from basic cell death mechanisms to novel cancer treatments a5... · a matter of life or death:...

Programme Book

01 - 03 February 2018Amsterdam, Netherlands

Scientific Programme CommitteeSimone Fulda || Verena Jendrossek (Chair) || Jan Paul Medema

4th EACR Conference

A Matter of Life or Death From Basic Cell Death Mechanisms to

Novel Cancer Treatments

A Matter of Life or Death: From Basic Cell Death Mechanisms to Novel Cancer TreatmentsAmsterdam, Netherlands, 01 - 03 February 2018

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Molecular Pathology Approach to Cancer

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

Coming soon...


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01 - 03 February 2018 • Amsterdam, Netherlands

A Matter of Life or DeathFrom Basic Cell Death Mechanisms to Novel Cancer Treatments

Day 1 - Thursday 01 February

11.00 – 13.00 REGISTRATION Rode Hoed foyer

12.00 – 13.00 WELCOME LUNCH & TRADE EXHIBITION including tea/coffee

13.00 – 13.05 CONFERENCE WELCOME Oosterhuiszaal Scientific Programme Committee

SESSION 1: MECHANISTIC INSIGHT IN THE REGULATION OF AUTOPHAGY AND THE ROLE OF SENESCENCE AS TUMOUR SUPPRESSORSession Chairs: Simone Fulda & Guillermo Velasco

13.05 – 13.35 OPENING KEYNOTE LECTURE Q&A: 13.35 – 13.50 Kevin Ryan Beatson Institute, UK ”Autophagy and cancer: issues we need to digest”

13.50 – 14.10 Tor Erik Rusten Oslo University Hospital, NorwayQ&A: 14.10 – 14.20 “Non-autonomous tumor growth support by autophagy”

14.20 – 14.30 Oriol de Barrios IDIBAPS, SpainQ&A: 14.30 – 14.35 Proffered Paper 1: ”The EMT-factor ZEB1 promotes tumor progression through inhibition of cell senescence”

14.35 – 14.45 EXHIBITOR INTRODUCTIONS

14.45 – 15.15 COFFEE BREAK & TRADE EXHIBITION


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SESSION 2: REGULATION OF CLASSICAL AND NON-CLASSICAL MODES OF APOPTOSISSession Chairs: Eugenia Piddini & Kevin Ryan

15.15 – 15.35 EMBO YOUNG INVESTIGATOR LECTURE Q&A: 15.35 – 15.45 Ana J. García-Sáez Universität Tübingen, Germany ”Bax and Bak at the single molecule level: telling the twins apart”

15.45 – 16.05 Jan Paul Medema AMC, NetherlandsQ&A: 16.05 – 16.15 ”Colorectal cancer, four subtypes with distinct biology”

16.15 – 17.45 POSTER DEFENCE SESSION 1 Lower Balcony Odd numbered posters (1, 3, 5, etc.) will be presented

17.45 – 19.00 NETWORKING RECEPTION & TRADE EXHIBITION With drinks & hot buffet dinner

19.00 – 19.30 EVENING KEYNOTE LECTURE Q&A: 19.30 – 19.45 Scott Dixon Stanford University, USA ”The regulation of ferroptosis in cancer cells”

Day 2 - Friday 02 February

08.30 – 09.00 POSTER VIEWING & TRADE EXHIBITION Coffee available to purchase

SESSION 3: ROLE OF CELL SURVIVAL AND DEATH SIGNALING IN CANCER SUSCEPTIBILITY, TUMOUR GROWTH AND THERAPY RESPONSESession Chairs: Jan Paul Medema & John Silke

09.00 – 09.20 Eugenia Piddini University of Bristol, UK Q&A: 09.20 – 09.30 “The role of cell competition in tumour promotion”

09.30 – 09.50 Peter de Keizer UMC Utrecht, NetherlandsQ&A: 09.50 – 10.00 “Targeted apoptosis of senescent cells against aging and cancer”

10.00 – 10.10 Yaron Fuch Technion, IsraelQ&A: 10.10 – 10.15 Proffered Paper 2: ”Caspase-3 Regulates YAP-dependent Cell Proliferation and Organ Size”

10.15 – 10.25 Michael Hahne CNRS / IGMM, FranceQ&A: 10.25 – 10.30 Proffered Paper 3: ”Primary cilia modulate susceptibility to colon carcinogenesis”


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11.00 – 11.20 Stephen Tait Beatson Institute, UKQ&A: 11.20 – 11.30 “Mitochondrial apoptosis and cancer: from phantom menace to a force awakens”

11.30 – 11.50 Giorgio Stassi Palermo University, ItalyQ&A: 11.50 – 12.00 “MAPK, PI3K and HER2 inhibition induces synthetic lethality in colorectal cancer stem cells regardless their mutational status”

12.00 – 13.00 BUFFET LUNCH & TRADE EXHIBITION

SESSION 4: IMPACT OF CELL DEATH ON IMMUNE RESPONSES TO CANCER AND CANCER THERAPIESSession Chairs: Ana J. García-Sáez & Manolis Pasparakis

13.00 – 13.20 Verena Jendrossek Universität Duisburg-Essen, GermanyQ&A: 13.20 – 13.30 “Normal tissue response to ionizing radiation and therapeutic targeting”

13.30 – 13.50 Sandra Demaria Weill Cornell Medical College, USA Q&A: 13.50– 14.00 “Mechanisms regulating the immunogenicity of radiation- induced cancer cell death”

14.00 – 14.10 Inge Verbrugge NKI, NetherlandsQ&A: 14.10 – 14.15 Proffered Paper 4: “Chemo-Radio-Immunotherapy: Cisplatin improves abscopal effects following radiotherapy and a-CD137/a-PD1 mAbs”

14.15 – 14.25 Jolien Bridelance VIB-UGent, BelgiumQ&A: 14.25 – 14.30 Proffered Paper 5: “Identification of molecular mechanisms essential for necroptosis execution by using a genome- wide CRISPR-Cas knockout screening”

14.30 – 14.50 Karin de Visser NKI, NetherlandsQ&A: 14.50 – 15.00 “The immune system: a double-edged sword in metastatic breast cancer”

15.00 – 16.30 POSTER DEFENCE SESSION 2 Lower Balcony Even numbered posters (2, 4, 6, etc.) will be presented

16.30 – 17.30 ROUND TABLE DISCUSSIONS Oosterhuiszaal

19.30 CONFERENCE DINNER Restaurant-Café In De Waag Pre-booked optional extra


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Day 3 - Saturday 03 February

08.30 – 09.00 POSTER VIEWING & TRADE EXHIBITION Coffee available to purchase

SESSION 5: CELL DEATH REGULATION IN CANCER AND THERAPEUTIC TARGETINGSession Chairs: Verena Jendrossek & Stephen Tait

09.00 – 09.20 Andreas Villunger Medical University of Innsbruck, AustriaQ&A: 09.20 – 09.30 “The PIDDosome – a signalling platform controlling p53 activation”

09.30 – 09.50 Simone Fulda UCT Frankfurt, GermanyQ&A: 09.50 – 10.00 “Novel opportunities to target cell death pathways for cancer therapy”

10.00 – 10.10 Prashanthi Ramesh AMC, NetherlandsQ&A: 10.10 – 10.15 Proffered Paper 6: “BCL-xL specific stem cell resistance is maintained through colon cancer transformation and allows identification of optimal combination strategies”

10.15– 10.25 Silvia Vega-Rubin-de-Celis DKFZ, GermanyQ&A: 10.25 – 10.30 Proffered Paper 7: “Inhibition of HER2 tumorigenesis by Beclin 1 and an autophagy-inducing peptide”


11.00 – 11.20 John Silke Walter and Eliza Hall Institute, AustraliaQ&A: 11.20 – 11.30 “Pump it Up - Will Smac-mimetics make it into the clinic? If so how and when?”

11.30 – 11.50 Guillermo Velasco Complutense University, SpainQ&A: 11.50 – 12.00 “Molecular mechanisms involved in the stimulation of autophagy-mediated cancer cell death”

12.00 – 12.30 CLOSING KEYNOTE LECTURE Q&A: 12.30 – 12.45 Manolis Pasparakis University of Cologne, Germany “KK/NF-kB and RIPK1 signalling in cell death and inflammation”


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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.

