nd eacr conference radiation break-through a5... · radiation (2) session chair: martin pruschy...

Programme Book

12 - 14 March 2018Oxford, UK

Scientific Programme CommitteeVerena Jendrossek || Martin Pruschy || Madalena Tarsounas

2nd EACR Conference

Radiation Break-through from DNA damage responses to precision

cancer therapy

ConferenceSeries

European Association for Cancer Research

www.eacr.org

ConferenceSeries


www.eacr.org

25th Biennial Congress of the European Association for Cancer ResearchFrom Fundamental Insight to Rational Cancer Treatment

EACR 2530 June - 03 July 2018 Amsterdam

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051968 - 2018YEARS

for this landmark congress celebrating 50 years of the EACRwww.eacr25.org #EACR25

Radiation Break-through: from DNA damage responses to precision cancer therapyOxford, UK | 12 - 14 March 2018

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12 - 14 March 2018 • Oxford, UK

Radiation Break-throughFrom DNA damage responses to precision cancer therapy

Day 1 - Monday 12 March

11.30 – 13.30 REGISTRATION Wolfson College foyer Tea/coffee will be available in the Haldane Room

12.00 – 13.30 LIGHT LUNCH Haldane Room

13.30 – 13.35 CONFERENCE WELCOME Leonard Wolfson Auditorium Scientific Programme Committee

SESSION 1: DDR & DNA REPAIR Session Chair: Jos Jonkers

13.35 – 14.05 OPENING LECTURE Q&A: 14.05 – 14.20 Steve Jackson University of Cambridge, UK ”Cellular responses to DNA damage: mechanistic insights and therapeutic applications”

14.20 – 14.40 Alessandro A. Sartori University of Zurich, SwitzerlandQ&A: 14.40 – 14.50 “Exploiting cell-penetrating peptide inhibitors of homologous recombination as research tools and as novel cancer therapeutics”

14.50 – 15.00 Sylvie Noordermeer Leiden University Medical CentreQ&A: 15.00 – 15.05 Netherlands Proffered Paper 1: ”Identification of the Shieldin complex as a novel effector of 53BP1-dependent DNA repair”

15.05 – 15.25 Karlene Cimprich Stanford University School of Medicine, USAQ&A: 15.25 – 15.35 “ATR couples DNA replication and mitosis through an S/G2 phosphorylation switch and transcription program”

15.35 – 16.05 COFFEE BREAK Hall


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16.05 – 16.15 Anton Gartner University of Dundee, UKQ&A: 16.15 – 16.20 Proffered Paper 2: ”Massive C. elegans whole-genome sequencing for profiling mutational signatures associated with IR, carcinogens and DNA repair deficiency”

16.20 – 16.40 Madalena Tarsounas CRUK/MRC Oxford Institute for Radiation Q&A: 16.40 – 16.50 Oncology, UK ”A pharmacological screen identifies drugs that selectively target BRCA1/2-deficiency and counteract drug resistance”

16.50 – 17.10 Christian Reinhardt University Hospital Cologne, Germany Q&A: 17.10 – 17.20 “Targeting defects in the DNA damage response to treat tumors driven by non-druggable oncogenes”

17.20 – 17.30 Nina Gustafsson Karolinska Institute, SwedenQ&A: 17.30 – 17.35 Proffered Paper 3: ”Protecting the genome through metabolic enzymes”

17.35 – 17.45 EXHIBITOR INTRODUCTIONS

17.45 – 18.45 WELCOME BUFFET & DRINKS Haldane Room & Hall Opening of the trade exhibition

18.45 – 20.45 POSTER DEFENCE SESSION 1 Odd numbered posters (1, 3, 5, etc.) will be presented

Day 2 - Tuesday 13 March

08.30 – 09.00 POSTER VIEWING Haldane Room & Hall Tea/coffee will be available

SESSION 2: BIOMARKERS & TISSUE RESPONSES TO RADIATION (1) Session Chair: Karlene Cimprich

09.00 – 09.20 Jos Jonkers NKI, Netherlands Q&A: 09.20 – 09.30 “Identification of cancer drivers and mechanisms of therapy resistance in mouse models of BRCA-deficient breast cancer”

09.30 – 09.50 Kirsten Lauber Ludwig-Maximilians-Universität München, Q&A: 09.50 – 10.00 Germany “Dying cell clearance and its impact on the outcome of cancer radiotherapy”


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10.00 – 10.10 Flavie Coquel Institut de Génétique Humaine, FranceQ&A: 10.10 – 10.15 Proffered Paper 4: ”SAMHD1 acts at stalled replication forks to prevent ssDNA-mediated induction of type I interferons”

10.15 – 10.35 Ludwig Dubois Maastricht University, NetherlandsQ&A: 10.35 – 10.45 ”Radiotherapy and immunotherapy: partners in crime”


11.15 – 11.25 Jason Parsons University of Liverpool, UKQ&A: 11.25 – 11.30 Proffered Paper 5: ”Complex DNA damage induced by high- LET protons triggers a specific cellular DNA damage response”

11.30 – 11.50 Verena Jendrossek University of Duisburg-Essen, GermanyQ&A: 11.50 – 12.00 “Radiation-induced normal tissue toxicity: new insight from damage signaling and tissue-specific immune defense strategies”

12.00 – 13.30 SEATED LUNCH Haldane Room & Buttery

SESSION 3: BIOMARKERS & TISSUE RESPONSES TO RADIATION (2) Session Chair: Martin Pruschy

13.30 – 13.50 Catharine West University of Manchester, UKQ&A: 13.50 – 14.00 “Radiobiology Biomarkers for Precision Radiotherapy”

14.00 – 14.20 Anderson Ryan University of Oxford, UK Q&A: 14.20– 14.30 “The impact of DNA damage response (DDR) modulators on normal tissues following thoracic irradiation in preclinical models”

14.30 – 14.40 Domenico Maiorano Institut de Génétique Humaine, FranceQ&A: 14.40 – 14.45 Proffered Paper 6: “Targeting the RAD18 ubiquitin ligase enhances sensitivity to therapy by inhibiting cancer stem cells proliferation”

14.45 – 15.05 Andrew Tutt The Institute of Cancer Research (ICR), UKQ&A: 15.05 – 15.15 “Understanding genomic instability and targeting defective DNA damage responses in breast cancer”


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15.15 – 16.00 MEET THE EXPERTS SESSION with Madalena Tarsounas, Verena Jendrossek and Karlene Cimprich

16.00 – 18.00 POSTER DEFENCE SESSION 2 Haldane Room & Hall Even numbered posters (2, 4, 6, etc.) will be presented

19.00 CONFERENCE DINNER Cherwell Boathouse Pre-booked optional extra

Day 3 - Wednesday 14 March

08.30 – 09.00 POSTER VIEWING Haldane Room & Hall Tea/coffee will be available

SESSION 4: TRANSLATIONAL Session Chair: Madalena Tarsounas

09.00 – 09.20 Timothy Humphrey CRUK/MRC Oxford Institute for Radiation Q&A: 09.20 – 09.30 Oncology, UK “Targeting histone H3K36me3-deficient cancers”

09.30 – 09.50 Frank Pajonk UCLA, USA Q&A: 09.50 – 10.00 “Cancer Therapies and Cancer Cell Plasticity”

10.00 – 10.10 Olivier De Wever Ghent University, BelgiumQ&A: 10.10 – 10.15 Proffered Paper 7: “Radiotherapy-activated cancer- associated fibroblasts promote tumor progression through paracrine IGF-1R activation”


11.00 – 11.20 Brita Singers Sorensen Aarhus University, DenmarkQ&A: 11.20 – 11.30 “Implementation and Validation of a 15-Gene Hypoxia Classifier in Head and Neck Cancer”

11.30 – 11.50 Martin Pruschy University Hospital Zürich, SwitzerlandQ&A: 11.50 – 12.00 “Targeting the secretome as part of combined treatment modalities with radiotherapy”


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Congratulations to the winners of the EACR-Worldwide Cancer Research Meeting Bursaries. Each winner received a full registration free of charge and funds of up to 500 Euros to assist with the cost of travel and accommodation.

EACR-Worldwide Cancer Research Meeting Bursary Award winners

Klaudia Al-Refae Germany

Julia Ketteler Germany

Cristina Espinosa-Diez USA

Patrícia Pereira USA

Purba Nag Australia

Amy Buckley Ireland

12.00 – 12.30 CLOSING LECTURE Q&A: 12.30 – 12.45 Amato Giaccia Stanford University School of Medicine, USA “Protecting normal tissue from DNA damage to enhance the efficacy of radiotherapy”

12.45 – 13.00 CLOSING SUMMARY AND PRESENTATION OF AWARDS Verena Jendrossek

13.00 LUNCH & DEPART Haldane Room

STEP UPTAKE YOUR RESEARCH TO THE NEXT LEVEL WITH CANCER RESEARCH UKOur project and programme awards offer funding across the entire research pipeline: from discovery science to drug discovery.

We also offer flexible fellowships and support for the brightest scientists and clinicians throughout their career.

VISIT OUR STAND TO FIND OUT MORE

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E4286 CRUK Research STEP UP Advert A5.indd 1 14/02/2018 14:05


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Meet the Exhibitors

AMSBIOWebsite: www.amsbio.comContact: 01235 828 200Represented at the conference by: Maja Petkovic

Describe AMSBIO in 5 words or lessAccelerating discovery through innovative life science

Tell us a little bit about AMSBIOFounded in 1987, AMS Biotechnology (AMSBIO) is recognised today as a leading company contributing to the acceleration of discovery through the provision of cutting-edge life science technology products and services for research and development in the medical, nutrition, cosmetics and energy industries.

Why are you attending the conference? Who would you like to meet at the conference?This EACR conference is a great opportunity for us to meet researchers working in DNA damage research and showcase our products that would drive this research forward. We would like to meet scientist working in basic and translational oncology research.

EnvigoWebsite: www.envigo.comContact: www.envigo.com/contactusRepresented at the conference by: Travis Rothrock, Mel Read, John Kennett

Describe Envigo in 5 words or lessYour Oncology Development Partner

Tell us a little bit about EnvigoEnvigo provides essential products and research services for pharmaceutical, crop protection, and chemical companies as well as universities, governments, and other research organizations. Envigo is committed to helping customers realize the full potential of their products and research.

Why are you attending the conference? Who would you like to meet at the conference?Envigo partners with customers to support their oncology research programs. Our team wants to meet oncology researchers and laboratory management staff involved in discovery and preclinical research programs.


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RPS Service LTDWebsite: www.rpsservice.co.ukContact: [email protected] Represented at the conference by: Robert Schmid

Describe RPS Service in 5 words or lessProfessional, Friendly, Efficient & Quick

Tell us a little bit about RPS ServiceRPS Service Ltd specialise in the sales and service of x-ray and gamma irradiators with more than 25 years’ experience in the market. RPS Service is the authorised distributor for Faxitron irradiators for the UK, Ireland and European markets.

XstrahlWebsite: www.xstrahl.comContact: [email protected] or +44 (0)1276 462696Represented at the conference by: Dr Andrew Lessey and Sara Chiblak

Describe Xstrahl in 5 words or lessAdvanced Preclinical X-ray Irradiators

Tell us a little bit about XstrahlXstrahl is a leading supplier of cabinet irradiators and advanced image-guided micro irradiators for preclinical research. Working with selected partners, Xstrahl is striving to improve the accuracy of preclinical dosimetry and are the undoubted experts within the field.

Why are you attending the conference? Who would you like to meet at the conference? As a leading supplier of preclinical X-ray irradiators for radiation research, Xstrahl are looking forward to catching up with our current users and meeting anyone interested in talking to us about their irradiation requirements. Come and see what we can do for you…

The conference will give plenty of opportunities for you to meet representatives from companies working in the same field.

To speak to our exhibitors and find out more about them, please visit the stands in the trade exhibition.


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Cancer Research UKWebsite: www.cancerresearchuk.org/our-researchContact: Gemma [email protected] at the conference by: Curtis Asante

Describe Cancer Research UK in 5 words or lessCutting-edge, innovative, pioneering

Tell us a little bit about Cancer Research UKCancer Research UK (CRUK) is the largest independent funder of cancer research in Europe and the world’s leading charity dedicated to cancer research. Our vision is to bring forward the day when all cancers are cured.

Why are you attending the conference? Who would you like to meet at the conference?We support many critical areas of translational research, including radiotherapy research. The CRUK/MRC Oxford Institute for Radiation Oncology is the world’s largest and most comprehensive centre for radiation oncology and biology. We want to speak to anyone who’d like to hear more about our research strategy or funding opportunities.

Register now...

Barcelona, Spain28 - 29 May 2018

LIF As We Know It: From Basic Science to Clinical Trials

Embryonic stem cellsImmunomodulation

Therapeutic target in cancerDrug development

TOPICSReduced registration rates and Meeting Bursary Awards for EACR members

www.eacr.org/conference/lif2018


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The organisers wish to express their appreciation for the support provided by sponsors at the EACR Conference Radiation Break-through: from DNA damage responses to precision cancer therapy. Their interest and enthusiasm for the conference has enabled the organisers to provide an impressive scientific programme.

Exhibitors

Partners and Sponsors

Media Partner


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Grants

EACR Industry Partners

The European Association for Cancer Research gratefully acknowledges the organisations that support the Association as Industry Partners. Industry Partners offer ongoing support to the EACR and provide the means for the Association to develop important initiatives. The EACR Conference Series is an example of this.


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Interactive activities at the ConferenceAn important part of the EACR Conference Series is the range of opportunities we aim to provide for participants to interact, discuss, reflect and build relationships and collaborations.

We hope you enjoy the dedicated interactive activities, which are listed below.

Welcome Reception

17.45-18.45 Monday 12 March

A complimentary buffet dinner and drinks will be served for all participants and exhibitors to enjoy on Monday evening. The trade exhibition will be open at this time, and there is an opportunity to meet new colleagues and reconnect with friends.

Poster Defence Sessions

18.45 – 20.45 Monday 12 March Odd numbered posters (1, 3, 5, etc.) will be presented16.00 – 18.00 Tuesday 13 March Even numbered posters (2, 4, 6, etc.) will be presented

There are two dedicated Poster Defence Sessions in the programme. At these times, the presenters for that session are asked to stand by their posters to discuss their work with other participants and invited speakers.

Two EACR Poster Prizes worth €100 each will be awarded to the best poster presentations at the conference. The judging panel is comprised of speakers from the conference, and they will assess the top scoring abstracts based on the scientific content, the layout of the poster, and the verbal discussion. The winners will be announced during the Closing Summary on Wednesday.

Poster Viewing

08.30 – 09.00 Tuesday 13 March 08.30 – 09.00 Wednesday 14 March

Tuesday and Wednesday will begin with an optional poster viewing slot. Participants are invited to use this time for further discussion in the poster areas, but presenters are not required to be by their posters at these sessions. Coffee and tea will be available.


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Meet the Experts Session15.15 - 16.00 Tuesday 13 March

In this session Karlene, Madalena and Verena will share their personal and professional perspective on the path to scientific leadership and the importance of maintaining the family-work balance. This session will be a forum for open discussion.

Professor Karlene Cimprich, from Stanford University (US) has uncovered essential functions of ATR kinase in DNA replication. More recently her work focused on the impact of resolution of transcription/replication conflicts on genome integrity.

Prof. Madalena Tarsounas from University of Oxford (UK) and Member of the Scientific Programme Committee, has made important contributions to our understanding of the roles of BRCA1/BRCA2 tumour suppresors in telomere and genome integrity. Her recent work is focused on strategies for targeting BRCA1/2-deficient cells and tumours.

Prof. Verena Jendrossek from University of Duisburg-Essen (Germany) and Member of the Scientific Programme Committee, investigates biological approaches to improve the outcome of radiotherapy, with a particular focus on mechanisms of intrinsic and microenvironment-mediated radiation resistance and of normal tissue toxicity.

Conference Dinner

19.00 – 22.00 Tuesday 13 March

The conference dinner is an excellent opportunity for participants and speakers to get to know each other in a relaxed and informal environment. The dinner will take place at the Cherwell Boathouse.

Tickets for the conference dinner are not included in the registration fee and can be selected as an optional extra for £50. Tickets must be purchased in advance.

Don’t forget to let us have your feedback about these activities in the survey we will send after the conference!


16worldwidecancerresearch.org

Worldwide Cancer Research is a charity registered in Scotland, No: SC022918

From the world’s best research institutions and renowned specialists to unexpected and diverse projects by up and coming talent. We fund all types of research and for one very good reason – to gain a global perspective. Because research doesn’t happen in isolation. And the answers will not come from one scientist, in one lab, in one country. That’s why Worldwide Cancer Research are prepared for whatever it takes and wherever it takes us.

We fund research into any type of cancer anywhere in the world.


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Speaker abstracts

Speaker abstracts

Cellular responses to DNA damage: mechanistic insights and therapeutic applications

Stephen P. Jackson1

1 The Gurdon Institute and Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge, UK

At rest, each human cell sustains tens-of-thousands of DNA lesions per day. Furthermore, additional DNA damage can be induced by agents such as ionizing radiation, chemotherapeutic drugs and environmental chemicals. To combat such threats to genome stability, our cells have evolved elaborate ways to detect, signal the presence of and repair DNA damage. The importance of such processes is highlighted by inherited or acquired defects in them causing various human pathologies, including immune-deficiencies, neurodegenerative diseases, premature ageing and cancer.

Much work in my laboratory aims to decipher mechanisms of DNA repair and also to determine how these mechanisms impact on, and are influenced by, myriad aspects of cellular physiology. In this talk, I will explain how cells respond to highly toxic DNA double-strand breaks. I will then briefly review how research by my group and others has contributed to development of the drug, olaparib/LynparzaTM, which inhibits the DNA-repair enzyme PARP. In addition to sensitising cells to ionizing radiation and certain chemotherapeutic agents, olaparib and other PARP inhibitors exhibit striking cytotoxicity towards cancer cells deficient in BRCA1, BRCA2, or certain other DNA-repair genes. In my talk, I will explain how we are searching for additional DNA-repair vulnerabilities in cancer cells and how we are defining mechanisms by which cancers can evolve resistance to PARP inhibitors and other molecularly-targeted agents. Finally, I will explain how such knowledge is not only providing us with fundamental insights into DNA-repair mechanisms but is also identifying new opportunities that might be exploited clinically.


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Speaker abstracts

Exploiting cell-penetrating peptide inhibitors of homologous recombination as research tools and as novel cancer therapeutics

Alessandro Sartori11 Institute of Molecular Cancer Research, University of Zurich, Zurich, SWITZERLAND

Cancer cells rely on efficient DNA repair and replication fork protection mechanisms to survive and proliferate. By virtue of its ability to promote RAD51 loading onto single-stranded DNA (ssDNA), the human BRCA2 tumour suppressor protein is essential for the homology-directed of DNA double-strand breaks (DSBs) and for maintaining fork stability. Therefore, blocking the direct physical interaction between BRCA2 and RAD51 represents an attractive strategy for the development of novel therapeutic drugs, in particular when used in combination with DNA-damaging agents. Targeting protein-protein interaction (PPI) interfaces has proven challenging because flat and large PPI surfaces generally do not support binding of small molecule inhibitors (SMIs). Instead, peptide therapeutics offer an alternative way to target PPIs with key advantages over SMIs, including their direct similarity to protein fragments and the coverage of extensive PPI interfaces. Moreover, the modification of otherwise membrane-impermeable cargo molecules with cell-penetrating peptides (CPPs) has recently, expanded the use peptide-based inhibitors to intracellular targets.

In my presentation, I will discuss our most recent work on the rational design and functional analysis of a small BRCA2-derived peptide that is capable of inhibiting BRCA2-RAD51 interaction. Remarkably, we find that efficient non-cytotoxic uptake of the BRCA2 peptide prevents RAD51 loading onto ssDNA, resulting in defective homology-mediated repair of DSBs as well as increased nucleolytic degradation of stalled replication forks. Consequently, peptide incubation rendered cells hypersensitive to the PARP inhibitor olaparib, providing a potential use for BRCA2 targeting peptides in the treatment of certain types of cancer.


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Speaker abstracts

Proffered Paper 1

Identification of the Shieldin complex as a novel effector of 53BP1-dependent DNA repair

Sylvie Noordermeer1,2, Salomé Adam2, Dheva Setiaputra2, Marco Barazas3, Stephen Pettitt4, Alexanda Ling7, Michele Olivieri2, Alejandro Álvarez-Quilón2, Nathalie Moatti2, Michal Zimmermann2, Stefano Annunziato3, Dragomir Krastev4, Feifei Song4, Inger Brandsma4, Jessica Frankum4, Rachel Brough4, Alana Sherker2, Sébastien Landry2, Rachel Szilard2, Meagan Munro2, Andrea McEwan2, Théo Goullet de Rugy2, Zhen-Yuan Lin2, Traver Hart6, Jason Moffat7, Anne-Claude Gingras2, Alberto Martin7, Jos Jonkers3, Christopher Lord4, Sven Rottenberg3,5, Haico van Attikum1, Daniel Durocher2

1 Leiden University Medical Centre, Leiden, NETHERLANDS, 2 Lunenfeld-Tanenbaum Research Institute, Toronto, ON, CANADA, 3 Netherlands Cancer Institute, Amsterdam, NETHERLANDS, 4 The Institute of Cancer Research, London, UK, 5 University of Bern, Bern, SWITZERLAND, 6 University of Texas MD Anderson Cancer Center, Houston, TX, USA, 7 University of Toronto, Toronto, ON, CANADA

The 53BP1-RIF1 axis plays an important role in the repair of DNA double strand breaks (DSBs) by stimulating non-homologous end joining (NHEJ) and inhibiting the DNA end resection step of homologous recombination (HR). However, it remains unclear how 53BP1-RIF1 regulate this pathway choice during DSB repair.

In an attempt to better understand the activity of the 53BP1 pathway, we performed genome-wide CRISPR/Cas9 screens to identify novel genes whose mutation leads to PARP inhibitor (PARPi) resistance in BRCA1-deficient cells, a phenotype linked to concomitant loss of 53BP1 and its known downstream factors. This led to the identification of C20orf196, which we renamed Shieldin 1 (SHLD1). Affinity purification mass spectrometry experiments suggested that SHLD1 is part of a protein complex together with FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7, a known downstream factor of 53BP1. Loss of any of the complex members resulted in PARPi resistance in BRCA1-deficient cells by re-activation of HR, and in inhibition of NHEJ and class switch recombination.

Recruitment of the Shieldin complex to DSBs was dependent on 53BP1 and RIF1 and was mediated by SHLD3. Importantly, SHLD2 contains three OB-fold domains similar to those in the telomere binding protein POT1 and the single strand DNA binding protein RPA1. Mutations in these OB-fold domains were detrimental for the function of the Shieldin complex in HR inhibition. Furthermore, artificially targeting SHLD2 to DSBs in a 53BP1-independent way showed that this complex can inhibit DNA end resection in the absence of 53BP1.

Our data suggest that the Shieldin complex is the ultimate downstream effector of 53BP1 in DSB repair by protecting broken DNA ends against resection and thereby stimulating NHEJ. Clinically, it will be important to study whether mutations in the Shieldin complex are responsible for PARPi resistance in BRCA1-mutated tumours, a phenotype we observed in mouse tumour models.


