchenko, I., 48 (137) Shevlin, G., 150 (459) Shi, C., 84 (250) Shi, J., 78 (231), 181 (561) Shi, J.S., 85 (255)

224 Shi, Q., 11 (14), 12 (19) Shi, W., 49 (141), 73 (216) Shi, Y., 82 (246) Shiah, S.G., 147 (449) Shiau, C.W., 31 (82) Shibata, N., 52 (155) Shibata, T., 190 (589) Shih, J., 8 (5) Shiina, I., 185 (574) Shikata, Y., 176 (543) Shikuma, K., 188 (584) Shim, J.S., 36 (98) Shimamura, T., 191 (593) Shimizu, T., 97 (296), 198 (9LBA) Shimoga, P., 161 (493) Shimomura, T., 120 (373) Shin Ogawa, L., 87 (260) Shin-Sim, M., 150 (460) Shingler, W., 44 (126) Shinoda, A., 190 (589) Shitara, K., 72 (213) Shmulevich, I., 90 (271) Shnyder, S.D., 95 (289) Shoemaker, R., 143 (436) Shoemaker, R.H., 76 (225) Shoeneman, J.K., 9 (9) Shojaei, F., 118 (366) Shokat, K.M., 28 (73) Shore, N.D., 8 (4) Shrimali, K., 40 (113) Shrivastava, N., 85 (256) Shu, J., 157 (483) Shuch, B., 8 (5) Shui, S., 115 (358) Shum, D., 96 (293) Shyu, W.C., 161 (493) Siddiqui, M.M., 8 (5) Sidor, C., 71 (210) Sidransky, D., 63 (195) Siegel, P.M., 22 (51) Siegel, S., 185 (573) Siegmund, B., 149 (455) Siena, S., 23 (54) Silberring, J., 12 (17) Silence, K., 39 (108) Silva Junior, ´ E.N., 87 (262) Silveira, D.D., 162 (498) Silvers, T., 30 (78), 147 (450) Simamura, T., 98 (299) Simizu, S., 51 (150) Simon, B., 177 (546) Simon, I., 61 (188) Simon, N.L., 73 (216) Simon, R.M., 57 (171), 78 (233) Simonelli, M., 176 (542) Simpson, G.R., 41 (118) Simpson, K., 144 (437) Simpson, K.L., 88 (264) Sims, D., 36 (101), 78 (232) Sims, D.J., 78 (233) Sims, R., 31 (83) Sims III, R., 187 (581) Singer, E., 8 (5) Singh, K., 181 (558) Singh, O., 32 (86) Singh, R., 87 (260) Sircar, K., 172 (530)

Author Index Sirera, R., 43 (122), 46 (133) Sisinni, L., 60 (182) Sissung, T.M., 13 (21) Siu, L., 171 (526) Siu, L.L., 79 (237), 165 (508) Siziopikou, K., 14 (24) Skalkoyannis, B., 132 (413) Skegro, D., 48 (139) Skene, D.J., 148 (453) Skinner, H., 67 (205), 109 (337) Sklar, J., 78 (232) Skog, J., 102 (313), 149 (454) Skrzypek, K., 170 (524) Slamon, D.J., 162 (498) Slater, C., 8 (6) Sloane, R., 144 (437) Sloss, C., 52 (152) Small, H., 94 (284), 195 (2LBA) Smans, K., 141 (430) Smil, D., 95 (287) Smith, A., 132 (413) Smith, B.D., 115 (355) Smith, D., 188 (585) Smith, D.A., 7 (3) Smith, H., 22 (51) Smith, J., 91 (275) Smith, K., 94 (284), 118 (366) Smith, L., 152 (465) Smith, M., 131 (410, 412) Smith, P., 123 (384), 149 (456), 171 (527) Smith, P.D., 169 (520) Smith, S., 125 (391), 196 (4LBA) Smitheman, K., 72 (212) Smyth, A., 168 (518) Smyth, M.J., 40 (115) Snaar, E.B., 12 (20) Sneeringer, C., 92 (277) Snyder, J., 179 (554) So, H.S., 55 (164) Sobrado, V.R., 135 (423) Sobrevals Amieva, L., 87 (263) Socinski, M., 19 (41) Sokol, E.S., 179 (552) Sokolova, A., 120 (372) Sole, M., 75 (223) Soler, G., 187 (579) Solit, D., 29 (75) Solomon, B.J., 199 (10LBA) Solomon, L., 82 (246) Solon, M., 124 (387) Soltermann, A., 65 (199) Somasundaram, R., 34 (93) Somovilla-Crespo, B., 18 (39) Sonderfan, A., 87 (260) Song, C., 134 (419) Song, S., 166 (510) Song, Y.J., 98 (298) Sonobe, M., 188 (584) Sonoda, K., 32 (84) Soong, R., 29 (74) Sootome, H., 127 (398), 142 (433) Sorensen, P.H.B., 82 (245) Soria, J., 199 (10LBA) Soria, J.C., 44 (127), 74 (219), 197 (6LBA) Soriano, F., 180 (555) Sos, M., 28 (73) Sosman, J.A., 33 (90)

Soto-Cerrato, V., 15 (28) Soto-Matos, A., 13 (23) Soucheray, M., 191 (593) Soude, A., 180 (557), 191 (594) Soueidan, H., 31 (81) Souers, A.J., 130 (408) Soukupova, J., 122 (379) Sousa, F., 84 (252) Sowa, T., 188 (584) Soyer, H.P., 34 (93) Sozu, T., 188 (584) Sozzi, G., 56 (168) Spang, R., 32 (85) Spano, J.P., 188 (582) Specenier, P., 27 (68) Spigel, D., 19 (41) Spijkers-Hagelstein, J.A.P., 113 (349) Spira, A.I., 199 (10LBA) Spoerke, J., 122 (382) Spreafico, A., 199 (11LBA) Sridhar, S., 116 (361) Sridharan, L., 145 (443) Srimongkolpithak, N., 179 (554) Srinivasan, R., 8 (5) Stachnik, A., 178 (548) Stagg, J., 40 (115) Stagg, R., 55 (166) Stamatakis, L., 8 (5) Stanfield, K., 156 (481) Stannard, K., 40 (115) Stassi, G., 164 (505) Stathis, A., 184 (568), 196 (5LBA) Staunton, D., 106 (325) Steele, V.E., 75 (222), 76 (225, 226) Stefanich, E.G., 71 (209) Stehbens, S., 122 (381) Stein, E.L., 62 (190) Stein, E.M., 195 (1LBA) Stein, R.M., 104 (320) Steinberg, G., 67 (205) Steinberg, S., 102 (312) Stenke, L., 184 (570) Stepanek, V.M., 151 (461) Stephens, P.J., 84 (250), 104 (319), 132 (414, 415) Stephenson, N., 164 (504) Stephenson, N.L., 29 (76), 167 (512) Stephenson, S., 122 (381) Stevenson, A., 97 (294) Stewart, A., 58 (176), 144 (440) Stewart, C.F., 63 (193), 133 (416, 418) Stienen, S.K., 174 (535) Stockdale, M., 171 (527) Stolarik, D., 82 (246) Stoletov, K., 187 (578) Stone, E., 181 (559) Stone, R.M., 195 (1LBA) Stoodley, M., 66 (203) Storm, M., 161 (495) Stoudemire, J., 196 (4LBA) Stover, D., 80 (238) Stowell, A., 94 (284) Stratton, M.R., 56 (167) Strauss, S.J., 82 (245) Strickler, J.H., 152 (465) Stringer, B., 78 (231), 181 (561) Stringer, B.S., 85 (255)

Author Index Strozek, W., 47 (135), 118 (367), 152 (467), 169 (519) Struman, I., 53 (159) Su, B., 123 (385) Su, D., 8 (5) Su, F., 106 (327) Su, S.V., 144 (438) Su, T.L., 20 (45) Su, W., 17 (34) Su, X., 35 (95), 188 (582) Subbiah, V., 56 (169), 151 (461) Sudey, I., 189 (586) Sudo, K., 105 (323) Suen, C., 76 (225) Sugiura, M., 175 (538) Suh, S.S., 116 (360) Sukumar, S., 36 (98) Sukumaran, S., 71 (209), 136 (424) Sullivan, D., 74 (220) Sullivan, R.J., 102 (314) Sully, K., 98 (300) Summa, J., 19 (41) Sun, C., 54 (160) Sun, H., 89 (268) Sun, L., 18 (38) Sun, M., 172 (530) Sun, P., 52 (153) Sun, S.X., 60 (183) Sun, Y., 123 (385) Sundaramoorthy, E., 35 (95) Sunilkumar, K.B., 40 (113) Suominen, M.I., 25 (63) Surendranath, S., 104 (321) Sushmita, R., 104 (321) Sustic, T., 54 (160) Suter, M., 176 (542) Sutherland, M., 95 (289) Sutiman, N., 32 (86) Suzuki, T., 142 (433), 167 (515) Swarbrick, M., 91 (275) Swisher, E., 73 (215) Symeonides, S., 177 (546) Symmans, F., 173 (532) Szewczyk, B., 170 (524) Szydlowska, M., 27 (67) Szyldergemajn, S., 13 (23) Szymanik, M., 47 (135), 118 (367), 152 (467), 169 (519)

T Tabata, K., 17 (36) Tabernero, J., 124 (386), 171 (526), 199 (11LBA) Tagliabue, E., 56 (168) Taguchi, C., 58 (175) Tahiri, S., 148 (451) Takabe, N., 78 (232) Takahashi, T., 127 (396) Takayama, T., 166 (509) Takebe, N., 36 (101) Takiguchi, S., 127 (396) Talasaz, A., 148 (451), 151 (462) Tallman, M.S., 195 (1LBA) Tam, D., 157 (483) Tam, R., 152 (465) Tam, W.F., 146 (446)

Tamboli, P., 172 (530) Tamir, S., 83 (247), 160 (492) Tamura, K., 129 (403) Tan, A.C., 120 (375) Tan, D., 165 (508) Tan, D.S.W., 32 (86) Tan, E.H., 32 (86) Tan, G., 52 (152) Tan, L., 98 (297) Tan, T.T., 52 (154) Tanaka, K., 120 (373), 121 (376) Tanaka, N., 13 (22), 109 (337) Tancredi, R., 21 (48) Tanese, K., 150 (460) Tang, K., 33 (89) Tang, L., 123 (385) Tang, Q., 48 (138) Tang, R., 161 (496) Tang, X., 124 (388) Tangutoori, S., 116 (361) Tannir, N., 172 (530) Tap, W., 121 (378) Taplin, M.E., 8 (4) Tarantelli, C., 184 (568) Tarazi, J., 116 (359) Taron, M., 156 (478) Tarrant, J., 136 (424) Tashiro, E., 176 (543) Tateishi, U., 154 (473) Tatsuta, N., 87 (260) Tavares, L.C., 26 (64) Taverna, P., 47 (134) Taylor, H., 71 (209), 136 (424) Tazaki, K., 172 (531) Tazawa, H., 96 (292) Tchistiakova, L., 145 (443) Tedesco, D., 52 (153) Teicher, B., 89 (269) Teicher, B.A., 30 (78), 57 (171), 147 (450) Teixido, C., 85 (254), 156 (478), 167 (514) Tejedor, S., 115 (357) Tejpar, S., 61 (188) Tellez Gabriel, M., 62 (191) Tempero, A.H., 148 (451) Teng, M., 22 (53) Teng, M.W., 40 (115) Tenkerian, C., 148 (452) Teo, S.H., 35 (95) Terracciano, L.T., 41 (116), 178 (549) Terui, Y., 136 (426), 166 (509) Tervonen, T., 57 (173) Terwisscha van Scheltinga, A.G.T., 174 (535) Tesikova, M., 161 (495) Teska-Kaminska, M., 47 (135), 118 (367), 152 (467), 169 (519) Testa, J.R., 146 (446) Testar, R., 123 (384) Testoni, E., 164 (504), 167 (512) Teuber, L., 184 (569) Texido, G., 16 (31) Thaler, F., 185 (571) Thamm, D., 9 (9) Thatcher, G., 103 (316) Thatte, J., 50 (146) Theelen-Engelsman, W., 54 (160) Theus, A., 26 (65)