EACR-Worldwide Cancer Research Meeting Bursary Award winners

Oriol de Barrios Spain

Javier Fernández Díaz Spain

Irene Fernández-Duran UK

Lahiru Gangoda Australia

Lauana Lemos Brazil

Gabriela Nestal Brazil

Shiau-Ying Tham Malaysia

12.45 – 13.00 CLOSING SUMMARY AND PRESENTATION OF AWARDS Scientific Programme Committee

13.00 LUNCH & DEPART Rode Hoed foyer

BCCM/LMBP: plasmids at your fingertips

Otherneeds?•Plasmidprofile analysis•Sequenceanalysis of non-characterisedplasmids•…

[email protected]+32 9 33 13 843

Share andfindplasmidsPublic depositof plasmidsandhost strains•Share yourwork–Increaseyourvisibility–Off-site backup

Browse ourpublic catalogues•Cloning& expressionvectors–DNA libraries–Host strains

Secure yourmaterials•Safe deposits•Patent deposits•Storage thirdparty’s biologicalmaterial

Celllines–Hybridomas–Plasmids–Geneticresources

Departmentof BiomedicalMolecularBiologyGhentUniversityTechnologiepark 927B-9052 Gent-Zwijnaarde-BelgiumT +32 (09) 331 38 43 [email protected]

BCCM/LMBP: plasmids at your fingertips

Other needs?• Plasmid profile analysis• Sequence analysis of non-characterised plasmids• …

[email protected]+32 9 33 13 843

Share and find plasmidsPublic deposit of plasmids and host strains• Share your work – Increase your visibility – Off-site backup

Browse our public catalogues• Cloning & expression vectors – DNA libraries – Host strains

Secure your materials• Safe deposits• Patent deposits• Storage third party’s biological material

Cell lines – Hybridomas – Plasmids – Genetic resources

Department of Biomedical Molecular BiologyGhent UniversityTechnologiepark 927B-9052 Gent-Zwijnaarde - BelgiumT +32 (09) 331 38 43 - [email protected]

Cancers Editorial [email protected] AGSt. Alban-Anlage 66

4052 Basel, SwitzerlandTel: +41 61 683 77 34 Fax: +41 61 302 89 18 www.mdpi.com

▶ mdpi.com/journal/cancers

Editor-in-Chief:Prof. Dr. Samuel C. Mok

Associate Editors-in-ChiefProf. Dr. David WongDr. Deepak Nagrath

Message from the Editor-in-ChiefCancers is an international, online journal addressing both clinical and basic science issues related to cancer research. The journal will continue its open access format, which will certainly evolve to ensure that the journal takes full advantage of the rapidly changing world of information and knowledge dissemination. It publishes high-quality clinical, translational, and basic science research on cancer prevention, initiation, progression, and treatment, as well as other related topics, particularly to capture the most seminal studies in the rapidly growing area of immunology, immunotherapy, and tumor microenvironment.

Selected Special Issues: Molecular Stress Response Dysregulation in Cancer: Therapeutic Targets and Opportunities July 2018 (Open for Submission)

CAR-T Cell Therapy-Novel Approaches and Challenges June 2018 (Open for Submission)

FAK Signaling Pathway in Cancers May 2018 (Open for Submission)

Integrins in Cancer 2017 (17 papers published)

The Epithelial-to-Mesenchymal Transition (EMT) in Cancer 2017 (14 papers published)

An Open Access Journal by MDPI

Author Benefits

Open Access Unlimited and free access for readers

No Copyright Constraints Retain copyright of your work and free use of your article

Thorough Peer-Review

Rapid Processing and Immediate Publication upon Acceptance

Coverage by Leading Indexing Services SCIE-Science Citation Index Expanded (Clarivate Analytics), PubMed (NLM), Scopus (Elsevier)

No Space Constraints, No Extra Space or Color Charges No restriction on the length of the papers, number of figures or colors

Discounts on Article Processing Charges (APC) If you belong to an institute that participates with the MDPI Institutional Open Access Program (IOAP)

IMPACT FACTOR

TRACKED FOR


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Meet the Exhibitors

Cell Signaling Technology Website: www.cellsignal.comContact: [email protected] at the conference by: Janet Bewsher & Martijn Blommaart

Tell us a little bit about Cell Signaling Technology Cell Signaling Technology (CST) is a private, family-owned company, founded by scientists and dedicated to providing high-quality research tools to the biomedical research community.

Why are you attending the conference? Who would you like to meet at the conference?Cell Signaling Technology is looking forward to enhancing the knowledge and understanding of how cancer cells evade cell death and the mechanisms involved in addition to interacting with researchers working in this exciting field to better understand the challenges they face.

ChemoMetec Website: www.chemometec.comContact: [email protected], +45 31 25 64 56Represented at the conference by: Arjé Brilleman

Describe ChemoMetec in 5 words or lessCell counting, analysis and image cytometry

Tell us a little bit about ChemoMetec ChemoMetec provides high-quality instrumentation for fluorescence imaging-based cell counting and advanced cytometry. We develop intuitive and automated solutions to support cell research and therapies as well as bioindustries such as biopharma, brewing, animal breeding and dairy.

Why are you attending the conference? Who would you like to meet at the conference?We are attending the conference to create awareness of our technologies within image-based cell counting and cell analysis. We want people in research and biopharma industries to better understand the solutions we provide.


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Don Whitley Scientific Limited Website: www.dwscientific.co.ukContact: [email protected] at the conference by: Naomi Berkeveld; Alun Kitsell

Describe Don Whitley Scientific in 5 words or less Cell culture workstation manufacturer

Tell us a little bit about Don Whitley Scientific If your research involves the culture of cells and you need strict, accurate control over O2, CO2, temperature and humidity, visit the Don Whitley Scientific stand. The H35 Hypoxystation provides a range of features to help replicate an in vivo environment.

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.

EACR Sustaining Members

The European Association for Cancer Research gratefully acknowledges the organisations that support the Association as Sustaining Members. Sustaining Members 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.


12worldwidecancerresearch.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.

The organisers wish to express their appreciation for the support provided by sponsors at the EACR Conference A Matter of Life or Death: From Basic Cell Death Mechanisms to Novel Cancer Treatments. Their interest and enthusiasm for the conference has enabled the organisers to provide an impressive scientific programme.

Exhibitors

Partners and Sponsors

Grants


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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.

Networking Reception

17.45 – 19.00 Thursday 01 February

A hot buffet dinner will be served prior to the evening Keynote Lecture, and this will be an ideal opportunity for participants to get to know each other and enjoy the unique venue of De Rode Hoed. De Rode Hoed’s Great Hall is the largest and oldest remaining hidden church in the Netherlands.

Poster Defence Sessions

16.15 – 17.45 Thursday 01 February 15.00 – 16.30 Friday 02 February

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 and two EMBO Press 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 Saturday.

Poster Viewing

08.30 – 09.00 Friday 02 February 08.30 – 09.00 Saturday 03 February

Friday and Saturday 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 can be purchased from the bar.


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Round Table Discussions 16.30 - 17.30 Friday 02 February

This session enables participants and speakers to converse informally in smaller groups. Participants are free to ask any questions they like and to move freely between different speakers.

It will be followed later in the evening by the Conference Dinner (see below).

Conference Dinner

19.30 – 22.00 Friday 02 February

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 Restaurant-Cafe In de Waag.

Tickets for the conference dinner are not included in the registration fee and can be selected as an optional extra for €55. 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!

Conference Dinner at Restaurant-Cafe In de Waag at the last edition of the conference in 2016.


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Speaker abstracts

Speaker abstracts

Keynote Lecture

Autophagy and cancer: issues we need to digest

Kevin M. Ryan1

1 Tumour Cell Death Laboratory, Cancer Research UK Beatson Institute, Glasgow, UK

Macroautophagy serves to deliver cellular material to lysosomes for degradation. The process operates at basal levels in virtually all, if not all, cells and contributes to cellular integrity and viability. Macroautophagy is also highly adaptable responding to both intracellular and extracellular cues. In response to stimulation, the rates and also critically the cargoes of autophagy can be changes to bring about desired effects within cells and tissues.

Although we have gained considerable insight regarding the way in which the core autophagy machinery is controlled, our knowledge about the way in which autophagy is modulated in different situations is less well defined. This is incredibly important for example in therapeutic situations where it may be desired to target a specific form of autophagy while not affecting homeostatic and potentially cytoprotective autophagy in normal cells. This is also highly relevant to cancer where autophagy has been reported to be either tumour-promoting or tumour suppressive depending on the context. To address this issue, we have identified and characterized a number of new autophagy regulators. The implications of our discoveries in relation to the genesis and treatment of cancer will be discussed.