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Speaker abstracts

ATR couples DNA replication and mitosis through an S/G2 phosphorylation switch and transcription program

Joshua Saldivar1, Michael Bocek1, Stephan Hamperl1, Mingyu Chung1, Tobias Meyer1, Karlene Cimprich1

1 Stanford University, Stanford, CA, USA

The cell cycle phases are strictly ordered to ensure faithful genome duplication and chromosome segregation. Underlying this order are control mechanisms that dictate when a cell transitions from one phase to the next. Much is known about control of the G1/S, G2/M, and metaphase/anaphase transitions, but there is no known control mechanism for the S/G2 transition. Here, we show that at the end of S phase, cells transactivate the mitotic gene network through a CDK-directed FOXM1 phosphorylation switch. Intrinsic activation of the checkpoint kinase ATR by ongoing DNA replication blocks this switch until the end of S phase. Deregulation of this S/G2 transition leads to premature mitotic entry with under-replicated DNA and subsequent DNA damage. Thus, ATR couples DNA replication to mitosis and preserves genome integrity by controlling the S/G2 phosphorylation switch and G2/M transcription program.


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Speaker abstracts

Proffered Paper 2

Massive C. elegans whole-genome sequencing for profiling mutational signatures associated with IR, carcinogens and DNA repair deficiency

B Meier2, N Volkova5, V Gonzalez-Huici2, S Bertolini2, Y Hong2, B Wang2, SL Cooke1, J Weiss2, PJ Campbell1,4, M Gerstung5, A Gartner2

1 Cancer Genome Project, Wellcome Trust Sanger Institute, Hinxton, UK, 2 Centre for Gene Regulation and Expression, University of Dundee, Dundee, UK, 3 Department of Haematology, Addenbrooke’s Hospital , Cambridge, UK, 4 Department of Haematology, University of Cambridge, Cambridge, UK, 5 European Bioinformatics Institute (EMBL-EBI), Hinxton, UK

While ionizing radiation (IR) is used in the clinic for more than 100 years and accidental expose is a major concern, very little is known about the direct effect on ionizing radiation on the number and nature of mutations induced.

We developed C. elegans whole genome sequencing to investigate genetic and environmental contributions to mutational signatures. To date we have sequenced over 3000 genomes of wild-type and ~40 C. elegans DNA damage response mutants grown over 20-40 generations or following exposure to a variety of DNA damaging agents, including IR, UV, alkylating agents and DNA cross-linking agents.

Mutation accumulation over generations showed that we can recapitulate mutational signatures occurring in cancer cells. Chromosome fusions caused by telomere erosion led to complex chromosomal rearrangements initiated by breakage-fusion-bridge cycles and completed by simultaneously acquired, localized clusters of breakpoints, a process akin to chromotripsis and comparable to genome rearrangements in lymphoblastic leukemia (1, 2). Analysis of mutation spectra of C. elegans mismatch repair mutants allowed to define one highly conserved MMR signature prominent in colorectal and stomach cancers (3). Hypermutation is observed when defects in Polε replicative polymerase are combined with MMR deficiency (3) as in childhood cancers (4,5).

Upon IR treatment increased base substitutions occur with increasing doses. Interestingly, a subset of base substitutions in nucleotide excision repair mutants encompasses dinucleotide substitutions, previously also observed following cisplatin exposure (1). We do not observe a clustering of point mutations. An increased number of structural variants, whose nature we are currently investigating, occur in a number of mutants such as him-6/BLM and fanci-1/FANCI. Mutagenesis upon IR differs from mutational signatures induced by alkylating agents methyl methanesulfonate (MMS), dimethyl sulphate (DMS), and ethyl methane-sulfonate (EMS) the latter leading to distinct mutational profiles modified in translesion synthesis polymerase mutants and mutants of the O6-methylguanine methyltransferase.


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Speaker abstracts

A pharmacological screen identifies drugs that selectively target BRCA1/2-deficiency and counteract drug resistance

Sophie Badie1, Eliana Tacconi1, Cecilia Folio1, Xianning Lai1, Giuliana de Grigoriis1, Simon Dedic1, Jonathan Lee1, John Moore3, Anderson Ryan3, Arnaud Kopp2, Laurent Brino2, Manuela Porru4, Anna Maria Biroccio4, Madalena Tarsounas1

1 Genome Stability and Tumourigenesis Group, CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK, 2 Institut de Génétique et de Biologie Cellulaire et Moléculaire (IGBMC), Université de Strasbourg, Illkirch, FRANCE, 3 Lung Cancer Translational Science Research Group, CR-UK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK, 4 Regina Elena National Cancer Institute, Rome, ITALY

Homologous recombination repair is compromised in tumours carrying BRCA1 or BRCA2 inactivating mutations, which accumulate DNA double-strand breaks and genomic rearrangements. Remarkably, these defects can be exploited in the clinic by developing therapeutic strategies that target specifically cancer cells, with PARP inhibitors as a prominent example. However, most tumours acquire resistance to such therapies and therefore novel strategies for their elimination are needed. Here we report a comprehensive screening approach that enabled us to identify drugs with specific toxicity against BRCA1/2-deficient cells and tumours, including those that have acquired olaparib or cisplatin resistance. Mechanistically, treatment with these drugs caused replication stress and DNA double-strand break accumulation, which triggered apoptosis specifically in BRCA1/2-deficient cells. These findings support the concept that cells lacking BRCA1 or BRCA2 activity are particularly vulnerable to drugs that exacerbate the cell-intrinsic challenges that arise during replication. For some of these drugs, the cellular effects translated remarkably well to the pre-clinical setting. Our results are therefore highly relevant to the search for treatments that selectively target BRCA1/2-deficient tumours resistant to conventional therapies.


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Speaker abstracts

Targeting defects in the DNA damage response to treat tumors driven by non-druggable oncogenes

Christian Reinhardt1, Gero Knittel1, Arina Riabinska1, Anna Schmitt1, Felix Dietlein1

1 University Hospital Cologne, Cologne, GERMANY

In response to genotoxic stress, cells activate a complex, kinase-driven signaling network to arrest the cell cycle, recruit the DNA repair machinery, or, if the damage is beyond repair capacity, initiate cell death. This signaling network is collectively referred to as the DNA damage response (DDR). Genes encoding for components of this network are among the most frequently mutated genes in human cancer. Defects in these genes promote genome destabilization through defective DNA repair, leading to a mutator phenotype. Furthermore, alterations within the DDR network can promote resistance against apoptosis. However, these genomic aberrations also offer therapeutic opportunities, as the mutations clearly distinguish healthy tissue from cancerous cells. We employed pharmaco-genomic screens, in order to uncover molecular liabilities associated with cancer-specific defects in the DDR. We could show that ATM-defective cells and tumors display resistance against genotoxic chemotherapies. However, these cells also display an actionable sensitivity against PARP1 and DNA-PKcs inhibitors. We validated these observations in vivo and have translated our findings in the context of a clinical trial in CLL patients carrying genomically hard-wired ATM-defects. In a further line of experimentation, we could show that KRAS-mutant human tumors display endogenous genotoxic stress, likely mediated through replication stress. Using pharmaco-genomic screening, we could unravel a molecular dependence of KRAS-mutant cells and tumors on the activity of the checkpoint kinases CHK1 and MK2. Mechanistically, we could show that simultaneous inhibition of these kinases leads to the induction of mitotic catastrophe in KRAS-mutant cells. We also validated this observation in three distinct autochthonous mouse models and are currently recruiting patients into a clinical trial to verify our observations in the clinical arena. Thus, overall, we could demonstrate that mutations within the DDR network offer ample opportunity for therapeutic intervention.


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Speaker abstracts

Proffered Paper 3

Protecting the genome through metabolic enzymes

Nina Gustafsson3,2, Thomas Lundbäck3,1, Katarina Färnegård5,2, Petra Groth3, Nadilly Bonagas3, Elisee Wiita3, Mattias Jönsson2, Rosa Pennisi4, Anna Huguet Ninou 3,2, Carina Norström 2, Johan Schultz2, Petra Marttila3, Martin Norin2, Thomas Olin2, Thomas Helleday3

1 AstraZeneca R&D, Mölndal, SWEDEN, 2 Kancera AB, Stockholm, SWEDEN, 3 Karolinska Institutet, Stockholm, SWEDEN, 4 Roma Tre University, Rome, ITALY, 5 Stockholm University, Stockholm, SWEDEN

The glycolytic PFKFB3 enzyme is widely overexpressed in cancer and a potential target for anti-cancer therapy. Here we report that PFKFB3 is required for homologous repair (HR) of DNA double strand breaks (DSBs) caused by radiotherapy in cancer cells. PFKFB3 rapidly relocates into ionizing radiation (IR)-induced nuclear foci in an ATM-γH2AX-MDC1-dependent manner. PFKFB3 supports HR through recruitment of the homologous repair (HR) factors and its knockdown suppresses HR activity and delays recovery from IR-induced cell cycle arrest. We further develop a potent PFKFB3 inhibitor, KAN0438757, which reveal a phenotype identical to the PFKFB3 knockdown phenotype. Inhibition of PFKFB3 enzymatic activity by KAN0438757 selectively inhibits proliferation of cancer cells while sparing normal cells and impairs IR-induced nucleotide incorporation in the G2/M phase of the cell cycle. In line with a role for PFKFB3 in supporting localized dNTP supply, KAN0438757 blocks replication fork progression that is restored by nucleoside supplementation. In conclusion, we demonstrate a regulatory role for nuclear PFKFB3 enzymatic activity in HR selectively in cancer cells.


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Speaker abstracts

Identification of cancer drivers and mechanisms of therapy resistance in mouse models of BRCA-deficient breast cancer

Jos Jonkers1

1 Netherlands Cancer Institute, Amsterdam, NETHERLANDS

Heterozygous germline mutations in BRCA1 or BRCA2 strongly predispose to development of breast and ovarian cancer (as well as other cancer types) via loss of the remaining wildtype allele. BRCA1/2-deficient cancers are defective in DNA double-strand break (DSB) repair via homologous recombination (HR) and therefore hypersensitive to DNA-damaging agents, including platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors. However, these treatments do not result in tumor eradication and eventually resistance develops.

To study therapy response and resistance in a realistic in vivo setting, we have established several genetically engineered mouse models (GEMMs) and patient-derived tumor xenograft (PDX) models for BRCA-deficient breast cancer. These mice develop mammary tumors that are characterized by genomic instability and hypersensitivity to DNA-damaging agents, including platinum drugs and PARP inhibitors (PARPi). Using cross-species oncogenomics and reverse genetics, we have identified several cancer genes including p53, MYC and RB as critical drivers in BRCA1-associated breast cancer. In addition, we have used these mammary tumor models for preclinical evaluation of therapy response and elucidation of mechanisms of acquired drug resistance. Using functional genetic screens, reverse genetics and genomic analysis of therapy-resistant tumors, we found that therapy response and resistance of BRCA1-deficient mammary tumors to cisplatin and PARPi is affected by several factors, including drug efflux transporter activity, type of BRCA1 founder mutation and restoration of HR repair via loss of factors that block DSB end resection in the absence of BRCA1. Also BRCA1 re-activation via genetic or epigenetic mechanisms contributes to therapy resistance in PDX models of BRCA1-deficient breast cancer. Importantly, pharmacokinetic or HR-related mechanisms underlie PARPi-resistance in only a fraction of BRCA1/2-deficient mammary tumors, indicating the existence of additional, unknown resistance mechanisms. One such mechanism involves the loss of poly(ADP-ribose) glycohydrolase (PARG), the major enzyme responsible for the catabolism of poly (ADP-ribose).


26

Speaker abstracts

Dying cell clearance and its impact on the outcome of cancer radiotherapyKirsten Lauber1

1 Ludwig-Maximilians-Universität München, Germany

Abstract not available at the time of printing.


27

Speaker abstracts

Proffered Paper 4

SAMHD1 acts at stalled replication forks to prevent ssDNA-mediated induction of type I interferons

Flavie Coquel3, Maria-Joao Silva3, Hervé Técher5, Karina Zadorozhny1, Sushma Sharma1, Jadwiga Nieminuszczy1, Antoine Barthe3, Alexandra Cribier3, Wojciech Niedzwiedz2, Vincenzo Costanzo5, Lumir Krejci1, Monsef Benkirane3, Andrei Chabes4, Yea-Lih Lin3, Philippe Pasero3

1 Department of Biology and National Centre for Biomolecular Research, Brno, CZECH REPUBLIC, 2 Institute of Cancer Research, Division of Cancer Biology, London, UK, 3 Institute of Human Genetics, Montpellier, FRANCE, 4 Laboratory for Molecular Infection Medicine, Umeå , SWEDEN, 5 The FIRC Institute of Molecular Oncology, Milan, ITALY

SAMHD1 is a triphosphohydrolase and nuclease that protects quiescent cells from HIV-1 and other viruses by depleting intracellular dNTP pools and/or by degrading viral genomes. Mutations in SAMHD1 are implicated in inflammatory disease and cancer. Indeed, inherited SAMHD1 deficiency causes Aicardi-Goutières Syndrome (AGS) cases, a severe congenital autoimmune disease. Likewise, acquired SAMHD1 mutations are linked to chronic lymphatic leukemia and colon cancers. More precisely, studies highlight that SAMHD1 plays a role in genome instability and in intracellular nucleotide metabolism. Yet, molecular mechanisms linking SAMHD1 deficiency, inflammatory disease and cancer are lacking.

We show that SAMHD1 prevents the accumulation of DNA fragments in the cytosol and thus prevents induction of type I interferons, especially when cells are exposed to DNA replication stress. SAMHD1 executes this function by degrading newly replicated DNA at stalled forks and so preventing the release of ssDNA fragments into the cytosol. This resection activity of SAMHD1, which is distinct from its dNTPase activity, is also important to trigger the intra-S phase checkpoint and promote fork restart. Together, these data indicate that SAMHD1 is an important player in the DNA replication stress response that prevents the production of type I interferons by limiting the accumulation of nascent ssDNA fragments in the cytosol.


28

Speaker abstracts

Radiotherapy and immunotherapy: partners in crime

Ludwig J. Dubois1, Veronica Olivo Pimentel1, Damienne Marcus1, Alexander van der Wiel1, Natasja G. Lieuwes1, Rianne Biemans1, Jan Theys1, Ala Yaromina1, Philippe Lambin1

1 Department of Radiotherapy, GROW - School for Oncology and Developmental Biology, Maastricht Comprehensive Cancer Centre, Maastricht University Medical Centre, NETHERLANDS

Radiotherapy (RT) is one of the current standard of care treatments for cancer. It induces DNA damage lethal to cancer cells, but also immunogenic cell death by releasing tumour associated-antigens and damage-associated molecular patterns, thus enhancing immunity against cancer. Cancer immunotherapy reaches its height and immune cytokines as well as checkpoint inhibitors show a lot of promise. However, results are neither unambiguous nor optimal yet. Within our group, we have shown that the addition of the immunocytokine L19-IL2, specifically delivering IL2 to the tumour by interaction of the L19 antibody fragment with ectodomain-B (ED-B) expressed at the tumour neovasculature, to RT (single dose 10Gy) resulted in 75% of tumor remission in a heterotopic of C51 murine colon carcinoma model (Rekers et al, Clin Cancer Res 2015). The success rate of this treatment was shown to be dependent on 1) the trigger dose, 2) levels of ED-B expression and 3) CD8+ cytotoxic T-cells. Furthermore, we observed that this combination treatment resulted in abscopal effects and a protective immunity over a long period of time, a memory potential (Rekers et al, OncoImmunology 2018). Addition of L19-IL2 to a single dose of RT (15Gy) resulted in 20% tumour remission in the non-irradiated contralateral tumor, an effect dependent on T-cells. Also, only for this treatment combination, as compared to surgery with L19-IL2 or a high single dose RT (40Gy), a long-lasting protection associated with central and effector memory T-cells was observed for the responder tumors, which was confirmed by co-culture experiments in vitro. Currently, the most advanced immunotherapy treatment is based on checkpoint inhibition using anti-PD-1 or anti-PD-L1 antibodies to prevent T-cell exhaustion. Our preliminary results indicate a preference for immune cytokines over immune checkpoint inhibitors when combined with single dose radiotherapy (Olivo Pimentel et al, unpublished). Interestingly, addition of any checkpoint inhibitor to the combination of RT with the immunocytokine L19-IL2 increased therapeutic efficacy compared with any bimodal treatment. Currently, experiments are ongoing to confirm these results, which will be presented during the conference.


29

Speaker abstracts

Proffered Paper 5

Complex DNA damage induced by high-LET protons triggers a specific cellular DNA damage response

Rachel Carter2, Catherine Nickson2, James Thompson3, Andrzej Kacperek1, Mark Hill3, Jason Parsons2

1 Clatterbridge Cancer Centre, Bebington, UK, 2 University of Liverpool, Liverpool, UK, 3 University of Oxford, Oxford, UK

Complex DNA damage (CDD), where several lesions are induced in close proximity within DNA, is a signature of ionising radiation (IR) and contributes significantly to cancer cell killing due to the difficult nature of its repair. This is particularly important following proton beam therapy (PBT), which is increasingly being utilised for cancer treatment, as this generates protons with differing energies therefore varying the amount and complexity of CDD. Despite this, very little is known about the mechanism of recognition and processing of CDD in cells, particularly within the context of chromatin. We have examined the cellular DNA damage response to proton irradiation using the 60 MeV cyclotron at the Clatterbridge Cancer Centre, the only clinically operating PBT in the UK, specifically analysing low-LET (60 MeV, 1 keV/μm) protons generated at the Bragg peak, versus high-LET (11 MeV, 12 keV/μm) protons generated at the distal end. We demonstrate that high-LET protons induce a significant decrease in cellular survival post-irradiation versus low-LET protons, due to an induction of CDD. By analysis of a large panel of histone post-translational modifications (eg. acetylation, methylation and ubiquitylation) post-IR, we have identified that histone H2B ubiquitylation on lysine 120 in particular is specifically induced by high-LET protons several hours post-IR where CDD exists, which is independent of DNA transcription. Data has also been reproduced with low-LET x-rays/γ-irradiation and high-LET α-particle irradiation, respectively. We have identified that histone H2B ubiquitylation is mediated by two E3 ubiquitin ligases, RNF20/40 and MSL2, that play vital roles in co-ordinating CDD repair and ultimately in controlling cellular radiosensitivity following high-LET protons. Finally, utilising an siRNA screen of enzymes controlling ubiquitylation (~94 deubiquitylating enzymes) following high- versus low-LET IR, we have identified novel enzymes that are specifically involved in modulating the radiosensitivity of cells to CDD-induced by high-LET radiation.


30

Speaker abstracts

Radiation-induced normal tissue toxicity: new insight from damage signaling and tissue-specific immune defense strategies

Verena Jendrossek1

1 Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, Medical School, Essen, GERMANY

About 60% of all cancer patients receive radiotherapy (RT) during the course of their disease and good results in terms of long-term survival and tumour cure are achieved in various tumours by multimodal combinations of surgery, RT and chemotherapy. Yet despite substantial advances in surgery and RCT cure rates remain still unsatisfactory, for common types of cancer with high loco-regional failure-rates or frequent development of metastases highlighting an urgent need for further innovations in RT practice.

Apart from biological factors adversely affecting the response of tumour cells to treatment, acute and late toxicity to normal tissues also limits the radiation dose that can be applied to the tumour thereby contributing to failures. Instead tolerable doses are often linked to suboptimal tumour control - despite accepting side-effects that decrease quality of life. Normal tissue toxicity also precludes therapy intensification efforts for many locally advanced tumours through the combination with cytotoxic chemotherapy. Therefore there is high interest in the development of effective strategies for a biological optimization of RT by modulating critical biological processes that determine the radiation response of normal tissues and thus allow to avoid or treat radiation-induced pulmonary disease.

The presentation will present new findings obtained in preclinical murine in vivo models of radiation-induced pneumopathy about the cellular and molecular mechanisms that link the initial radiation-induced damage to the DNA of resident cells and its perception to immune activation, lung inflammation (pneumonitis), excessive tissue remodelling and pulmonary fibrosis after >7 months. It will also discuss recently discovered novel strategies for the prevention or treatment of these unwanted side effects by targeting radiation-induced changes in the lung tissue that participate in shaping a disease-promoting microenvironment. An improved knowledge of the normal tissue response to injury is particularly important in view of the increasing interest in combining radiotherapy with immune checkpoint blockade or immunotherapies to avoid exacerbation of radiation-induced normal tissue toxicity.


31

Speaker abstracts

Radiobiology Biomarkers for Precision Radiotherapy

Catharine West1

1 The University of Manchester, Manchester, UK

Advances in technology and imaging improve the precision and individualisation of radiotherapy delivery but schedules remain population based with no/little consideration of individual biology. Two radiobiological factors that impact on radiotherapy outcomes are hypoxia and intrinsic radiosensitivity. There is an unmet need to develop biomarkers and to move away from research aimed at discovery to focus on validation, qualification and application. The importance of collaborative efforts for biomarker development will be emphasised. An overview will be given of the author’s research developing gene signatures reflecting hypoxia. Gene expression signatures have been developed for a number of cancers (head and neck, bladder, sarcoma, prostate) and shown to validate in multiple cohorts and, where data are available, to predict benefit from giving hypoxia-modifying treatments with radiotherapy. The trials required for biomarker qualification and clinical application are either underway or under development. The importance of showing that radiobiology biomarkers are independent from emerging disease-specific classifiers will be stressed. A brief update will also be given of international collaborative efforts aimed at identifying genetic variants that predict whether a patient undergoing radiotherapy has an increased risk of developing late toxicity. The latter involves the recent analysis of ~4,000 samples from prostate cancer patients with genotyping carried out via the OncoArray network that has identified several new variants. Ongoing work within the EU-funded REQUITE consortium will be mentioned to stress the feasibility of international collaboration in generating resources for biomarker validation.


32

Speaker abstracts

The impact of DNA damage response (DDR) modulators on normal tissues following thoracic irradiation in preclinical models

Anderson J. Ryan1, Luiza Madia Lourenco1, Jennifer Martin1, Yanyan Jiang1

1 CRUK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK

Background: Approximately half of cancer patients will receive DNA-damaging radiotherapy as part of their treatment, many with curative intent. However, the effectiveness of radiation is limited in large part by normal tissue toxicities. For thoracic irradiation, oesophagitis, pneumonitis and pulmonary fibrosis are clinically important side effects. To improve the effectiveness of radiation several approaches to modulate the DDR have been developed including inhibitors of PARP, WEE1, ATR and ATM. Methods: We examined the impact of DDR inhibitors in nude mice bearing established Calu-6 human non-small cell lung cancer xenografts and in C57BL6 mice, a well-established model of radiation-induced pulmonary fibrosis. We also evaluated normal tissue responses in tumour-bearing A/J mice. Results: Inhibition of PARP, ATR or ATM (but not WEE1) significantly increased the effectiveness of fractionated radiation (4 x 5 Gy) against established Calu-6 xenografts. In C57BL6 mice, PARP inhibition in combination with fractionated radiation (4 x 5 Gy) induced marked oesophageal and skin toxicity. In contrast, ATR inhibition combined with radiation was well tolerated with no evidence for increased normal tissue effects compared with radiation treatment alone. ATM inhibition combined with radiation did not produce any observable toxicity, although there evidence of increased levels of collagen in the lungs. In tumour-bearing A/J mice, ATM inhibition was associated with a significant increase in anti-tumour activity but was also associated with decreased tolerability compared with radiation (5 x 5 Gy). Conclusions: A newly established A/J mouse model has the potential to evaluate the therapeutic window of novel therapies in combination with thoracic radiation by allowing for simultaneous assessment of both normal and tumour tissues within the field of irradiation in the context of a fully functioning immune system.