225 Thibault, A., 39 (108) Thieblemont, C., 162 (499), 196 (5LBA) Thijssen, B., 34 (91) Thin, T.H., 81 (241) Thomas, A., 102 (312) Thomas, M., 149 (456) Thomas, S., 120 (372) Thomas, X., 196 (5LBA) Thomason, A., 150 (457) Thompson, N., 122 (380), 168 (518) Throm, S.L., 133 (416, 418) Thurmond, J., 151 (461) Thurston, D.E., 19 (42) Thyparambil, S., 159 (489), 175 (537) Tian, L., 155 (477) Tian, S., 61 (188) Tibes, R., 196 (4LBA) Timmers, C., 151 (461) Timms, K.M., 72 (214) Tirado, O.M., 110 (341) Tiriac, H., 63 (194) Tiron, C., 117 (362) Tiziani, S., 181 (559) Tjulandin, S., 155 (476) To, K.K.W., 93 (280, 281) Tobin, J., 181 (560) Todaro, M., 164 (505) Todic, A., 94 (285) Tognolini, M., 90 (270) Tolcher, A., 104 (320), 111 (342), 125 (389) Tolcher, A.W., 7 (2), 12 (18), 35 (96, 97), 37 (102), 156 (481) Tomaro, J., 179 (551) Tomaszewski, J.E., 77 (229) Tomek, P., 91 (273) Tomilo, M., 166 (511) Tomlins, S.A., 61 (187) Tonetti, D., 103 (316) Tong, P., 143 (435) Tonnesen, B., 184 (569) Tookman, L.A., 38 (106) Topp, C.F., 75 (222) Tormo, J.R., 24 (59) Torrecilla, S., 75 (223) Torres, R., 128 (402) Tortorella, C., 161 (494) Tosi, F., 23 (54) Toska, E., 28 (71) Toth, R., 55 (165) Totpal, K., 45 (128), 71 (209) Tovar, C., 106 (327) Tovar, V., 75 (223) Towbin, H., 108 (333) Towle, M.J., 20 (46) Tran, N.T., 10 (12) Traore, T., 78 (231), 85 (255), 181 (561) Trapani, F., 88 (264) Travassos, L.R., 26 (64), 43 (124) Travica, S., 95 (289) Treinies, I., 91 (275) Tremayne, J., 124 (387) Trent, J., 120 (372) Triana-Baltzer, G., 160 (490) Tricoli, J., 78 (232) Trifiro’, P., 185 (571) Trippett, T., 131 (411)

226 Trojer, P., 125 (390) Trombino, G., 55 (165) Troncone, M.J., 67 (205) Tronel, V., 115 (356), 128 (400) Trotter, E., 164 (504) Trotter, E.W., 29 (76), 167 (512) Trueblood, E., 40 (112) Truong, T., 131 (411) Trusko, S., 154 (471) Tsai, M., 19 (43) Tschuch, C., 10 (11) Tsiani, E., 67 (205) Tsimafeyeu, I., 155 (476) Tsimberidou, A.M., 56 (169) Tsuji, T., 100 (306), 150 (459) Tsujikawa, L., 181 (560) Tu, Y., 144 (440) Tuang, S., 54 (161) Tuffal, G., 121 (378) Tumati, V., 68 (208) Turdo, F., 56 (168) Turkki, R., 47 (136) Turkson, J., 93 (282) Turley, E.A., 187 (578) Turner, B., 188 (583) Turner, B.A., 115 (355) Turner, D.C., 133 (416, 418) Turner, J., 74 (220) Turnham, D., 122 (379) Tuveson, D.A., 63 (194) Tyler, S., 120 (374)

U Uboldi, S., 190 (590) Uchino, H., 58 (175) Uegaki, K., 198 (9LBA) Uehara, F., 96 (292), 121 (376) Uenaka, T., 20 (46) Ueno, D., 154 (473) Ueno, H., 15 (27) Ueno, N., 31 (80) Uhlig, P.C., 151 (464) Uitdehaag, J.C.M., 37 (104), 113 (349) Ukath, J., 66 (203) Ulivi, P.U., 168 (516) Ullal, A., 54 (161) Ullmann, C.D., 21 (50) Umansky, V., 48 (137) Underwood, C.R., 184 (569) Unger, F.T., 151 (464) Ungerechts, G., 48 (137) Uno, Y., 112 (346) Urano, S., 175 (538) Urick, M.E., 166 (511) Uso, ´ M., 43 (122), 46 (133) Utsugi, T., 13 (22), 15 (27, 29), 103 (315), 120 (373), 127 (398), 128 (401), 142 (433), 167 (515), 176 (541), 191 (592) Uzzell, J., 175 (537)

V Vader, P., 147 (448) Vakkalanka, S., 108 (332) Valaire, C., 180 (557) Valente, S., 185 (571)

Author Index Valenzuela, C.M., 38 (107) Valera, S., 185 (572) Valladares, M., 17 (37) Valle, E., 180 (555) Van Allen, E., 141 (428) Van Aller, G., 72 (212) Van Brussel, T., 11 (15) van de Merbel, A.F., 12 (20) van den Bent, M.J., 53 (157) Van den Bossche, J., 27 (68) van den Heuvel, M., 54 (160) Van den Neste, E., 162 (499) van der Heijden, M.S., 54 (160) van der Horst, G., 12 (20) van der Meel, R., 147 (448) van der Pluijm, G., 12 (20) Van Der Steen, K., 112 (347) Van Der Steen, N., 65 (200), 112 (347) van Dommelen, S.M., 147 (448) van Doornmalen, A.M., 37 (104), 113 (349) van Geel, R., 199 (11LBA) van Gerwen, S., 37 (104) Van Laere, S., 31 (80) van Marion, R.M., 53 (157) van Rhijn, B.W., 54 (160) van Schaik, R.H.N., 26 (66) van Solinge, W.W., 147 (448) van ’t Veer, L., 154 (472) van ’t Veer, L.J., 173 (532) van Wageningen, S., 51 (148) VanBrocklin, H.F., 145 (442) Varasi, M., 185 (571) Varda, J., 42 (120) Varga, A., 74 (219), 121 (378), 142 (431), 199 (10LBA) Vasilevskaya, I., 154 (474) Vaslin Chessex, A., 177 (547) Vasquez, D., 63 (195) Vassilev, L., 47 (136), 106 (327) Vaught, T., 12 (18), 35 (96, 97), 37 (102), 156 (481) Vautier, M., 90 (272) Vazquez, R., 182 (564) Vazquez, ´ R., 187 (580) Vecchione, L., 61 (188) Vedadi, M., 179 (554) Veenstra, T., 175 (537) Veeraraghavan, S., 108 (332) Veinalde, R., 48 (137) Veith, J., 40 (114) Velasco-Miguel, S., 110 (339), 128 (402) Velculescu, V., 74 (218) Velez-Bravo, V., 151 (461) Vena, F., 146 (445) Venesio, T., 23 (54) Venkatakrishnan, K., 21 (50), 105 (324), 109 (336) Venkitaraman, A., 35 (95) Venook, A.P., 148 (451) Ventura, R., 190 (591) Verhulst, T., 115 (356), 128 (400) Verlicchi, A.V., 168 (516) Verma, A., 108 (335) Verma, C., 27 (69) Vermette, J., 171 (526) Verner, E., 157 (483)

Verschuren, E., 57 (173) Verwaal, V., 59 (177) Vessella, R.L., 25 (63) Vey, N., 183 (566), 196 (5LBA) Vialard, J., 115 (356), 128 (400) Viale, G.V., 41 (116), 178 (549) Vianello, P., 185 (571) Vibat, C.R.T., 51 (149) Vicente, F., 24 (59) Vidal, C.M., 146 (446) Vidal Rodriguez, J., 31 (81), 34 (91) Viktorsson, K., 184 (570) Villa, M., 185 (571) Villacanas, ˜ O., 156 (478) Villalona-Calero, M., 151 (461) Villanueva, A., 152 (466) Vincent, M.D., 21 (49) Vincent, S., 120 (374) Vincent-Salomon, A., 186 (575) Visakorpi, T., 12 (20) Vishwantahan, K., 73 (217) Visscher, D., 73 (215) Viswanadha, S., 108 (332) Viteri, S., 85 (254), 167 (514) Vitolo, U., 162 (499) Vivero, L., 87 (263) Vo, P., 84 (253) Vogeti, S., 115 (355) Vojnovic, B., 77 (228) Vokes, E., 111 (342) Volkmer, H., 107 (331) Volland, R., 130 (409) Voloshanenko, O., 32 (85) Volova, S., 89 (266) Volpe, S., 164 (505) von Euler, M., 47 (136) von Heyking, K., 134 (420) Von Hoff, D., 195 (3LBA) Von Hoff, D.D., 179 (551) von Kalle, C., 48 (137) Vormoor, B., 133 (417) Vu, T., 155 (477) Vuagniaux, G., 177 (547) Vuaroqueaux, V., 9 (7), 62 (192) Vuidepot, A., 44 (126) Vultur, A., 34 (93) Vuolanto, A., 47 (136) Vuong, V., 65 (202) Vutukuri, D., 87 (260)

W Waaijer, S., 174 (535) Wabler, M., 154 (471) Waddell, I., 94 (284), 195 (2LBA) Waddell, N., 35 (94) Wadham, C., 135 (421) Wagle, M.C., 124 (387) Wagle, N., 141 (428) Wagman, A., 190 (591) Wagner, A., 121 (378) Wagner, K., 34 (93), 132 (415) Wagoner, M., 123 (383) Wahle, C., 47 (136) Wai, P., 186 (575) Wainberg, Z., 171 (526), 199 (11LBA) Wakasa, T., 15 (29)

Author Index Wakefield, A., 122 (379) Wakelee, H.A., 199 (10LBA) Waksal, S., 46 (132) Waldron, E., 185 (572) Walker, I., 172 (529) Walker, M., 90 (272) Wallace, B., 152 (465) Wallace, R., 125 (391) Wallis, K., 135 (423) Wallis, N., 168 (518) Walsh, C., 118 (366) Walsh, W.D., 78 (233) Wan, X., 101 (311) Wan-Ching, Y., 55 (166) Wang, B., 81 (242), 105 (322) Wang, C., 8 (6) Wang, F., 54 (162) Wang, G., 89 (268), 109 (338), 162 (497), 176 (544) Wang, H., 109 (338), 123 (383), 143 (434), 162 (497), 176 (544) Wang, J., 66 (204), 81 (243), 109 (337), 124 (388), 143 (435), 180 (555) Wang, J.Y., 123 (385) Wang, K., 132 (414, 415), 145 (443) Wang, K.A.I., 104 (319) Wang, L.Z., 106 (325) Wang, M., 152 (465) Wang, Q., 179 (552) Wang, S., 32 (87), 89 (268), 98 (297), 109 (338), 148 (452), 162 (497), 176 (544) Wang, W., 115 (358) Wang, X., 106 (326), 114 (352), 120 (375), 124 (387), 164 (503) Wang, Y., 18 (38), 38 (106), 43 (123), 72 (214), 80 (239), 103 (316) Wantuch, S., 175 (540) Ward, G., 122 (380) Ward, L., 90 (272), 177 (546) Ware, J., 122 (382) Wargo, J.A., 102 (314) Warholic, N., 92 (277) Warin, N., 90 (272) Waring, M., 91 (275) Warmuth, M., 149 (456) Warnders, F.J., 174 (535) Wartenberg, M., 89 (266) Wasney, G.A., 95 (287) Waszczuk, J., 190 (591) Waszkowycz, B., 94 (284), 195 (2LBA) Watanabe, K., 176 (541) Watanabe, M., 129 (403) Watari, K., 32 (84), 190 (589) Watson, A.F., 91 (276) Watson, M., 195 (2LBA) Watters, J., 96 (291) Weaver, D., 144 (439) Weaver, D.T., 99 (302) Webber, H., 135 (422) Webber, J.T., 28 (73) Weber, J.S., 48 (140) Wedge, S., 106 (325) Wegiel, B., 18 (38) Wei, G., 143 (436) Wei, Y.M., 93 (280) Weigelt, B., 186 (575)

Weigman, V., 74 (218) Weiguo, S., 125 (392) Weinmann, H., 185 (573) Weiss, G., 19 (41) Weiss, G.J., 165 (506) Weissleder, R., 54 (161) Weitsman, G., 77 (228) Wen, J., 89 (268), 109 (338), 162 (497), 176 (544) Wendel, H., 181 (558) Weng, Z., 120 (374) Werner-Wasik, M., 49 (141), 73 (216) Wessels, L.F., 31 (81), 34 (91) Westermann, F., 130 (409) Westwell, A., 122 (379) Whelan, J.S., 82 (245) Wheler, J.J., 56 (169) White, A.J., 37 (102) Wæhre, H., 161 (495) Wick, M.J., 12 (18), 35 (96, 97), 37 (102), 156 (481) Wickenden, J., 171 (527) Wigle, T., 92 (277) Wilcoxen, K., 165 (506) Wilcoxen, K.M., 72 (214), 80 (239) Wilkins, D., 136 (424) Wilkinson, B., 154 (471) Wilkman, H., 106 (328) Willems, S.M., 54 (160) Willetts, L., 147 (447) William, W.N., 143 (435) Williams, J.A., 116 (359) Williams, K., 170 (525) Williams, M., 36 (101), 78 (232), 147 (450) Williams, P., 61 (187) Williams, P.M., 78 (233) Williams, V., 169 (520) Williamson, D., 132 (413) Wilson, H.L., 98 (299) Wilson, J., 99 (301) Wilson, R., 188 (583) Wilson, W.R., 66 (204) Winski, S., 125 (391) Winter, P., 161 (494) Wirtz, D., 60 (183) Wise, S.C., 115 (355) Wisell, J., 59 (180) Wishka, D.G., 89 (269) Wisman, G.B., 145 (441) Wistuba, I., 159 (489) Wistuba, I.I., 124 (388) Witte, L., 46 (132) Witteveen, P.O., 185 (572) Wnup-Lipinska, K., 117 (362) Woessner, R., 163 (501) Wohlfahrt, G., 142 (432) Wolchok, J.D., 48 (140) Wolf, B., 52 (152), 107 (329), 162 (498) Wolf, D.M., 173 (532) Wolfe, A., 181 (558) Wolsztynski, E., 64 (197), 79 (234, 235) Womack, C., 163 (501), 198 (8LBA) Won, H.H., 29 (75) Wong, A., 141 (429) Wong, D.J.L., 162 (498) Wong, K., 120 (375) Wong, K.K., 98 (299), 191 (593)