Work in The Tumour Cell Death Laboratory is supported by Cancer Research UK and Worldwide Cancer Research.


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Speaker abstracts

Non-autonomous tumor growth support by autophagy

Nadja Katheder1, 2, Rojyar Khezri1, 2,, Fergal O´Farrell1, 2,, Sebastian W. Schultz1,

2, Ashish Jain1, 2, 3, Mohammed M. Rahman1, 2, Kay O. Schink1, 2, Theodossis A. Theodossiou4, Terje Johansen3, Gabor Juhász5, David Bilder6, Andreas Brech1, 2, Harald Stenmark1, 2, Tor Erik Rusten1, 2 1 Department of Molecular Cell Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, Oslo, NORWAY, 2 Centre for Cancer Biomedicine, Faculty of Medicine, University of Oslo, Montebello, Oslo, NORWAY, 3 Molecular Cancer Research Group, Institute of Medical Biology, UiT - The Arctic University of Norway, Tromsø, NORWAY, 4 Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital, Montebello, Oslo, NORWAY, 5 Department of Anatomy, Cell and Developmental Biology, Eötvös Loránd University, Budapest, HUNGARY, 6 Department of Molecular and Cell Biology, University of California, Berkeley, USA

Malignant tumors develop in intimate interaction with the microenvironment and can activate autophagy, a catabolic process which provides nutrients during starvation. How autophagy is regulated by tumors in vivo and if it impacts tumor growth is controversial. We demonstrate, using a well-characterized Drosophila malignant tumor model, that non-cell-autonomous autophagy is induced in the tumor microenvironment and systemically in distant tissues. Tumor growth can be pharmacologically restrained using autophagy inhibitors, and genetic analyses reveal that early-stage tumor growth and invasion are dependent on autophagy within the local tumor microenvironment. Induction of autophagy is mediated by Drosophila Tumor Necrosis Factor-a and Interleukin-6-like signaling from metabolically stressed tumor cells, and tumor growth depends on active amino acid transport. Importantly, dormant growth-impaired tumors that stem from autophagy-deficient animals reactivate tumorous growth when transplanted into autophagy-proficient hosts. We conclude that transformed cells engage surrounding normal cells as an active and essential microenvironmental contributor to early tumor growth through nutrient-generating autophagy.


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Speaker abstracts

Proffered Paper 1

The EMT-factor ZEB1 promotes tumor progression through inhibition of cell senescence

Oriol de Barrios5, Balázs Györffy8, María Jesús Fernández-Aceñero3, Ester Sánchez-Tilló5, Lidia Sanchez-Moral5, Laura Siles5, Anna Esteve-Arenys9, Gaël Roué9, José Ignacio Casal1, Douglas S. Darling2, Antoni Castells7,4, Antonio Postigo5,6

1 Dept of Cellular and Molecular Medicine, CIB-CSIC, Madrid, SPAIN, 2 Dept of Oral Immunology and Infectious Diseases and Center for Genetics and Molecular Medicine, University of Louisville, KY, USA, 3 Dept of Pathology, Hospital Clínico San Carlos, Madrid, SPAIN, 4 Gastrointestinal and Pancreatic Oncology Team, CIBERehd, ISCIII, Barcelona, SPAIN, 5 Group of Transcriptional Regulation of Gene Expression. Dept of Oncology and Hematology, IDIBAPS, Barcelona, SPAIN, 6 ICREA, Barcelona, SPAIN, 7 Institute of Metabolic and Digestive Diseases, Hospital Clínic, Barcelona, SPAIN, 8 Lendület Cancer Biomarker Research Group, MTA TTK and 2nd Dept of Pediatrics, Semmelweis University, Budapest, HUNGARY, 9 Lymphoma Group. Dept of Oncology and Hematology, IDIBAPS, Barcelona, SPAIN

The entrance of tumor cells in senescence, a common mechanism of tumor suppression, is prevented by the activation of the Wnt signaling pathway. A key target of Wnt in activating cancer progression is the transcription factor ZEB1, widely known by its role in triggering an epithelial-to-mesenchymal transition (EMT). However, ZEB1 also mediates the induction of additional hallmarks of cancer. As we report here, the poor survival determined by ZEB1 in colorectal carcinomas (CRC) depends on its capacity to inhibit cancer cell senescence through the activation of a newly identified molecular pathway.

We found that ZEB1 induces the expression of Wnt-antagonist DKK1 and that both genes jointly repress a senescence-associated signature in CRC patients, thus promoting a worse prognosis. In p53-mutant cells, ZEB1 blocks the assembly of senescence-associated heterochromatin foci (SAHF) through the activation of a regulatory cascade that involves the sequential activation of DKK1, mutant p53, MDM2 and CtBP, to ultimately inhibit histone macroH2A1. Given the key role of macroH2A1 in the formation of SAHF, its ZEB1-mediated transcriptional repression prevents the onset of senescence in CRC cells. In a murine model of colon cancer, the partial deficiency of ZEB1 proved to be sufficient to trigger senescence in vivo through the upregulation of macroH2A1 histone, resulting in a lower formation of tumors and an improved survival. Moreover, we demonstrated that the ability of ZEB1 to induce tumorigenesis in a xenograft model requires the absence of macroH2A1 expression and that the poor outcome of CRC patients determined by ZEB1 depends on a concomitant low expression of macroH2A1.

In summary, these data establish a new mechanism through which ZEB1 mediates tumor progression and expands its range of functions beyond the classical role as an EMT-inducer, setting it as a potential target in the development of therapeutic strategies aiming at senescence induction.


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Speaker abstracts

EMBO Young Investigator Lecture

Bax and Bak at the single molecule level: telling the twins apart

Ana J. Garcia-Saez1

1 University of Tübingen, Tübingen, GERMANY

Bax and Bak are key regulators of apoptosis and mediate the permeabilization of the outer mitocondrial membrane that leads to cytochrome and Smac release. Although it is widely accepted that Bax and Bak function and molecular mechanism largely overlap, there is limited evidence how Bak works. In previous studies, we have used single molecule microscopy to characterize the oligomerization of Bax in the membrane and its organization at the nanoscale in the mitochondria of apoptotic cells. We now extended these approaches to Bak and identified key structural differences between the two proteins that may have functional implications.


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Speaker abstracts

Colorectal cancer, four subtypes with distinct biology

Jan Paul Medema1,2

1 LEXOR, Academic Medical Center, Amsterdam, NETHERLANDS, 2 Oncode Institute, Academic Medical Center, Amsterdam, NETHERLANDS

Colorectal cancer (CRC) is one of the leading forms of cancer and remains a deadly disease when diagnosed at late stage. Prognosis is relatively good when diagnosed early, but still a percentage of the patients display recurrences and we currently have no good tools to identify patients at risk. Using gene expression profiling we have, together with a large consortium, defined 4 distinct subgroups in CRC that differ both in their biology and clinical course. These so called consensus molecular subtypes (CMS) are not defined by specific mutations, yet appear to define entities with distinct prognosis and response to therapy.

To better understand the underlying biological principles we have set out to identify the 4 consensus molecular subtypes in colorectal cancer cell lines, primary cultures and patient-derived xenografts. These analyses reveal that commonly used cell lines belong to distinct subtypes and indeed respond differently to classical chemotherapy. In addition, distinct dependencies can be identified using a drop –out and compound screen and indicate that this stratification approach will aid the development of novel subtype-specific therapies. Novel data regarding these dependencies as well as novel compounds targeting specific subtypes will be presented.


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Speaker abstracts

Keynote Lecture

The regulation of ferroptosis in cancer cells

Scott Dixon1

1 Stanford University, Stanford, CA, USA

Cancer cells accumulate high levels of reactive oxygen species (ROS) and as a consequence are particularly sensitive to perturbation of intracellular antioxidant networks compared to non-transformed cells. The tripeptide glutathione (γ-L-glutamyl-L-cysteinylglycine) is a crucial intracellular antioxidant required to prevent oxidative damage and cell death. We find that blockade of the cell surface cystine/glutamate antiporter system xc

- results in the rapid depletion of intracellular glutathione and the onset of a non-apoptotic, oxidative, iron-dependent form of cell death termed ferroptosis. Interestingly, unlike apoptosis and other forms of cell death, ferroptosis does not involve activation of caspase or cathepsin proteases. Rather, cell death is due to the destruction of plasma membrane lipids by a poorly understood oxidative mechanism. We developed scalable time-lapse analysis of cell death kinetics (STACK) as a method to monitor the induction of ferroptosis over time and have used this method, together with small molecule library screening, to pinpoint metabolite-sensing and tumor suppressor pathways that regulate ferroptosis. These studies indicate that ferroptosis is a highly regulated form of metabolic stress-induced cell death.