33

Speaker abstracts

Proffered Paper 6

Targeting the Rad18 ubiquitin ligase enhances sensitivity to therapy by inhibiting cancer stem cells proliferation

Chames Kermi3, Lenka Stefancikova3, Aurore Siegfried-Vergnon1, Jean-Marc Pascussi2, Julie Pannequin2, Jean-Philippe Hugnot4, Emmanuelle Uro-Coste1, Elizabeth Cohen-Moyal1, Domenico Maiorano3

1 Cancer Research Center of Toulouse, INSERM, Toulouse, FRANCE, 2 Institute of Functional Genomics, CNRS, INSERM, Montpellier, FRANCE, 3 Institute of Human Genetics, CNRS, University of Montpellier, Montpellier, FRANCE, 4 Institute of Neurosciences of Montpellier, INSERM, Montpellier, FRANCE

Rad18 is a non-essential gene coding for an E3 ubiquitin ligase that has been so far mainly implicated in DNA damage tolerance, by facilitating the progression of DNA replication in the presence of DNA damage. Rad18 is also involved in the two major double strand break repair pathways, non-homologous end joining and homologous recombination.

We have previously shown that high levels of Rad18 are sufficient to shut down the DNA damage checkpoint and induce resistance to DNA damaging agents in mammalian cells. In addition, we have shown that Cancer Stem Cells (CSCs) of the radiation-resistant brain cancer glioblastoma express a high level of Rad18 compared to the their differentiated counterparts, and that Rad18 down regulation sensitizes glioblastoma to cisplatin (Kermi et al., Dev Cell 2015).

Here we report an unexpected essential role for Rad18 in the proliferation of CSCs of divers origins. Rad18 down regulation strongly affects the growth and self-renewal of glioblastoma CSCs and reduces the fraction of the stem cells population, resulting in cell cycle arrest in the absence of external damage. Exposure to DNA damaging agents additionally exacerbated this phenotype. Further, tumor growth of glioblastoma xenografts under expressing Rad18 in mice was severely reduced and survival was increased. Consistent with these results, high Rad18 expression in glioblastoma is correlated with a bad patient prognosis. Conversely, ectopic Rad18 expression provided a proliferation advantage and induced loss of cell-to-cell contact inhibition. The molecular mechanisms underlying this novel function will be presented. Altogether these results show that Rad18 is an essential gene required for CSCs proliferation and put forward this gene as a novel target to sensitize CSCs.


34

Understanding genomic instability and targeting defective DNA damage responses in breast cancer

Andrew Tutt1

1 The Institute of Cancer Research (ICR), London, UK

Breast cancer has now been subjected to extensive molecular characterisation. Although much attention has focused on transcriptional profiling, with consequent identification of a number of biological sub-types, it is clear that breast cancers show significant variability in their somatic genome. Breast cancers are diseases that are largely dominated by copy number aberrations (CNAs) rather than single nucleotide variants (SNVs). Although HER2 is the poster child CNA driven therapeutic target a number of other mutation and CNA candidates are being pursued. The mutational processes that drive the different forms of genomic instability in breast cancer are increasingly understood and have been described. These mutational and rearrangement signatures may reveal aberrations in the DNA damage and DNA Replication Stress response that themselves represent vulnerability and a therapeutic target. A number of new novel candidates drug targets have emerged. I will discuss a range of strategies and clinical trials being employed to challenge the DNA damage response in specific ways, target DNA repair, relevant cell cycle check points, genome instability and consequent replicative stress in breast cancer. I will also discuss the state of development of and inherent limitations of assays that categorise a range of forms of mutational signature and genome instability, transcriptional responses to DNA damage and replicative stress which may act in future as predictive biomarkers of therapy response or resistance to DNA damage response targeting therapies with utility in the clinic. I will describe the increasing understanding of how the DNA damage and mutation in the somatic genome may stimulate immune response to cancers and some of the rational for combination strategies that target both the DNA damage response and active immune check points in cancer.

Speaker abstracts


35

The impact of DNA repair and nutrition on cancer and aging

Jan Hoeijmakers1,2

1 Dept of Molecular Genetics, Erasmus Medical Center, Rotterdam, NETHERLANDS, 2 Princess Máxima Center for Pedriatic Oncology, Utrecht, NETHERLANDS

The molecular basis underlying ageing and ageing-related diseases, including cancer, is one of the main unsolved questions in biology. Ageing in various model organisms appears remarkably plastic: e.g. suppressing insulin signalling extends lifespan in numerous species. However, virtually all premature aging syndromes in man link with genome instability. We have generated mouse models which strikingly mimic human DNA repair deficiency syndromes and display wide-spread accelerated aging with or without cancer predisposition. For instance, Ercc1- mice defective in four repair pathways show numerous accelerated aging features limiting lifespan to 4-6 month. Simultaneously they exhibit an anti-aging ‘survival response’, which suppresses growth and enhances maintenance, resembling the longevity response induced by dietary restriction (DR). Interestingly, subjecting these progeroid mutants to 30% DR tripled median and maximal lifespan, and drastically retarded accelerated aging, e.g. DR animals retained 50% more neurons and maintained full motoric function. The same findings were made in repair-deficient Xpg-/- mice, extending this observation beyond Ercc1∆/-. The DR response in Ercc1∆/- mice resembled DR in wild type animals including reduced insulin signaling. Interestingly, ad libitum Ercc1∆/- liver expression profiles showed gradual preferential extinction of expression of long genes, consistent with genome-wide accumulation of stochastic, transcription-blocking lesions, which affect long genes more than short ones. DR largely prevented this decline of transcriptional output, indicating that DR prolongs genome function. We will present phenotypes of conditional DNA repair models targeting aging to selected organs, striking parallels with Alzheimer’s disease. Our findings strengthen the link between DNA damage and aging, establish Ercc1∆/- mice as powerful model for identifying interventions to promote healthy aging, reveal untapped potential for reducing endogenous damage and hence also cancer, provide new venues for understanding the molecular mechanism of DR, and suggest a counterintuitive DR-like therapy for human progeroid genome instability syndromes and DR-like interventions for preventing neurodegenerative diseases.

Speaker abstracts


36

Targeting the secretome as part of combined treatment modalities with radiotherapy

Sabine Bender1, Ashish Sharma1, Martin Pruschy1

1 University Hospital Zurich, Zurich, SWITZERLAND

Initial biopsy material and fine needle aspirates are often the only biological materials available for direct molecular analysis of the tumor. Therefore molecular analysis of the tumor often only includes histological and cytogenetic analysis and DNA-extraction followed by mutational analysis. However, it is also of high importance to have access to tumor material during and in response to radiotherapy to gain insights into the treatment response on the molecular and cellular level and to develop putative (surrogate) markers. As such, biomarker analysis of tumor-derived blood serum factors in tumor patients represents an additional minimally invasive approach to identify predictive and prognostic factors, which can be analyzed prior to therapy start (basal level), following single high dose irradiation but also consecutively during the time course of a fractionated treatment regimen. The basal level and radiotherapy-induced serum factors also affect the radiation resistance in an auto- and/or paracrine way via the tumor microenvironment and might act as potential targets for combined treatment modalities with ionizing radiation. As part of an own secretome analysis we recently identified that radiotherapy significantly activates ADAM17 (A Disintegrin and metalloprotease domain 17) in non-small cell lung cancer (NSCLC) , which results in shedding of survival factors, growth factor pathway activation, and contributes to treatment resistance in NSCLC. Additionally, our data point towards a novel link between ADAM17, regulation of the chromatin state and the DNA damage response. Here we will discuss recent preclinical and clinical approaches to analyze the treatment-induced secretome from lung and head&neck carcinoma and to exploit specific secretome factors as part of a combined treatment modality with radiotherapy. We will further illustrate these approaches with experiments derived from our own research platform with antisignaling agents, overcoming EGFR-linked and microtubule-targeting agent-linked treatment resistances.

Speaker abstracts


37

Proffered Paper 7

Radiotherapy-activated cancer-associated fibroblasts promote tumor progression through paracrine IGF-1R activation

Joke Tommelein1, Olivier De Wever1

1 Ghent University, Gent, BELGIUM

Preoperative radiotherapy (RT) is a mainstay in the management of rectal cancer (RC), a tumor characterized by desmoplastic stroma containing cancer-associated fibroblasts (CAF). Although CAF are abundantly present, the effects of RT to CAF and its impact on cancer cells are unknown. We evaluated the damage responses of CAF to RT and investigated changes in colorectal cancer (CRC) cell growth, transcriptome, metabolome and kinome in response to paracrine signals emerging from irradiated CAF. RT to CAF induced DNA damage, p53 activation, cell-cycle arrest and secretion of paracrine mediators, including insulin-like growth factor-1 (IGF-1). Subsequently, RT-activated CAF promoted survival of CRC cells, as well as a metabolic switch favoring glutamine consumption through IGF-1 receptor (IGF-1R) activation. RT followed by IGF-1R neutralization in orthotopic CRC models reduced the number of mice with organ metastases. Activation of the downstream IGF-1R mediator mTOR was significantly higher in matched (intrapatient) samples and in unmatched (interpatient) samples from RC patients after neoadjuvant chemoRT. Taken together, our data support the notion that paracrine IGF-1/IGF-1R signaling initiated by RT-activated CAF worsen CRC progression, establishing a preclinical rationale to target this activation loop to further improve clinical responses and patient survival.

Tommelein et al., Cancer Res in press

Speaker abstracts


38

Implementation and Validation of a 15-Gene Hypoxia Classifier in Head and Neck Cancer

Brita Singers Sørensen1

1 Aarhus University Hospital, Århus C, DENMARK

Hypoxia is a well-known feature of the microenvironment in solid tumors, and it is associated with reduced therapeutic response, increased aggressiveness, and negative effect on the clinical outcome. Therefore, predictive and clinically applicable methods for determining hypoxic status has been a focus point. A number of hypoxia gene expression signatures have been developed and have shown promising prognostic impact when tested retrospectively in historical clinical cohorts. In head and neck squamous cell carcinomas (HNSCC), hypoxic radioresistance can be reduced by use of the hypoxic modifier nimorazole, as shown in the DAHANCA 5 trial. A 15-gene hypoxia profile, based on genes preferentially upregulated by hypoxia in vitro and further developed on formalin fixed paraffin embedded tissue, demonstrated to have both prognostic and predictive impact for hypoxic modification in head and neck squamous cell carcinomas. This presentation will go through the development and the validation of the 15- gene hypoxia classifier, tested in the prospective multicentre EORTC-1219 study.

Speaker abstracts


39

Cancer Therapies and Cancer Cell Plasticity

Frank Pajonk1

1 University of California, Los Angeles, USA

Glioblastoma (GBM) is currently incurable and poor overall survival in GBM has remained largely stagnant for the past 60 years. Radiotherapy (RT) is the most effective treatment option for GBM but despite the moderate radioresistance of GBM cells or GBM xenografts, RT cannot control the disease. Based on our published data we hypothesized that radiation induces a phenotype conversion of differentiated glioma cells into therapy-resistant induced glioma-initiating cells (iGICs) and that interfering with this process will increase the efficacy of RT.

Patient-derived GBM specimen demonstrated a remarkable cellular plasticity that allowed cell populations depleted from GICs to spontaneously repopulate the GIC population from differentiated GBM. Using sphere-formation assays, in-vivo limiting dilution assays and qRT-PCR we found that in primary GBM specimen, depleted of GICs, irradiation caused a dose-dependent, up-to 10-fold increase in radiation-induced GICs (iGICs) when compared to iGICs observed under spontaneous phenotype conversion. iGICs grew more aggressively in vivo and were more radioresistant than intrinsic GICs. Phenotype conversion coincided with re-expression of Yamanaka factors (SOX2, Oct4 and Nanog), down-regulation of histone-modifying enzymes and acquisition of on open chromatin state. Employing a high-throughput screen using a library of 83,000 chemical compounds, we identified several classes of drugs that prevented RT-induced phenotype conversion of non-tumorigenic glioma cells into iGICs either with or without toxicity for the bulk cell population. Treatment of patient-derived GBM specimen with inhibitors of phenotype conversion prevented spontaneous and RT-induced conversion of non-tumorigenic GBM cells into iGICs, reduced the self-renewal capacity of existing GICs and prevented re-expression of Yamanaka factors in-vitro. Treatment of animals bearing orthotopic GBMs with these compounds lead to preferential drug uptake into the tumor (assessed by LC-MS/MS), reduced tumor volumes in bioluminescence imaging and caused a reduction in the number of marker-positive as well as functional GICs. When combined with radiation, applied using an image-guided small animal irradiator (SmART, PXI), these compounds significantly prolonged median survival in a syngeneic murine model of GBM (GL261) in C57BL/6 mice as well as in PDOX in NSG mice. In conclusion, we provide strong evidence that combining RT and compounds that prevent radiation-induced phenotype conversion can improve the efficacy of radiotherapy in murine models of GBM.

Speaker abstracts


40

Protecting normal tissue from DNA damage to enhance the efficacy of radiotherapy

Amato Giaccia1

1 Stanford University School of Medicine, USA

Abstract not available at the time of printing.

Speaker abstracts


41

Poster abstracts

1

Effects of Low Dose Radiation on miRNA Expression and Its Role in Immunity/ Tumorigenicity Responses in Triple Negative Breast Cancer (TNBC)

Sama’ Abderahman1, Ahlam Abdunnabi1, Jonathan Coulter1, Fiona Furlong1

1 Queen’s University of Belfast, Belfast, UK

TNBC is known as an aggressive subtype of breast cancer which lacks a definite tailored therapeutic approach. While radiation has been proposed as an inducer of immunity response, the specific parameters of this induction have not yet been identified, especially in TNBC. This study aims to investigate the outcome of inducing minimal DNA damage on the tumorigenicity/ immunogenicity balance in TNBC following an exposure to a low dose of radiation. Studying this outcome at the miRNA molecular level and correlating it with NFkB-p65 levels and pro-inflammatory/ anti-inflammatory end points could provide us with answers to this dilemma.

Results: An in silico analysis identified three potential miRNAs involved in DNA damage induced immunity response in TNBC. The characterisation of miRNAs in our current data revealed differences in their profile expression in the different TNBC cell lines exposed to low dose radiation. Results indicated a high base line level of miR-21, miR-15a and miR29a in the relatively radio-resistant MDA-MB-231 cell line compared to the more radio-sensitive cell line, HCC1937 and MDA-MB-468. However, these miRNAs tended to decrease in MDA-MB-231 post 0.5 Gy radiation with miR-21 statistical reduction at 8 hr post radiation exposure (p≤ 0.05). In contrast, an increase in miRNA expression was measured in miR- 29a along with a significant increase of miR-15a in HCC1937 after the same radiation dose (p≤ 0.01). Higher radiation dose of 2 Gy significantly increased miR-21 expression in MDA-MB-231 (p≤ 0.01) opposing a reduction that was detected at the same time point, 8 hr, post 0.5 Gy dose (p≤ 0.05).

Conclusion: Low-dose radiation initiated changes in the profile expression of miR-21, miR-29a and miR-15-a, a change that is suspected to reflect a DNA- induced immune signalling pathway.

Poster abstracts


42

Poster abstracts

2

Response to DNA-PKcs inhibitor KU60648 is linked to DNA mismatch repair status

Anne Ackermann1, Inga Hinrichsen1, Tonja Düding1, Natalie Filmann2, Angela Brieger1

1 1 Medical Clinic I, Biomedical Research Laboratory, Goethe University, Frankfurt a.M., GERMANY, 2 2 Institute of Biostatistics and Mathematical Modeling, Goethe University , Frankfurt a.M, GERMANY

Inactivation of DNA mismatch repair (MMR) genes cause 15% of sporadic colorectal cancer (CRC). Most often, hypermethylation in the promotor region of MMR protein MLH1 leads to loss of DNA repair capacity, microsatellite instability and tumorigenesis. Germline mutations of MLH1 and other MMR genes cause Lynch syndrome, the most common hereditary form of CRC, accounting for up to 5% of all cases.

Although Lynch syndrome patients have an increased extracolonic tumor risk, they show better overall survival and less metastasis than those with sporadic CRC. However, MMR deficient tumors do not benefit from standard chemotherapy and better therapy options are needed.

It was shown by Dietlein et al. (2014) that the sensitivity to the DNA-PKcs inhibitor KU60648 seems to be linked to loss of the MMR protein MSH3 and suggested to screen tumors for MSH3 deficiency before therapy. However, loss of MSH3 is rather secondary as a consequence of MMR deficiency, and frequently detectable in MLH1 deficient tumors.

Therefore, we analyzed the expression of MLH1, MSH2, MSH3 and MSH6 in a panel of MMR deficient and proficient CRC cell lines and determined their KU60648 sensitivity by cell viability and survival. MLH1 dependent ability of double strand break (DSB) repair was monitored after irradiation via γH2AX detection.

In summary, we found that MLH1 and/or MSH3 deficient cells exhibited a significantly higher sensitivity to KU60648 than MMR proficient cells and that overexpression of MLH1 in deficient cells resulted in a decrease of cell sensitivity. KU60648 efficiency seems to be associated with reduced DSB repair capacity. Since the molecular testing of colon tumors for MLH1 expression is a clinical standard we believe that MLH1 is a much better marker and a greater number of patients would benefit from KU60648 treatment.


43

3

Impact of Akt1 mutants on the cellular response to ionizing radiation

Klaudia Al-Refae2, Sebastian Oeck2,1, George Iliakis3, Verena Jendrossek2

1 Department of Therapeutic Radiology, Yale University, New Haven, USA, 2 Institute of Cell Biology, University Hospital Essen, Essen, GERMANY, 3 Institute of Medical Radiation Biology, University of Duisburg-Essen, Essen, GERMANY

The survival kinase Akt participates in the regulation of essential subcellular processes, e.g. proliferation, growth, survival and apoptosis and is aberrantly activated in various human solid tumors. Akt may promote resistance against genotoxic stress including radiotherapy presumably by influencing the DNA damage response, DNA repair or both. We recently demonstrated that Akt1 is a direct target of DNA-PKcs in the presence of damaged DNA. Furthermore, to study the role of active Akt1 in the cellular radiation response and DNA repair we established murine prostate cancer cells (TRAMPC1) stably overexpressing various human Akt1-mutants including the clinically relevant Akt1-E17K mutant with increased membrane recruitment and activating phosphorylation at Threonine308 (T308) and Serine473 (S473), the phosphomimicking mutant Akt1-T308D/S473D as well as -T308A, -S473A and -T308A/S473A phosphoablating mutants. We showed that overexpression of the dominant active Akt1-E17K mutant and the constitutively active phosphomimicking mutant significantly improved DNA repair and cell survival upon ionizing radiation (IR) whereas overexpression of Akt1-WT was without effect (Oeck et al., 2017). Our novel data reveal that only expression of human Akt1-T308A/S473A but not of the single phosphoablating mutants induces a significant delay in the kinetics of DNA repair in irradiated TRAMPC1 cells compared to Akt1-WT expressing cells as determined by the γH2A.X-assay and comet assay. This delay caused pronounced reduction in long-term survival after IR compared to Akt1-WT expressing cells. Since the different Akt1 variants differently affect the repair of IR-induced DNA damage, we strive to identify Akt1 target proteins involved in DSB repair processes. Notably, we observed a pronounced radiosensitizing effects upon RNAi of a recently discovered novel Akt1 target protein - an ubiquitin-conjugating enzyme with a proposed function in DNA repair by non-homologous end joining. Summarized our new data might be crucial for understanding the unclear role of Akt in the repair of IR-induced DNA damage.

Poster abstracts


44

Poster abstracts

4

Targeting replication stress to selectively kill cancer cells

N. Badra1, M. Petropoulos1, A. Kanellou1, S. Taraviras2, Z. Lygerou1

1 Department of General Biology, School of Medicine, University of Patras, GREECE, 2 Department of Physiology, School of Medicine, University of Patras, GREECE

Faithful control of DNA replication is crucial to maintain genome integrity. Cdt1 plays a pivotal role in DNA replication licensing by assembling the MCM2-7 complex onto origins. Aberrant expression of Cdt1 or its inhibitor Geminin is related to replication stress and DDR activation in higher eukaryotes. Moreover Cdt1 and Geminin are proposed as important markers for malignant transformation and poor survival. Chemical compounds targeting Cdt1 and Geminin in DDR defective cells are promising for cancer treatment based on synthetic lethality approaches. Our aim was to unravel promising synthetic lethal interactions by targeting the DDR signaling pathways in several cell lines that face replication stress. We depleted Geminin in cancerous and non-cancerous cell lines to induce rereplication, a form of replication stress. We then evaluated the viability and γH2AX expression in the presence and in the absence of key factors for the DDR and the DSBs repair pathways. Cdt1 inhibition by Geminin is crucial for the prevention of rereplication in mammalian cells and here we demonstrate that Geminin depletion alone is enough to promote rereplication and DSB formation in cancer cells. Blocking pathways such as ATM and ATR signaling results in an additive effect in terms of cell death in cancer cells. This is due to an abrogation of the checkpoint elicited by rereplication, manifested by decreased γH2AX levels. We aim to use high throughput screening approaches to identify novel factors and chemical compounds which will selectively kill cancer cells harboring replication licensing aberrations.


45

Poster abstracts

5

Silver nanoparticles: A novel radiosensitizer for the treatment of oral cavity cancer cell line

Yasemin Baskin4,6, Seniz Inanc5, Tarik Bugra Elcitepe2, Dogukan Akcay3, Gizem Calibasi Kocal4, Lutfiye Zumre Alicikus3, Gorkem Eskiizmir1, Kerim Yapici71 Celal Bayar University, Department of Otorhinolaryngology, Head and Neck Surgery, Manisa, TURKEY, 2 Cumhuriyet University, Department of Energy Science and Technology, Sivas, TURKEY, 3 Dokuz Eylul University, Department of Radiation Oncology, Izmir, TURKEY, 4 Dokuz Eylul University, Institue of Oncology, Department of Basic Oncology, Izmir, TURKEY, 5 Dokuz Eylul University, School of Medicine, Department of Medical Biochemistry, Izmir, TURKEY, 6 Personalized Medicine and Pharmacogenomics/Genomics Centre-BIFAGEM, Izmir, TURKEY, 7 Suleyman Demirel University, Department of Chemical Engineering, Isparta, TURKEY

Radiotherapy is the workhorse for the treatment of head and neck cancers despite of its serious side effects. Therefore, de-escalating radiation dose without jeopardizing treatment outcome is of utmost important for lowering toxicities and complications. Currently, it is known that high-Z materials are effective radiosensitizers. Therefore, silver nanoparticles (AgNPs), a high-Z nanomaterial, are good candidates for the increment of radiation efficacy and nano-drug carrier systems due to their unique physicochemical properties. Herein, the purpose of this study was to evaluate the cytotoxic effect of functionalized (PEG-NH2) AgNP for radiotherapy combined treatment of oral cavity cancer cell line (UPCI-SCC-131).