227 Wong, S., 7 (3), 180 (555) Wood, C., 172 (530) Wood, J., 132 (413) Wood, K., 158 (484) Wood, R., 91 (275) Woodman, N., 77 (228) Workman, P., 148 (453), 175 (540) Worland, P., 150 (459) Wortmann, A., 35 (94) Wouters, A., 27 (68), 97 (295) Wouters, B.G., 66 (204) Wright, G., 117 (363) Wright, J., 67 (205) Wright, K.D., 63 (193), 133 (416, 418) Wright, Q., 144 (439) Wroblowski, B., 115 (356), 128 (400) Wu, C.H., 86 (257) Wu, G., 76 (227) Wu, H., 95 (287) Wu, H.C., 86 (257) Wu, J., 20 (46), 131 (410, 412), 181 (560) Wu, M., 162 (497), 176 (544) Wu, M.H., 20 (45) Wu, S., 156 (480) Wu, Y., 46 (132) Wulf-Goldenberg, A., 10 (10) Wustrow, D., 180 (555) Wuthrick, E., 49 (141) Wyatt, M.D., 18 (40)

X Xia, C., 181 (561) Xia, C.Q., 54 (162) Xia, C.X., 85 (255) Xiao, S.Y., 175 (537) Xiao, Y., 130 (408) Xie, L., 99 (301) Xie, T., 109 (337) Xie, Y., 84 (253) Xiong, H., 111 (342) Xiong, R., 103 (316) Xu, G.W., 95 (286) Xu, J.F., 123 (385) Xu, L., 7 (2) Xu, P., 175 (537) Xu, Q., 91 (276), 99 (302), 144 (439), 146 (446) Xu, S., 100 (306) Xu, W., 150 (459)

Y Yaeger, R., 199 (11LBA) Yakes, F., 180 (555) Yamada, T., 112 (346), 150 (458), 188 (584) Yamada, Y., 199 (11LBA) Yamade, M., 84 (252) Yamamoto, M., 121 (376) Yamamura, K., 13 (22), 15 (27) Yamanaka, T., 58 (175) Yamasaki, M., 127 (396) Yamashita, R., 30 (77) Yamin, T.T., 145 (443) Yamori, T., 185 (574) Yan, Y., 52 (154) Yang, C.Y., 32 (87), 109 (338)

228 Yang, D., 89 (268), 109 (338), 162 (497), 176 (544) Yang, H., 115 (358), 161 (496), 195 (1LBA) Yang, J., 17 (34) Yang, J.C.H., 72 (213) Yang, L., 89 (267) Yang, M., 112 (348) Yang, P., 99 (301) Yang, S., 19 (43), 55 (164) Yang, S.X., 77 (229) Yang, W., 122 (379) Yang, X.Q., 52 (154) Yang, Y., 78 (231), 181 (561) Yang, Y.Y., 85 (255) Yano, S., 96 (292), 121 (376) Yano, W., 15 (29) Yao, B., 180 (555) Yao, M., 154 (473) Yates, J., 84 (251), 98 (300), 99 (301) Yau, C., 173 (532) Yaylaoglu, M., 179 (553) Ye, X., 123 (385) Ye, X.K., 52 (154) Yee, D., 173 (532) Yee, K., 196 (5LBA) Yeh, T.K., 92 (279) Yelensky, R., 84 (250), 132 (414, 415) Yen, C.J., 73 (217) Yen, K., 195 (1LBA) Yetil, A., 172 (530) Yeung, C., 101 (311) Yi, S.A., 153 (470) Yin, W., 155 (476) Yin, X., 198 (8LBA) Ying, B., 115 (358) Ying, W., 87 (260) Yogurtcu, O., 60 (183) Yokogawa, T., 15 (27, 29) Yonekura, K., 120 (373), 128 (401), 167 (515), 176 (541) Yoo, N., 49 (143) Yoshida, S., 190 (589) Yoshimatsu, K., 185 (574) Yoshimura, C., 191 (592) Yoshino, T., 199 (11LBA) Yoshisue, K., 15 (29) Young, A., 40 (115) Youngblood, V., 98 (297) Ysebrant de Lendonck, L., 39 (108) Yu, H.A., 199 (10LBA) Yu, K., 63 (194) Yu, L., 149 (456) Yu, T.F., 52 (154) Yu, Y., 155 (475), 156 (481), 167 (513), 172 (531)

Author Index Yuan, A., 106 (326) Yuan, C.C., 125 (390) Yuan, Z., 117 (364) Yun, S., 174 (534) Yunokawa, M., 129 (403) Yvonnet, V., 58 (174)

Z Zais, J., 89 (269) Zaman, G., 37 (104) Zaman, G.J.R., 113 (349) Zambelli, A., 21 (48) Zanetta, S., 189 (586) Zangarini, M., 110 (340) Zanna, C., 146 (444), 177 (547) Zapata, A.G., 187 (579) Zapata, J.M., 18 (39) Zarour, H., 48 (140) Zecca, M., 21 (48) Zeitouni, B., 62 (192) Zender, L., 152 (466) Zeng, J., 159 (488) Zerek, B., 47 (135), 118 (367), 169 (519) Zerek, B.M., 152 (467) Zhai, Y., 89 (268), 109 (338), 162 (497), 176 (544) Zhang, B., 21 (50), 109 (336) Zhang, C., 71 (209), 136 (424), 152 (465), 188 (585) Zhang, G., 34 (93) Zhang, H., 46 (132) Zhang, H.P., 123 (385) Zhang, J., 11 (14), 12 (19), 44 (127), 60 (181), 87 (260), 123 (385), 188 (582) Zhang, L., 12 (19), 60 (181), 123 (385) Zhang, M., 88 (265), 156 (479) Zhang, M.Z., 123 (385) Zhang, N., 111 (343) Zhang, Q., 155 (477) Zhang, R., 60 (181), 96 (293), 155 (477) Zhang, V., 163 (500) Zhang, W., 114 (352) Zhang, X., 172 (530) Zhang, Y., 61 (187), 96 (292), 106 (327), 111 (343), 121 (376), 123 (383), 162 (497) Zhang, Z., 46 (132), 68 (208), 179 (553) Zhao, C., 170 (523) Zhao, F., 125 (390) Zhao, G., 110 (339) Zhao, J., 103 (316) Zhao, L., 175 (537) Zhao, M., 96 (292), 109 (337), 111 (343), 121 (376) Zhao, S., 161 (495)

Zhao, W., 93 (282) Zhao, Y., 57 (171), 78 (233), 91 (276), 133 (417) Zhao, Z., 66 (203) Zheng, D., 123 (385) Zheng, W., 121 (378) Zhi, D., 43 (123) Zhong, Y., 181 (558) Zhong, Z., 46 (132) Zhongwu, L., 198 (8LBA) Zhou, D., 87 (260) Zhou, H., 109 (338) Zhou, H.J., 180 (555) Zhou, L., 152 (465), 172 (530) Zhou, T., 150 (457) Zhou, W., 115 (358) Zhou, X., 21 (50), 54 (162), 105 (324), 109 (336), 117 (364) Zhou, Y., 84 (253) Zhoui, L., 74 (220) Zhu, C., 108 (335) Zhu, G.S., 123 (385) Zhu, J., 45 (128), 71 (209) Zhu, P., 149 (456), 171 (527) Zhu, Y., 39 (111) Zhu, Z., 46 (132) Zhukova-Harrill, V., 195 (3LBA) Ziane, I., 99 (303) Ziazadeh, D., 32 (87) Ziegelbauer, K., 25 (63) Ziegler, D., 135 (421, 422) Zimmermann, A., 164 (503) Zimon, D., 62 (190) Zinda, M., 123 (383) Zinner, R.G., 56 (169) Zinzani, P.L., 162 (499) Zipelius, A., 11 (16) Ziyeh, S., 148 (451) Zoli, W.Z., 168 (516) Zong, D., 184 (570) Zoni, E., 12 (20) Zopf, C., 105 (324) Zopf, C.J., 161 (493) Zou, Y., 108 (335) Zucali, P., 176 (542) Zucca, E., 184 (568), 196 (5LBA) Zuccari, D.A.P.C., 169 (522) Zucchetti, M., 110 (340) Zucht, H.D., 107 (331) Zucman-Rossi, J., 152 (466) Zukiwski, A., 142 (431) Zurita, A., 172 (530) Zwaenepoel, K., 39 (108), 65 (200), 112 (347)

229

Subject Index A A2AR inhibitors, 40 (115) AAV6-DTA, 22 (53) Abiraterone, 8 (4), 101 (308), 143 (434), 150 (457) ABL inhibitors, 167 (512) ABL1 mutations, 167 (512) AccretaMab, 98 (300) Acquired resistance, 9 (9), 27 (69), 29 (74, 75, 76), 30 (78, 79), 31 (80, 82, 83), 32 (84, 87), 33 (89, 90), 34 (93), 35 (94), 59 (180), 80 (238), 88 (264), 93 (281), 100 (304), 101 (311), 102 (314), 112 (347), 123 (385), 128 (401), 141 (428), 143 (434), 151 (461, 463), 160 (491), 165 (506) Actionable alterations, 3 (1), 10 (12), 27 (69), 36 (101), 37 (103), 61 (187), 78 (232, 233), 102 (312), 104 (319), 108 (335), 132 (414, 415), 147 (450), 151 (462), 159 (488), 165 (506), 167 (512) Activated leukocyte adhesion molecule (ALCAM, CD166), 55 (165), 60 (182), 67 (206) Acute lymphoblastic leukaemia (ALL), 10 (11), 19 (43), 114 (352), 134 (419) Acute myeloid leukaemia (AML), 10 (11), 48 (138), 72 (212), 77 (230), 114 (352), 126 (394), 183 (566), 184 (570) Adenoid cystic carcinoma (ACC), 7 (2), 35 (97), 51 (150) Adjuvant therapy, 14 (25), 20 (44), 25 (62), 40 (114), 43 (124), 89 (267), 166 (510) Advanced lung adenocarcinoma, 102 (312) Advanced solid tumours, 7 (2), 17 (34), 21 (50), 38 (107), 40 (114), 72 (213), 84 (253), 87 (261), 104 (320), 109 (336), 111 (342), 117 (364), 122 (382), 124 (386), 125 (389), 136 (424), 148 (453), 158 (484), 165 (506, 508), 171 (526), 181 (561), 186 (577), 189 (586), 197 (6LBA, 7LBA) Afatinib, 32 (84), 112 (348) Aflibercept, 96 (291), 130 (406) AKT3 activation, 27 (69) AKT2 amplification, 54 (161), 120 (373) AKT inhibitors, 104 (320), 120 (373), 155 (475), 156 (481), 167 (513), 172 (531) AKT kinase, 7 (3) Akt/mTOR inhibitors, 67 (205) Akt/mTOR pathway alterations, 104 (319) Alectinib, 168 (518) ALK inhibitors, 126 (393), 165 (506), 168 (518), 182 (564) Allografts, 11 (16), 115 (355), 181 (559) AMP kinase, 67 (205) AMP kinase activation, 76 (224) Androgen deprivation Bipolar androgen therapy, 143 (434) Androgen receptor antagonists, 8 (4), 101 (308), 103 (315), 150 (457) Androgen receptor expression, 14 (24)