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Speaker abstracts

The role of cell competition in tumour promotion

Eugenia Piddini11 School of Cellular and Molecular Medicine, University of Bristol, UK

Cell competition is a fundamental phenomenon that allows fitter cells (called ‘winners’) to kill less fit cells (called ‘losers’) and colonize tissues in their stead. The goal of the research we carry out in my group is to understand cell competition, its mechanism of action, and how it modulates cell colonization in tissues and organs. For our studies, we use an integrated approach that combines genetics and whole tissue in vivo studies in Drosophila with mammalian cell culture, which together facilitate manipulation, live-cell imaging and translation of our findings to mammalian models.

We are particularly interested in the role that cell competition may have in tumour promotion. Indeed it has been suggested that tumour cells may compete with and kill neighbouring host tissue to clear space and fuel their expansion. I will present our work in Drosophila tumour models showing that tumour and host tissues in fact compete and that clearance of host cells is required for tumour growth.

I will then describe our efforts to identify the mechanisms that cells use to compete. For example our group has discovered that mammalian cells in culture can compete using mechanical insults. We found that mutations can lead cells to become hypersensitive to compaction and be killed by their fitter neighbours, through cell crowding. We have then shown that it is the activation of the tumour-suppressor p53 that confers hypersensitivity to crowding and is both necessary and sufficient to induce mechanical cell competition. Since p53 is very frequently mutated in cancer, our work suggests that mutations in p53 may help cancer cells to evade mechanical cell competition in the crowded tumour micro-environment and to kill host cells to free space for their expansion.


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Speaker abstracts

Targeted apoptosis of senescent cells against aging and cancer

Ron de Bruin1, Marjolein Baar1, Renata Brandt1, Diana Putavet1,5, Julian Klein1, Kasper Derks1, Benjamin Bourgeois3, Sarah Stryeck3, Yvonne Rijksen1, Hester van Willigenburg1, Danny Feijtel1, Ingrid van der Pluijm1, Jeroen Essers1, Wiggert van Cappellen1, Wilfred van IJcken1, Adriaan Houtsmuller1, Joris Pothof1, Tobias Madl3, Jan Hoeijmakers1, Judith Campisi4,2, Peter de Keizer1,5

1 Erasmus University Medical Center, Rotterdam, NETHERLANDS, 2 Lawrence Berkeley National Laboratories, Berkeley, USA, 3 Medical University of Graz, Graz, AUSTRIA, 4 The Buck Institute for Research on Aging, Novato, USA, 5 University Medical Center Utrecht, Utrecht, NETHERLANDS

Population age continues to increase in the foreseeable future. Many diseases, including cancer, manifest themselves at older age. As we age, we accumulate cellular damage, which can eventually result in cells becoming “senescent”. Senescent cells cease to divide, but chronically secrete a wide range of factors that permanently alter their environment. As such, they are thought to impair tissue function and promote cancer progression, migration and therapy resistance by permanently enforcing a state of stemness. Therapies to effectively eliminate senescent cells and senescent-like cancer cells would be very valuable to extend human healthspan and overcome cancer therapy resistance.

Here, I will highlight how DNA-damaging radio- and chemotherapy can invoke a senescence response in healthy cells, which paradoxically can be deleterious to tumor burden. I will show how we identified the damage-associated protein FOXO4 as a pivot in senescent cell viability and how we designed a drug, the D-Retro-Inversed FOXO4 peptide FOXO4-DRI, which selectively eliminates senescent cells by forcing nuclear exclusion of active p53 and triggering canonical apoptosis. Under conditions where it was well tolerated in vivo, FOXO4-DRI could restore fitness, fur density and renal function of fast aging XpdTTD/TTD and, where applicable, naturally aged mice.

We now observed that therapy-surviving cancer cells can also activate a FOXO4-p53 response, making them sensitive to FOXO4-DRI. Current research is focused on better understanding the potential long-term dangers of senescence-clearance, optimization of FOXO4-DRI for better clinical translation and identifying the molecular mechanisms that dictate sensitivity or resistance. Thus, therapeutic targeting of senescent cells is feasible under conditions where loss of health has already occurred and such treatment may hold promise against tumors that activate FOXO4-p53 signaling due to their survival of damaging initial cancer therapies.


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Speaker abstracts

Proffered Paper 2

Caspase-3 Regulates YAP-dependent Cell Proliferation and Organ Size

Yahav Yosefzon1, Despina Soteriou1, Lana Kostic1, Elle Koren1, Egor Sedov1, Fabian Glaser1, Yaron Fuchs1

1 Technion, Haifa, ISRAEL

Apoptosis culminates in the activation of caspase-3, which plays an important role in implementing the cell death program. Here, we reveal a surprising non-apoptotic role of caspase-3 as a key regulator of cell proliferation and organ size. Caspase-3 is specifically activated in the proliferating cells of the epidermis, but does not instruct cellular elimination. Deletion or chemical inhibition of caspase-3 diminishes cellular proliferation, decreases cell number and reduces sebaceous gland size in vivo. Exploring the underlying mechanism, we demonstrate that α-Catenin is cleaved by caspase-3, thus facilitating the activation and nuclear translocation of YAP, a vital regulator of organ size. Accordingly, activation of caspase-3 leads to YAP-dependent organ size augmentation. Finally, we show that XIAP serves as an endogenous feedback antagonist for the caspase-3-YAP signaling module.

Taken together, here we unravel a unique molecular mechanism where the apoptotic machinery is refocused to regulate cell proliferation and orchestrate organ size.


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Speaker abstracts

Proffered Paper 3

Primary cilia modulate susceptibility to colon carcinogenesis

Michael Hahne1, Carsten Janke2, Rossano Lattanzanio3, Benedicte Lemmers1, Conception Paul1, Valerie Pinet1, Sophie Poupeau1, Ruizhi Tang1

1 IGMM, CNRS, Univ. Montpellier, Montpellier, FRANCE, 2 Institute Curie, Orasay, FRANCE, 3 University “Gabriele d’Annunzio”, Chieti, ITALY

Primary cilia (PC) are an organelle assumed to extrude most mammalian cells. They act as a sensory antennae and control diverse signaling pathways such as Hedgehog and Wnt. The latter are important regulators of cell proliferation, differentiation and crucial to maintain colon homeostasis. A recent report described a correlation between the frequency of colonic PC and disease outcome in CRC patients with notably a significant longer overall survival in CRC patients with higher frequency of PC. To test for a potential role of PC in colon homeostasis and pathology we analyzed PC expression in murine colon by immunohistochemistry and observed that the number of colonic primary cilia decreases during colon carcinogenesis in mice. Moreover, we found that mainly colonic mesenchymal cells, but only few epithelial cells express PC. We next established mouse models in which PC are deleted in either colonic epithelial or mesenchymal cells. For this we used mouse strains carrying the conditional knockout (KO) alleles for either the kinesin family member 3A (Kif3aflx/flx) or intraflagellar transport protein 88 (Ift88flx/

flx), two proteins essential for cilia formation. Cell specific deletion of conditional alleles was obtained by crossing the conditional KO mice with transgenic mice for cre-recombinase targeting either intestinal epithelial cells or mesenchymal cells, including a subset of colonic fibroblasts. Analysis of those conditional knock-out mice revealed that cell-type specific deletion of PC in the colon does not affect colon homeostasis. Partial depletion of PC in colonic mesenchymal cells, however, increases significantly the susceptibility to chemically induced colon carcinogenesis. This appears to be associated with an increased beta-catenin activity in PC-deficient colons.

All together, these findings suggest a link between the presence of primary cilia and colon carcinogenesis, and we are presently investigating whether PC expression could serve as a biomarker in CRC.