Silver nanoparticles were synthesized by using sodium borohydride solution as a reducing agent and characterised using SEM. UPCI-SCC-131 was cultured in MEM at 370C with 5% CO2. Cells were seeded at 1x104 cells/well into 96-E plates and exposed to various concentrations (100-50-25µg/mL) of PEG-NH2/Ag nano-complex. Cell viability was monitored by xCELLigence RTCA SP system for 48 h. The percentage of cell viability was calculated by the ratio of cell index of control cells to PEG-NH2/Ag nano-complex applied cells. Thereafter, 25 mg/ml of PEG-NH2/Ag nano-complex combined radiotherapy with different doses was applied for colony formation assay.

In vitro studies demonstrated that PEG-NH2/Ag nano-complex had no cytotoxic effect at all concentrations. In colony formation assay, a significant decrease in surviving fractions was determined after application of PEG-NH2/Ag nano-complex combined RT with a dose of 0.5Gy. In contrast, similar surviving fractions were obtained at 6Gy in control group.

In conclusion, it has been obviously demonstrated that PEG-NH2/Ag nano-complex is non-toxic and likely to provide high amount of reduction in surviving fractions by a significant radiotherapy dose reduction. Therefore, PEG-NH2/Ag nano-complex may be considered as a radiosensitizer for the treatment of oral cavity cancer. However, in vivo studies are required.

This study was supported by TUBITAK-115S483.


46

6

Biomarkers of radiotherapy response in Head and Neck Cancer: the role of miR-196a and its targets in radiation-induced DNA damage repair

Katheryn Begg2, Yae-Eun Suh2, Eleanor Fewings2, Nina Raulf2, Hersi Hersi2, Elham Alsahafi2, Teresa Gerrero-Urbano1, Mahvash Tavassoli21 Department of Clinical Oncology, King’s College London, London, UK, 2 Department of Molecular Oncology, King’s College London, London, UK

For head and neck squamous cell carcinomas (HNSCCs) the major means of treatment is radiotherapy. However, 50% of patients fail radiation treatment and suffer recurrent disease. Treatment programmes for HNSCC are harsh and all modalities carry significant morbidity. Notably, despite the life-limiting side-effects of treatment and well-known heterogeneity between patients, patients are still treated with the same treatment programmes meaning that many undergo unnecessarily severe treatment. Unfortunately, despite commendable efforts to characterise genetic signatures and biomarkers of HNSCC, none of those identified so far (Human Papilloma Virus infection, EGFR overexpression) allow for tailoring of treatment regimens.

Microarray data generated by this lab recently identified a signature of microRNAs differentially expressed in patient groups classed as good or bad responders to radiotherapy. Of this signature, miR-196a was found to be highly expressed in patients with recurrent disease. In vitro, HNSCC cell lines modulated for expression of miR-196a showed altered invasion, migration and proliferation potential and expression of EMT markers. Overexpression cells showed an increased ability to repair DNA following exposure to radiation, using the comet assay and γH2AX staining, and better survival of these cells using clonogenic and MTT assays. Concurrently, knockdown of miR-196a showed the reverse effect. However, changes in expression of previously validated targets of this miRNA could not alone account for the altered phenotype with respect to radiosensitivity. Thus, it was hypothesized that miR-196a in its regulation of a network of targets, perhaps some not previously validated, could be altering DNA damage repair pathways. To investigate this, RNA-sequencing was performed on HNSCC cell lines modulated for overexpression and knockdown of miR-196a. Analysis of this data using advanced bioinformatics techniques will allow us better insight into current molecular understanding of HNSCC, and pave the way for development of biomarkers or even therapeutics for sensitisation to radiotherapy.

Poster abstracts


47

7

A unique, robust and scalable co-expression approach for high yield bacterial production of recombinant Human Tousled-like Kinase 1B involved in cancer

Siddhant Bhoir1, Althaf Shaik1, Vijay Thiruvenkatam1, Sivapriya Kirubakaran1

1 Indian Institute of Technology Gandhinagar, Gujarat, INDIA

Cancer is considered to be a severe health concern worldwide. Deregulation of the kinase activity has emerged as a primary mechanism by which cancer cells evade normal physiological constraints on the growth and survival. Such aberrant functions of the kinases in a cancer cell have highlighted them as one of the most successful families of drug targets. Human Tousled-like kinases (TLK1 and TLK2) are highly conserved serine/threonine protein kinases responsible for cell proliferation, DNA repair, and genome surveillance. Their discernible role in cancer development via efficient DNA repair mechanisms have made them clinically relevant molecular targets for anticancer therapy. Human TLKs are frequently overexpressed in breast cancer, prostate cancer and cholangiocarcinoma, and often correspond to reduced sensitivity towards chemo- and radiomimetic therapies. In response to genotoxic stress, TLK1B, a splice variant of TLK1, becomes translationally upregulated and interacts specifically with Asf1 and Rad9, and promotes chromatin-remodelling coupled DNA repair at the double-strand break (DSB) ends. Innovative approaches in chemical biology have played a key role in validating the importance of kinases as molecular targets. However, the detailed understanding of the protein structure and the mechanisms of protein–drug interaction through biochemical and biophysical techniques demands a method for the production of an active protein of exceptional stability and purity on a large scale. In our work, we have designed a bacterial expression system to express and purify biologically active, wild-type Human Tousled-like Kinase 1B (hTLK1B) by co-expression with the protein phosphatase from bacteriophage λ. We have obtained remarkably high amounts of the soluble and homogeneously dephosphorylated form of biologically active hTLK1B with our unique, custom-built vector design strategy. The recombinant hTLK1B can be used for the structural studies and may further facilitate the development of new TLK inhibitors for anti-cancer therapy using a structure-based drug design approach.

Poster abstracts


48

8

Single agent activity of ATR inhibitor, VE-821, in a panel of ovarian cancer cell lines

Alice Bradbury1, Yvette Drew1, Nicola Curtin1

1 Northern institute for cancer research, Newcastle University, Newcastle upon Tyne, UK

Ovarian cancer is the leading cause of death from gynaecological malignancy. Despite the development of improved surgical techniques and delivery of chemotherapy agents, the 5-year survival rate remains poor at around 30% justifying the need for novel therapeutic agents. Dysregulation of the DNA damage response (DDR) is common in cancers and can be exploited therapeutically by targeting other components of the DDR on which the cancer has come to rely. ATR is a key DDR protein, responsible for signalling to S and G2/M cell cycle checkpoints. Loss of G1 checkpoint control is a common event in ovarian cancer, e.g. by upregulation of cyclin E, making the ATR and G2/M checkpoint an attractive target for anticancer therapy. In addition, other determinants of sensitivity to ATR inhibition have been identified including ATM, BRCA2 and Ku80 loss and DNA-PKcs overexpression. Here the single agent activity of ATR inhibitor (ATRi) VE-821 has been investigated in a panel of ovarian cancer cell lines characterised for selected mutations.

VE-821 cytotoxicity was assessed by clonogenic survival assays in ovarian cancer cell lines (IGROV1, NIH-OVCAR3, CAOV3, COV318, OAW42, ES-2, COV362, A2780, CP70 A2 and CP70 B1) and this data was related to the baseline expression of DDR proteins and other known determinants of sensitivity to ATRi. Cells exhibited a range of sensitivities to VE-821, from 0.76 ± 0.09 µM (CAOV3) to 11.64 ± 1.90 µM (NIH-OVCAR3). The most sensitive cell line, CAOV3, had the second lowest expression of ATM and second highest expression of DNA-PKcs but otherwise there was no correlation between VE-821 sensitivity and DDR protein expression. Studies are ongoing in additional cell lines to determine the underlying mechanisms and the importance of replication stress, genetic and molecular drivers of the response to VE-821 with a hope to identify potential biomarkers to stratify patients for ATRi therapy.

Poster abstracts


49

9

The action of a novel radiosensitiser within the Oesophageal Adenocarcinoma tumour microenvironment

Amy Buckley2, Niamh Lynam-Lennon 2, Susan Kennedy2, Aoife Cannon2, Róisín Byrne2, Alison Reynolds3, David Gomez-Matallanas1, Dermott O’Toole2, John V Reynolds2, Stephen Maher2, Breandán Kennedy2, Jacintha O’Sullivan2

1 Systems Biology Ireland, University College Dublin, Dublin, EIRE, 2 Trinity Translational Medicine Institute, Department Of Surgery, St. James’s Hospital, Trinity College Dublin, Dublin, EIRE, 3 UCD Conway Institute; UCD School of Biomolecular and Biomedical Science, University College Dublin, Dublin, EIRE

Background: Oesophageal Cancer (OAC) is an aggressive disease with survival rates of ~15-20%. Current therapeutic regimes focus on neo-adjuvant therapy (chemo-radiation) prior to surgery. Unfortunately, only 20-30 % of patients show a beneficial response. This major clinical challenge of treatment resistance reinforces the need for the identification of novel treatments which can act as radio-sensitisers in the neo-adjuvant setting.

Methods: Through a drug screening approach in-vivo, we have identified a novel anti-angiogenic and anti-metabolic compound, 11B_CC8 with radiosensitising activity. The ability of 11B_CC8 to act as an anti-metabolic agent under hypoxia was evaluated using the XFe24 Seahorse analyser and the Don Whitley i2 workstation. The ability of 11B_CC8 to radiosensitise under hypoxic conditions was assessed by clonogenic assay. The effect of 11B_CC8 on inflammatory, metabolic and angiogenic protein secretions from OAC treatment naïve tumour conditioned media (TCM) was evaluated by multiplex ELISA. Fresh treatment naïve patient biopsies were screened for their metabolic activity using the XFe24 seahorse analyser at baseline and post- treatment with 11B_CC8. The elucidation of the possible mechanism of action of 11B_CC8 was evaluated by Mass-Spectrometry.

Results: Our novel anti-angiogenic and anti-metabolic agent can enhance radiosensitivity in our isogenic model of OAC under both normoxic and hypoxic conditions. 11B_CC8 significantly reduces oxygen consumption rate (OCR) under normoxic but not hypoxic conditions. Ex-vivo, 11B_CC8 significantly reduced the secretion of IL1β (p=0.0117). Real-time ex-vivo metabolic analysis of our pre-treatment OAC biopsies showed significantly elevated rate of oxidative phosphorlation when compared to glycolysis (p=0.0059). Treatment with 11B_CC8 produced a reduction in OCR (p=0.0039).

Conclusion: Our novel anti-angiogenic and anti-metabolic agent can enhance radiosensitivity in-vitro under both normoxic and hypoxic conditions. Ex-vivo, treatment naïve OAC human patient samples, 11B_CC8 can significantly reduce the secretion of IL1β and altered metabolic programming, specifically oxidative phosphorylation in human explants.

Poster abstracts


50

10

Enhancement of homology-directed DNA repair by polyamines

Chih-Ying Lee1, Guan-Chin Su1, Wen-Yen Huang3, Min-Yu Ko1, Hsin-Yi Yeh1, Geen-Dong Chang1, Sung-Jan Lin3, Peter Chi1,2

1 Institute of Biochemical Sciences/National Taiwan University, Taipei, TAIWAN ROC, 2 Institute of Biological Chemistry/ Academia Sinica, Taipei, TAIWAN ROC, 3 Institute of Biomedical Engineering/ National Taiwan University, Taipei, TAIWAN ROC

Elevated levels of polyamines are found in cancer cells, and there is evidence that these naturally occurring compounds help sustain cancer cell growth and proliferation. Here, we investigate whether and how polyamines affect genome integrity. Importantly, we find a novel function of these compounds in DNA double-strand break (DSB) repair. First, we reveal the importance of polyamines in DSB repair in a mouse hair follicle model. We then show that polyamines facilitate homologous recombination-mediated DSB repair without affecting repair via non-homologous DNA end-joining. Biochemical reconstitution and functional analyses demonstrate that polyamines significantly enhance the DNA strand exchange activity of RAD51. The stimulatory effect of polyamines by RAD51 stems from their ability to enhance the capture of homologous duplex DNA and synaptic complex formation by the RAD51-ssDNA nucleoprotein filament. Our findings furnish valuable insights into the role of polyamines in genome maintenance via homology-directed DNA repair.

Poster abstracts


51

11

Using functional genetic screens to understand resistance to PARPi in BRCA-deficient tumors

Mariana Dias2, Ewa Gogola2, Alexandra Duarte2, Stefano Annunziato2, Peter Bouwman2, Jinhyuk Bhin2, Sven Rottenberg1,2, Jos Jonkers2

1 Institute of Animal Pathology, Vetsuisse Faculty, University of Bern, Bern, SWITZERLAND, 2 Netherlands Cancer Institute (NKI-AvL), Amsterdam, NETHERLANDS

Error-free repair of DNA DSBs is achieved by homologous recombination (HR), and both BRCA1 and BRCA2 are crucial for this process. Inactivating germline mutations of Brca1 and Brca2 genes predispose to breast and ovarian cancers and result in HR deficiency. This defect can be specifically targeted by inhibition of Poly-(ADP-ribose) polymerase (PARP) 1, which leads to the selective killing of HR-deficient tumor cells. These observations have recently resulted in the approval of the first PARP inhibitors (PARPis) for the treatment of patients with germline BRCA-mutated tumors. Although this approach has shown promise, the efficacy of PARPis is limited due to drug resistance, with only a fraction of the BRCA1/2 mutation carriers responding to this therapy. Those who do respond eventually develop resistance and relapse. Although some drug resistance mechanisms have been characterized, many other mechanisms remain to be elucidated. Further investigation is needed to achieve a strategy to overcome drug resistance in order to improve this promising targeted therapy. Therefore, our aim is to conduct in vitro functional genetic screens to uncover novel mechanisms of resistance and to find promising therapeutic targets able to revert the resistant phenotype. We will perform the functional genetic screens in PARPi-sensitive and PARPi-resistant cell lines and we will combine 2D and 3D in vitro cultures. Furthermore, we will use multi-omics analysis of BRCA1/2-deficient mammary tumors that acquired PARPi resistance in vivo. Taken together, both approaches will yield a list of candidate genes that we further intend to validate and characterize, both in vitro and in vivo.

Poster abstracts


52

12

DNA damage induced by proton beam radiation in human uveal melanoma cells

Katarzyna Jasinska-Konior1, Krzysztof Berniak4, Paweł Olko3, Bozena Romanowska-Dixon2, Krystyna Urbanska1, Jerzy Dobrucki4, Martyna Elas1

1 Department of Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, POLAND, 2 Department of Ophthalmology and Ophthalmic Oncology, Jagiellonian University Medical College, Krakow, POLAND, 3 Institute of Nuclear Physics, PAS, Krakow, POLAND, 4 Laboratory of Cell Biophysics, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, Cracow, Poland, Krakow, POLAND

Purpose: Uveal melanoma is successfully treated with brachytherapy or proton beam irradiation. Differences in cellular response to photon and proton beam irradiation has been shown, such as differences in DNA repair mechanisms, angiogenesis and metastatic potential. Our aim was to evaluate DNA damage by assessing cellular markers of SSB and DSB and their co-localization in cells irradiated with low doses of proton beam irradiation.

Materials/methods: Mel270 human uveal melanoma cells, derived from a primary tumor, were irradiated with 1 – 5 Gy of X ray (300 kVp Phillips, 1Gy/min) or proton beam (SOBP, 60 MeV) from Proteus C-235 cyclotron. Cells were analysed for survival using both rate of proliferation and clonogenic assay. DNA damage was evaluated using comet assay and using immunofluorescence staining of γ-H2AX, 53BP1 (for double strand breaks) and XRCC1 (for single strand breaks) and detection with confocal microscopy.

Results and conclusions: Mel270 proliferation was slightly inhibited after 1 Gy, and strongly inhibited after 5 Gy. RBE value for Mel270 cells was 1.075. Both types of radiation caused a dose dependent increase of the percent of DNA in the tail of the comet. The number of damage foci increased with dose of proton beam irradiation. The size of γ-H2AX foci were bigger than other foci. Two distinct groups of gH2AX and 53BP1: co-localized and randomly localized were seen. High number of 53BP1 foci was seen after X-rays. Low doses of proton beam irradiation and X rays damage DNA affecting the formation of foci indicating single and double strand breaks.

Poster abstracts


53

13

Radiation-induced microRNA targets DNA repair complex and reprograms the tumor microenvironment

Cristina Espinosa-Diez1, RaeAnna Wilson1, Namita Chatterjee1, Rebbeca Ruhl1, Christina Hipfinger1, Erin Helms1, Sudarshan Anand1,2

1 Department of Cell, Developmental & Cancer Biology/Oregon Health & Science University, Portland/Oregon, USA, 2 Department of Radiation Medicine/Oregon Health & Science University, Portland/Oregon, USA

Preclinical and clinical studies have revealed that tumor endothelium is abnormal, resistant to genotoxic stress, and as such, functions as a key determinant of therapeutic responses to radiation and chemotherapy. While it is well established that radiation and chemotherapy cause DNA damage in tumor vasculature, the molecular mechanisms leading to subsequent cell cycle arrest, apoptosis or senescence in vascular cells are poorly understood. Therefore, identifying and understanding factor(s) that mediate DNA damage responses in tumor endothelial cells will provide potential targets for sensitizing tumor vasculature to radiation and other DNA damaging agents and improve their therapeutic efficacy in cancer.

We recently discovered a microRNA, miR-494 that is induced after DNA damage stressors in the endothelium and in senescent endothelial cells. Our results demonstrate that miR-494 drives EC senescence, affects telomerase activity and inhibits pathological angiogenesis in vitro and in vivo. In contrast, loss of function of this miR decreases DNA damage and cell cycle arrest after radiation exposure. We identified that MRE11a-RAD50-NBN (MRN) complex—a critical DNA Repair node—is a primary target for miR-494.

Since TCGA data shows that MRE-11a is upregulated in breast cancer patients, we asked if there was any differential expression of miR-494 in either the tumor ECs or tumor cells. Interestingly, ISH of a breast cancer tissue array revealed a significant reduction in tumor miR-494 levels compared with the adjacent normal tissue. Furthermore, ectopic expression of miR-494 on tumor cells or vascular-targeted delivery of miR-494 diminished tumor growth and angiogenesis in vitro and in vivo.

Our observations indicate that miR-494 behaves as a tumor suppressor, targeting the MRN complex, inducing senescence, cell cycle arrest and decreasing angiogenesis. Therefore, we propose that restoration of this miR and diminishing the function of the MRN complex in breast cancer is likely to synergize with radiation therapy.

Poster abstracts


54

14

Biopsy- and imaging-based hypoxia classification of cervical cancer patients

Christina S. Fjeldbo1, Tord Hompland1, Eva-Katrine Aarnes1, Gunnar B. Kristensen1, Eirik Malinen1, Heidi Lyng1

1 Norwegian Radium Hospital, Oslo University Hospital, Oslo, NORWAY

We have previously constructed and validated a prognostic 6-gene classifier associated with tumor hypoxia in cervical cancer patients, as evaluated by the dynamic contrast enhanced MR imaging (DCE-MRI) parameter ABrix. The classifier may give an early indication of a patient’s risk of hypoxia-related recurrence after chemoradiotherapy and suggest the optimal hypoxia-modifying drugs for combination trials. A benefit of this biopsy-based classifier is the possibility to integrate it with DCE-MRI to assess the intra-tumor heterogeneity in hypoxia and potentially improve response prediction. The aim of this work was to compare gene-based and DCE-MRI-based hypoxia classification of cervical cancer patients.

A cohort of 69 patients was included. For 64 patients, pairwise data of ABrix and 6-gene classifier were available. For the remaining 5 patients, the 6-gene classifier was assessed in 2-3 biopsies from each tumor by RT-qPCR. Totally 49 out of 64 patients were classified correctly with the 6-gene classifier according to ABrix, giving an accuracy of 0.77 in gene-based classification. The correlation between ABrix and the 6-gene classifier was improved by using ABrix only from the lower part of the tumor where the biopsies were taken from, suggesting that the incorrect classification was caused by intra-tumor heterogeneity. The effect of the intra-tumor heterogeneity for the 6-gene classifier was further evaluated by comparing classification between different biopsies from the same patient. All biopsies were classified to either the more or the less hypoxic group for four patients, showing robustness of the 6-gene classifier. However, different classification of biopsies from the fifth patient strongly indicates that the intra-tumor heterogeneity may result in misclassification. These results support the use of a combined gene- and imaging-based classification tool for hypoxia, providing means to control for intra-tumor heterogeneity in addition to obtaining biological knowledge of pathways involved in the aggressive phenotype.

Poster abstracts


55

15

64CuCl2, a simple tool for prostate cancer theranostics

Joana F. Guerreiro1, Vítor Alves2, Antero J. Abrunhosa2, António Paulo1, Octávia Monteiro Gil1, Filipa Mendes1

1 C2TN - Centro de Ciências e Tecnologias Nucleares, Instituto Superior Técnico, Universidade de Lisboa, Bobadela LRS, PORTUGAL, 2 ICNAS - Instituto de Ciências Nucleares Aplicadas à Saúde, Universidade de Coimbra, Pólo da Saúde, Coimbra, PORTUGAL

Perturbations of cellular copper homeostasis are associated to various pathologies, including cancer, and several copper isotopes are currently used for cancer imaging and therapy1,2. Among those, 64Cu is one of the few known radionuclides to have the potential to be used simultaneously for diagnosis and therapeutics (theranostics)2. This radionuclide might thus be used for targeted radionuclide therapy, through the combined emission of beta minus particles and Auger electrons (with therapeutic potential) and for imaging via the emission of positrons (allowing high resolution PET imaging)2. In particular, the simple ionic form, 64CuCl2 has been shown to have potential as a theranostic agent in several human malignancies, including prostate cancer3.

In this work, the preclinical evaluation of 64CuCl2 was performed in human normal and prostate cancer cell lines, focusing on the mechanisms involved in copper transport homeostasis. In addition, the genotoxicity of exposure to ionising radiation (64Cu) was assessed using the γ-H2AX and micronuclei assays.

The results obtained have revealed that 64CuCl2 exhibits higher uptake in prostate cancer cell lines in comparison with the non-tumoural cell line, which might correlate with the different expression of the main copper cellular transporters exhibited by those cell lines. In addition, early DNA damage was also higher in prostate cancer cells compared with non-tumoural cells. Some of the tumoural cell lines have also revealed deficient DNA-damage repair ability as observed with the γ-H2AX assay performed at 24h post-exposure.

Overall, the results herein obtained suggest that 64CuCl2 deserves to be further evaluated for cancer theranostics, exploring for clinical purposes the possible defective DNA damage repair responses exhibited by some of these prostate cancer cell lines.

1. Brewer (2005) Drug Discov Today 10:1103-9. 2. Niccoli Asabella et al. (2014) Biomed Res Int 2014:786463. 3. Duatti (2015) Nucl Med Biol 42:215-8.

Poster abstracts


56

16

Understanding platinum chemotherapy combinations with the ATR inhibitor, AZD6738.