Angiogenesis inhibitors, 16 (30), 39 (111), 55 (166), 67 (205), 75 (223), 97 (296), 100 (307), 111 (344), 115 (355), 118 (367), 150 (458), 172 (530), 190 (589) Animal models, 9 (9), 11 (13, 14, 16), 22 (52), 25 (63), 39 (111), 50 (145), 63 (195), 75 (223), 76 (226), 98 (300), 100 (307), 114 (352), 118 (365), 152 (466), 158 (486), 164 (503), 174 (535), 190 (589) Annexin A1 (ANXA1), 144 (440) Anthrax lethal toxin, 126 (394) Anti-CSC antibodies, 118 (366), 152 (465) Anti-CTLA-4 antibodies, 40 (115), 47 (134), 48 (137), 49 (141) Anti-CXADR antibody, 175 (538) Anti-EGFR antibodies, 52 (152), 106 (326), 107 (329) Anti-ErbB3 antibodies, 71 (210), 120 (374), 130 (407), 164 (505) Anti-IGF antibodies, 152 (466) Anti-Met antibodies, 98 (298) Anti-Netrin-1 antibody, 118 (368) Anti-OX40 antibody, 45 (128), 71 (209), 136 (424) Anti-PD-1 antibodies, 40 (115), 44 (127), 46 (132), 48 (137) Anti-PD-L1 antibodies, 48 (140) Anti-SialylTn antibodies, 156 (479) Anti-uPA antibodies, 145 (442) Anti-VEGF antibodies, 41 (117) Anti-VEGF vaccine, 38 (107) Antibody-dependent cell cytotoxicity (ADCC), 12 (19), 39 (108), 98 (300), 107 (329), 113 (351), 175 (538) Antibody–drug conjugates, 22 (51), 41 (116), 106 (326), 111 (342), 145 (443), 164 (502) Anticancer agents under development A-1155463, 130 (408) A-1210477, 130 (408) Abexinostat, 189 (586) ABT-199, 130 (408) ABT-414, 111 (342) AC-3-019, 94 (285) AD-056.9, 152 (467) AD-O53.2, 169 (519) AD-O54.9, 118 (367) AD-O64.3, 47 (135) Afuresertib, 7 (3) AG-120, 195 (1LBA) Alisertib, see MLN8237 Alpelisib, see BYL719 Altiratinib (DCC-2701), 115 (355) AMG 110, 174 (535) AMG 386, 165 (508) AMG 900, 197 (7LBA) Anti-DLL4, see Demcizumab APG-1252, 109 (338), 176 (544) APG-1387, 89 (268), 162 (497)

230 Anticancer agents under development (cont’d) ARQ 087, 50 (145), 125 (389), 155 (475), 156 (481), 167 (513) ARQ 092, 104 (320), 155 (475), 156 (481), 167 (513), 172 (531) ARQ 761, 84 (253) ASP8273, 198 (9LBA) AT13387, 168 (518) AU-2121, 104 (321) AV-203, 120 (374) AZ-1775, 109 (337) AZD1208, 144 (437) AZD2014, 177 (546) AZD4547, 10 (12), 122 (381), 157 (483), 169 (520) AZD6738, 84 (251) AZD7762, 71 (211), 81 (240) AZD8055, 34 (91), 150 (458) AZD8186, 90 (272), 177 (546) AZD8931, 160 (491) AZD9150, 73 (217), 163 (501) AZD9291, 99 (301) AZD6094 (HMPL-504, volitinib), 125 (392), 126 (395) AZD6244 (selumetinib), 131 (412), 148 (451) BAL101553, 65 (202) BAY6356, 185 (573) BAY 80-6946 (copanlisib), 162 (499) BEZ235, 34 (91) BGB324, 117 (362) BGJ398, 35 (94), 122 (381), 157 (483) BI836845, 152 (466) BI 853520, 72 (213) BI6727 (volasertib), 27 (68), 131 (412) BIND-014, 19 (41) BIS-1602, 107 (330) BKM120, 81 (243) BM-1252, see APG-1252 BMN 673, 33 (88), 80 (238), 81 (243) Nanoformulation, 116 (361) BMS-936558, 11 (14) BNC101, 118 (366) BO-2094, 20 (45) BYL719 (alpelisib), 28 (71), 29 (75), 199 (11LBA) C-1305, 26 (65), 27 (67) C-1311, 27 (67) 5C-Z, 94 (283) CB-5083, 180 (555) CBL0137, 135 (422) CC-115, 100 (306), 150 (459), 170 (523) CC-486, 179 (551) CDX011, 22 (51) CEP-32496, 154 (471) CIGB-247, 38 (107) CO-1686 (rociletinib), 199 (10LBA) Copanlisib, see BAY 80-6946 Coxsackievirus A21 (CVA21), 41 (118) D-BLOCK, 106 (326) D07001-F4, 17 (34) DBPR114, 92 (279) DCC-2701, see Altiratinib Debio 1143, 146 (444) Debio 1347, 177 (547) Defactinib, see VS-6063 Demcizumab (Anti-DLL4), 55 (166) Dovitinib, 35 (97) DT-1154, 158 (486), 178 (548) E7050, see Golvatinib E7080, see Lenvatinib

Subject Index E7438 (EPZ-6438), 92 (277), 197 (6LBA) EC-70124, 159 (487) Enadenotucirev, 41 (117) Encorafenib, see LGX818 Entolimod, 40 (114) EPZ-6438, see E7438 EV20, 164 (505) 6G10A, 175 (538) Galeterone, 8 (4) GBR1302, 48 (139) GDC-0980, 122 (382) GDC-0994, 124 (387) GNS396, 183 (566) Go6976, ¨ 71 (211) Golvatinib (E7050), 158 (484) GSK2849330, 98 (300) GSK2879552, 72 (212) HMPL-504, see AZD6094 hz515H7, 113 (351) IMCgp100, 44 (126) Indotecan, see LMP-400 JQ1, 191 (593) KML001, 23 (56), 24 (60), 25 (62) KRCA-386, 126 (393) KTN3379, 71 (210), 130 (407) LEE011, 163 (500) Lenvatinib (E7080), 20 (46), 158 (484) LGX818 (encorafenib), 199 (11LBA) LMP-400 (indotecan), 83 (248) LOXO-101, 125 (391) Lucitanib, 110 (340) Lurbinectidin, see PM01183 LY2603618, 71 (211) LY2835219, 162 (498), 165 (507) LY2874455, 122 (381) M-COPA, 185 (574) MGD006, 48 (138) MI130004, 164 (502) MK1775, 71 (211) MK-8242, 131 (410) MLN2480, 54 (162), 105 (324), 117 (364), 161 (493) MLN4924, 95 (286), 189 (588) MLN7243, 78 (231), 85 (255), 186 (577) MLN8237 (alisertib), 21 (50), 97 (294), 109 (336), 131 (412) MM-131, 144 (438) MM-151, 52 (152), 107 (329) MM-398, 87 (261) MOXR0916, 45 (128), 71 (209), 136 (424) MRX34, 196 (4LBA) MSK-777, 96 (293) NDD0005, 133 (417) Nintedanib, 75 (223) NP137, 118 (368) NPD926, 14 (26) NU6102, 106 (325) NU7441, 68 (208), 100 (306) NU8406A, 91 (276) ODM-203, 142 (432) OMP-52M51, 7 (2) ONCOS-102, 47 (136) OTX015, 153 (469), 183 (565, 567), 184 (568), 187 (580), 189 (587), 196 (5LBA) PAC-1, 171 (528) Palbociclib, see PD0332991 Panobinostat, 73 (216), 183 (567), 185 (572) PD173074, 35 (94), 122 (381) PD0332991 (palbociclib), 86 (259), 131 (411)

Subject Index Anticancer agents under development (cont’d) PF-05212384, 171 (526) Pimasertib, 146 (445) Plitidepsin, 108 (334), 111 (345) PM00128, 24 (57) PM060184, 23 (55) PM01183 (lurbinectidin), 13 (23), 20 (47), 21 (48), 24 (57), 82 (244) PRI-724, 121 (377) PRN-1109, 157 (483) ProCervix, 45 (130) Protein–membrane anchors, 93 (282) Pt-Erlotinib, 93 (281) Pt-Imatinib, 93 (280) PYZD-4409, 95 (286) REDX04988, 123 (384) Rociletinib, see CO-1686 RP6530, 108 (332) RPT835, 155 (476) RXDX-101, 101 (310), 143 (436) SAIT301, 98 (298) SAR405838, 32 (87), 121 (378) Selinexor, 83 (247), 100 (305), 160 (492) Selumetinib, see AZD6244 SG-110, 47 (134) SJG-136, 19 (42) SMK-17, 176 (543) SN30000, 66 (204) Tarextumab, 152 (465) TAS3681, 103 (315) TAS-102, 13 (22), 15 (27) TAS-114, 15 (29) TAS-115, 167 (515), 176 (541) TAS-116, 191 (592) TAS-119, 127 (398), 142 (433) TAS-121, 128 (401) TRC-794, 158 (486), 178 (548) TSR-011, 165 (506) TVB-2640, 195 (3LBA) TVB-3166, 190 (591) UNC2025, 114 (352), 120 (375), 164 (503) UniPR129, 90 (270) VE-821, 71 (211), 81 (240), 83 (248) Volasertib, see BI6727 Volitinib, see AZD6094 VS-5584, 144 (439), 146 (446) VS-6063 (defactinib), 97 (296), 99 (302), 146 (446) W014A, 40 (113) ZEN3365, 181 (560) APC mutations, 25 (61), 32 (85), 60 (181), 123 (383) Apoptosis induction, 9 (9), 14 (25), 16 (30), 18 (38, 39), 20 (47), 21 (48), 22 (52), 24 (59), 26 (64), 31 (80), 39 (111), 42 (119), 52 (155), 78 (231), 84 (253), 98 (297), 105 (323), 110 (341), 118 (367, 368), 122 (380), 125 (390), 146 (444), 148 (452), 158 (486), 169 (519), 176 (543), 178 (548, 550), 183 (566, 567) Apoptotic signalling, 31 (83), 101 (308), 109 (337), 135 (423), 152 (467) ARID1A mutations, 103 (318) Astrocytic Elevated Gene-1 (AEG1) disruption, 156 (478) ATR inhibitors, 71 (211), 81 (240), 83 (248), 84 (251) Atypical teratoid/rhabdoid tumours, 42 (121) Auranofin, 9 (9) Aurora A kinase inhibitors, 16 (31), 19 (43), 21 (50), 97 (294), 109 (336), 127 (398), 129 (405), 131 (411), 142 (433) Aurora B kinase inhibitors, 131 (411) Axitinib, 116 (359)

231

Axl signalling, 117 (362)

B B-cell lymphoma, 39 (108), 104 (321), 118 (365), 136 (426), 181 (560), 197 (6LBA) BABA chemotherapeutics, 89 (269) Bcl-3, metastasis mediator in breast cancer, 122 (379) Bcl-2/Bcl-xL dual inhibitor, 109 (338), 176 (544) BCL-2 status, 130 (408) BClnc-X, 101 (309) BCR pathway, 182 (563) BCR–ABL fusion protein, 28 (70), 56 (167) BD1 blockade, 184 (569) Bendamustine, prediction of skin rash, 136 (426) BET inhibitors, 31 (83), 153 (469), 181 (560), 182 (564), 183 (565, 567), 184 (568), 185 (573), 187 (580), 188 (583), 189 (587), 191 (593), 196 (5LBA) Biological aspects, 184 (569) Response prediction, 187 (581) b-Catenin, 25 (61), 32 (85), 60 (181), 112 (346), 113 (349), 115 (358), 121 (377), 123 (383), 128 (399), 142 (433), 153 (468), 176 (543) b-Lapachone, 55 (164), 84 (253), 87 (262) Bevacizumab, 8 (5), 13 (22), 96 (291), 111 (344), 115 (355), 121 (376), 122 (382) Bicalutamide, 103 (315) Bifunctional protein, 46 (132) Biliary tract cancer (BCT), 191 (592) Biologics, 35 (95), 44 (126), 76 (225), 181 (559) Biomarker identification, 33 (90), 62 (192), 76 (225), 82 (244), 105 (322), 107 (331), 150 (460), 166 (511), 190 (591) Bispecific antibody, 48 (139) Bisphosphonates, 55 (165), 114 (353) BiTE molecules, 40 (112), 174 (535) Bladder cancer, 11 (13), 17 (37), 41 (118) Blood biomarkers, 111 (344) Blood–brain barrier (BBB), 10 (12), 23 (56), 50 (145), 79 (234), 86 (259), 93 (281) Bone marrow failure, 136 (427) Bone marrow stromal cells (BMSC), 187 (579) Bone metastases, 114 (353) Bone metastases from breast cancer, 182 (562) Bone metastases from lung cancer, 114 (353), 167 (515) Bone metastases from prostate cancer, 12 (20), 25 (63), 176 (541) Bortezomib, 74 (220), 85 (255), 108 (332) BRAF/CRAF dual inhibitors, 123 (384) BRAF/EGFR dual inhibitors, 154 (471) BRAF inhibitors, 33 (90), 59 (180), 102 (314), 114 (354), 151 (461), 154 (471), 175 (540), 199 (11LBA) BRAF mutations, 13 (22), 28 (73), 29 (76), 33 (90), 41 (116), 56 (169), 57 (170), 59 (180), 61 (188), 100 (304), 114 (354), 117 (364), 123 (384), 124 (387), 131 (412), 141 (428), 149 (454), 151 (461, 463), 154 (471), 164 (505), 175 (540), 176 (543) Brain metastases, 128 (399) Brain metastases from breast cancer, 111 (343), 128 (399) Brain metastases from lung cancer, 23 (56), 93 (281), 99 (301) Brain metastases from melanoma, 49 (141) BRCA expression analysis, 17 (35) BRCA mutations, 11 (16), 16 (33), 35 (95), 72 (214), 73 (215), 80 (238), 81 (241, 242), 82 (244), 83 (249), 173 (532) BRCAness, 173 (532) Breast cancer Benign–malignant transformation, 119 (370)