26

Speaker abstracts

Mitochondrial apoptosis and cancer: from phantom menace to a force awakens

Stephen Tait1

1 Cancer Research UK Beatson Institute, Glasgow, UK

Apoptosis is largely considered a beneficial process in cancer, where it serves important roles in tumour suppression and cancer treatment. Nevertheless, various data suggest that apoptosis signalling may also be oncogenic in some contexts. Addressing this, we have found that sub-lethal caspase activity - what we call failed-apoptosis- can promote DNA-damage, genomic instability and cellular transformation. I will discuss our recent work further investigating the role of failed apoptosis as a cancer driver and its possible implications for physiological and therapeutic engagement of apoptosis. Secondly, I will discuss how we can subvert this dark-side of apoptosis by inhibiting caspase function. Surprisingly, we found that this leads to an immunogenic form of cell death that can elicit anti-tumour immunity. Using castration-resistant prostate cancer as a clinical paradigm, the potential benefits of engaging caspase-independent cell death in the clinic will be highlighted.


27

Speaker abstracts

MAPK, PI3K and HER2 inhibition induces synthetic lethality in colorectal cancer stem cells regardless their mutational status

Laura Rosa Mangiapane2, Annalisa Nicotra2, Tiziana Apuzzo2, Simone Di Franco2, Matilde Todaro1, Giorgio Stassi21 Department of DIBIMIS, University of Palermo, Palermo, ITALY, 2 Surgical, Oncological and Stomatological Sciences, University of Palermo, Palermo, ITALY

The resistance mechanisms responsible for the failure of target-oriented combination therapies against colorectal cancer (CRC), are still poorly understood. Here, we show that the HER2 expression demarcates CRC stem cell (CR-CSC) population, whose activation is needed as an escape mechanism for EGFR and BRAF inhibitors. The blockade of HER2 in combination with BRAF inhibitor, enriches those cells expressing CD44v6/PI3K, which are endowed with Wnt/b-catenin pathway activation. PI3K pathway inactivation in combination with BRAF and HER2 inhibitors, leads to the shrinkage of BRAF mutated and KRAS wild-type tumors and to stabilization or slow progression of KRAS mutated tumors. We found that, following this combination treatment, PIK3CA-mutated cells up-regulate c-Myc, which sustains tumor growth over serial in vivo passages. The replacement of the BRAF inhibitor with a MEK inhibitor, ameliorates the effect of the combination therapy by causing a marked reduction of CD44v6 expression in PIK3CA mutated cells. The MEK-based therapeutic combination prevents the tumorigenic activity of CR-CSCs and induces a massive regression of xenograft tumors, regardless their mutational background. The plethora of mechanisms involved in CRC resistance supports the execution of other studies to establish alternative therapeutic strategies to sensitize CSCs to standard therapy. Of note, a stable BMP molecule (BMP7v) gradually decreases CD44v6 expression and β-catenin activity of CR-CSCs, impairing their capabilities to sustain the tumor growth. Our results provide a solid preclinical rationale for to design an effective therapy targeting CR-CSCs.


28

Speaker abstracts

Normal tissue response to ionizing radiation and therapeutic targeting

Verena Jendrossek1

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

Radiotherapy is an integral part of standard treatment for thorax-associated neoplasms. But radiation-induced pneumonitis and fibrosis represent severe and dose limiting side effects in the radiotherapy of thorax-associated neoplasms leading to decreased quality of life or - as a consequence of treatment with suboptimal radiation doses - to fatal outcomes by local recurrence or metastatic disease. It is assumed that the initial radiation-induced damage to the DNA of resident cells triggers a cascade of events that can lead within weeks to a pronounced lung inflammation (pneumonitis) or after months to excessive deposition of extracellular matrix molecules and tissue scarring (lung fibrosis). The use of preclinical in vivo models of DNA damage-induced pneumopathy in genetically modified mice has helped to substantially advance our understanding of cellular and molecular mechanisms as well as signalling molecules that participate in the pathogenesis of radiation-induced adverse late effects in the lung. The presentation will highlight our new findings on radiation-induced changes in the lung tissue that participate in shaping a disease-promoting microenvironment. It will also discuss their potential as new biomarkers and targets for future treatments, particularly in view of the increasing interest in combining ionizing radiation with immune checkpoint blockade or immunotherapy to enhance the immune response against the tumor systemically.


29

Speaker abstracts

Mechanisms regulating the immunogenicity of radiation-induced cancer cell death

Sandra Demaria1

1 Weill Cornell Medical College, New York, USA

Most cancer cells exposed to ionizing radiation do not undergo immediate death even when the damage is lethal but remain within the tumour mass for some time during which they generate molecular signals that modify the cross-talk of the tumour with the host immune system. Central to this process is the activation of the DNA damage repair (DDR) response, which is often dysregulated in neoplastic cells, and associated with DNA displacement to the cytosol. The presence of dsDNA in the cytosol is sensed by the cyclic GMP-AMP synthase, cGAS, which catalyzes the formation of the second messenger cGAMP and activates STING, leading to the production of type I interferon (IFN-I). IFN-I is a critical effector of cell-mediated anti-viral and anti-tumour immunity and is primarily responsible for the recruitment and activation of BATF3-dendritic cells (DCs), and downstream activation of anti-tumour CD8 T cells. Accumulation of cytosolic IFN-stimulatory dsDNA is regulated by the single radiation dose size, with an optimal window ranging between 4-12 Gy in most human and mouse carcinoma cells tested. Above these doses, upregulation of the DNA exonuclease TREX1 results in clearance of cytosolic dsDNA, abrogating radiation immunogenicity. Fractionation, i.e., repeated (three times) daily delivery of radiation therapy at doses within this window, amplifies the IFN-I pathway activation in the carcinoma cells, an effect that requires induction of IFNRA. Furthermore, the synergy of RT with immune checkpoint blockers (ICBs) anti-CTLA-4 or anti-PD-1 and the induction of abscopal effects (i.e., immune-mediated rejection of non-irradiated synchronous tumours) are completely dependent on the ability of radiotherapy to induce cancer cell-intrinsic IFN-I. These findings have critical implications for the use of radiotherapy to increase the response to ICBs, a combination currently being tested in many clinical trials.

Supported by NIH 1R01CA201246, Breast Cancer Research Foundation, and The Chemotherapy Foundation.


30

Speaker abstracts

Proffered Paper 4

Chemo-Radio-Immunotherapy: Cisplatin improves abscopal effects following radiotherapy and a-CD137/a-PD1 mAbs

Elselien Frijlink 3, Paula Kroon3, Victoria Iglesias-Guimarais3, Roel Kluin3, Marit van Buren 3,2, Mireille Toebes3, Carsten Linnemann3,1, Lorenzo Fanchi3, Ton Schumacher3, Jannie Borst3, Inge Verbrugge3

1 Kite Pharma Europe, Amsterdam, NETHERLANDS, 2 Neon Therapeutics, Boston, USA, 3 Netherlands Cancer Institute, Amsterdam, NETHERLANDS

Accumulating evidence indicates that radiotherapy can act as an in situ vaccine and, in combination with immunotherapy, can result in tumor regression of non-irradiated disseminated disease. However, despite observed preclinical synergistic effects with radiotherapy and immunotherapy, improved control of non-irradiated tumors following combined treatment remains a relatively rare phenomenon in the clinic. Here, we examined the mechanisms that underlie combined responses to irradiated mammary tumors following radio-immunotherapy with (α)-CD137 and α-PD-1 mAbs. We subsequently exploited these findings to identify and overcome bottlenecks that impeded control of non-irradiated tumors in the same experimental setting.

We found that T-cell priming in this radio-immunotherapy approach was strictly required to eliminate the irradiated tumor. However, non-irradiated tumors were not eliminated in the same experimental setting, despite robust T cell infiltration into both the irradiated and non-irradiated tumor and blockade of PD-1 signaling in place. Therefore, these data suggest that radiotherapy has an auxiliary function that allows T-cell-mediated tumor eradication by modulating the suppressive tumor-microenvironment beyond of PD-1 signaling. To mimic features of localized radiotherapy that allowed T-cell-mediated tumor rejection systemically, we combined radio-immunotherapy with low-dose cisplatin and observed significantly improved T-cell-mediated control of non-irradiated tumors and increased overall survival.

In conclusion, our data highlight that control of disseminated disease with radio-immunotherapy approaches should not only result in enhanced T-cell priming, but also modulate the tumor-microenvironment in favor of T-cell immunity. Rational approaches to achieve the latter may include low-dose cisplatin, particularly in a setting where PD-1 blockade by mAbs alone is insufficiently effective.