Sally Hall5, Emma Dean4, Alan Lau1, Elizabeth Martin3, Christopher Ottley2, Yvette Drew5, Gareth Veal51 Bioscience, Oncology, IMED Biotech Unit, AstraZeneca, Cambridge, UK, 2 Department of Earth Sciences, Durham University , Durham, UK, 3 Drug Safety and Metabolism, IMED Biotech Unit, AstraZeneca, Cambridge, UK, 4 Early Clinical Development, IMED Biotech Unit, AstraZeneca, Cambridge, UK, 5 Northern Institute for Cancer Research, Newcastle University, Newcastle , UK

Background: AZD6738 is an oral inhibitor of the ataxia telangiectasia and rad3 related (ATR) protein kinase, a key constituent of the DNA damage response (DDR). In a Phase I trial of AZD6738 and carboplatin (NCT02264678), the recommended Phase 2 dose was AZD6738 40 mg OD D1-2 + carboplatin AUC5 in a 3-weekly cycle. Whilst 3 confirmed RECIST partial responses were observed, overlapping ≥G3 toxicities of anaemia, neutropenia and thrombocytopenia were demonstrated across a range of doses and schedules. A greater understanding of the interaction between AZD6738 and carboplatin at a molecular level, particularly with regards to the formation and repair of platinum (Pt) drug-induced DNA adducts, and whether this is modulated by AZD6738, is needed to inform the optimum therapeutic dose and schedule in the clinic.

Methods: Two human non-small cell lung cancer cell lines, H23 and H460, were chosen for the preliminary stages of this work. Sulphorhodamine B (SRB) colorimetric assays were performed to test the growth inhibition of cisplatin, carboplatin and oxaliplatin, each in combination with AZD6738. Data was evaluated by median effect analysis using CalcuSyn software (Biosoft, Cambridge, UK). Pt-DNA adduct formation in H460 cells treated with cisplatin (10 µM) in combination with AZD6738 (1.5 µM and 10 µM) was assessed using inductively-coupled plasma mass spectrometry (ICP-MS).

Results: AZD6738 in combination with cisplatin and carboplatin respectively was synergistic in both cell lines. The combination of AZD6738 with oxaliplatin was antagonistic in H23 cells, although the reason for this remains unclear. Co-incubation of H460 cells with AZD6738 and cisplatin for 4 hours compared to cisplatin alone did not increase Pt-DNA adduct formation. This work is ongoing and we are currently exploring further time-courses of Pt-DNA adduct formation under a range of conditions including exposure of cells to AZD6738 prior to, and following, cisplatin dosing.

1 Abstract 1LBA (Plenary Session 3), 28th EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics.

Poster abstracts


57

17

Targeting radiation-induced cell death and Senescence by perturbations of programmed cell death proteins in lung cancer

Liat Hammer1, Adi Kimchi11 The Weizmann Institute of Science, Rehovot, ISRAEL

Radiation therapy has a major role is the treatment of Non-small cell lung cancer (NSCLC). Yet, the efficacy of radiation is limited by cancer cell resistance, resulting from mechanisms that are not fully understood. Irradiation (IR) of cancer cells can induce multiple death pathways including apoptosis, necrosis, autophagy, mitotic catastrophe and cellular senescence. Senescence in known to involve p53 activation and is defined as proliferation arrest with preserved metabolic activity and distinct morphology.

In this study we show that IR of NCI-H460 cells (NSCLC cell line) induces senescence as the main cellular response, as evident by morphology, biochemical changes and decreased viability. We hypothesize that IR-induced senescence in NCI-H460 is interconnected with proteins involved in the programmed cell death network, comprising three main signaling modules (apoptosis, autophagy and programmed necrosis). To this end we developed a platform in which we measured the cellular viability following the combination of IR and siRNA perturbation of each of 98 genes involved in the programmed cell death network. We have identified potential targets which effect cellular viability post-IR and their mechanisms are currently being studied. These targets may have a clinical relevance in increasing radiation therapy efficiency and elucidate novel mechanisms connecting senescence with different cell death pathways.

Poster abstracts


58

18

Developing novel models to study radiobiological response in Prostate Cancer

Charles Haughey2, Sharon Eddie-Parkinson2, Lara Dura-Perez1, Umbreen Syed2, Ian Mills 2, Suneil Jain2

1 Almac Pharmaceutical Company, Belfast, UK, 2 Queens University Belfast , Belfast , UK

Introduction: In the United Kingdom, prostate cancer is the most commonly diagnosed cancer in males. Recent sequencing studies have shown considerable intra-tumoral heterogeneity in the spectrum of somatic mutations associated with prostate cancer. Current pre-clinical models inadequately reflect these changes and there is an urgent need for tissue-derived models to support drug development/optimisation of therapies. To address some of these challenges we have set out to derive new pre-clinical models from mouse transgenic tumours and human tumour samples.

Methods: Biopsies are obtained from pre and post brachytherapy treated prostate cancer patients as well as PTEN/p53 knock out mice. Prostate cells are then dissociated and co-cultured on a fibroblast feeder layer which has been pre-treated with Mitomycin C, with subsequent enrichment of luminal, basal and progenitor/stem-like cell using tailored media conditions. The efficacy of the approach is assessed by flow cytometry and imaging using a panel of cellular markers.

Results: The culture conditions have allowed us to obtain viable primary cells from both mouse (PTEN/p53 knockout) and human tumours. The various media conditions used downstream to propagate them have enabled us to enrich/de-enrich for EpCAM, CD44 and other markers. Currently the different media conditions have been capable of changing the expression of EpCAM and CD44 in primary cultures derived from PTEN/p53 knockout mice. Primary human prostate cells cultures are also capable of growing in certain culture conditions.

Discussion: Our methods for deriving and propagating primary cells from tissue samples have generated novel prostate cancer models which will now be subjected to further characterisation including genomic profiling and assessments of their responses to radiotherapy, anti-androgens. Ultimately it will be important to assess this in an in vivo setting to account for the impact of the tumour microenvironment.

Poster abstracts


59

19

Cell-cycle checkpoints and cell-death modes after irradiation of lymphoblastoid cell lines with different p53 status

Carsten Herskind1, Xiaolei Liu1, Elsa Angelie1, Frank A. Giordano1, Frederik Wenz1, Marlon R. Veldwijk1

1 Dept. of Radiation Oncology, Universitaetsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, GERMANY

Background: Cells lacking p53 expression show a different phenotype from cells mutated in p53. The purpose was to test if differences in radiosensitivity and apoptosis may be related to differences in cell-cycle regulation.

Materials and Methods: The related human lymphoblastoid cells lines TK6 (p53-wildtype), TK6E6 (p53-suppressed), and WTK1 (p53-mutated) were used. Cell-cycle progression was monitored by flow cytometry of BrdU puls-labelled cells. The PLK1-CHK1 switch controlling mitotic entry from the G2-phase was detected by Western blotting using phospho-specific antibodies. Irradiation was performed with 6 MV X-rays.

Results: TK6 cells are radiosensitive with a linear survival curve and undergo radiation-induced apoptosis. Progression of BrdU-negative G1-cells into S-phase after irradiation with a dose of 3 Gy was blocked demonstrating an intact G1/S checkpoint. TK6E6 cells showed a defective G1/S checkpoint and a reduced rate of progression into and through S phase. The radiosensitivity of TK6E6 was similar to TK6 but evidence for a shift in cell death mode from apoptosis and cell-cycle arrest towards mitotic cell death was observed. By contrast, genomically instable WTK1, which are resistant with a shouldered survival curve and show little apoptosis, progressed almost unperturbed into and through S. All cell lines showed an intact G2/M checkpoint. Transient down-modulation of the p-PLK1/p-CHK1 ratio returned to normal within 8-12h in TK6 and TK6E6. In WTK1, it stayed at a moderately reduced level for 24h although cells exited G2 and appeared in G1 during 12-24h.

Conclusion: Suppression of p53 lead to a shift in cell-death mode but little change in radiosensitivity. Increased survival of the p53-mutated WTK1 cell line was not caused by a leaky G2/M checkpoint. Instead radioresistant WTK1 cells seemed to progress normally through M and into G1 with genomic instability, possibly due to a gain-of-function mutation in p53 or a change in another gene.

Poster abstracts


60

20

MDC1 and TopBP1 interact to maintain genome stability in mitotic cells

Samuel E Jones3,1, Pia-Amata Ahorner2, Manuel Stucki2, Andrew N Blackford3,1

1 Cancer Research UK and Medical Research Council Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK, 2 Department of Gynecology, University of Zurich, Zurich, SWITZERLAND, 3 Department of Oncology, Weatherall Institute of Molecular Medicine, University of Oxford , Oxford, UK

MDC1 is an adaptor protein that is recruited to DNA double-strand breaks (DSBs) via its interaction with phosphorylated histone H2AX. Once recruited, it acts as a scaffold to recruit multiple proteins required for DSB repair such as NBS1 and RNF8. Here, we identify a novel, highly conserved interaction between MDC1 and the mediator protein TopBP1. We establish that MDC1 phosphorylation by casein kinase 2 (CK2) on two highly conserved N-terminal serine residues create a binding site for tandem BRCT domains of TopBP1. Unexpectedly, radiation-induced TopBP1 foci formation was defective in cells expressing an MDC1 variant that cannot bind TopBP1 only in mitotic but not interphase cells, indicating that the MDC1-TopBP1 interaction plays a specific role during mitosis. In line with this, such mutant cells displayed hypersensitivity to ionizing radiation in mitosis, increased micronuclei formation and frequency of telomere loss. These results suggest an important function of the MDC1-TopBP1 interaction in maintaining genome stability following DNA damage in mitotic cells, possibly by holding broken DNA ends together to promote DNA repair in the following G1 phase.

Poster abstracts


61

21

The role of miRNAs in the transcriptional program regulated by hypoxia with and without lactic acidosis in cervical cancer

Marte Jonsson2, Anja Nilsen2, Unn Beate Salberg2, Malin Forårsveen Forsberg2, Gunnar Balle Kristensen1, Cathinka Halle Julin2, Heidi Lyng2

1 Department of Gynaecologic Oncology, Oslo University Hospital, Oslo, NORWAY, 2 Department of Radiation Biology, Oslo University Hospital, Oslo, NORWAY

Hypoxia may promote tumor aggressiveness through downregulation of microRNAs (miRNAs). Lactic acidosis often coexists with hypoxia in solid tumors, but the understanding of how these metabolic stressors combined affect miRNA expression and cancer progression is limited. We aimed to investigate the effect of lactic acidosis on hypoxia induced miRNA regulation and its relevance for cervical cancer aggressiveness. Samples from HeLa and SiHa cervical cancer cells exposed to hypoxia (Hyp) or hypoxia combined with lactic acidosis (HypLA), and tumor biopsies from two cervical cancer cohorts of 110 and 90 patients, were subjected to Illumina gene expression profiling and miRNA sequencing. The database miRGate was used to predict miRNA target genes based on the stress responsive miRNAs and mRNAs found in cell line experiments. A correlation analysis of the clinical data was performed to retrieve the predicted miRNA-mRNA pairs in patients.

Unique miRNA signatures consisting of 26 (Hyp) and 91 (HypLA) miRNAs were found in the cell line experiments. In addition, 31 miRNAs were common for the two stressors (Hyp-HypLA). One or more target genes were predicted for most miRNAs. Of the cell line derived miRNA signatures, 7 (Hyp), 46 (HypLA) and 18 (Hyp-HypLA) miRNAs with corresponding predicted targets were retrieved in patient tumors. A score based on the HypLA predicted target genes exhibited prognostic impact in both cohorts. Gene set analysis of the HypLA target genes showed significant overlap with inflammatory hallmarks like TNFA signaling via NFKB. This hallmark was mainly covered by the miR-99a-5p, miR-125b-2-3p and let-7c-3p cluster. A high score based on the predicted targets of these three miRNAs; NAMPT, IRS2, PTGS2, SERPINE1 and SDC4, was associated with poor outcome in both cohorts. Our finding reveals a link between miRNA regulation by hypoxia combined with lactic acidosis and tumor aggressiveness that may involve activation of an inflammatory response.

Poster abstracts


62

22

Caveolin-1-deficiency in stromal fibroblasts influences DNA damage response and repair in vitro

Julia Ketteler2, Patrick Maier1, Carsten Herskind1, Verena Jendrossek2, Diana Klein2

1 Department of Radiation Oncology, University Hospital Mannheim, Heidelberg University, Mannheim, GERMANY, 2 Institute of Cell Biology (Cancer Research), University of Duisburg-Essen, University Hospital, Essen, GERMANY

Although there are good treatment options for benign prostate carcinoma, advanced stages still display high therapy resistance to radio- and chemotherapy. The membrane protein caveolin-1 (Cav1) functions here as an important oncogene. A characteristic switch in Cav1 expression occurs where prostate epithelial cells gain Cav1 expression in advanced and metastatic cancer stages whereas a decrease of stromal Cav1 can be observed in fibroblasts of the tumor microenvironment at advanced stages. Here, we investigated the influence of differential Cav1 expression levels on the radiosensitivity of prostate carcinoma cells and stromal fibroblasts, especially focusing on DNA damage repair and cell cycle in vitro. Cav1-silenced PC3 cells [PC3 Cav1(-)] showed an increased sensitivity to IR in vitro, while Cav1(-) fibroblasts showed increased radioresistance. In order to study the influence of stromal Cav1 for the radiation response of prostate carcinoma cells we started with an indirect approach using cell culture supernatants (SN) derived from the differentially Cav1 expressing HS5 fibroblasts. Here, epithelial PC3 cells cultured with HS5 Cav1(-) SN resulted in an increase in radioresistance. These results could be confirmed in vivo. Concerning the repair of radiation-induced DNA damage we observed a pronounced DNA repair in Cav1-deficient fibroblasts. We further investigated DNA repair proteins and could observe increased UBE2S expression in Cav1-deficient fibroblasts upon irradiation. Interestingly, UBE2S as well as LigaseIV were also upregulated in Cav1-proficient cancer cells indicating improved non-homologous end joining (NHEJ). In addition, downregulated Cav1 increased the ATP production of fibroblasts, which might be linked to an enhanced DNA damage repair. We further plan to investigate the DNA damage response and repair in indirect co-culture of fibroblasts and cancer cells. Loss of stromal Cav1 expression in advanced tumor stages may thus contribute to resistance of these tumors to radiotherapy.

Poster abstracts


63

23

Radiation therapy potentiates the efficacy of immune checkpoint blockade in a syngeneic murine hepatocellular carcinoma model

Hee Chul Park1,3, Shin-yeong Kim1, Wonseok Kang2,3, Changhoon Choi1, Sung-won Shin1,3

1 Department of Radiation Oncology / Samsung medical center, Seoul, SOUTH KOREA, 2 Division of Gastroenterology and Hepatology, Department of Medicine / Samsung medical center, Seoul, SOUTH KOREA, 3 Sungkyunkwan University School of Medicine, Seoul, SOUTH KOREA

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Recently, PD-1 blockade has shown promising results in the treatment of advanced HCC. However, only 15-20% of patients showed objective response, suggesting the need for development of an effective combination therapy. To explore the therapeutic potential of radiation therapy in combination with PD-1 blockade, we studied its anti-tumor efficacy in a syngeneic mouse model of HCC. Ionizing radiation upregulated MHC class I expression in murine hepatoma cells, which may facilitate tumor antigen presentation to effector T cells. Tumor growth was significantly suppressed by either radiation or PD-1 blockade alone, compared to isotype control. However, combination of radiation and PD-1 blockade significantly suppressed tumor growth compared with radiation alone or PD-1 blockade alone group. Furthermore, survival was significantly improved in the combination group compared to radiation alone or PD-1 blockade alone group. Collectively, our findings demonstrate that radiation therapy effectively synergizes with PD-1 blockade therapy and induce a maximal anti-tumor immune response.

Keywords: hepatocellular carcinoma, radiation therapy, PD-1 blockade

Poster abstracts


64

24

Radiosensitization of novel DNMT-inhibiting phthalimido-alkanamide derivatives in human cancer cells in vitro and in vivo by inhibition of DNA damage repair

Hyun-Cheol Kang3,1, Chan Woo Wee3,1, Soo Youn Suh1, Jin Ho Kim3,1, Hak Jae Kim3,1, Eui Kyu Chie3,1, Beom Soo Shin5, Eunsook Ma2, Kwangsoo Kim4, Il Han Kim3,1

1 Cancer Research Institute, Seoul National University , Seoul, SOUTH KOREA, 2 College of Pharmacy, Catholic University of Daegu, Daegu, SOUTH KOREA, 3 Dept. of Radiation Oncology, Seoul National University College of Medicine, Seoul, SOUTH KOREA, 4 Division of Clinical Bioinformatics, Biomedical Research Institute, Seoul National University Hospital, Seoul, SOUTH KOREA, 5 School of Pharmacy, Sungkyunkwan University, Suwon, SOUTH KOREA

Background & purpose: We synthesized a series of new promising inhibitors of DNA methyltransferase (DNMT) with biological availability, and elucidated its radiosensitizing effect and underlying mechamism.

Methods: A novel non-nucleoside compound, designated as MA17, was newly derived from a phthalimidoalkanamide structure. Four human glioblastoma (U373MG, U87MG, T98G, U138MG), one lung cancer (A549), and normal astrocyte cells were cultured for radiosensitizing effects of MA17 using clonogenic assay. Pharmacokinetic characteristics for MA17 were examined in ICR mice. Quantitative assay for the inhibition of DNMT1 was performed. Tumor growth delay was examined in Balb/c-nude mice in vivo with single radiation dose of 8Gy. Effect of MA17 on cell cycle, apoptosis, DNA double-strand break repair, and differentially expressed genes were assayed.

Results: MA17 significantly radiosensitized U373MG, U87MG, T98G, U138MG, and A549 cells with mean sensitizer enhancement ratios (SER) of 1.441, 1.196, 1.350, 1.152, and 1.896 at the surviving fraction of 0.2, respectively (all p<0.05 by one-tailed ratio paired t-test). MA17 did not affect normal astrocytes (mean SER2, 1.016; p=0.420). MA17 demonstrated a mean half-life of 1.0 hour in vivo and a mean inhibition rate of 60.29% for DNMT1 activity. Pre-irradiation MA17 increased sub-G1 fraction and prolonged γH2AX expression induced by irradiation. MA17 also down-regulated the DNA homologous recombination and Fanconi anemia pathway (FANCA, BRCA1, and RAD51C) in A549 cells. In nude mice model, tumor growth was significantly delayed (18.60±4.84 vs. 12.25±2.96 days, p=0.009).

Conclusion: A novel phthalimidoalkanamide derivative, a DNMT inhibitor, possessed both biostability and preferential and substantial radiosensitizing effects by augmenting apoptosis or inhibiting DNA damage repair.

Key word: DNA methyltransferase, radiosensitizer, glioblastoma, lung cancer

Poster abstracts


65

25

Different effectiveness of integrin antagonists -/+ irradiation in 2D monolayer and 3D spheroid culture of melanoma cells

Verena Kopatz1,2, Wolfgang Dörr1, Edgar Selzer1

1 1Medical University of Vienna, Department for Radiation Oncology, Vienna, AUSTRIA, 2 2Medical University of Vienna, Christian Doppler Lab. for Medical Radiation Research for Radiooncology, Vienna, AUSTRIA

Preclinical evaluation of novel biology-based therapeutic substances, as well as the assessment of radiation effects, is frequently performed under standard 2D cell culture conditions. However, such monolayer cultures may fail to adequately represent morphological in vivo conditions and (radio)biological consequences of different treatments. Tumor cells grown in 3D aggregates show more “in vivo-like” properties, such as oxygen, nutrient and metabolite gradients, as well as complex cellular interactions and gene expression profiles. For investigations of cell surface receptors such as integrins, microenvironmental factors are critical, as these receptors transduce signals from the microenvironment to the intracellular space (regarding cell survival, proliferation and invasion signaling). Therefore, we evaluated the response of two integrin antagonizing compounds - cilengitide and a novel integrin antagonist (NIA) – in 518A2 and other melanoma cell lines using conventional 2D monolayer and 3D spheroid culture models. Also, the effects of radiation, alone or in combination with the drugs, were assessed. Twodimensional cultures of 518A2 melanoma cells were more sensitive to cilengitide (IC50 value of 0.65 μM vs. 8.2 µM), whereas 3D spheroid cultures displayed no inhibitory effect up to 50 µM. NIA had a similar efficacy in 2D as well as in 3D cultures. Spheroids treated with NIA initially showed growth retardation, followed by spheroid disintegration and cell death. The radiosensitivity of 518A2 melanoma cells was similar under 2D as well as 3D culture conditions, as assessed by clonogenic survival. Similar differences in treatment responses between 2D and 3D cell culture environments have been reported for various other anti-cancer substances as well as for some radiation exposure endpoints. We plan to perform further, comparative studies on survival-related aspects, such as proliferation, apoptosis, anoikis, and clonogenic cell death, with integrin antagonists alone as well as in combination with irradiation in 2D cell culture versus 3D spheroids.

Poster abstracts


66

26

Functional characterisation of BRIP1/FANCJ in tumour suppression and therapy response

Arne Nedergaard Kousholt1,2, Peter Bouwman1,2, Jos Jonkers1,2

1 Oncode Institute, Amsterdam, NETHERLANDS, 2 The Netherlands Cancer Institute, Amsterdam, NETHERLANDS

The helicase BRIP1 is critically required for genomic maintenance, and BRIP1 helicase deficient germline variants are associated with Fanconi Anemia (bi-allelic) and various cancers (mono-allelic), in particular ovarian and breast cancer. Cellular based assays and biochemical studies have elucidated the function of BRIP1 in replication stress responses, including interaction partners and enzymatic substrate specificity. However, functional characterisation of BRIP1 for these processes in vivo are largely unexplored, including the importance of BRIP1 as a tumour suppressor for breast cancer, and therapy responses.

This project aims to elucidate the functional roles of BRIP1 as a tumour suppressor for breast cancer and in therapy responses. We will develop breast tissue-specific knockout mice for BRIP1, and monitor the development of breast tumours. In addition, we will develop mice deficient for BRIP1 helicase activity (K52R) to further investigate the functional role of BRIP1 as a tumour suppressor. In addition, we will use the mouse models for intervention studies, testing known and novel therapies for BRIP1 deficient breast cancer. Moreover, we will investigate BRIP1 germline variants identified in high-risk breast cancer families by large scale sequencing efforts. This comprehensive list of patient derived mutations will be introduced endogenously in a primary epithelial cell line using a CRISPR-CAS9 approach. To monitor the effect on BRIP1 activity, these cell lines will be tested in cancer relevant functional assays for which BRIP1 deficient cells are known to have a phenotype. This approach will give a comprehensive knowledge of pathogenic BRIP1 variants, and give insight into functional important domains of BRIP1. Hits from the functional assay testing, will be further investigated using in-vitro and in vivo approaches.

Importantly, this project is expected to yield novel insight into the regulation of critical genomic stability pathways, and provide important knowledge for developing improved germ line variant risk prediction and treatment opportunities for patients.