232

Subject Index

Breast cancer (cont’d) Bone metastases, 182 (562) Brain metastases, 111 (343), 128 (399) BRCA-related, 82 (244) Cell migration inhibition, 95 (288) ER + , 28 (71), 36 (100), 58 (176), 186 (575) ER + , aggressiveness, 158 (485) ER status, 35 (96), 103 (316) Hepsin expression, 57 (173) HER2 + , 8 (6), 20 (46), 22 (51), 27 (69), 36 (98, 100), 74 (218), 167 (514) Her2 + , 155 (477) HER2-positive, 160 (491) HER2 status, 77 (228) Inflammatory (IBC), 31 (80), 37 (103), 74 (218) Invasive, 144 (440) Luminal, 99 (303), 188 (583) Metastatic, see Metastatic breast cancer Molecular analysis, 85 (254) Neoadjuvant therapy, 53 (159) Role of cohesin, 57 (172) Role of Jab1/Csn5 in therapy resistance, 155 (477) Role of Jagged1 in metastatic progression, 106 (328) Role of resolvin D2, 58 (176) Tamoxifen-resistant, 103 (316) Target identification, 101 (309) Targeted small molecules, 36 (100) Therapy resistance, 28 (72) Triple negative, see Triple negative breast cancer Triple therapy, 163 (500) Tumour-associated macrophages (TAM), 59 (179) 3-Bromopyruvate, 42 (119)

C Cancer cell stemness, 33 (89), 46 (131), 60 (182), 117 (362), 174 (536), 179 (552), 188 (584) Cancer stem cells (CSCs), 7 (2), 12 (20), 65 (201), 99 (302), 115 (357), 118 (366), 144 (439), 146 (446), 153 (470), 168 (517) Capecitabine, 15 (29), 17 (36), 122 (382) Carbohydrate-restriction diet, 76 (224) Carboplatin, 7 (3), 14 (24), 30 (78), 146 (444), 173 (532) Carcinogenesis, 65 (199), 75 (223), 76 (224), 112 (346), 141 (430), 162 (498) Carfilzomib, 74 (220), 184 (568) Castration-resistant prostate cancer (CRPC), 8 (4), 101 (308), 103 (315), 143 (434), 178 (548) CD70 (TNFSF7) tissue expression, 39 (108) CDC7 inhibitors, 96 (293) CDCP1, 56 (168) CDK inhibitors, 80 (238), 86 (259), 87 (260), 106 (325), 110 (341), 130 (408), 131 (411), 162 (498), 163 (500), 165 (507) Cell cycle arrest, 15 (28), 16 (31), 18 (40), 24 (59), 78 (231), 91 (274), 183 (565) Cell cycle disruption, 16 (32), 18 (38), 20 (47), 42 (121), 82 (245), 96 (293), 106 (325), 108 (332), 110 (341), 134 (419) Cell cycle distribution, 9 (9), 16 (30), 27 (68), 36 (100), 68 (208), 101 (309) Cell cycle induction, 96 (292) Cell cycle models, 84 (251) Cell differentiation, 50 (147), 103 (318), 128 (400), 130 (409), 141 (429), 149 (456), 180 (556), 190 (589) Cell free DNA (cfDNA), 76 (227), 102 (313), 149 (454), 151 (462) Cell free tumour DNA (ctDNA), 51 (149), 114 (354)

Cell migration, 41 (116), 51 (151), 60 (183), 64 (196), 75 (221), 87 (263), 95 (288), 128 (400), 130 (406) Cell proliferation, 16 (31), 26 (65), 31 (81), 36 (98), 41 (116), 60 (183), 65 (199), 66 (203), 77 (228), 79 (235), 87 (263), 89 (267), 91 (275), 92 (279), 94 (283), 103 (318), 110 (339), 128 (400), 141 (429), 147 (447), 164 (504), 190 (589) mechanisms, 179 (553) Cell signalling observation, 62 (191) Cell–cell interactions, 17 (37), 50 (147), 51 (150), 62 (191) Ceritinib, 126 (393), 168 (518) Cervical cancer, 45 (130), 74 (219), 97 (294), 119 (369), 179 (551) Cetuximab, 10 (10), 13 (22), 62 (192), 106 (326), 112 (348), 147 (448), 199 (11LBA) CHEK1 inhibition, 117 (363) Chemoradiotherapy, 67 (207), 74 (219), 109 (337), 132 (414) Chemosensitisation, 21 (49), 25 (62), 28 (72), 31 (81), 96 (292) Chemosensitivity, 62 (190) Childhood cancers, 135 (421) CHK inhibitors, 16 (32), 71 (211), 81 (240), 82 (245) Choline uptake in gliobastoma, 58 (175) Chromatin-modifying genes as targets in ccRCC, 188 (582) Chronic inflammation, 150 (460) Chronic lymphocytic leukaemia (CLL), 18 (39), 39 (109), 149 (456), 187 (579) Chronic myelomonocytic leukaemia (CMML), 21 (48) cIAP1/XIAP dual antagonist, 122 (380) Cidofovir, 74 (219) Circulating epithelial tumour cells (CETCs), 62 (190) Circulating microRNAs, 53 (159) Circulating tumour cells (CTCs), 45 (129), 106 (328), 127 (397), 129 (403), 166 (509) Cisplatin, 30 (78), 80 (239), 87 (263), 97 (295), 119 (371), 178 (550), 189 (586) Acquired resistance, 34 (92) Attenuation of hearing impairment, 55 (164) CK2 inhibition, 134 (419) Clear cell renal cell carcinoma (ccRCC), 41 (116), 188 (582) cMet expression in meningioma, 174 (534) cMET inhibitors, 65 (200) cMet inhibitors, 107 (331), 125 (392), 126 (395), 144 (438), 174 (534) cMET pathway, link with EGFR pathway, 112 (347) CNKSR1 inhibitors, 160 (490) Cobimetinib, 100 (304), 106 (327), 124 (387) Cohesin, role in breast cancer, 57 (172) Colorectal cancer (CRC), 16 (30), 20 (45), 26 (65), 29 (74), 31 (82), 32 (85), 42 (119), 49 (143), 51 (149), 59 (177), 60 (182), 61 (188), 62 (192), 65 (201), 86 (257), 96 (291), 106 (327), 118 (366), 120 (375), 122 (382), 123 (383, 384), 130 (406), 141 (428), 153 (468), 154 (471, 474), 164 (505), 171 (526), 199 (11LBA) Aggressiveness, 39 (110) Lymph node metastases, 64 (196) Metastatic, see Metastatic colorectal cancer Combination therapies, 8 (5), 13 (22), 14 (25), 16 (32, 33), 18 (39, 40), 20 (45, 46), 22 (51), 23 (56), 24 (60), 25 (61, 62), 29 (74), 30 (78), 31 (81), 32 (84), 35 (97), 36 (98), 38 (106), 41 (118), 43 (123), 47 (134), 49 (141), 54 (161), 57 (171), 63 (193), 65 (200, 202), 68 (208), 73 (216), 74 (219, 220), 80 (238), 81 (240, 242, 243), 83 (248), 84 (251), 97 (295), 100 (305, 306), 101 (311), 102 (314), 106 (327), 107 (329), 108 (332), 112 (347), 113 (349), 115 (355), 117 (362), 119 (371), 121 (376), 124 (386, 387), 125 (390), 127 (398), 130 (408), 131 (411), 135 (422), 141 (428), 146 (444, 445, 446), 148 (451, 452), 151 (461,

Subject Index Combination therapies (cont’d) 463), 152 (465), 154 (474), 155 (475), 156 (481), 157 (482), 158 (484), 163 (500), 164 (502, 505), 165 (508), 166 (511), 167 (513), 171 (526, 528), 173 (532), 176 (542), 178 (550), 179 (554), 183 (567), 184 (568), 187 (580), 189 (586), 192 (595), 199 (11LBA) Drug screening, 36 (99) ComboPredictor, 166 (511) Complement dependent cytoxicity (CDC), 98 (300), 107 (329), 113 (351), 175 (538) CRAF, 84 (250), 171 (527) Crizotinib, 65 (200), 107 (331), 125 (391), 132 (414, 415), 165 (506), 167 (515), 168 (518), 176 (541), 182 (564) Cyclodepsipeptides, 16 (30), 24 (58), 108 (334), 111 (345) CYP17 inhibitors, 8 (4), 101 (308), 143 (434), 150 (457) Cysteine derivatives, 91 (274) Cytarabine, 77 (230) Cytochrome P450 enzymes as drug targets, 95 (289) Cytosolic p21, 117 (363)

D Dabrafenib, 33 (90), 151 (461) Danusertib, 16 (31) DART molecules, 48 (138) Dasatinib, 32 (84), 103 (318), 167 (512) Death receptor signalling, 186 (576) Deforolimus, 131 (411) Denosumab, 114 (353) Destruxins, 16 (30) Determinants of sensitivity, 33 (88), 34 (91), 62 (192), 66 (204), 84 (252) Diffuse large B-cell lymphoma (DLBCL), 10 (11), 125 (390), 130 (408), 162 (499), 182 (563) Dinaciclib, 110 (341), 131 (411) DNA barcoding, 33 (88), 54 (161) DNA damage response, 184 (570) DNA methylation, 23 (54), 77 (229), 94 (284), 99 (303), 145 (441), 152 (466), 174 (536), 184 (570), 192 (595) DNA-PK/mTOR dual inhibitors, 100 (306), 150 (459), 170 (523) DNA repair, 100 (306) DNMT inhibitors, 77 (229) Docetaxel, 9 (8), 89 (268), 127 (398), 188 (585) Docetaxel-titanate nanotubes, 67 (207) Nanoformulation, 19 (41) Docosahexaenoic acid (DHA), 75 (221) Dosing regimens, 72 (213), 104 (320), 133 (416), 179 (551), 190 (590) BRAF/MEK inhibitor combination, 102 (314) EGFR inhibitors, 76 (226) Naproxen, 76 (226) PM01183, 13 (23) Doxorubicin, 17 (35), 28 (72), 37 (102), 38 (105), 74 (220), 82 (245), 86 (257), 118 (368), 131 (411), 153 (470) Drug availability, 143 (436) Drug characterisation, 15 (27), 17 (36), 24 (57, 58), 52 (152), 71 (209), 82 (246), 90 (270), 98 (300), 105 (324), 106 (325), 115 (356), 125 (389, 391), 128 (400), 136 (424), 150 (459), 161 (493), 176 (544), 181 (561), 185 (572) Drug combination screening, 36 (99) Drug delivery, 41 (117), 66 (203), 85 (256), 86 (257, 259), 87 (260, 262, 263), 88 (264, 265), 89 (266) Drug design, 141 (430) Drug discovery, 15 (28), 36 (101), 90 (270, 271), 92 (279), 95 (287), 156 (479), 180 (557), 195 (2LBA) Drug distribution imaging, 174 (535) Drug reactions in Phase I studies, 136 (425)

233

Drug screening, 30 (78), 91 (275), 131 (411), 147 (447), 160 (490) Drug synthesis, 141 (429) Drug–drug conjugates, 20 (45), 87 (260) Drug–drug interaction, 57 (171) Drug–drug interactions, 54 (162) Duocarmycin pharmacophore, 95 (289) dUTPase/DPD inhibitor, 15 (29)