31

Speaker abstracts

Proffered Paper 5

Identification of molecular mechanisms essential for necroptosis execution by using a genome-wide CRISPR-Cas knockout screening

Jolien Bridelance1, Wim Declercq1, Peter Vandenabeele1

1 VIB-UGhent Center for Inflammation Research, Zwijnaarde, BELGIUM

Since it has been shown that MLKL (Mixed lineage kinase domain-like) is essential for necroptotic cell death, multiple research groups discovered different “pieces of the puzzle” on how MLKL can result in cell death execution (incl. conformational changes, membrane recruitment, interaction with ion channels, …) and even on the role of MLKL in other events, such as endosomal trafficking and exosome formation. Despite this, it is still not known how these pieces fit together and as such how MLKL results in cell death.

An important research question to help solve the puzzle, is if there are still other proteins or signaling events necessary downstream or at the level of MLKL phosphorylation, to assist MLKL in its cell-death executing function (cfr. conformational changes, plasma membrane recruitment, oligomerization, ion channel formation, …)?

In this project we try to identify the molecular mechanisms essential for cell death execution downstream or at the level of MLKL phosphorylation by using a genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas knockout screening. For this we made use of Mlkl-/- L929 cells reconstituted with an inducible WT or phosphomimetic (S345D) MLKL construct. By inducing MLKL expression in these cell lines it is possible to study MLKL-dependent cell death, without the interference of the complex upstream TNF (tumor necrosis factor) signalling pathway. As a control we made use of TNF-induced necroptosis in wild type L929 cells.

These cell lines were screened using a genome-wide CRISPR-Cas knockout screening. In this screen a pooled lentiviral guide RNA library is used to knock out thousands of genes throughout the genome. A selection pressure is then applied to these knockout cells by inducing MLKL expression. Comparison of the integrated guide RNA amounts of stimulated versus unstimulated cells shows which gene knockouts influence survival upon the cell death trigger.


32

Speaker abstracts

The immune system: a double-edged sword in metastatic breast cancer

Karin E. de Visser1

1 Division of Tumor Biology & Immunology, The Netherlands Cancer Institute, Amsterdam, NETHERLANDS

Chemoresistance is one of the key challenges in cancer patient care. Besides targeting cancer cells directly, chemotherapeutic drugs elicit a number of changes in immune-related parameters including the composition, phenotype and function of immune cells. There is a growing realization that these changes in the immune system influence the success of chemotherapy. Using the K14cre;Cdh1F/F;Trp53F/F mouse mammary tumor model, we study the impact of the immune system on the anti-cancer efficacy of chemotherapy. We found that different types of chemotherapeutic drugs have a differential effect on the tumor immune contexture. Against the prevailing dogma, we have demonstrated that adaptive immune cells do not affect the outcome of chemotherapy against established spontaneous mammary tumors. Instead, components of the innate immune system were found to counteract the anti-cancer efficacy of chemotherapy. Targeting macrophages by CSF-1R blockade markedly improved the anti-cancer efficacy of cisplatin in a Type I IFN dependent manner, but not of docetaxel. We discovered that depletion of macrophages through CSF-1R blockade together with cisplatin treatment evoked a compensatory neutrophil response limiting the synergistic anti-cancer effect.

These data highlight the importance for optimally matching chemotherapeutics with immunomodulatory compounds and indicate that the inherent flexibility and redundancy of the immune system lends itself to deleterious feedback mechanisms in which the function of a depleted population is reinstated by another population. The goal of our current projects is to understand the underlying mechanisms by which myeloid cells counteract the efficacy of conventional anti-cancer therapies, and to dissect the resistance pathways arising from within the immune system upon treatment with immunomodulatory therapies. Besides providing mechanistic insights, this research line has clear relevance for the clinic, and we foresee that findings from this project may contribute to the rational design of combinatorial strategies aimed at maximizing clinical success of chemotherapy for breast cancer patients.


33

Speaker abstracts

The PIDDosome – a signalling platform controlling p53 activation

Luca Fava1, Valentina Sladky1, Fabian Schuler1, Andreas Villunger1

1 Division of Developmental Immunology, Medical University of Innsbruck, AUSTRIA

The PIDDosome is often used as the alias for a multi-protein complex that includes the p53-induced death domain protein 1 (PIDD1), the bipartite linker protein CRADD (also known as RAIDD) and the pro-form of an endopeptidase belonging to the caspase family, i.e. caspase-2. Yet, PIDD1 variants can also interact with a number of other proteins that include RIPK1 (also known as RIP1) and IKBKG (also known as NEMO), PCNA and RFC5, as well as nucleolar components such as NPM1 or NCL. This promiscuity in protein binding is facilitated mainly by autoprocessing of the full-length protein into various fragments that contain different structural domains. As a result, multiple responses can be mediated by protein complexes that contain a PIDD1 domain. This suggests that PIDD1 acts as an integrator for multiple types of stress that need instant attention. Examples are various types of DNA lesion but also the presence of extra centrosomes that can foster aneuploidy and, ultimately, promote DNA damage. Here, we review the role of PIDD1 in response to DNA damage and also highlight novel functions of PIDD1, such as in centrosome surveillance and scheduled polyploidisation as part of a cellular differentiation program during organogenesis.


34

Novel opportunities to target cell death pathways for cancer therapy

Simone Fulda1

1 Institute for Experimental Cancer Research in Pediatrics, Goethe-University Frankfurt, GERMANY

Evasion of cell death is a hallmark of human cancers and contributes to tumor formation and progression as well as resistance to current cancer therapies, which largely rely on intact cell death pathways in cancer cells. Cell death resistance can be caused by aberrant expression of antiapoptotic proteins, for example “Inhibitor of Apoptosis” (IAP) proteins, which are expressed at high levels in many human cancers. Among the therapeutic approaches that have been developed to target IAP proteins, the most widely used strategy is based on mimicking the IAP-binding motif of second mitochondria- derived activator of caspase (Smac), which functions as an endogenous IAP antagonist. Current and future perspectives on neutralizing IAP proteins by Smac mimetics for therapeutic intervention in human cancers will be discussed.

Speaker abstracts


35

Proffered Paper 6

BCL-xL specific stem cell resistance is maintained through colon cancer transformation and allows identification of optimal combination strategies

Prashanthi Ramesh2, Maartje van der Heijden2, Danielle Dekker2, Jarno Drost1, Louis Vermeulen2, Jan Paul Medema2

1 Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht, Utrecht, NETHERLANDS, 2 Laboratory of Experimental Oncology and Radiobiology, Center for Experimental and Molecular Medicine, Cancer Center Amsterdam, Amsterdam, NETHERLANDS

Cancer stem cells (CSCs), often considered the drivers of cancer progression and metastasis, are found to be particularly resistant to cell death and respond poorly to therapy, thereby contributing to relapse. In colon cancer, our previous work has shown that CSC-specific resistance to chemotherapy relies strongly on BCL-xL, as addition of the BH3 mimetic WEHI-539 along with chemotherapeutic agents dramatically increases CSC death, while the BCL-2-targeting agent ABT-199 has no effect. More recently, we have shown that mouse intestinal stem cells selectively express BCL-2 which is required for the formation of APC mutation-driven early adenomatous lesions. Thus, a potential shift is observed in anti-apoptotic BCL-2 family protein reliance as CSCs progress through the adenoma-to-carcinoma sequence. To address this, CRISPR engineered human colon organoids with specific mutations modelling the classical colon cancer progression pathway were analyzed for their BCL-2 family dependence. Clonogenic outgrowth of the organoids was measured following treatment with BH3 mimetics that specifically target either BCL-2 or BCL-xL. Preliminary results suggest that APC mutant human colon organoids that model early adenomas are dependent on BCL-xL, thus pointing towards either a mouse-human or a small intestine – large intestine difference. However, BCL-2 could play an important role solely during the first steps of transformation i.e. acquisition of the APC mutation, after which CSCs switch to BCL-xL for protection. BCL-xL specific resistance is maintained throughout CRC progression as modeled in the CRISPR organoids. Based on this finding, we performed a drug screen to identify targets that synergize with low dose BCL-xL inhibition in CRC stem cell spheroid cultures which allows for the selection of optimal combinatorial strategies and moreover, to gain a better understanding of the establishment of this resistance mechanism.