Poster abstracts


67

27

The membrane-targeted antineoplastic alkylphosphocholine erufosine modulates the cellular radiation response by interfering with the lipid compartment

Adam Krysztofiak3, Sebastian Oeck3,1, Johann Matschke3, René Handrick2, Justine Rudner3, Verena Jendrossek3

1 Department of Therapeutic Radiology, Yale School of Medicine, New Haven, CT, USA, 2 Institute of Applied Biotechnology, University of Applied Sciences Biberach, Biberach, GERMANY, 3 Institute of Cell Biology (Cancer Research), University Hospital Essen, University of Duisburg-Essen, Essen, GERMANY

Introduction: Erufosine (erucylphosphohomocholine) is a synthetic phospholipid analogue with antineoplastic activity. In contrast to classical anticancer drugs and radiotherapy, erufosine primarily interferes with cellular membranes. Own previous reports revealed that erufosine and related drugs (alkylphosphocholines) interfere with PI3K/Akt-dependent survival signals and trigger cell death in various cancer cell lines. Furthermore, these drugs increase cytotoxic efficacy of ionizing radiation in vitro and in vivo. However, molecular mechanisms of their antineoplastic and radiosensitizing actions are insufficiently understood. Herein, we aimed to identify the presumed unique molecular changes initiated by drug treatment alone and in combination with IR, focused on alterations in the lipid compartment.

Methods: We used NCI-H460 and A549 human lung cancer cells as well as drug-resistant A549 cells obtained by repeated exposure to increasing drug concentrations. Cytotoxic effects of the erufosine, IR or combination were determined in short- and long-term survival assays. Alterations in lipid trafficking and utilization were analyzed by flow cytometry and fluorescence microscopy. Effects of erufosine on cell signaling were assessed by western blot, whereas changes in cell metabolism were determined using Seahorse XF-Technology®.

Results: Acute and chronic erufosine treatment significantly reduced clonogenic survival of lung cancer cells applied alone or combined with IR. Cytotoxic activity was associated with inhibition of Akt-phosphorylation and impairment of fatty acids subcellular trafficking by blocking their translocation from lipid droplets into mitochondria. Acute erufosine treatment evoked reduced mitochondrial respiration and energy production. Interestingly, these effects were compensated in drug-resistant cells by increased glycolysis rate.

Conclusions: Our study reveals novel mechanistic aspects of the cytotoxic action of membrane-active compounds of the alkylphosphocholines family in cancer cells. We speculate that chronic changes in Akt-dependent survival signaling, cellular lipid composition and metabolism contribute to increased radiation sensitivity of the drug-resistant cells compared to the non-selected cells and might indicate novel therapeutic targets for radiosensitisation.

Poster abstracts


68

28

Radiosensitization effect of 5-hydroxymethyl-2-furfural on human hepatoma cells

Yi-Hsien Lin4,5, Wei-Hsun Wang3,2, Yu-Chen Chang1, Wei-Hsiang Weng1, Jeng-Jong Hwang1

1 Department of Biomedical Imaging and Radiological Sciences, National Yang-Ming University, Taipei, TAIWAN ROC, 2 Department of Golden-Ager Industry Management, Chaoyang University of Technology, Taichung, TAIWAN ROC, 3 Department of Orthopedic Surgery, Changhua Christian Hospital, Changhua, TAIWAN ROC, 4 Division of Radiotherapy, Cheng Hsin General Hospital, Taipei, TAIWAN ROC, 5 School of Medicine, National Yang-Ming University, Taipei, TAIWAN ROC

Cancer has been the top cause of death in Taiwan since 1982. More than 50% of cancer patients are treated with radiotherapy (RT), and many of these patients have also been administered with traditional Chinese medicine (TCM). The influence of herbal medicine on RT, however, is ambiguous. Hence, the interaction between TCM and RT, as well as the underlying mechanism is important for both therapeutics and safety issues. It is well known that radioresistance may be induced during RT. In this case, NF-kB plays an important role. Here, the NF-kB promoter/firefly luciferase reporter system was used as a molecular imaging platform to detect the expression of NF-kB in real time. 5-Hydroxymethyl-2-furfural (5-HMF), a common compound produced during the preparation of TCM, was used in combination with radiation in the human Huh7/NF-kB/luc2 hepatocellular carcinoma cell line to investigate the combination effects and underlying mechanism. Results show that 5-HMF decreases radiation-induced NF-kB activity and expression of its downstream effector proteins. Cells were arrested at G2/M after 5-HMF treatment. Radiation combined with 5-HMF increases the apoptotic protein,cleaved caspase-3, and invasive protein,MMP-9. Together, these results suggest that 5-HMF could be a radiosensitizer, and may be beneficial for cancer patients treated with RT.

Poster abstracts


69

29

Individualized prophylactic radiotherapy in cN0 head and neck cancer patients based on sentinel lymph node(s) identification: a prospective phase II study

Eleonore Longton4, Georges Lawson1, Benoit Bihin5, Stephanie Deheneffe4, Isabelle Mathieu3, François-Xavier Hanin3, Thierry Vander Borght2, Jean-François Daisne4

1 CHU UCL Namur, site Godinne, Head and Neck Surgery, Yvoir, BELGIUM, 2 CHU UCL Namur, site Godinne, Nuclear Medicine, Yvoir, BELGIUM, 3 CHU UCL Namur, site Sainte-Elisabeth, Nuclear Medicine, Namur, BELGIUM, 4 CHU UCL Namur, site Sainte-Elisabeth, Radiation Oncology, Namur, BELGIUM, 5 Namur Research Institute for Life Sciences Narilis, Unit of Biostatistics, Namur, BELGIUM

Introduction: Due to a risk of occult nodal metastases in clinically node-negative (cN0) head and neck cancer patients, prophylactic bilateral neck irradiation is often mandatory. Anyway, it leads to a large irradiation of normal tissues and some tumors drain in unpredicted nodal basins. Sentinel lymph nodes (SLN) SPECT/CT lymphoscintigraphy could help to individualize prophylactic neck irradiation and, hence, to reduce the irradiated volume, which could improve patient’s quality of life. This ongoing phase II study investigates this oncological safety.

Material and Methods: Thirty-nine patients with newly diagnosed cN0 squamous cell carcinoma of the oral cavity, oropharynx, larynx or hypopharynx were included. All patients were imaged with SPECT/CT after 99mTc nanocolloid injection around the tumor. The neck levels containing up to four hottest SLN were selected for prophylactic irradiation (CTVn-LS). A comparative virtual planning was performed based on current international guidelines (CTVn-IG).

Results: Migration was observed in all of the 39 patients with an average of 3.4 SLN detected per patient. Four patients presented an unpredicted drainage and a half of the patients had only an unilateral drainage. CTVn-LS and related PTV were systematically smaller than IG ones, by a factor of two on average. This led to a significant dose decrease in identified organs at risk (OAR). With a median follow-up of 65 months, 3 regional relapses (8,5%) were observed, 2 in an irradiated area and only one in a non-irradiated area.

Conclusion: SLN SPECT/CT lymphoscintigraphy allows individualizing prophylactic node volumes in cN0 head and neck cancer patients eligible for definitive radiotherapy. Both CTV and PTV are significantly reduced, which results in a significant dose decrease in OAR. At a median follow-up of 65 months, 3 regional relapses were observed with only one in a non-irradiated area. However, more follow-up and recruitment are necessary to ensure the oncological safety.

Poster abstracts


70

30

Comparative genomic profiling between microsatellite instable type and chromosomal instable type colorectal cancer

Meng-Fu Lu3, Nicolas Limthong5, Hong M. Chen4, Jeremy C. Chen2, Yahya Elshimali6,1

1 Charles R. Drew University, Los Angeles, CA, USA, 2 JC Acupuncture, Rosemead, CA, USA, 3 La Sierra University, Riverside, CA, USA, 4 Maternal and Child Health Center, Taipei City, TAIWAN ROC, 5 Private Practice, Santa Barbara, CA, USA, 6 University of California, Los Angeles, Los Angeles, CA, USA

Adenocarcinoma of colon and rectum is one of the highest cause of cancer occurrence and death for men and women in the world. Eighty-five percent of colorectal cancer are sporadic type with significant chromosomal abnormality. The remaining fifteen percent of colorectal cancer are microsatellite instability (MSI) type without significant chromosomal abnormality. Colorectal cancer of MSI type can be further divided into sporadic and Lynch syndrome type. The hallmark of Lynch syndrome type is the presence of pathogenic germline mutations in mismatch repair proteins. One hallmark of chromosomal abnormality is the loss-of-heterozygosity (LOH) observed in the cancer genome. To determine whether percent LOH can correlate with 2 types of colorectal cancer, analyzed 32 colorectal cancer cases with FFPE blocks. The cases have prior MSI results. 7 cases are MSI type (high or low) and the remaining 25 are microsatellite stable. DNA extraction is conducted from each of the 32 tissue blocks. We then use a short-tandem repeat (STR) marker panel (91 totals) to determine the percent LOH across the entire genome. 7 cases of MSI type colorectal are found to have no LOH observed. In contrast, 4.2% markers are detected to have LOH in the microsatellite stable group. The results suggest %LOH can potentially be used as a parameter for Microsatellite stable CRC tumor.

Poster abstracts


71

31

Alpha radiation as a way to target heterochromatic and gamma radiation-resistant breast cancer cells

Maja Svetličič1, Anton Bomhard1, Lovisa Lundholm1

1 Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, SWEDEN

Compact chromatin has been linked to a poor tumour prognosis and is a factor responsible for therapy resistance of cancer cells to sparsely ionising photon radiation. We investigated the therapeutic potential of alpha emitters by evaluating the response of therapy resistant cells to densely ionising alpha radiation, and the relative importance of DNA damage induction and repair in the context of chromatin structure.

Methods: Cell culture, radiation exposure, Western blot, clonogenic survival, γH2AX foci and real time PCR analysis was used.

Results: Levels of the heterochromatin marker trimethylated lysine 9 of histone H3 (H3K9me3) were higher in the aggressive, triple negative breast cancer cell line MDA-MB-231 which is more resistant towards gamma radiation compared to the hormone receptor positive MCF7 cell line. Pretreatment with the histone deacetylase inhibitor trichostatin A (TSA) for 18 h opened the chromatin as determined by elevated acetylated lysine 8 of histone H4 (H4K8ac), and resulted in reduced clonogenic survival after gamma irradiation, while it improved survival after alpha irradiation. Gamma radiation-induced γH2AX foci were increased after TSA pretreatment, which suggests increased damage by free radicals due to chromatin opening. In contrast, alpha radiation-induced foci were not influenced by TSA which suggests an increased DNA repair due to chromatin opening. MDA-MB-231 cells were made resistant to gamma radiation by exposing them to 12 Gy of radiation in 2 Gy fractions. The radioresistant cells expressed high levels of cancer/normal stem cell markers CD44, CD133, Sox2, Oct4 and Nanog, elevated heterochromatin marker H3K9me3 and a trend towards reduced clonogenic survival in response to alpha radiation.

Conclusions: The results show that heterochromatin makes cells resistant to gamma radiation, but less so to alpha radiation. We show that gamma radiation-resistant cells can be targeted using alpha radiation and provide a mechanistic basis for the involvement of chromatin in these effects.

Poster abstracts


72

32

Cancer-associated fibroblast iNOS regulate therapeutic response of pancreatic cancer cells to radiation therapy

Patricia M.R. Pereira1, Kimberly J. Edwards1, Komal Mandleywala1, Lolkje Abma1, Freddy E. Escorcia2, Luis F. Campesato1, Sadna Budhu1, Christine A. Iacobuzio-Donahue 1, Taha Merghoub1, Jason S. Lewis1

1 Memorial Sloan-Kettering Cancer Center, New York, USA, 2 Molecular Imaging Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD , USA

Introduction: In pancreatic ductal adenocarcinomas (PDACs), the vast majority of the tumor bulk is comprised of cancer-associated fibroblasts (CAFs). Although studies demonstrate the importance of CAFs in PDAC and the effect of radiation therapy (RT) on CAFs, how they contribute to the RT resistance of neighboring cancer cells is not well characterized. Previous studies have shown that PDAC growth is better controlled after depletion of CAFs. Others have demonstrated PDACs resistance to therapy due to increased nitric oxide (NO) release by the CAFs.

Methods: Inducible nitric oxide synthase (iNOS) expression in normal and tumor tissue of pancreatic cancer patients or orthotopic PDACs was studied by immunohistochemistry (IHC). To establish an orthotopic pancreatic tumor model, FC1245 murine pancreatic cancer cells were injected into the pancreas of C57BL/6 mice and tumor growth was monitored by bioluminescence. Tumors were also visualized with contrast (Omnipaque) in an image-guided small animal microirradiator. Therapeutic radiation was delivered in a continuous arc at doses of 12 Gy or 3x 3 Gy. CAFs were isolated from CT or RT treated mice and co-cultured with FC1245 cells. Co-cultures were irradiated at 5 Gy with a 137Cs cell irradiator.

Results: Here, we show an increase of NO and iNOS in human and orthotopic PDACs after RT. Although undetectable in tumor cells, iNOS expression and NO secretion were significant in CAFs that could be further induced by the tumor cells. If co-cultured with fibroblasts, tumor cells were less sensitive to RT. This resistance-inducing effect of the CAFs was significantly abolished when the iNOS inhibitors, 1400W or dexamethasone, were added during co-culture. iNOS inhibition resulted in improved therapeutic response to RT and survival outcomes when compared with mice treated only with RT.

Conclusions: Collectively, these findings show the potential for iNOS inhibitors as treatment options alongside RT for overcoming PDAC resistance to therapy.

Poster abstracts


73

33

The miRNA classifier as the ancillary prognostic biomarker for radiation therapy

An-Lun Li2, Tao-Sang Chung1, Yao-Ning Chan2, Chien-Lung Chen1, Yun-Ru Chiang2, Nianhan Ma2

1 Land-seed hospital, Taoyuan, TAIWAN ROC, 2 National Central University, Taoyuan, TAIWAN ROC

Radiotherapy, surgery and chemotherapy are the three primary modalities used in cancer treatment. More 50% cancer patients will receive the radiation treatment. However, only some cancer patients would have poor response to radiation. Recent studies reported that miRNA profiles displayed potential and novel biomarkers for diagnosis and prognosis of diseases. In view of the limited knowledge of cancer resistance to radiation therapy in plasma, we set up to the miRNA screen to investigate the miRNA expressions before and after radiotherapy to distinguish between poor response and response after six months of radiation therapy for head-neck cancer and colorectal patients. In our research, we had developed the ratio of miRNA expressions for ancillary diagnosis predication and prognosis for radiation. In before radiation cohort, three ratios of miRNA expressions showed significant difference (p=0.0012 and 0.0419 and 0.0087). In after radiation cohort, one ratios of miRNAs showed significant difference (p=0.03147). Next, we utilized multiple logistic regression analysis to combine the ratios of miRNAs with clinical data. The values of area under curve (AUC) are 0.8923 and 0.9405 respectively for before and after cohorts. In our study, the miRNA classifier produced the significant difference between poor response and response patients and thus these may be developed the potential non-invasive ancillary tool for prognosis of radiation therapy.

Poster abstracts


74

34

New role for DNA Damage Response pathway in the tumour microenvironment

Massimiliano Mellone3, Megan Lloyd3, Giulia Venturi3, Christopher Hanley3, Aleksandra Bzura1, Kirsty Ford3, Lija James3, Sara Waise3, Toby Mellows3, Jason Parsons2, George D. Jones1, Emre Sayan3, Gareth Thomas3

1 University of Leicester, Leicester, UK, 2 University of Liverpool, Liverpool, UK, 3 University of Southampton, Southampton, UK

Tumours are complex tissues composed of carcinoma cells and various stromal cell types including cancer-associated fibroblasts (CAF). Historically, cancer research has focused mainly on tumour cells with little attention being paid to ‘normal’ stromal components. However accumulating evidence indicates that the tumour-host cross-talk affects the behaviour of malignant cells so contributing critically to tumour progression. We have previously shown that CAF with myofibroblastic phenotype are significantly associated with poor outcome in different solid tumours and promote tumour progression by inducing cancer cell invasion/metastatic dissemination and immune evasion. Why myofibroblasts accumulate only in aggressive tumours remains unclear though.

DNA Damage Response pathway (DDR) has been extensively studied in cancer cells because of their intrinsic genomic instability. However, its role in the myofibroblastic stroma of aggressive solid tumours has been ignored so far. We investigated whether the DDR signalling plays a role in myofibroblast phenotype.

We used TGF-β1 to induce myofibroblast differentiation in primary fibroblasts from different tissues, and found that TGF-β1 triggers DNA damages and the canonical double strand break (DSB) response signalling via the generation of Reactive Oxygen Species (ROS) due to the NADPH oxidase 4 (NOX4). Bioinformatic analysis and immunohistochemistry confirmed DDR activation in the stroma of a number of tumours.

Inhibition of DDR pathway (ATM, CHK1-2 and p53) using specific inhibitors/siRNAs impaired myofibroblast phenotype in TGF-β1-treated fibroblasts and in CAFs isolated from different tumours.

Myofibroblastic CAF are key players in tumour progression. We found that DDR inhibition in fibroblasts impaired myofibroblast-dependent cancer cell invasion in vitro and tumour growth in different animal models.

This study has identified the DDR signalling as a novel crucial pathway regulating myofibroblast differentiation. Given the importance of myofibroblasts in cancer progression, the inhibition of myofibroblast phenotype using DDR targeting drugs already in use in clinical practice could be a novel potential clinical strategy.

Poster abstracts


75

35

The role of mitochondria in the non-targeted effect of ionizing radiation

Silvana Miranda1,2, Marcelo Correia1, Ana Pestana1, AG Dias2, Paula Boaventura1

1 i3S/ipatimup, Porto, PORTUGAL, 2 IPO-Porto, Porto, PORTUGAL

Emerging evidences show radiobiological effects in cells not traversed by radiation. These effects have been described as bystander effects and the term comprises a variety of changes occurring in cells which were not directly irradiated. Recently, it has been shown that the production of a bystander effect does not occur when the irradiated cells do not have mitochondrial DNA (mtDNA). We propose to investigate the role of mitochondria in the response to direct irradiation and in the production of a bystander effect using 143B cell line manipulated in order to obtain cybrids with different mitochondrial status.

mtDNA-normal (Cy143Bwt), mtDNA-mutated (A3243T tRNALeu(UUR) gene - MELAS syndrome), cells without mtDNA (143B-ρ0) were used. Cells were irradiated with megavoltage X-ray (6Mv) (0.2Gy and 2.0Gy). Response to direct irradiation was assessed through cellular growth, ROS production, mitochondrial membrane potential, DNA double-strand breaks, and apoptosis. Media from irradiated cells was used to treat bystander non-irradiated cells (irradiated conditioned media - ICCM). The bystander effect was analysed through DNA double-strand breaks and apoptosis.

Mitochondria dysfunction (mutaded mtDNA or absence of mtDNA) seems to change the response to direct irradiation, observed through DNA damage, without increase in apoptosis. ICCM increased DNA damage in non-irradiated cells differently, according to the cell line it derived from: 0.2 Gy Cy143bwt ICCM induced more damage in Cy143Bwt, whereas 2.0Gy Cy143BMELAS ICCM induced more damage in this cell line. No effect was observed when 143B-ρ0 conditioned media was used. ICCM did not increase apoptosis in the cells.

These results point to a possible role of mitochondria in the radiation-induced bystander effect which needs to be better clarified. Late DNA damage, and other radiation response mechanisms other than apoptosis, such as cell cycle arrest, should be evaluated Mitochondrial function may be an interesting modulator of the response to radiotherapy.

Poster abstracts


76

36

Caspase-10: a molecular switch from cell-autonomous apoptosis to communal cell death in response to DNA damage

Andrea Mohr1, Ralf Zwacka1

1 University of Essex, Colchester, UK

The mechanisms of how chemotherapeutic drugs lead to cell cycle checkpoint regulation and DNA damage repair are well understood, but how such signals are transmitted to the cellular apoptosis machinery is less clear. We identified ATR as the crucial signalling factor for the formation of an apoptosis-initiating complex in response to chemotherapeutic drugs causing DNA single-strand breaks. Formation of this complex, we termed FADDosome, is driven by ATR-dependent caspase-10 upregulation. During FADDosome-induced apoptosis, cFLIPL is ubiquitinated by TRAF2, leading to its degradation and subsequent caspase-8 activation. Cancer cells lacking caspase-10, TRAF2 or ATR switch from this cell-autonomous suicide to a more effective, autocrine/paracrine mode of apoptosis initiated by a different complex, the FLIPosome, which leads to processing of cFLIPL to cFLIPp43, TNF production and consequently, contrary to the FADDosome, p53-independent apoptosis. Thus, targeting the molecular levers that switch between these mechanisms can increase efficacy of treatment and overcome resistance in cancer cells.

Poster abstracts


77

37

Somatic engineering of mammary gland epithelial cells using CRISPR/Cas9 for rapid testing of breast cancer susceptibility genes in mouse models

Ana Carolina Moisés da Silva1, Emilia Pulver1, Peter Bouwman1, Stefano Annunziato1, Jos Jonkers1

1 The Netherlands Cancer Institute, Amsterdam, NETHERLANDS

Breast cancer is one of the most commonly diagnosed malignancies in women. In Western countries it is the second leading cause of cancer death among women. Five to ten percent of all breast cancers have a hereditary component, and approximately twenty five percent of all hereditary breast and ovarian cases are attributable to mutations in the breast cancer 1 or breast cancer 2 (BRCA1 or BRCA2) genes. In vivo studies represent a highly comprehensive and relevant setting for studying the complexity of this human disease. Genetically engineered germline mouse models of breast cancer paved the way for improved basic and translational research, but their generation is slow and expensive. To overcome these limitations, our lab previously developed CRISPR/Cas9-mediated somatic genome editing approaches to test the role of candidate factors in the adult mammary tissue. In particular, a conditional Cas9-expressing mouse model was used for intraductal injection of sgRNA encoding lentiviruses (LV-sgRNA), which allowed for somatic gene disruption of tumor suppressor genes in mammary epithelial cells and for evaluating the effect on tumor formation and disease progression (Annunziato et al. 2016). We will extend this approach to analyze candidate susceptibility genes in triple negative breast cancer. Using a K14Cre;p53F/F;Cas9 mouse model, this LV-sgRNA intraductal platform provides the opportunity to test in vivo several new (candidate) susceptibility genes, distinguish passenger from driver mutations, and study the effects of tumor suppressor domain deletions.

Poster abstracts


78

38

Tumour Microenvironment Inhibition of Dendritic Cells is Differentially Inhibited by Gastrointestinal Tract Tumour Type and Radiation Treatment

Maria Morrissey1, Roísín Byrne1, Niamh Lynam-Lennon1, Clare Butler1, Celina Nulty1, Margaret Dunne1, Susan Kennedy1, Niamh McCabe1, John V. Reynolds1, Jacintha O’Sullivan1

1 Trinity Translational Medicine Institute, Department of Surgery, St. James’ Hospital, Trinity College Dublin, Dublin, Ireland, UK

Oesophageal adenocarcinoma (OAC) and rectal adenocarcinoma are treated with neoadjuvant chemoradiotherapy in order to reduce tumour size prior to surgery however only 10-30% of patients have a complete pathological response. Inflammatory and angiogenic mediators in the tumour microenvironment (TME) have many functions, such as enabling evasion of anti-tumour immune responses by disabling infiltrating dendritic cells (DCs) and have been linked with radioresistance. Tumour Conditioned Media (TCM) from colonic cancer has been shown to strongly inhibit DC maturation. Our aim was to understand if this DC inhibition extends to other cancers of the gastrointestinal tract and to investigate if radiotherapy may influence this.