E 4E-BP1, 34 (91) E-Cadherin, 50 (147), 51 (150) eEF1A, 111 (345) Efficacy assessment, 154 (472) EGFR expression in TNBC, 169 (521) EGFR inhibitors, 32 (84), 65 (200), 93 (281), 112 (347, 348), 123 (385), 128 (401), 147 (448), 168 (516), 198 (9LBA), 199 (10LBA) Dosing regimens, 76 (226) EGFR/MEK dual inhibitors, 148 (451) EGFR mutations, 32 (86), 112 (348), 198 (9LBA) T790M, 93 (281), 123 (385), 128 (401) EGFR status, 51 (151) EGR1, 57 (171) EGR-1 inhibitors, 98 (298) eIF2aP inhibitors, 148 (452) EML4–ALK fusion protein, 182 (564) Endometrial cancer, 35 (94), 110 (340), 156 (481), 167 (513) Enzalutamide, 8 (4), 101 (308), 103 (315), 143 (434), 150 (457) Ependymoma, 63 (193), 133 (416, 418) EPH–ephrin system, 90 (270), 187 (579) EPHA2 inhibition, 90 (270), 98 (297) Epigenetics, 42 (121), 47 (134), 50 (147), 81 (241), 95 (287), 134 (419), 145 (441), 177 (545), 180 (556) Epithelial cell adhesion molecule, 144 (438), 166 (509), 174 (535) Epithelial–mesenchymal transition (EMT), 17 (37), 39 (110), 51 (150, 151), 65 (199), 117 (362), 129 (404), 143 (435), 174 (536), 175 (539), 179 (552), 188 (584) ER stress, 51 (148), 94 (283), 148 (452), 161 (495), 180 (555) Eribulin, 14 (24), 17 (36) In combination therapies, 20 (46) ERK1/2 translocation, 34 (92) ERK inhibitors, 124 (387), 141 (429) Erlotinib, 8 (5), 30 (79), 57 (171), 93 (281), 112 (348), 147 (448), 148 (451), 168 (516) Esomeprazole, 21 (50) Etirinotecan pegol, 16 (33) Etoposide, 30 (78), 80 (239), 178 (550) Everolimus, 10 (10), 163 (500), 187 (580) Ewing’s sarcoma, 33 (88), 84 (252), 133 (417), 134 (420) EWS–FLI1 fusion protein, 84 (252), 134 (420) Exemestane, 163 (500) EXN32, 178 (549) EXN36, 41 (116) EXN91, 41 (116) exoRNA, 102 (313), 149 (454) Exosomes, 96 (290), 102 (313), 149 (454) Exportin 1 (XPO1) inhibitors, 74 (220), 83 (247), 100 (305), 160 (492) Extracellular matrix (ECM), 60 (183), 89 (266), 179 (552) Extracellular vesicles, 147 (448) Extravasation, 12 (20), 111 (343), 187 (578) EZH2/EHMT2 dual inhibitors, 179 (554) EZH2 inhibitors, 92 (277), 104 (321), 125 (390), 191 (594)

234

Subject Index

F FAK inhibitors, 72 (213), 97 (296), 99 (302), 146 (446) FASN inhibitors, 141 (430), 190 (591), 195 (3LBA) Ferumoxytol MRI, 87 (261) FGF inhibitors, 100 (307) FGFR, 134 (420) FGFR inhibitors, 35 (94), 115 (356), 122 (381), 125 (389), 128 (400), 155 (475, 476), 156 (481), 157 (483), 167 (513), 169 (520) FGFR mutations, 35 (94) FGFR/VEGFR dual inhibitors, 142 (432) FGRF mutations, 122 (381) Flavopiridol, 130 (408) 5-Fluorouracil, 13 (22), 15 (27, 29), 20 (45), 130 (406), 133 (416, 418) Follicular adenoma, 42 (120) FoxO activation, 24 (59) Fusion genes, 54 (160), 173 (533) Fusion molecules, 169 (519) Fusion proteins, 24 (59), 28 (70), 47 (135), 56 (167), 84 (252), 111 (345), 118 (367), 134 (420), 152 (467), 182 (564)

G Ganetespib, 188 (585) Gastric cancer, 20 (44), 81 (242), 110 (340), 115 (356), 166 (509), 188 (585), 198 (8LBA) Gastro-enteropancreatic neuroendocrine neoplasms, 149 (455) Gastro-intestinal stromal tumours (GISTs), 127 (396) Gastro-oesophageal cancer, 175 (537) Gefitinib, 62 (189), 65 (200), 112 (348) Gemcitabine, 16 (32), 21 (49), 57 (171), 63 (193), 100 (305), 121 (376), 146 (445), 152 (465), 176 (542) Gemcitabine HCl, 17 (34) Gene editing, 147 (450) Gene expression analysis, 169 (520) Gene expression profiling, 85 (254), 99 (303), 119 (370) Gene expression signatures, 31 (80), 61 (188), 173 (532) Gene–gene interactions, 188 (583) Genetic profiling, 168 (516) Genetically engineered mouse models (GEMMs), 11 (16), 152 (466), 158 (486), 164 (503) Genomic profiling, 61 (187), 79 (236), 151 (462) Genomics, 3 (1), 28 (73), 36 (101), 50 (147), 56 (167), 74 (218), 84 (250) Data integration, graph analysis, 90 (271) GFP–FOXO3a fusion protein, 24 (59) GHRH antagonist, 107 (330) Glioblastoma, 25 (62), 170 (523) Glioblastoma multiforme (GBM), 10 (12), 23 (54), 51 (151), 58 (175), 86 (259), 153 (469), 166 (510), 171 (528) Prognostic/predictive biomarker, 23 (54) Gliomas, 53 (157), 64 (197), 73 (216) Glutathione depletion, 14 (26), 16 (30), 86 (258) Glutathione S-transferases, role in reducing breast cancer aggressiveness, 158 (485) Glycolysis, 8 (5), 42 (119), 60 (182), 175 (540) Glycoprotein expression, 12 (17) Glycoprotein NMB, 22 (51) Glycosylation, 12 (17), 55 (165), 95 (288), 156 (479) Golgi apparatus, 185 (574)

H Haematologic malignancies, 195 (1LBA), 196 (5LBA) Haematological malignancies, 10 (11), 39 (108), 48 (138), 50 (145), 79 (237), 83 (247), 91 (274), 94 (285), 96 (293),

118 (365), 144 (437), 176 (544), 177 (546), 180 (555), 181 (560), 183 (567), 184 (570), 185 (571, 573), 189 (587) Haematopoietic stem cells, 136 (427) Haematotoxicity assessment, 136 (427) HDAC inhibitors, 35 (97), 73 (216), 185 (572), 189 (586) HDC platform, 87 (260) HDM2 antagonist, 121 (378) Head and neck cancer (HNC), 10 (10), 109 (337), 119 (371), 179 (551) Hedgehog inhibitor, 157 (482) Hepatoblastoma, 25 (61) Hepatocarcinoma, 108 (333) Hepatocellular carcinoma (HCC), 61 (186), 73 (217), 75 (223), 141 (429), 150 (458), 152 (466), 196 (4LBA) Metastatic, 73 (217) Pathogenesis, 161 (494) Hepsin expression, 57 (173) HER2 + cancers, 48 (139) HER2 gene status, 166 (509) HER2 inhibitors, 74 (218) HER3 signalling, 98 (300) HIF1a expression, 167 (514) High-throughput screening, 37 (104), 54 (160), 60 (184), 63 (193), 168 (517) Hippo signalling, 180 (557) Histone methylation, 192 (595) Homologous recombination deficiency (HRD), 72 (214), 73 (215), 83 (249) HPV, 97 (294) HPV-related malignancies, 74 (219), 97 (294), 109 (337), 119 (369), 179 (551) HPV vaccines, 45 (130) HSP90 inhibitors, 65 (200), 87 (260), 168 (518), 182 (563), 188 (585), 191 (592) HT-KAM assay, 154 (472) Hybrid drugs, 93 (280, 281) Hypoxia models, 53 (156) Hypoxia targetting, 66 (204)

I Ibrutinib, 182 (563), 184 (568) Idarubicin, 77 (230) IDH1 inhibitor, 195 (1LBA) IDH1 mutations, 128 (402), 195 (1LBA) IDO1 inhibitors, 37 (104) Binding sites, 91 (273) IFN-g–TRAIL fusion protein, 47 (135) IGF-1R inhibitors, 31 (81), 35 (97), 101 (311) IKK inhibition, 103 (317), 117 (363) IL-6/STAT3/Fra-1 signalling, 39 (110) IL1R2 expression, 31 (82) Imaging Tracers, 92 (278) Imaging biomarkers, 154 (473) Imatinib, 56 (167), 127 (396), 167 (512) Therapy resistance, 28 (70), 93 (280) Immune cells, 46 (133) Immune checkpoints, 39 (111), 40 (113), 47 (134), 48 (137), 63 (195), 136 (424), 143 (435) Immune modulation, 39 (108) Immune response markers in NSCLC, 43 (122) Immune-stimulating cytokines, 46 (132) Immune-stimulating transgenes, 47 (136) Immunohistochemistry, 77 (229) Immunophenotyping, 8 (6), 115 (357)

Subject Index Immunotherapy, 8 (6), 11 (14), 12 (19), 37 (104), 40 (112, 113, 114), 42 (119), 43 (124), 45 (128, 129), 46 (132), 47 (134), 49 (142), 71 (209), 136 (424), 169 (521) Indole-3-carbinol, 18 (39), 89 (269) Induced drug tolerant cells (IDTC), 34 (93) Inhibitor of apoptosis proteins (IAPs), 89 (268), 122 (380), 162 (497) Inhibitor of differentiation 1 (Id1), 65 (199) Integrin inhibitors, 12 (20), 38 (106) Interleukin 17, 49 (142), 50 (144) Invadopodia, 75 (221), 187 (578) Ionising radiation, 65 (202), 66 (203), 67 (206), 68 (208), 74 (219), 81 (241), 84 (251) Ipilimumab, 49 (141) Irinotecan, 25 (61), 62 (192), 83 (248), 107 (329), 135 (422), 171 (526) Genetic biomarker for neutropenia, 26 (66) Nanoliposomal (MM-398), 87 (261) Iron chelators, 175 (539) Ischaemia, effect on specimen quality, 151 (464) Isoindolinones, 91 (276), 133 (417) Isomalyngamide A analogs, 95 (288)

J JAK inhibitors, 163 (501) JNK inhibitors, 154 (474), 186 (576) JNK pathway, 164 (504) Juvenile myelomonocytic leukemia (JMML), 21 (48)

K KDM1A inhibitors, 185 (571) Kinome capture, 59 (177), 82 (245), 98 (299) Kinome capture enrichment strategy, 54 (160) KIT mutations, 127 (396) KRAS mutations, 13 (22), 19 (41), 41 (116), 51 (149), 64 (198), 96 (291), 98 (297), 102 (312), 103 (317), 106 (327), 114 (353), 124 (387), 129 (405), 158 (486), 160 (490), 171 (527), 177 (545), 191 (593) KSP inhibitors, 91 (274)

L Lapatinib, 22 (51), 27 (69), 36 (98), 74 (218), 76 (226), 188 (585), 191 (592) Leiomyomatosis, 8 (5) Leiomyosarcomas, 104 (319) Letrozole, 35 (96) Leukaemia Acute lymphoblastic, see Acute lymphoblastic leukaemia Acute myeloid, see Acute myeloid leukaemia Chronic lymphocytic, see Chronic lymphocytic leukaemia Chronic myelomonocytic (CMML), 21 (48) Paediatric, 131 (411) Therapy resistance, 93 (280) Linsitinib, 35 (97) Liquid biopsy, 51 (149), 102 (313), 127 (397), 149 (454) Lithocholic acid (LCA), 90 (270) Liver metastases from NSCLC, 65 (199) Liver toxicity, 13 (21) Long non-coding RNAs, 101 (309) Loss-of-function mutations, 7 (2), 35 (95), 52 (153), 81 (241), 82 (245), 103 (318), 151 (463), 164 (504) LPA6–Pim-3 axis in HCC, 161 (494) LSD1 inhibitors, 72 (212) Lung adenocarcinoma, 124 (388), 188 (584), 195 (2LBA) Lung cancer, 15 (28), 129 (405) Bone metastases, 167 (515)

235

Brain metastases, 23 (56), 93 (281) Metastatic, 23 (56), 39 (111) Radiosensitisation, 68 (208) Squamous, 188 (583) Tumour-suppressive microRNAs, 30 (77) Lung cancer initiating cells, 103 (317) Lymph node metastases, 145 (441) Lymphoma, 9 (9), 10 (11), 17 (34), 21 (50), 39 (108), 77 (229), 88 (265), 92 (277), 104 (320, 321), 109 (336), 113 (351), 118 (365), 125 (390), 130 (408), 136 (426), 143 (436), 162 (499), 181 (558, 560), 182 (563), 184 (568), 191 (594) Lysosome disruption, 183 (566)