Speaker abstracts


36

Proffered Paper 7

Inhibition of HER2 tumorigenesis by Beclin 1 and an autophagy-inducing peptide

Silvia Vega-Rubin-de-Celis2,1, Zhongju Zou2, Alvaro F. Fernandez2, Guanghua Xiao2, Min Kim2, Beth Levine2

1 German Cancer Research Center (DKFZ), Molecular Genome Analysis Division. Im Neuenheimer Feld 580. 69120 Heidelberg, GERMANY, 2 University of Texas Southwestern Medical Center, Dallas, Texas, USA

HER2 is a receptor tyrosine kinase whose gene is amplified in ~20% human breast cancer patients. Allelic loss of the autophagy gene, beclin 1/BECN1, is associated with HER2 amplification in breast cancer; low beclin 1 mRNA expression is associated with increased risk of HER2-positive breast cancer; and overexpressed HER2 and Beclin 1 interact in cultured cells. However, the functional significance of HER2/Beclin 1 interaction and of altered autophagy in HER2-driven tumorigenesis and whether autophagy induction may be beneficial in preventing HER2-positive breast tumor growth is unknown. We explored the regulation of autophagy in breast cancer cells by HER2 in vitro and the effects of genetic and pharmacological approaches to increase autophagy on HER2-driven breast cancer growth in vivo. We show that endogenous HER2 interacts with Beclin 1 in HER2+ breast cancer cells and it inhibits autophagy. Mice with an increased basal autophagy due to a knock-in mutation in Becn1 (Becn1F121A) that results in a decreased Bcl-2 binding to Beclin 1 are protected from mammary tumorigenesis when crossed with mammary-specific HER2 transgenic mice, and HER2 fails to inhibit autophagy in primary cells derived from these mice. Furthermore, HER2-positive human breast cancer xenografts treated with the autophagy-inducing peptide Tat-Beclin 1 inhibit tumor growth as effectively as a clinically used HER2 tyrosine kinase inhibitor (TKI). This inhibition of tumor growth is associated with a robust induction of autophagy, a disruption of HER2/Beclin 1 binding, and a transcriptional signature in the tumors that is distinct from that observed with HER2 TKI treatment.

Taken together, these findings indicate that the HER2-mediated inhibition of Beclin 1 and autophagy likely contributes to HER2-mediated tumorigenesis. They also suggest that strategies to block HER2/Beclin 1 binding and/or increase autophagy may represent a new therapeutic approach for HER2-positive breast cancers.

Speaker abstracts


37

Pump It Up - will Smac-mimetics make it into the clinic? If so, how and when?

Emma Morrish1, Gabriella Brumatti1, John Silke1

1 The Walter and Eliza Hall Institute, Parkville, Victoria, AUSTRALIA

Smac-mimetics are anti-cancer drugs that function by inhibiting Inhibitor of APoptosis proteins. The most clinically advanced Smac-mimetic is birinapant. Birinapant is very specific and we have shown, in many diverse scenarios, in vitro and in vivo, that it induces almost identical effects to those induced by genetic deletion of IAPs. This specificity is probably due to the fact that, like the Bcl-2 antagonist, Venetoclax, it targets a protein protein interaction interface; rather than a catalytic activity. Furthermore birinapant is also very well tolerated, and some patients have taken birinapant for over two years without untoward effects. Unfortunately, despite these great positives, to date it has not been very effective in the clinic. There are salutary lessons to learn from the journey thus far, which I will touch on in my talk, and I will discuss why I think Smac-mimetics, as a class of anti-cancer drugs, still have enormous potential. In particular in the areas of inducing non-apoptotic cell death and the ability to synergise with other well tolerated drugs and therapies that harness the potential of the immune system.

Speaker abstracts


38

Molecular mechanisms involved in the stimulation of autophagy-mediated cancer cell death

Guillermo Velasco1,2

1 Complutense University, Madrid, SPAIN, 2 Instituto de Investigaciones sanitarias San Carlos, Madrid, SPAIN

Autophagy is the main cellular degradation pathway for the clearance of damaged or superfluous proteins and organelles and represents the principle catabolic process regulating cellular homeostasis and organelle and protein turnover. The final outcome of autophagy activation seems to be highly dependent on the cellular context and the strength and duration of the stress-inducing signals. Thus, besides its role in cellular homeostasis, autophagy can be a form of programmed cell death or play a cytoprotective role, for example in situations of nutrient starvation. Accordingly, autophagy plays a dual role in cancer as this cellular process may help to overcome the stress evoked at the initial steps of tumorigenesis or work as a tumor suppressor mechanism. Moreover, different anticancer treatments activate autophagy in tumor cells, which either enhance cancer cell death or act as a mechanism of resistance to chemotherapy. Prior work by our group showed that Δ9- tetrahydrocannabinol (THC, the main active component of marijuana) triggers autophagy-mediated cancer cell death. In this presentation I will summarize several recent findings supporting that the modification of the sphingolipid metabolism of cancer cells by cannabinoids plays a pivotal role in the stimulation of autophagy-mediated cancer cell death.

Speaker abstracts


39

Keynote Lecture

IKK/NF-kB and RIPK1 signalling in cell death and inflammation

Manolis Pasparakis1

1 Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD) and Centre for Molecular Medicine (CMMC), University of Cologne, GERMANY

Cell death is an essential physiological process during development and for the maintenance of tissue homeostasis. Deregulation of the balance between cell proliferation and cell death can cause hyperplasia and cancer or tissue damage and degeneration. However, the molecular processes that regulate cell survival and cell death and contribute to the maintenance of tissue homeostasis and the pathogenesis of inflammatory diseases and cancer remain poorly understood. Multiple studies in recent years identified cell death as a critical trigger of inflammation. Necroptosis, a type of regulated necrotic cell death regulated by RIPK3 and its substrate MLKL, was identified as a highly inflammatory cell death process. Apoptosis was also shown to trigger inflammation in some settings, suggesting that the concept of apoptosis as a non-immunogenic type of cell death needs to be revised. Our studies revealed that the interplay between IKK/NF-kB and RIPK1 signalling is critical for the regulation of tissue homeostasis, inflammation and cancer in different tissues. We found that IKK subunits maintain tissue homeostasis by inhibiting RIPK1 kinase activity-dependent cell death by NF-kB-dependent and –independent mechanisms. RIPK1 also exhibits kinase independent functions that are essential to inhibit apoptosis and necroptosis of epithelial cells and prevent inflammation in the intestine and the skin respectively. Our experiments revealed important, though opposing, functions of RIPK1 in liver cancer. RIPK1 acts in a kinase activity-dependent fashion to trigger hepatocyte apoptosis, chronic liver inflammation and ultimately hepatocellular carcinoma in mice with liver-specific NEMO knockout. On the other hand, RIPK1 exhibits kinase-independent functions that promote chemically induced liver carcinogenesis. Therefore, elucidating the molecular processes controlling the interplay of IKK/NF-kB and RIPK1 signalling will be critical in order to better understand the mechanisms causing inflammation and cancer, and may lead to new therapeutic approaches.

Speaker abstracts


40

Poster abstracts

Poster abstracts

1

Novel anti-cancer therapeutics for multiple myeloma which target the STAT3 signaling pathway

Rebecca Amet3,2, Viola Previtali4, Paul Browne1, Isabel Rozas4, Tony McElligott2, Daniela Zisterer3

1 Department of Haematology, St James’s Hospital, Dublin, EIRE, 2 John Durkan Leukaemia Laboratories, Trinity Translational Medicine Institute, Trinity College Dublin, Dublin, EIRE, 3 School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, EIRE, 4 School of Chemistry, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin, EIRE

Multiple myeloma (MM) is the second most common haematological malignancy. A better understanding of the pathophysiology of myeloma and advancements in the development of novel therapeutics have allowed the median survival of patients to increase from 3 to 6 years in the past decade. However, despite the effectiveness of the first-line treatments, patients invariably relapse and become drug refractory; therefore, novel therapeutics which target this incurable disease are still required.

Signal transducer and activator of transcription 3 (STAT3) is activated by the MM growth and survival factor IL-6 secreted by the bone marrow stromal cells within the tumour microenvironment. STAT3 upregulates expression of genes involved in apoptosis, proliferation and angiogenesis. Consequently, STAT3 has emerged as a therapeutic target in various cancers including MM (Hua et al., 2014).

We have previously rationally designed and synthesized guanidinium based novel compounds to target the RAS/RAF/MEK/ERK signalling pathway which demonstrated anti-tumour activity in leukaemia cells (Diez-Cecilia et al., 2014). In the present study, we evaluated the anti-cancer activity of an optimised series of these compounds in MM cell lines NCI-H929 and U266B1, by AlamarBlue© assay and examined the ability of the lead compound, VP79s, to induce apoptosis and to target both the ERK and STAT3 signalling pathways by western blot analysis.