TCM from 0Gy or 2Gy-irradiated cell lines (in vitro TCM) or tumour explants (ex vivo TCM), was used to pre-treat monocyte-derived DCs before stimulation with LPS to measure inhibition of DC maturation based on DC cell surface markers (HLA-DR, CD86, CD54, CD80, CD83 and PD-L1) and cytokine levels (IL12 p70 and TNF alpha) in DC supernatants.

For in vitro TCM, only low levels of inhibition was induced by OAC lines, whereas extensive DC inhibition was induced by 0Gy or 2Gy-irradiated CRC cell lines. Correspondingly with ex vivo TCM, 0Gy and 2Gy-irradiated OAC TCM induced significant DC activation and rectal adenocarcinoma induced moderate levels of DC activation. Interestingly, 2Gy-irradiated rectal adenocarcinoma induced significant inhibition of DC maturation. Whereas colonic cancer TCM induced extensive DC inhibition based on DC cell surface markers and IL12 p70 and TNF alpha levels. Levels of inflammatory and angiogenic mediators in ex vivo TCM correlate with DC maturation levels.

In summary, TCM from different gastrointestinal tumour types induce different effects on DC maturation with OAC inducing significant activation, rectal cancer inducing minor activation and colonic cancer inducing inhibition, with 2Gy-irradiated TCM enhancing DC inhibition.

Poster abstracts


79

39

A DNA Repair Enzyme Signature to characterize the DNA Damage Response of patients treated for Head and Neck Cancer

Giovanna Muggiolu5, Sarah Libert5, Alexandra Lauret2, Philippe Ceruse3, Christian Adrien Righini4, Claire Rodriguez_Lafrasse1, Sylvie Sauvaigo5

1 Civil Hospital of Lyon, Lyon-Sud Hospital, Pierre-Bénite, FRANCE, 2 CNRS/UMR 5822, University of Medicine Lyon-Sud, Oullins, FRANCE, 3 Head Neck Department, University Claude Bernard Lyon 1, Lyon, FRANCE, 4 Head Neck Surgery Department, CHU of Grenoble, Grenoble, FRANCE, 5 LX Repair, Grenoble, FRANCE

Worldwide, 14 million of new cancer cases occur per year and more than 8 million of cancer deaths are registered. Treatment for about half patients entails radiation and chemotherapy which can be associated with tumor resistance over time. In addition, about 5-10% of patients also develop acute toxicity after radiotherapy.

Defining sub-group of patients according to their sensitivity/resistance is one of the main developments for increasing therapeutic effects. Therefore there is a need for markers identification able to predict the occurrence of adverse effects and thus, adapt the treatment for each patient.

Intrinsic tumors resistance and individual sensitivity are determined by both the ability of chemo/radiotherapy to induce DNA damage and cells capability to sense and repair DNA lesions. The objective of this project consists of profiling the DNA Damage Response to characterize effective individuals DNA repair capacity. We proposed innovative biochip-based technologies to investigate the contribution of several DNA repair pathways, simultaneously. Multiplexed biochips are functionalized with a panel of specific DNA lesions which are repaired by the enzymes present in the cells extract. Enzyme activities lead to fluorescent changes which are quantified. Then, the enzymatic signatures obtained can be associated with treatment resistance or hypersensitivity.

Blood samples and tumor biopsies of patients afflicted with head and neck cancers were collected before and in the time course of treatment. Whole cell extract was prepared from all samples. The glycosylases and AP endonucleases activities were evaluated with the Glyco-SPOT assay; excision/synthesis repair mechanisms were measured with the ExSy-SPOT assay.

The preliminary results of this approach show an inter-individual variability in DNA repair capacities at basal level in both PBMCs and tumor cells. We believe that these assays help stratify patients and tumours sensitivity in according to DNA Repair Enzyme signature.

The study was supported by a grant from Cancéropôle Lyon Auvergne-Rhône-Alpes CLARA « Preuve du Concept » program.

Poster abstracts


80

40

Role of Ssb1 and Ssb2 in maintenance of genomic stability

Purba Nag2,1, Amanda Bain2, Alejandro Lopez1,2, Kum Kum Khanna2

1 Griffith University, Brisbane, QLD, AUSTRALIA, 2 QIMR Berghofer Medical Research Institute, Brisbane, QLD, AUSTRALIA

Single strand DNA (ssDNA) binding proteins (SSBs), are known key players of the DNA damage response (DDR) and play an essential role in stabilising fragile ssDNA generated during DNA replication, transcription and repair. Our lab recently described two new SSBs, termed Ssb1 and Ssb2, which form independent co-complexes with two additional proteins- IntS3, which is a subunit of the multiprotein complex called Integrator and a small acidic protein named C9orf80. Previously, we demonstrated that whilst Ssb1 knockout caused perinatal lethality, Ssb2 knockout did not lead to any phenotypic abnormalities. Interestingly however, knockout of Ssb1 led to stabilisation of Ssb2 and vice-versa, indicating functional redundancy between these two proteins. This was recently demonstrated in-vivo by the generation of Ssb1/Ssb2 double-knockout mice, which caused early embryonic lethality in a constitutive model and acute bone marrow failure and intestinal atrophy using the inducible Rosa26-CreERT2 system.

To delineate the functional redundancy between these two proteins at the molecular level, we have generated inducible double-knockout mouse embryonic fibroblast (MEFs) using the Rosa26-CreERT2 system. We found that cumulative loss of SSBs in immortalised MEFs led to acute proliferation arrest and cell death following tamoxifen administration. This was associated with accumulation of genomic instability via endogenous replication stress. Interestingly, although ectopic overexpression of Ssb1 could restore this phenotype, expression of a mutant defective in IntS3 binding was not able to, illustrating the critical importance of the Ssb-Integrator interaction. Indeed, we observed defective processing and accumulation of polyadenylated U2 small nuclear RNA (snRNA) in Ssb1/Ssb2 double knockout cells, suggesting that the canonical Integrator function is perturbed. Supporting this hypothesis, RNA-seq analysis has also revealed a novel role of SSBs in regulation of alternative splicing. Together, these findings reveal the critical role of Ssbs and their association with the Integrator complex in regulating cellular proliferation, genomic stability and spliceosomal function.

Poster abstracts


81

41

RNF8 homeostasis: an attractive target for cancer treatment

Abhay Narayan Singh1, Judith Oehler1, Ignacio Torrecilla1, Benedikt Kessler2, Kristijan Ramadan1

1 CRUK/MRC Oxford Institute for Radiation Oncology, Department of Oncology, University of Oxford, Oxford, UK, 2 Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK

DNA damage response (DDR) is a robust cellular response initiated after DNA damage and routinely targeted for an effective genotoxic cancer therapy. Due to high therapeutic importance, identification of novel DDR factors is always in demand. DDR is regulated by a series of post translational modifications (PTMs) and ubiquitination emerged as a key PTM. RNF8 is a ring E3 ubiquitin ligase which initiates ubiquitination at the site of DNA damage. High cellular RNF8 level is tumour-promoting and positively correlates with genome instability, cancer cell invasion, metastasis and poor patient prognosis. Although it is obvious that the spatiotemporal regulation of RNF8 is essential for genome stability and protection from cancer, there is hardly anything known about the mechanisms that regulate RNF8 level. Here, we identify the cellular machinery to regulate RNF8 homeostasis under physiological and genotoxic conditions. RNF8 is degraded by ubiquitin proteasome system (UPS) and the rate of degradation is regulated by ubiquitin dependent segregase p97/VCP in cooperation with a deubiquitinating enzyme ATX3. This cellular machinery regulates the homeostasis of RNF8 soluble pool under physiological condition. However, after genotoxic stress RNF8 quickly recruits at the site of DNA damage and p97-ATX3 system stimulates its chromatin extraction to prevent RNF8 hyper accumulation at damage site. Pathological hyper accumulation of RNF8 at damage site attenuates downstream signalling which specifically leads to a defect in non-homologous end joining (NHEJ) repair pathway. We propose that the p97-ATX3 complex is the essential cellular machinery for regulation of RNF8 homeostasis and targeting this complex is a promising strategy to radio-sensitise homologous recombination defective (BRCA deficient) cancers.

Poster abstracts


82

42

MiR-105-5p, miR-767-5p and miR-6499-5p expression is associated with chemoradiotherapy failure and shows oncogenic potential in cervical cancer

Anja Nilsen2, Torleif Tollefsrud Gjølberg2, Eva-Katrine Aarnes2, Marte Jonsson2, Gunnar B. Kristensen1, Heidi Lyng2

1 Department of Gynecologic Oncology, Oslo University Hospital, Oslo, NORWAY, 2 Department of Radiation Biology, Oslo University Hospital, Oslo, NORWAY

Patients with locally advanced cervical cancer (LACC) are treated with chemoradiotherapy. Many patients experience recurrence after treatment, and identification of prognostic biomarkers is important. We aimed to investigate the potential of microRNAs (miRNAs) as prognostic biomarkers.

The miRNA transcriptome was measured in an explorative cohort of 90 LACC tumors by Illumina deep sequencing of biopsies taken before the start of treatment. Cluster analysis revealed a set of 3 co-regulated miRNAs (miR-105-5p, miR-767-5p, miR-6499-5p), for which elevated expression was associated with higher risk of recurrence. A score calculated from the expression data was associated with recurrence both in the explorative cohort and a validation cohort of 110 patients. The three miRNAs were therefore considered to be candidate prognostic biomarkers. In the merged cohort of 200 patients, the score had prognostic impact independent on traditional clinical markers like tumor stage, tumor size or lymph node status, suggesting that the score provides information about tumor progression that is not covered by the clinical markers.

Cell culture experiments were conducted to investigate roles of the miRNAs in carcinogenesis and radiation response. Upregulation of miR-6499 and miR-767, but not miR-105-5p, by transfection with miRNA mimics, was found to increase clonogenic survival after 4 Gy in the SW756 cervical cancer cell line, suggesting that miR-6499 and miR-767 promote radiation resistance. Assessment of H2Ax phosphorylation by flow cytometry up to 48 hours after irradiation revealed no difference in the level of DNA damage or in double-strand break repair capacity between transfected and control cells. Upregulation of miR-105-5p and miR-767-5p increased proliferation of SW756 cells, as measured with the IncuCyte® confluence proliferation assay.

These data suggest that miR-105, miR-767 and miR-6499 may serve as prognostic markers for cervical cancer, and indicate potential oncogenic roles of the miRNAs in radiation response and cell proliferation.

Poster abstracts


83

43

Trisomy 21 and double-strand breaks: a causative relationship and implications for cancer profile in Down Syndrome?

David Koschut1, Reinhard Brunmeir1, Debleena Ray1, Aoife Murray1, Yee-Jie Yeap1, Segey Nikolaev2, Stylianos Antonarakis2, Jurgen Groet3, Emanuela Giarin4, Giuseppe Basso4, Dean Nizetic1,3

1 1Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore, SINGAPORE, 2 2Dept. of Genetic Medicine and Development, University of Geneva Medical School, Geneva, SWITZERLAND, 3 3The Blizard Institute, Barts&The London School of Medicine, Queen Mary University of London, London, UK, 4 4Dipartimento di Salute della Donna e del Bambino, IRP—Istituto di Ricerca Pediatrica—Fondazione Citta` della Speranza, University of Padova, Padua, ITALY

Though paradoxically having a much lower incidence and mortality from a range of solid tumours, people with Down Syndrome (DS) (caused by trisomy 21 (T21)) have an approximately 50-100 fold higher overall incidence of leukaemias in childhood than normal children, including all types of acute myeloid leukaemia (AML) and B-cell acute lymphoblastic leukaemia (ALL). Children with DS are prone to suffer a relapse and have a higher risk of death from therapy-related side effects. Paradoxically though, individuals with DS have a substantially reduced incidence of second malignancies following radiation therapy, even at a juvenile age. We previously reported (Nikolaev et al. Nature Comm. 2014) the discoveries that 36% of DS-ALLs have RAS mutations and that RAS mutations are mutually exclusive with JAK2 mutations (Pval = 0.016) in DS-ALL. A discussion of unpublished new data on molecular mechanisms behind this mutual exclusion will be presented. Another unpublished observation made from exome-sequencing data is that there appears to be a significantly increased “passenger” mutation rate per year of age in DS haematopoietic cells in vivo. Though this result could be partly biased by an increased number of cell divisions during pre-leukaemic clonal evolution, the number of passenger mutations showed a highly significant correlation with patient age, even in the short age-span (1-16 years, Kendall-tau non-parametric rank coefficient=0.45, p=0.023). We also detected that T21 causes a significantly increased number of DNA double strand breaks (γH2AX foci) in undifferentiated proliferating hiPSCs, post-mitotic neurons derived from hiPSCs, as well as in a transchromosomic mouse model of DS. Experiments aimed at identifying the chromosome 21 genes whose trisomic overdose is responsible for this increased DNA damage are on-going and will be presented.

Poster abstracts


84

44

Radiobiology of targeted radionuclide therapy to improve treatment effects

Danny Feijtel1,2, Gabriela N. Doeswijk2, Winnie van den Boogaard1, Joost C. Haeck2, Mark W. Konijnenberg2, Dik C. van Gent1, Marion de Jong2, Julie Nonnekens1,2

1 Erasmus MC, Department of Molecular Genetics, Rotterdam, NETHERLANDS, 2 Erasmus MC, Department of Radiology and Nuclear Medicine, Rotterdam, NETHERLANDS

Radiobiological principles of external beam- and brachy radiotherapy have been studied for decades, while the radiobiology of radionuclide therapy is still in its infancy. During peptide receptor radionuclide therapy (PRRT) of metastasized neuroendocrine tumors (NETs) with overexpression of somatostatin receptors (SSTR), Lutetium-177 is targeted to the tumor via coupling to the somatostatin analogue DOTA-[Tyr3]octreotate (177Lu-DOTA-TATE). Lutetium-177’s b-particles (mean energy 0.133MeV) will induce DNA damage leading to tumor cell death with limited harm to healthy tissues. Patient treatment strongly increases progression-free survival and life quality. There is nevertheless still room for improvement, as very few patients are cured at this stage of disease. For possible future therapy optimizations, it is essential to have a better understanding of local treatment effects, both in tumor and healthy tissues.

To gain insight in the underlying radiobiological principles, we characterized the PRRT induced DNA damage response (DDR) in cell lines, ex vivo cultured human NET slices and xenografted mice. PRRT induces DNA double strand break (DSBs) which are repaired over time. Our results show that DDR inhibitors (PARPi, DNA-PKi and ATMi) differentially impair DNA repair (radiosensitizers) and vastly increase cell death in SSTR-positive cells and NET slices, while SSTR-negative cells are not sensitized to PRRT. Xenografted mice were followed for 14 days post PRRT and scanned with SPECT/MRI. Dosimetric calculations were performed based on both in vivo imaging and ex vivo biodistribution data. Our analyses show that PRRT produces DSBs in the tumor and dose limiting organs; the kidneys and bone marrow. DSBs in the tumor (dose 10.5Gy over 14days) are observed until at least 14days post treatment (also massive apoptosis induction), while DSBs in the bone marrow and kidneys (dose 3.7Gy over 14days) are only observed transiently until 3 days after treatment. This nicely illustrates the window of opportunity for combination therapy with DDR inhibitors.

Poster abstracts


85

45

Mechanistic insights into cellular responses to chromosomal damage induced by protons, a-particles and accelerated C-ions as studied by means of the Premature Chromosome Condensation assay

Antonio Pantelias3, Roberto Cherubini1, Demetre Zafiropoulos1, Lucia Sarchiapone1, Viviana De Nadal1, Laura Baggio2, Gabriel Pantelias3, Georgia Terzoudi31 INFN-Laboratori Nazionali di Legnaro, Legnaro-Padova, ITALY, 2 Istituto Oncologico Veneto, Padova, ITALY, 3 NCSR “Demokritos”, Athens, GREECE

For precision cancer therapy based on DNA and chromosomal damage induction and repair, it is crucial to elucidate the mechanism underlying the effectiveness of different qualities of radiation and develop biomarkers to minimize tumour resistance and optimize therapeutic strategies. In particular, proton irradiation offers a substantial clinical advantage over conventional photons because of its unique depth-dose characteristics, which can be exploited to achieve significant reductions in normal tissue doses. However, current data on chromosomal damage as analysed at metaphase vary significantly due to the complications introduced by cell cycle kinetics and the difficulties of irradiated cells to reach mitosis. To overcome these issues, the initial number and repair kinetics of excess chromosome fragments were investigated using the fusion premature chromosome condensation (PCC) methodology. In the present work, G0-human lymphocytes isolated from whole blood were exposed to a proton beam with an incident energy of 2.2 MeV and incident LET of 28.5 keV/μm in the dose range 0-6 Gy, and repair kinetics as quantified by the number of residual fragments were carried out for 2,6,12 and 24 hours post-irradiation. A significant increase in the initial excess chromosome fragments was observed in comparison to those obtained by γ-rays, alpha-particles and accelerated Carbon-ions. The distribution of residual G0-PCC breaks as well as the mean break number for protons differed from those for α-particles and C-ions and all were significantly higher when compared to those obtained for γ-rays. This result reinforces the notion that the LET- dependent structure in the irradiated lymphocytes is reflected in the repair processes. The different RBE values obtained for protons, α-particles and C-ions, based on chromatin breakage and formation of chromosomal aberrations, are indicative of their toxicity and crucial for micronuclei formation and chromothripsis, which are at the basis of both cell death and radiation induced carcinogenesis.

Poster abstracts


86

46

Histone H2B phosphorylation at Serine 14 marks nucleolar chromatin in response to DNA damage

Dafni Eleftheria Pefani1, Eric O’ Neill11 CRUK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Oxford, UK

Cells are continuously exposed to endogenous and environmental conditions that cause lesions in DNA which can lead to genomic instability. As DNA is organized to chromatin, dynamic changes of histone modifications are critical for regulating double strand break repair. Recent studies have shown that the position of a DNA break relative to chromatin influences the impact of the break on genomic stability.

The heavily transcribed rDNA repeats that give rise to the ribosomal RNA are clustered in a unique chromatin structure, the nucleolus. Due to its highly repetitive nature and transcriptional activity the nucleolus is considered a hotspot of genomic instability. Recent studies show that cells are highly sensitive to rDNA breaks, uncovering the nucleolus as an attractive target for cancer treatment design.

rDNA breaks induce a transient transcriptional shut down to conserve energy and promote rDNA repair. Recent observations show that perturbed rDNA transcription rates associate with DNA repair defects and genome instability, however how nucleolar chromatin is modified under these conditions and how these modifications impact on rDNA remains largely unknown.

Here we uncover that phosphorylation of Serine 14 on Histone2B marks transcriptionally inactive nucleolar chromatin in response to DNA damage. We identified that the MST2 kinase localizes at the nucleoli and targets phosphorylation of H2BS14 in an ATM dependent manner. We show that establishment H2BS14p is necessary for damage induced rDNA transcriptional shut-down and genomic integrity maintenance. Absence of MST2 kinase or upstream activators results in defective establishment of nucleolar H2BS14p, perturbed DNA damage repair, sensitisation to rDNA damage and increased cell lethality. We highlight the impact of chromatin regulation in the rDNA damage response and targeting of the nucleolus as an emerging cancer therapeutic approach.

Poster abstracts


87

47

OGG1 inhibitors: A new way of exploiting DNA damage to treat cancer

Armando Cázares-Körner4, Torkild Visnes4, Wenjing Hao3, Olov Wallner4, Elisee Wiita4, Oliver Mortusewicz4, Tove Bekkhus4, Sofia Henriksson4, Saeed Eshtad4, Judith Unterlass4, Aleksandra Pettke4, Olga Loseva4, Antonio Sarno3, Stella Karsten4, Alexandr Manoilov2, Juan Astorga-Wells2, Kumar Sanjiv2, Thomas Lundbäck1, Hans Einar Krokan2, Roman Zubarev2, Istvan Boldogh3, Thomas Helleday4

1 Chemical Biology Consortium Sweden, Science for Life Laboratory, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, SWEDEN, 2 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, SWEDEN, 3 Department of Microbiology and Immunology, Sealy Center for Molecular Medicine, University of Texas Medical Branch at Galveston, Galveston, Texas, USA, 4 Sciliefelab, Division of Translational Medicine and Chemical Biology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, SWEDEN

High levels of reactive oxygen species (ROS) which are present in most cancer cells, lead to accumulation of oxidative DNA damage. Base excision repair processed by glycosylases is the main mechanism to repair it.

It has become increasingly clear in recent years, that targeting DNA repair is an interesting approach to fight cancer, especially in combination with DNA damaging therapies. However, there are only a few inhibitors of DNA damage repair enzymes.

We present a novel selective inhibitor of 8-oxoguanine-DNA glycosylase 1 (OGG1), a DNA glycosylase that excises 8-oxo-dG. Our inhibitor binds at the active site of the enzyme and thus prohibits OGG1 from binding to its substrate in cells and in vitro.

The inhibitor generated in our lab engages with its intended target in cells. It also impairs recruitment of OGG1 to damaged chromatin in cells, while at the same time increasing overall DNA damage in cancer cell lines.

A hallmark of cancer is the accumulation of DNA damage, followed by genomic instability and high sensitivity to blockage of DNA damage repair pathways. In our studies, we were able to show that indeed, non-transformed cells tolerate treatment with our compound, while it is toxic to cancer cell lines. This demonstrates cancer phenotype lethality. We were also able to show synergistic effects of our compound with tubulin poisons and MTH1 inhibitors.

In summary, we suggest that OGG1 inhibition may be a valuable addition to current anti-cancer therapies, especially after priming with DNA damaging therapies like radiation.

Poster abstracts


88

48

Effects of actin manipulation on the recruitment of nuclear DNA damage repair factors

Lisa Pfitzer1, Rebecca Smith2, Gyula Timinszky2, Angelika M Vollmar1, Stefan Zahler1

1 Department of Pharmacy, Ludwig Maximilian University Munich, Munich, GERMANY, 2 Department of Physiological Chemistry, Ludwig Maximilian University Munich, Munich, GERMANY

The role of the actin cytoskeleton is well characterized in the cytoplasm but only recently it has been shown that actin polymerization can also occur in the nucleus, upon stress signals such as DNA damage. However, up to now the exact functions of actin for DNA repair processes are not well understood.

We apply actin binding substances, such as the actin polymerizer Jasplakinolide and the depolymerizer Latrunculin B (LB) to investigate the role of actin in DNA double strand break (DSB) repair. We want to uncover the underlying mechanisms behind the involvement of actin in DNA repair and evaluate the application of actin binding substances as a novel strategy for combination therapy.

We could show that actin manipulation inhibits DSB repair by influencing specific signalling cascades in both homologous recombination and non-homologous end joining.

In homology derived repair (HDR) actin manipulation affected the recruitment of replication protein A (RPA) to the site of DNA damage, a process that is crucial for the initiation of both HDR and single strand annealing (SSA). LB treatment inhibited chromatin association of RPA upon damage induction and thus the ability to repair the break with HDR or SSA. As chromatin decondensation was not influenced by LB, we suggest that actin is directly involved in the recruitment of repair factors. This hypothesis was strengthened by our finding that RPA is bound to nuclear actin under control conditions and released after DNA damage induction.