M Malignant melanoma, 44 (126), 136 (426), 149 (454) Mantle cell lymphoma (MCL), 162 (499), 184 (568) MAPK inhibitors, 28 (73), 150 (458), 151 (463) MAPK pathway, 113 (350), 117 (364), 151 (461) MAPK pathway alterations, 141 (428) Marine-derived anticancer agents, 20 (46), 23 (55), 24 (57, 58, 59), 108 (334), 111 (345), 164 (502) Mass spectrometry, 159 (489), 175 (537) MCF-7 cell lines, glycoprotein expression, 12 (17) MDM2 inhibitors, 106 (327) MDR1 inhibitors, 54 (161) Mebendazole, as anticancer agent, 157 (482) Medullary thyroid carcinoma, 195 (2LBA) MEK/ERK pathway, 29 (76) MEK inhibitors, 29 (74), 33 (90), 54 (161), 102 (314), 106 (327), 124 (386, 387), 131 (412), 146 (445), 176 (543) MEK/PI3K dual unhibitors, 166 (511) Melanoma, 20 (46), 29 (76), 34 (93), 43 (124), 48 (140), 54 (162), 100 (304), 117 (364), 122 (380), 150 (460), 154 (471), 161 (493), 164 (503), 175 (540) Brain metastases, 49 (141) Metastatic, see Metastatic melanoma Melatonin, 169 (522) MELK inhibitors, 168 (517) Melphalan, 74 (220) Meningioma, 174 (534) MER inhibitors, 120 (375) Merkel cell carcinoma, 12 (18) MerTK/Flt3 dual inhibitor, 114 (352) Mesothelin expression, 102 (312) Mesothelioma, 62 (189), 99 (302), 146 (446), 176 (542) MET addiction, 185 (574) MET expression, 161 (496) MET mutations, 161 (496) MET quantitation, 175 (537) MET signalling, 170 (524) MET/VEGFR dual inhibitors, 167 (515), 176 (541) Metastasis mechanisms, 187 (578) Metastasis regulation, 147 (447) Metastatic biliary tract cancer, 191 (592) Metastatic breast cancer, 17 (36), 29 (75), 36 (100), 82 (244), 111 (343), 163 (500) mechanisms, 106 (328), 122 (379), 144 (440), 169 (522), 182 (562) Metastasis mediator, 122 (379) Subtypes, 37 (103) Metastatic colorectal cancer (mCRC), 13 (22), 15 (27), 31 (82), 59 (177), 64 (196), 65 (201), 96 (291), 120 (375), 130 (406), 164 (505), 171 (526) prognostic/predictive biomarker, 23 (54) Metastatic hepatocellular carcinoma, 73 (217) Metastatic lung cancer, 23 (56), 39 (111)

236

Subject Index

Metastatic melanoma, 26 (64), 43 (124), 44 (126), 49 (141), 59 (180), 123 (385), 164 (503) prognostic biomarker, 33 (90) Metastatic pancreatic cancer, 152 (465), 175 (539) Metastatic prostate cancer, 18 (38), 25 (63), 33 (89), 101 (308), 107 (331), 143 (434), 147 (447), 176 (541) Metastatic renal cell carcinoma (mRCC), 98 (299), 116 (359), 172 (530) Metformin, 67 (205) as anticancer treatment after gastrectomy, 20 (44) mFOLFOX6, 122 (382) Microcapsules, 87 (262) Microcystin-LR, 86 (258) MicroRNAs, 12 (20), 51 (151), 108 (333), 116 (360), 147 (449), 177 (545), 180 (556), 181 (558) microRNAs, 174 (536) MicroRNAs Shortening, 105 (323) Tumour suppression in lung cancer, 30 (77) Microsatellite instability (MSI), 49 (143), 62 (192), 153 (468) Microtubules, 14 (24), 18 (38), 20 (46), 23 (55), 52 (155), 65 (202), 106 (326), 111 (342), 127 (398), 145 (443) Minicells, 85 (256) Mithramycin, 13 (21) Mitotic catastrophe, 18 (38), 68 (208) Mixed lineage kinases, 29 (76) Mixeno model, 11 (14) MLK4 mutations, 164 (504) MLN7243, 78 (231) Model validation, 11 (14, 15), 99 (301) Molecular profiling, 60 (184), 78 (233), 96 (291), 102 (312), 103 (318), 124 (388), 127 (397), 135 (421) Morphologic assays for predicting tumour outcome, 75 (222) Mouse clinical trial, 9 (7) mTOR inhibitors, 10 (10), 34 (91), 131 (411), 150 (458), 177 (546) mTOR/PI3K dual inhibitors, 34 (91) Multi-drug testing in single tumour, 38 (105) Multikinase inhibitors, 92 (279), 110 (340) Multiple myeloma, 74 (220), 108 (332, 334), 111 (345), 184 (568) Multispectral imaging, 8 (6) Mutation detection, 76 (227) MYC mutations, 16 (31), 113 (349), 142 (433), 153 (468) Myeloid-derived suppressor cells (MDSCs), 33 (90), 43 (123) Myxoid liposarcomas (MRCLs), 190 (590)

N NAMPT inhibitor, 110 (339) NAMPT inhibitors, 82 (246) Nanomolar compounds, 94 (285) Nanoparticles, 19 (41), 87 (263), 88 (265), 89 (267), 116 (361) NanoString platform, 161 (496), 169 (520) Nanotubes, 67 (207) Naproxen dosing regimen, 76 (226) Nasopharyngeal carcinoma (NPC), 98 (298), 179 (551) Navitoclax, 102 (314), 130 (408) NCI60 cell line panel, 36 (101) NCI-MATCH screening assay, 78 (232) NDRG1, 175 (539), 190 (589) Neddylation, 189 (588) Neoadjuvant chemotherapy, 14 (24), 53 (159), 85 (254), 167 (514), 173 (532) Neoadjuvant therapy, 40 (114) Neuregulin 1 (NRG1), 120 (374)

Neuroblastoma, 130 (409), 135 (422, 423) Neurofibromatosis, 127 (396) Neuropilin 2, 64 (196) Next-generation sequencing (NGS), 10 (12), 11 (13), 33 (88), 37 (103), 51 (149), 53 (157), 55 (163), 57 (170), 59 (178), 61 (187), 73 (215), 76 (227), 78 (232, 233), 84 (250), 96 (290), 102 (313), 103 (318), 132 (413, 414, 415), 134 (420), 135 (421), 149 (454), 150 (458), 163 (500), 172 (529), 173 (533) NeyGcGM3 expression in TNBC, 169 (521) NF-úB, 31 (80) NF-úB inhibitors, 19 (42), 114 (353), 135 (422), 175 (539), 178 (550) Nifuroxazide halogenic derivatives, 26 (64) Niraparib, 72 (214), 80 (239) Non-Hodgkin lymphoma (NHL), 10 (11), 88 (265), 92 (277), 104 (321), 125 (390), 130 (408), 162 (499) Non-small cell lung cancer (NSCLC), 19 (41), 20 (46), 24 (60), 27 (68), 30 (79), 32 (84, 86), 39 (111), 43 (122, 123), 44 (127), 45 (129), 46 (133), 60 (185), 64 (198), 67 (205, 206), 97 (295), 98 (297), 111 (344), 112 (347), 114 (353), 115 (357), 116 (360), 123 (385), 127 (397), 146 (444), 158 (486), 161 (496), 165 (506), 167 (512), 168 (516, 518), 171 (527), 177 (545), 182 (564), 191 (593), 198 (9LBA) Brain metastases, 99 (301) Liver metastases, 65 (199) Squamous, 169 (520) Notch pathway, 152 (465) Notch signaling inhibitors, 145 (443) NRAS mutations, 117 (364), 147 (450) NSCLC, 199 (10LBA) Nutlin-3, 91 (276), 97 (295), 133 (417), 146 (445), 149 (455)

O Oesophageal cancer, 92 (278) Olaparib, 33 (88), 82 (244), 198 (8LBA) Nanoformulation, 116 (361) Onapristone, 142 (431) Oncogene addiction, 56 (167), 165 (507) Oncogene-induced senescence (OIS), 128 (402) Oral cancer, 147 (449) Oral squamous cell carcinoma (OSCC), 145 (441) Organic anion transporting polypeptides (OATP), 86 (258) Organoid cultures, 59 (177) Oropharyngeal squamous cell carcinoma (OPSCC), 109 (337) Orthotopic tumour models, 53 (158) Orthotopic xenografts, 62 (189) Osteosarcoma, 82 (245) Ototoxicity, 55 (164) Ovarian cancer, 20 (46), 34 (92), 37 (102), 38 (106), 72 (214), 117 (363), 146 (444), 167 (513) platinum- and anthracycline-based therapies, 37 (102) Recurrent, 7 (3) Ovarian clear cell carcinoma (OCCC), 103 (318) Oxaliplatin, 62 (192) Potentiation in colorectal cancer, 154 (474) Pt(IV) derivatives, 22 (52) Oxidative stress, 31 (80)

P p53, 153 (470) p53 activation, 135 (422) p97 inhibitors, 180 (555) p53 mutations, 167 (514)

Subject Index p53 pathway, 91 (276), 121 (378), 133 (417) p53 status, 27 (68) p53–MDM2 inhibitors, 91 (276), 97 (295), 131 (410), 133 (417), 149 (455) Paclitaxel, 7 (3), 54 (162), 65 (202), 127 (398), 146 (444), 147 (447) nab-Paclitaxel, 55 (166), 152 (465), 179 (551) Paediatric malignancies, 21 (48), 25 (61), 42 (121), 63 (193), 131 (410, 411, 412), 133 (416, 418), 134 (419), 135 (422), 189 (588) Pediatric Preclinical Testing Program (PPTP), 131 (410, 412) PAK4 allosteric modulators (PAMs), 156 (480) PAK4 mutations, 156 (480) pan-Aurora kinase inhibitor, 197 (7LBA) Pancreatic acinar cell carcinoma (PACC), 84 (250) Pancreatic cancer, 22 (53), 55 (166), 79 (236), 83 (249), 86 (258), 87 (263), 100 (305), 118 (366) Metastasis mechanisms, 175 (539) Metastatic, 152 (465), 174 (535) Pancreatic ductal adenocarcinoma (PDAC), 63 (194), 146 (445), 148 (451) Panitumumab, 13 (22), 106 (326) Papillary renal cell carcinoma (PRCC), 125 (392), 126 (395) Papillary thyroid carcinoma (PTC), 42 (120), 50 (144) PARG inhibitors, 94 (284) PARP inhibitors, 16 (33), 33 (88), 35 (95), 72 (214), 73 (215), 80 (238, 239), 81 (243), 82 (244), 83 (249), 94 (284), 173 (532), 198 (8LBA) Mechanisms, 82 (246) Patient-derived xenografts (PDX), 9 (7, 8), 10 (10, 11, 12), 11 (15), 12 (18), 35 (96, 97), 37 (102), 50 (146), 52 (154), 58 (174), 59 (178), 60 (185), 63 (195), 82 (244), 96 (291), 112 (348), 121 (376), 155 (475), 156 (481) Characterisation, 62 (192) Orthotopic setting, 9 (8) Patient stratification, 25 (61), 27 (69), 58 (174), 90 (271), 112 (348), 151 (464), 172 (529, 531) Pazopanib, 12 (18), 151 (461) PBD inhibitors, 18 (40) PD-1 expression, 45 (129) PD-1 inhibitors, 39 (109) PD-L1 expression, 48 (140), 159 (489) PDGFRA mutations, 127 (396) Pediatric Preclinical Testing Program (PPTP), 131 (410, 412) Pembrolizumab, 44 (127), 48 (140) Penile squamous cell carcinoma (PeSCC), 61 (187) Pentraxin-3, 100 (307) Peritoneal seeding, 186 (576) PERK–eIF2a axis, 179 (552) Personalised therapy, 56 (169), 57 (170), 58 (174), 59 (177), 79 (236), 84 (250), 90 (271), 95 (289), 119 (369), 151 (462, 464) PET imaging approaches, 64 (197), 79 (234, 235) Pharmacodynamics, 40 (113), 54 (161), 71 (209), 78 (231), 105 (324), 117 (363), 121 (377), 143 (436), 152 (465), 161 (493), 189 (587) Pharmacogenomics, 13 (21), 55 (163) Pharmacokinetics, 21 (50), 71 (209), 73 (217), 93 (281), 97 (296), 105 (324), 109 (336), 111 (342), 133 (418), 142 (431), 161 (493), 185 (572), 189 (587), 195 (3LBA) Pharmacoproteomics, 182 (563) Phase I/II studies, 23 (56), 24 (60), 83 (247), 111 (342), 160 (492), 165 (506) Phase I studies, 7 (2), 15 (29), 17 (34), 23 (55), 32 (87), 38 (107), 40 (114), 41 (118), 44 (126), 45 (130), 49 (141), 55 (166), 65 (202), 71 (210), 72 (212, 213), 73 (216, 217), 74 (219), 78 (231), 84 (253), 89 (268), 90 (272), 95 (286),