Functionally, we saw synergistic effects of low dose combination therapy of Doxorubicin and LB on proliferation in different cancer cell lines.

Our findings imply a direct involvement of actin in DNA damage repair and propose a possible application of actin binding substances for combination therapy with DNA damaging agents.

Poster abstracts


89

49

Statistical re-evaluation of in vivo and ex vivo irradiated tumour samples revealed pronounced differences on DNA damage response

Treewut Rassamegevanon4, Steffen Löck4,1, Mechthild Krause4,3, Michael Baumann4,2, Cläre von Neubeck4,2

1 Department of Modeling and Biostatistics in Radiation Oncology, OncoRay, Dresden, GERMANY, 2 German Cancer Research Center (DKFZ), Heidelberg, GERMANY, 3 Helmholtz-Zentrum Dresden - Rossendorf, Institute of Radiooncology – OncoRay, Dresden, GERMANY, 4 OncoRay – National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technische Universität Dresden, Helmholtz-Zentrum Dresden - Rossendorf, Dresden, GERMANY

Determination of phosphorylated histone H2AX (γH2AX), a DNA double strand break marker, demonstrates a great potential to be further developed as a predictive assay, by which treatment individualisation and improvement of therapeutic outcome could be achieved. Tumour heterogeneity, however, causes a strong deviation in radiation-response estimation [1]. This study aims to determine tumour heterogeneity in γH2AX foci assay in two previously published datasets with comparable experimental designs exempt sampling and radiation exposure condition i.e. in vivo [2] and ex vivo [1]. Data were processed and subjected to linear mixed-effects model, where fixed- and random effects were incorporated in the analyses. The statistical reanalysis revealed significant intra-tumoural heterogeneity of initial γH2AX foci (30 minutes post irradiation) for the in vivo set-up in most of evaluated tumour lines. Residual γH2AX foci (24 hours post irradiation) of the ex vivo setup exhibited a higher degree of intra-tumoural heterogeneity compared to the in vivo setup. In both experimental settings, a slight enlargement of nucleus size post irradiation was found in most of the analysed tumour lines. Difference of nucleus size was present between the in- and ex vivo setups depending on tumour lines. In conclusion, the greater degree of heterogeneity in γH2AX foci for the ex vivo setup and the difference in nucleus size between the experimental settings indicated a strong influence of the ex vivo culturing in cellular adaptation and DNA damage management. An automated foci evaluation platform has been developed to reduce inter-observer variations, increase sample throughput and further enhance clinical applicability of γH2AX foci assay.

Acknowledgement: This work was supported by a grant of the Federal Ministry of Education and Research (BMBF 02NUK035C).

References: [1] Rassamegevanon T. et. al., Radiother. Oncol. 124(3):379-385 (2017) [2] Koch U. et al., Radiother. Oncol. 108:434–39 (2013)

Poster abstracts


90

50

Molecular hydrogen as a neuroprotective cytoprotective agent and a therapeutic antioxidant

Amir Roland1

1 Centre de Santé des Fagnes, CHIMAY, BELGIUM

Emerging evidence by different publications with, more often, converging data, has consistently demonstrated that molecular hydrogen (H2) is a promising option for a variety of diseases and the underlying comprehensive mechanism is, among others ones, hydroxyl radicals scavenging.

The non-toxicity at high concentrations and rapid cellular diffusion features of H2 ensure the feasibility and readiness of its clinical translation to human patients.

There is a growing body of evidence based on the results of animal experiments and clinical observations that H2 may represent an effective antioxidant for the prevention of oxidative stress-related diseases.

Application of H2 in situations, by several pathologies at different localisations, with excessive protection of free radicals, in particular, hydroxyl and nitrosyl radicals, has been well documented and, on the basis on preliminary results, relatively conclusive.

Al though most neurological disorders are currently incurable, recent studies suggest the clinical potential of H2 administration for their prevention, treatment and mitigation. Several of the potential effectors of H2 will also be discussed, including cell signalling molecules and hormones that are responsible for preventing oxidative stress and inflammation. Nevertheless, further investigations will be required to determine the direct target molecule of H2.

Many recent studies confirm more and more than H2 may become in the future a novel therapeutic treatment or an adjuvant tool which may be useful when associated with other treatment or may have a protective effect on liver function of colorectal patients treated with mFolfox6 chemotherapy.

Moreover, radiation may induces tissue injury at the cellular level. To protect healthy tissue surrounding tumor, H2 may afford effective protection.

Poster abstracts


91

51

Radiotherapy combined with immune checkpoint blockade immunotherapy and theranostic nanoparticles in treatment of malignant brain tumors

Maxim Shevtsov4,5, Stephan Stangl4, Emil Pitkin6, Boris Nikolaev3, Ludmila Yakovleva3, Yaroslav Marchenko3, Vyacheslav Ryzhov2, Boris Margulis1, Irina Guzhova1, Gabriele Multhoff4

1 Institute of Cytology of the Russian Academy of Sciences (RAS), St.Petersburg, RUSSIAN FEDERATION, 2 NRC “Kurchatov Institute”, Petersburg Nuclear Physics Institute, Gatchina, RUSSIAN FEDERATION, 3 Research Institute of Highly Pure Biopreparations, St.Petersburg, RUSSIAN FEDERATION, 4 Technische Universität München, Klinikum rechts der Isar, Munich, GERMANY, 5 University College of London, London, UK, 6 Wharton School, University of Pennsylvania, Philadelphia, USA

Combinatorial regimens based on the radiotherapy, theranostic tumor-targeting nanoparticles and blocking of immune checkpoint inhibitors could further enhance the therapeutic benefit in the management of multiforme glioblastoma. We have designed superparamagnetic nanoparticles coupled with granzyme B (GrB-SPIONs) that specifically target membrane Hsp70-positive cancer cells. In vitro studies utilizing confocal and electron microscopies demonstrated that nanoparticles accumulated in the cytoplasm (via the endocytosis pathway) after specific binding to Hsp70 that is presented on the membrane of cancer cells, but not on corresponding normal cells. Intracellular uptake of the magnetic conjugates resulted in apoptic tumor cell death (due to the pro-apoptotic activity of granzyme B). Due to the magnetic properties of the nanocarriers we could track GrB-SPIONs (using magnetic resonance imaging and high sensitive non-linear magnetic response measurements NLR-M2) following intravenous injection in a murine glioblastoma model and their specific retention in the tumor. Monotherapy with GrB-SPIONs in the orthotopic models of human U87 glioma in NMRI nu/nu mice, GL261 glioma in C57/Bl6 mice and C6 glioma in rats resulted in a significantly delayed tumor progression (as shown by magnetic resonance imaging) and an increased overall survival. Subsequent combination of single dose (10 Gy) ionizing radiation of brain tumors and therapy with pro-apoptotic GrB-SPIONs further increased the therapeutic benefit. Combination therapy of anti-CTLA-4 and anti-PD-1 antibodies with radiotherapy and targeted nanoparticles increased long-term survival in animals. Inhibitory immune checkpoint blockade quantitatively enhanced infiltration of glioma with activated CD8+ and natural killer (NK1.1) cells and decreased suppressive PD-1+ cells in the tumor microenvironment. In conclusion, our results support the preclinical proof-of-concept of synergistic activity in combination of radiotherapy, nanotechnology and immunotherapy in the treatment of glioblastoma.

Poster abstracts


92

52

DNA double-strand break repair pathway regulates PD-L1 expression in cancer cells

Hiro Sato1, Atsuko Niimi1, Takaaki Yasuhara3, Tiara Bunga Mayang Permata11, Yoshihiko Hagiwara1, Takahiro Oike1, Kathryn D. Held2,1, Takashi Nakano1, Atsushi Shibata1

1 Gunma University, Maebashi, JAPAN, 2 Massachusetts General Hospital/Harvard Medical School, Boston, USA, 3 The University of Tokyo, Tokyo, JAPAN

Immune checkpoint therapy has recently emerged as a promising next-generation cancer treatment. Anti-PD-1 antibody restores antitumor immune responses by disrupting the interactions between PD-1 and its ligand, PD-L1, thereby providing effective antitumor effects. Since high PD-L1 expression is related to poor prognoses for several types of tumours, it is important to elucidate the molecular mechanisms underlying PD-L1 expression. Recent evidence suggests that exogenous cellular stress upregulates PD-L1 expression in cancer cells. DNA double-strand break (DSB) is the most critical type of genotoxic stress; however, the involvement of DSB repair in PD-L1 expression has not been investigated to date. In this study, we show that PD-L1 expression in cancer cells is upregulated in response to DSB. This upregulation requires ATM/ATR/Chk1 kinase activities. Using an siRNA library targeting DSB repair genes, we show that BRCA2 depletion enhances Chk1-dependent PD-L1 upregulation after X-ray or PARP inhibition. Moreover, Ku70/80 depletion substantially enhances PD-L1 upregulation after X-ray. The upregulation by Ku80 depletion requires Chk1 activation following DNA end-resection by Exonuclease 1. DSBs activate STAT1 and STAT3 signalling, and IRF1 is required for DSB-dependent PD-L1 upregulation. Thus, our findings reveal the involvement of DSB repair in PD-L1 expression and provide mechanistic insight into how PD-L1 expression is regulated after DSB (Sato et al., Nature Communications, 2017). In this presentation, our latest data on the mechanism of PD-L1 upregulation after oxidative stress will be also presented. Understanding the molecular mechanisms underlying PD-L1 expression in cancer cells may help the development of strategies to predict prognosis. It may also contribute to improving the efficacy of therapies in DNA-repair-defective cancer cells, particularly when anti-PD-1 therapy and radiotherapy are combined.

Poster abstracts


93

53

Proton-sensitizing Effect of C646, a small molecule inhibitor of p300 histone acetyltransferase in human paNcreatic cancer cells

Hee Chul Park1,2, Sung-won Shin1,2, Changhoon Choi1, Shin-yeong Kim1

1 Department of Radiation Oncology/Samsung Medical Center, Seoul, SOUTH KOREA, 2 Sungkyunkwan University School of Medicine, Seoul, SOUTH KOREA

Histone acetylation plays an important role in chromatin remodeling and is tightly regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs). Recent studies have demonstrated that inhibitors of these chromatin-remodeling proteins can modulate cellular responses to other cytotoxic modalities including ionizing radiation and chemotherapeutic drugs. In contrast to well-studied HDAC inhibitors, little is known about the cancer therapeutic potential of HAT inhibitors. In this study, we investigated the proton beam-sensitizing effect of C646, a selective small molecule inhibitor of p300 HAT in human pancreatic cancer cells in vitro and in vivo. C646 sensitized BxPC-3 and MiaPaca-2 cells to proton beam irradiation by enhancing apoptotic cell death through the down-regulation of anti-apoptotic molecules. Pretreatment with C646 led to a steady increase in sub-G1 population and abolishment of proton-induced G2/M arrest. In addition, the combination of C646 and proton beam therapy was most effective in inhibiting tumor growth in BxPC-3 xenograft tumor models. These results suggest that C646 may be a potential agent to increase proton sensitivity of human pancreatic cancer cells.

Poster abstracts


94

54

Kinome-wide synthetic lethality screen to identify the genetic dependencies in DIPG survival to irradiation

Claudia Carolina Silva Evangelista4, Emilie Barret4, Coralie Werbrouck4, Jane Merlevede4, Ambre Saccasyn4, Stephanie Puget3, Jacques Grill4,2, David Castel4,2, Marie-Anne Debily4,1

1 Département de Biologie, Université Evry Val-d’Essonne, 91037, Evry, FRANCE, 2 Département de Cancérologie de l’Enfant et de l’Adolescent, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay, 94805, Villejuif, FRANCE, 3 Département de Neurochirurgie Pédiatrique, Hôpital Necker-Enfants Malades, Université Paris V Descartes, Paris, 75015, FRANCE, 4 UMR8203 «Vectorologie and Thérapeutiques Anticancéreuses», CNRS, Gustave Roussy, Univ. Paris-Sud, Université Paris-Saclay, 94805, Villejuif, FRANCE

Diffuse Intrinsic Pontine Glioma (DIPG) is the most fatal form of pediatric brain tumor, killing almost every child within two years after diagnosis. These neoplasms are inoperable due to their infiltrative nature and their deep-seated location in the brainstem. They are chemoresistant, and radiotherapy is only transiently effective. Consequently, these tumors are considered one of the biggest challenges of pediatric oncology. A specific somatic mutation (H3K27M) in the H3 histone gene was discovered recently and found in around 95% of DIPG and is considered as the initial oncogenic event. Two subgroups of patients, with distinct oncogenic programs and response to radiotherapy can be defined according to the H3 histone gene mutated encoding H3.1 and H3.3 protein variants.

To identify genes that can sensitize DIPG to radiotherapy, we conducted a kinome-wide shRNA screen. Four H3K27M DIPG cells derived from patients’ biopsies harboring either H3.1 (n=2) or H3.3-mutation (n=2) were analysed to cover the diversity observed in patients. An integrative lentiviral pooled library of 7450 shRNAs was used and the entire set of shRNAs was identified by next-generation sequencing 22 days after transduction, with or without irradiation at lethal dose 25%. The target genes with at least 3 distinct shRNAs presenting a significant decrease in their abundance in irradiated vs. untreated samples were selected. Preliminary analysis indicates that only few genes increase the radiosensitivity of H3.3-K27M cells when knocked-down and none was shared by the 2 cell-lines. In contrast, 16 target genes were found as impairing similarly cell survival in conjunction with radiation in the 2 H3.1-K27M models. We are currently investigating the mechanisms underlying these differences in radioresistance, never described in DIPG. Moreover, a second screening at higher dose is currently ongoing in H3.3-K27M models. This screen will provide valuable information on DIPG radioresistance, and identify potential new adjuvants to radiotherapy.

Poster abstracts


95

55

67Ga-trastuzumab: proof of principle of targeted Auger electron-emitting radiotherapy

Muhamad Faiz bin Othman2, Margaret S Cooper2, Cinzia Imberti2, Michelle T Ma2, Valerie J Lewington1, Philip J Blower2, Samantha YA Terry2

1 Guy’s & St Thomas’ NHS Foundation Trust, London, UK, 2 King’s College London, London, UK

Purpose: Previously, we showed that Auger electron emitter 67Ga causes DNA damage in cell-free systems and cell kill in non-targeted cell studies. Here, we target 67Ga to breast cancer cells using trastuzumab and compare toxicity to well-described Auger electron emitter, 111In.

Methods: THP- and DOTA-trastuzumab were prepared and labeled with 67Ga and 111In, respectively. Radiopharmaceuticals (0.19 MBq/μg) were tested for their internalisation and effects on viability (dye exclusion) and clonogenicity of Her-2-positive (HCC1954) and –negative (MDA-MB-231) cell lines. Microautoradiography of cells in 18% gelatin was also performed.

Results: Radiopharmaceuticals specifically bound Her2-positive HCC1954 cells. At 4nM, 67Ga-THP-trastuzumab showed significantly higher cell binding uptake (10.69±1.32%) than 111In-DOTA-trastuzumab (6.15±1.64%; p=0.01) although internalised fractions were equal as were cell-binding percentages at 100nM. Microautoradiography showed radioactivity bound individual cells to varying degrees (from <10 to >90 silver grains per cell). Viability and clonogenicity decreased with increasing radiolabeled trastuzumab concentration. In HCC1954 cells, the surviving fraction after treatment at approximately 0.1 Bq/cell 67Ga-THP-trastuzumab (average) reduced to 0.38±0.13, with fewer cells surviving than for 111In-DOTA-trastuzumab (0.55±0.16; p=0.03). Radiopharmaceutical treatment of MDA-MB-231 cells or non-internalised activity in HCC1954 cells did not affect cell viability or clonogenicity.

Conclusion: 67Ga-THP-trastuzumab and 111In-DOTA-trastuzumab both specifically bind HER2-positive cells and reduce their viability and clonogenicity. This shows 67Ga holds promise as a therapeutic radionuclide as a targeted radiopharmaceutical however non-homogeneous uptake amongst cells needs further investigation.

Acknowledgments: The project was in part supported by the Academy of Medical Sciences and Malaysian Ministry of Education.

Poster abstracts


96

56

Therapeutic efficacy through intravenous administration of the DNA repair inhibitor AsiDNA in Triple Negative Breast Cancer

Hélène Toussaint1, Wael Jdey1, Richard Tripelon1, Vincent Hayes1, Jean-Louis Labernardière1, Françoise Bono1

1 Onxeo, Paris, FRANCE

Background: AsiDNA, a double stranded DNA molecule, acts as a bait for DNA repair enzymes and abrogates DNA repair by inducing a false DNA damage signaling. AsiDNA molecules are recognized by the DNA-PK proteins as double-strand DNA breaks (DSBs) allowing a broad repair inhibitor activity, rather than a specific protein targeting. AsiDNA has previously demonstrated anti-tumor efficacy in vitro and in vivo in genetically instable tumor models through either intratumoral, subcutaneous or intraperitoneal administration pathways.

Methods: Antitumor efficacy and biomarkers correlation was evaluated in nude mice bearing orthotopic human breast tumor cells treated by intravenous (IV) 1-hour infusion injection of AsiDNA alone and following different administration schedule. Because DSBs induce the phosphorylation of histone H2AX and the phosphorylation of HSP90 protein, AsiDNA activity can be followed using these two biomarkers.

Results: Biomarkers of DNA damage analyses using immunohistochemistry after repeating treatment demonstrate AsiDNA sequesters and sustainably hyperactivates the DNA repair proteins in the tumor. AsiDNA-induced false signaling is more sustained after 3 consecutive days of repeated treatments, compared to one single administration. Moreover, plasmatic exposure and biomarker activation is associated to an anti-tumor efficacy. Finally, repeating AsiDNA treatment, either daily during three consecutive days or once weekly gives a higher tumor control.

Conclusions: These results demonstrate that in a highly resistant model AsiDNA administered through IV/infusion route displays significant anti-tumor efficacy in monotherapy. This protocol will be shortly tested in Phase I, open label, dose escalating trial by 1 hour administration IV in patients with solid tumor.

Poster abstracts


97

57

Radiation-induced synthetic lethality to sensitise NSCLC cells to proton and X-ray irradiation: Combined effects of PARP and RAD51 inhibitors

Anne-Catherine Wéra1,2, Alison Lobbens1,2, Stéphane Lucas1,2, Carine Michiels1,2

1 NAmur Research Institute for LIfe Sciences, Namur, BELGIUM, 2 University of Namur, Namur, BELGIUM, 3 URBC-NARILIS, Namur, BELGIUM

During the course of their treatment, about 50% of cancer patients will undergo radiotherapy. An emerging radiotherapy technique is the use of high energy charged particles like protons or carbon ions instead of X-rays as they present a clear benefit thanks to their depth dose profile. Although improvements were made over the years, radioresistance is still a major challenge and a lot of work is performed to find ways to radiosensitise cancer cells. In this context, the fact that cancer cells are often deficient for DNA repair response may be exploited.

In this work, we studied the combination of poly(ADP-ribose) polymerase (PARP) and RAD51 inhibitors to induce synthetic lethality after proton and X-ray irradiation. In that case, the inhibitors were used at a concentration that led to limited toxicity, but to an increased cell death after irradiation.

For that purpose, the response of A549 cells was studied, in vitro, after irradiation with 225kV X-rays or 25keV/µm proton broad beam in the presence of two different inhibitors: Olaparib, a PARP inhibitor and B02, a RAD51 inhibitor. The cells were exposed to 0.5 µM of Olaparib and/or 10 µM of B02 for a total duration of 24 hours.

24h after irradiation, protein and mRNA level analysis indicated cell cycle arrest and persistent γ-H2AX foci were observed. 7 days after irradiation, cells exhibiting a senescent phenotype associated to increased beta-galactosidase activity were highlighted. Survival fractions were determined using clonogenic assay after irradiation. The inhibitors used in combination showed additive or synergistic effect after X-rays or protons, respectively. At 50% survival, the sensitivity enhancement ratio was around 1.4 for both irradiations and the relative biological effectiveness was equal to 3.8.

This work highlights promising alternatives for targeting cancer cell resistance to therapy while taking advantage of the ballistic of charged particles.

Poster abstracts


98

58

Downregulation of glycogen metabolism sensitises cancer cells to ionizing radiation

Christos Zois3,6, Syed Haider5, Elisabete Pires2, Dimitra Kalamida1, Christoffer Lagerholm6, John Morris4, James Mccullagh2, Adrian Harris3,6

1 Democritus University of Thrace, Thrace, UK, 2 Department of Chemistry/University of Oxford, Oxford, UK, 3 Department of Oncology/University of Oxford, Oxford, UK, 4 DPAG/University of Oxford, Oxford, UK, 5 Institute Cancer Research, London, UK, 6 WIMM/University of Oxford, Oxford, UK

Metabolic reprogramming is a hallmark of cancer cells and contributes to their adaptation within the tumour microenvironment and resistance to anticancer therapies. Glycogen metabolism has major role within tumor microenvironment and has become a recognized feature of cancer cells. It is upregulated in many tumour types, suggesting it is an important aspect of cancer cell pathophysiology. In this study, we found that downregulation of glycogen phosphorylase liver isoform (PYGL) significantly led to a decrease in cellular growth in breast and glioblastoma cancer cell lines. Further, we found that inhibition of PYGL greatly sensitises tumour cells to higher doses of radiation producing marked increases in senescence. This is not related to increased DNA damage or differences in DNA repair in response to radiation, but damage to other subcellular compartments. Inhibition of PYGL induces dysfunction of autophagy flux and display different mitochondria morphologies following ionizing radiation. Also, we found that inhibition of PYGL alters the metabolic profile following ionizing radiation as analysed by seahorse and metabolomics.

We are now investigating a series of novel compounds targeted to PYGL, previously developed for treating diabetes. Interactions with higher dose selectively targeted radiation may provide a critical development for treating primary tumours and low volume metastasis as a part of management of oligo metastatic disease.

This work was supported by Cancer Research UK and the Breast Cancer Research Foundation.

Poster abstracts


99

59

Development and Characterization of the Ultra Immunodeficient B6;129 Rag2tm1FwaIL2rgtm1Rsky/DwlHsd (R2G2) Mouse Model

Sheryl J. Wildt1, Jamie McClellan1, Mandy Horn1, Krishnan Kolappaswamy1

1 Envigo, Huntingdon, UK

The “R2G2” (B6;129-Rag2tm1FwaIL2rgtm1Rsky/DwlHsd) knockout mouse is the latest advancement to provide a different option in the highly immunodeficient mouse model category for the oncology and immunology research communities. This model was generated by backcrossing an IL2rg (common gamma) knockout model to a RAG2 (recombinase activating gene) knockout model. The resulting mouse lacks various cytokines including IL-2, IL-4, IL-7, IL-9 and IL-15. In addition, this model lacks B cells, T cells, NK cells and has a deficit in lymphocyte development. The R2G2 model is not only ultra immunodeficient, but provides a model that is less leaky and more tolerant to gamma radiation than traditional SCID models. This model is particularly useful for both tumor transplantation and humanization studies. Herein we describe the development and characterization of the R2G2 model, including breeding history, growth curve, CBC/Chemistry, flow cytometry and both huHSC and huPMBC humanization studies.

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Molecular Pathology Approach to Cancer

8th EACR-OECI Joint Course 04 - 06 June 2018AMSTERDAM NETHERLANDS

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