237

96 (293), 98 (300), 100 (306), 104 (320), 107 (329), 113 (351), 118 (366), 120 (374), 121 (378), 122 (382), 125 (389, 391), 130 (407), 133 (416, 418), 142 (431), 143 (436), 146 (444), 150 (459), 151 (461), 162 (497), 163 (500), 165 (508), 171 (526), 177 (547), 179 (551), 181 (561), 186 (577), 189 (586), 195 (1LBA, 3LBA), 196 (4LBA, 5LBA), 197 (6LBA, 7LBA), 199 (11LBA) Drug reactions, 136 (425) Phase II studies, 8 (4, 5), 14 (24), 61 (186), 73 (215), 79 (237), 83 (249), 125 (392), 162 (499), 172 (529), 198 (8LBA), 199 (10LBA) Phase III studies, 13 (22), 17 (35), 108 (334), 111 (345), 116 (359) Phenotypic plasticity, 117 (362) Phosphatidylserine externalisation, 66 (203) Phospho-reactome measurements, 154 (472) Phospholipase C (PLC) inhibition, 113 (350) PI3K/AKT/mTOR pathway, 120 (373) PI3K/AKT pathway, 158 (485), 167 (513), 172 (531) PI3K inhibitors, 24 (59), 28 (71), 29 (74, 75), 31 (81), 81 (242, 243), 87 (260), 90 (272), 100 (304), 108 (332), 124 (386), 129 (404), 148 (453), 162 (499), 177 (546), 199 (11LBA) PI3K/mTOR dual inhibitors, 122 (382), 144 (439), 146 (446) PI3K/mTor dual inhibitors, 171 (526) PI3K/mTOR pathway, 74 (218) PI3K mutations, 10 (10), 129 (404) PI3K pathway, 28 (71) Effects of radiotherapy, 170 (525) PIM-1 inhibitors, 64 (198) PIM kinase, 144 (437) PIP signalling, 91 (275) Piperlongumine, 119 (371) PIP5K inhibition, 91 (275) Pirin, 51 (150) PKC activation, 113 (350) Plasma metabolomics, 148 (453) Platelet-derived growth factor (PDGF), 118 (367) Platinum resistance, 7 (3) Platinum(IV) compounds, 22 (52), 53 (156) Plitidepsin, 108 (334) PLK inhibitors, 18 (40), 27 (68) Plucked hair culture platform, 121 (377) PP2A activators, 158 (486), 178 (548) PP2A inhibitors, 86 (258) Preclinical studies, 9 (7, 8), 11 (15), 67 (205), 75 (223), 144 (437) Predictive biomarkers, 17 (35), 19 (43), 23 (54), 26 (66), 44 (127), 52 (154), 59 (180), 61 (186), 62 (192), 75 (221), 78 (232), 83 (248), 87 (261), 107 (331), 111 (344), 116 (359), 120 (374), 136 (426), 152 (465), 173 (532), 178 (549), 187 (581) PREVENT programme, 76 (225) Prognostic factors, 17 (35), 23 (54), 46 (133), 77 (228), 102 (312), 167 (514) Prognostic markers, 116 (360) Prostate cancer, 12 (20), 33 (89), 67 (207), 87 (262), 105 (322), 107 (331), 143 (434), 159 (487), 161 (495), 180 (556), 181 (559) Bone metastases, 25 (63), 176 (541) Castration-resistant (CRPC), 8 (4), 101 (308), 103 (315), 143 (434), 178 (548) Metastatic, see Metastatic prostate cancer Prevention, 49 (142) Proteasome pathway, 20 (47) Protein–membrane anchor, 93 (282) Proteomics, 14 (26), 28 (73), 30 (79), 51 (150), 159 (489), 160 (491), 182 (563)

238

Subject Index

PRPF6 inhibition, 179 (553) PTEN loss, 29 (75), 90 (272), 100 (304), 177 (546) PTPN11, 151 (463) Public knowledge base for actionable genes, 159 (488) Pyrido[1,2-a]pyrimidin-4-ones, 90 (272) Pyrido[2,3-b]pyrazine, 115 (356), 128 (400)

Q Quantitative textural analysis (QTA), 61 (186)

R Radioimmunotherapy (RIT), 88 (264), 145 (442) Radiosensitisation, 23 (56), 65 (202), 67 (206), 68 (208), 74 (219), 116 (361), 166 (510) Radiosurgery, 49 (141) Radiotherapy, 60 (185), 170 (525) High single dose, mechanism, 81 (241) Radium-223 dichloride, 25 (63), 167 (515) RAF/EGFR dual inhibitors, 141 (428) RAF inhibitors, 29 (76), 54 (162), 105 (324), 117 (364), 161 (493) RAF/MEK dual inhibitors, 141 (428) Ras-MAPK pathway, 57 (173) RAS mutations, 113 (350) Reactive oxygen species (ROS), 9 (9), 14 (26), 26 (64), 31 (80), 81 (241), 86 (258), 119 (371), 158 (485), 189 (588) Redox modulation, 31 (80) Regorafenib, 31 (82) Regulatory aspects, 44 (125) Renal cell carcinoma (RCC), 8 (5), 98 (299), 126 (395), 154 (473) Metastatic, see Metastatic renal cell carcinoma Resolvin D2, role in breast cancer, 58 (176) Response database, 52 (154) Response monitoring, 149 (454) Response prediction, 73 (215), 77 (230), 111 (344), 142 (433) Retinoblastoma, 162 (498) Retinoic acid, 130 (409) Reverse phase protein array profiling (RPPA), 34 (93), 81 (243), 83 (247), 102 (314), 124 (388), 150 (458), 160 (491) Rhabdomyosarcoma, 101 (311), 170 (524) Rho-associated kinase, 169 (522) RNA barcoding, 60 (184) Rucaparib, 16 (33), 73 (215), 83 (249)

S Salmonella typhimurium A1-R Chemotherapy enhancement, 96 (292), 121 (376) Effect on brain metastases of breast cancer, 111 (343) Sarcomas, 147 (450) Schlafen 11 gene, 84 (252), 134 (420) Secretome analysis, 67 (206) Selective estrogen mimic (SEM), 103 (316) Selective inhibitors of nuclear export (SINE), 83 (247), 100 (305), 160 (492) Sentinel lymph nodes, 92 (278) SF3B1 mutations, 149 (456), 186 (575) SFK inhibitors, 32 (84), 101 (311) shRNAs, 52 (153), 85 (256), 147 (447) Single Cell Network Profiling (SCNP), 39 (109) Single-nucleotide polymorphisms (SNPs), 11 (13), 50 (144), 53 (157), 72 (214) siRNAs, 21 (49), 115 (358) Small cell lung cancer (SCLC), 30 (78), 59 (178), 80 (239), 81 (242, 243), 88 (264), 144 (437, 439), 183 (565)

SNIPER(TACC), 52 (155) Sodium metaarsenite, 23 (56), 24 (60), 25 (62) Soft-tissue sarcomas, 17 (35), 110 (341), 121 (378), 134 (420), 170 (524) Sorafenib, 61 (186), 73 (217), 150 (458), 154 (473) Specimen quality, 151 (464) Splice variants, 105 (322) Spliceosome aberrations, 179 (553) Spliceosome inhibition, 149 (456) Spliceostatin A, 186 (575) Squamous cell lung cancer (SQCLC), 129 (404) Src inhibitors Effects of radiotherapy, 170 (525) SRC/mTOR dual inhibition, 98 (299) Stable isotope labeling by amino acids (SILAC), 156 (480) STAT3 inhibitors, 18 (39), 73 (217), 89 (269), 93 (282), 163 (501), 166 (510) STAT5 inhibitors, 94 (285) STAT3/NF-úB dual inhibitor, 159 (487) STAT pathway, 147 (449) Stereotactic radiosurgery, 49 (141), 66 (203) Stereotactic radiotherapy, 73 (216) Stratified Medicine Programme, 172 (529) Structure–activity relationships, 91 (274, 275, 276), 92 (279), 115 (356), 128 (400), 141 (430) Sunitinib, 12 (18), 89 (267), 127 (396), 154 (473), 167 (515), 172 (530), 176 (541) Survivin, 19 (42), 22 (53), 65 (201), 100 (305), 119 (369) Syngeneic models, 11 (14, 16), 12 (19), 26 (64), 40 (113, 114), 43 (124), 45 (128), 47 (134), 48 (137), 71 (209), 100 (307), 118 (368), 136 (424), 163 (501), 190 (589) Synthetic lethality, 35 (95), 50 (147), 51 (148), 61 (188), 71 (211), 97 (294), 109 (337), 113 (349)

T Tambjamine analogs, 15 (28) Tamoxifen, 31 (81), 35 (96), 103 (316), 142 (431) Tankyrase inhibitors, 123 (383) Target discovery, 41 (116), 56 (168), 82 (245), 120 (375), 122 (379), 124 (388), 129 (403), 143 (435), 152 (466), 153 (470), 155 (477), 156 (478), 178 (549), 180 (556), 182 (563) TDO inhibitors, 37 (104) Telomerase reverse transcriptase (hTERT) expression, 119 (369), 120 (372) Temozolomide (TMZ), 10 (12), 25 (61, 62), 81 (242), 82 (246), 94 (284), 131 (412), 135 (422), 153 (469), 166 (510), 171 (528) Temsirolimus, 61 (186), 131 (412), 165 (508) Test platforms, 12 (18), 38 (105), 40 (112), 48 (139), 49 (143), 50 (146), 54 (161), 59 (177, 178), 60 (185), 63 (194), 102 (313), 129 (403), 136 (427), 147 (447), 169 (520) TFAB2B expression, 130 (409) Therapy resistance, 21 (49), 28 (72, 73), 48 (139), 52 (153), 80 (238), 82 (244), 85 (254), 93 (280), 149 (454), 151 (463), 155 (477), 164 (505), 165 (506), 168 (516, 518), 191 (593), 197 (7LBA) Acquired, see Acquired resistance Thoracic malignancies, 13 (21), 102 (312), 176 (542) Tie-2, 33 (89) Tivantinib, 176 (542) TKI inhibitors, 32 (86) TLR agonists, 40 (114) TNF signalling, 46 (131) TNIK inhibitors, 112 (346) Topoisomerase inhibitors, 16 (33), 83 (248), 84 (252)

Subject Index Topotecan, 16 (33), 83 (248), 131 (412), 135 (422) Trabectedin, 17 (35), 21 (48), 24 (57), 190 (590) TRAIL, 21 (48), 47 (135), 118 (367), 152 (467), 162 (497), 169 (519), 186 (576) Trametinib, 22 (51), 33 (90), 35 (94), 84 (250), 105 (324), 109 (338), 113 (349), 147 (450) Transcriptional regulation, 29 (74), 84 (252), 153 (470) Transcriptional silencing, 120 (372), 179 (554) Transitional cell carcinoma (TCC), 11 (13) Translation regulatory elements (TREs), 181 (558) TRAP1, 60 (182) Trastuzumab, 27 (69), 31 (81), 36 (98), 37 (103), 155 (477), 164 (502), 166 (509) Trial assessment, 44 (125) Trial design, 96 (290), 166 (511) Trial enrollment, 3 (1) Triple negative breast cancer (TNBC), 14 (24), 20 (46), 22 (51), 31 (81), 34 (91), 36 (100), 50 (146), 80 (238), 111 (342), 128 (399), 146 (444), 162 (497), 169 (521), 179 (554), 187 (580) Adjuvant therapy, 89 (267) Aggressiveness marker, 56 (168) Triptolide, 36 (98) TRK inhibitors, 101 (310), 125 (391), 143 (436), 165 (506) Tryptophan metabolism, 37 (104), 43 (123) TumorGraft, 63 (195) Tumour-associated carbohydrate antigens (TACAs), 156 (479) Tumour-associated macrophages (TAM), 59 (179), 116 (359) Tumour characterisation, 60 (183, 184), 62 (189) Tumour-infiltrating lymphocytes, 42 (120), 43 (124), 46 (133), 47 (136), 116 (359) Tumour microenvironment, 8 (6), 17 (36), 33 (90), 42 (120), 46 (132, 133), 64 (196), 89 (266), 110 (339), 143 (435), 172 (530) Tumour spheroids, 62 (190) Tumour visualisation, 8 (6), 9 (8), 53 (158), 187 (578)

Ubiquitin–proteasome system (UPS), 52 (155), 85 (255), 181 (561), 186 (577) UFM1 pathway, 94 (283) UGT expression, 26 (65), 27 (67) Unfolded protein response (UPR), 51 (148), 161 (495), 179 (552), 186 (577) Uterine sarcomas, 11 (15), 104 (319), 152 (467)

V Vaccines, 38 (107), 45 (130), 76 (225) Vaccinia virus, 42 (121) Validation of PKPD efficacy model, 99 (301) Validation of xenograft models, 11 (14, 15) Vascular targetting, 66 (203) Vasculogenic mimicry, 88 (264) Veliparib, 80 (238), 82 (246), 173 (532) Vemurafenib, 29 (76), 105 (324), 106 (327), 123 (384), 124 (387), 132 (415), 154 (471), 164 (505), 175 (540) Vinflunine, 17 (37) Virotherapy, 22 (53), 38 (106), 41 (117, 118), 42 (121), 44 (125), 47 (136), 48 (137) Vorinostat, 35 (97), 184 (568, 570)

W WDR5–MLL interaction inhibitors, 95 (287) WEE1 inhibitors, 71 (211) Wilms tumour protein 1 (WT 1), 43 (124) Wnt signalling, 32 (85), 60 (181), 112 (346), 115 (358), 121 (377), 128 (399)

X Xenografts, 20 (46), 25 (61), 47 (135), 52 (153), 115 (355), 181 (559) patient-derived, see Patient-derived xenografts

Y YAP–TEAD interaction inhibitors, 180 (557)

U UAE inhibitors, 78 (231), 85 (255), 95 (286), 181 (561), 186 (577) UBA1, see UAE UBA5 inhibitors, 94 (283)

239

Z ZAK kinase, oncogenic isoform, 179 (553) Zalypsis, 24 (57) Zfra, 46 (131)