Our data have shown that VP79s potently reduced the viability of MM cells. VP79s induced apoptosis in a dose and time-dependent manner resulting in caspase-3 activation. Interestingly, VP79s induced a sustained ERK activation in MM cells. VP79s rapidly inhibited both constitutive and IL-6 induced STAT3 activation, and was found to decrease expression of STAT3 mediated anti-apoptotic gene products, Mcl-1 and survivin, and the cell cycle protein cyclin D1.

In conclusion, the novel compound VP79s can target deregulated STAT3 activation and induce apoptosis of MM cells suggesting, it’s potential as a novel anti-cancer therapeutic.


41

2

Uncovering the mysteries under the effectiveness of 2OHOA.

Laura Arbona-González2, Raquel Rodríguez.Lorca2, Maitane Ibarguren1, Victoria Lladó2, Xavier Busquets2

1 . Lipopharma Therapeutics, S.L., Palma, SPAIN, 2 University of the Balearic Islands, Palma , SPAIN

2-hydroxyoleic acid (2OHOA), a lipid compound, has demonstrated efficacy against cancer by inducing cell cycle arrest in lung cancer cells, apoptosis in leukemia cells and differentiation followed by autophagy in glioma cells.

Glioma is the most common type of primary brain tumor and is associated with very high rates of mortality and a high resistance to chemotherapy. The reference drug in glioma treatment is temozolomide (TMZ), which increases the life expectancy of patients to about 2.5 months. 2OHOA has already passed with success the phase I/IIa clinical trial and will start the next stage shortly. Moreover, due to the greater efficacy than TMZ in animal models and the lack of toxicity at therapeutic doses has been designated orphan drug for the glioma treatment by the European Medicines Agency (EMA).

Despite its promising effectiveness, its mechanism of action is not yet fully elucidated, especially with respect to the first series of molecular events triggered by this molecule. Our studies have shown that changes in membrane lipid content fulfils a critical role in the propagation of tumorigenic signals. The novel mechanism of action of 2OHOA is associated with important changes in membrane-lipid composition, primarily a recovery of sphingomyelin (SM) levels, which is markedly low in glioma cells before treatment. All these changes influence the location, activity or interactions of amphitropic membrane proteins that are involved in proliferation/differentiation signaling.

The aim of this work is to deepen the lipid and protein changes produced by 2OHOA in different cell lines of human glioma and nontumor human cells to find connections among them and shed light on its mechanism of action. Indeed, we have observed a different molecular pattern triggered in responders vs non-responders cells to 2OHOA that could help figuring out which is the critical mechanism responsible of 2OHOA’s antitumoral effect.

Poster abstracts


42

Poster abstracts

3

Camalexin induced cell death and apoptosis of SW480 colon cancer cells through MMP-9 related pathway

Filiz Bakar Ates1, Nuri Özmen1

1 Ankara University, Faculty of Pharmacy, Department of Biochemistry, Ankara, TURKEY

Phytoalexins are low molecular weight compounds and abundant amounts of this compound are present in plants. In addition to the antioxidant and antibacterial effects of phytoalexins, they are also suggested to modulate cellular processes on cancer including cell proliferation, invasion and metastasis and hormonal regulation. Camalexin is one of the most studied compound that belongs to phytoalexins. It’s been reported that camalexin accumulates in cruciferae plants through the exposure to environmental stress and plant pathogens. Camalexin has also been reported to have antiproliferative effects in breast cancer and leukemia. At present study, we aimed to evaluate the effects of camalexin on proliferation and apoptosis of SW480 colon cancer cells. The cytotoxic doses were determined by MTT assay, while the apoptosis studies were performed via Annexin V binding assay. The effect of camalexin on MMP-9 mRNA expression has been determined by real-time pcr texhnique. This study was supported by Scientific and Technological Research Council of Turkey, Grant SBAG-216S129. According to the data camalexin significantly induced cell death at 25 uM and above concentrations and also an increase of Annexin V binding cell amount has been detected at 25 uM. The application of camalexin has significantly down-regulated mRNA expression of MMP-9 protein correlated with the increase of apoptotic cell amount. In conclusion, these results showed that camalexin display potential effects on colon cancer cells through inducing cell death and apoptosis and the future studies will illuminate the detailed mechanisms underlying these effects as well as potential utility of this compound in cancer treatments.


43

Poster abstracts

4

Differential role of TNF-α in multifactorial resistance in cancer cells

Tandressa Berguetti1, Paloma Souza1, Marcela Robaina1, Raquel Maia1

1 Instituto Nacional de Câncer, Rio de Janeiro, BRAZIL

Multidrug resistance phenotype (MDR) is characterized by P-glycoprotein (Pgp/ABCB1) overexpression and related to chemotherapy failure. However, MDR may be associated with apoptotic pathways deregulation. The Tumor Necrosis Factor-alpha (TNF-α) is an important cytokine that may trigger signal death or tumor growth. In addition, our group previously demonstrated a correlation between Pgp and TNF superfamily proteins. Therefore, this study aimed to investigate the TNF-α role in multifactorial cancer resistance phenotype associated with Pgp expression. We used two cervical cancer cells: KB-3-1, parental and KB-C1, Pgp-positive. The TNF superfamily members were analyzed by TaqMan Array Human Apoptosis 96-well. Cells were treated with recombinant TNF-α (rTNF-α) and apoptosis were measure through Annexin-V/PI staining and pro-caspase-3 levels. Pgp/ABCB1 and endogenous TNF-α/TNFA were analyzed by Western blot, immunofluorescence and qRT-PCR. Pgp efflux activity was analyzed by flow cytometry. The TNFA mRNA was up regulated in KB-C1 cells compared to the parental cells. We also observed low apoptosis rate following rTNF-α treatment in both cell lines by Annexin-V/PI staining, however KB-C1 cells showed pro-caspase-3 drop down protein levels. While rTNF-α induced an increase of ABCB1 mRNA levels in KB-3-1 cells, no changes were observed in protein levels. Instead, rTNF-α did not alter ABCB1 mRNA expression levels in KB-C1 cells, but induced downregulation in protein levels with no efflux activity changes. However, TNF-α mRNA and protein showed upregulated in both cell lines after rTNF-α treatment. In summary, our results suggest that rTNF-α does not significantly alter cell viability, independent of Pgp expression. However, the downregulation of pro-caspase-3 may be associated with Pgp expression. In addition, rTNF-α induces a differential regulation of Pgp/ABCB1, which suggests an epigenetic regulation of Pgp expression in KB-3-1 cells. The maintenance of Pgp activity even after downregulation of Pgp expression levels, suggest a possible role of TNF-α in supporting resistance phenotype.


44

5

LP226, a novel hydroxylated-derivative of DHA that triggers cell death in glioma cells

Roberto Beteta-Göbel2,1, Javier Fernández-Díaz2,1, Sebastià Parets2,1, Catalina Ana Rosselló2,1, Xavier Busquets2,1, Manuel Torres2,1, Victoria Lladó2

1 Lipopharma Therapeutics SL, Balearic Islands, SPAIN, 2 University of the Balearic Islands, Balearic Islands, SPAIN

Gliomas, a malignant tumor of the glial tissue of the nervous system, are the second highest cause of cancer death among young people with an average life expectancy of 14.4 months under Standard of Care (SoC, radiotherapy plus Temozolomide –TMZ–) for which reason is essential to develop new therapies. Lipopharma designs and develops disease-modifying synthetic bio-active lipids based on cutting edge technology, Membrane Lipid Therapy (MLT), which aims to regulate the membrane lipid composition and structure to control cell functions. In this sense, we designed LP226, a 2-hydroxylated -derivative of DHA, which showed a high anticancer activity.

In this study, we demonstrate that LP226 is actively incorporated into cultured cells to inhibit glioma cell proliferation. Such an anticancer activity was further confirmed in nude mice bearing human glioblastoma cells (U-118 MG) where LP226 decreased tumor growth as compared with untreated mice, with minimal toxic effects. Regarding the molecular mechanism of action, LP226 induced BiP expression in glioma cells, suggesting that LP226 treatment may stimulate reticulum stress via UPR (Unfolded Protein Response). Accordingly, we also observed the activation of JNK and eIF2α. In order to test if this pathway activation led to cell death, we also measured CHOP levels. As expected, CHOP was shown increased after LP226 treatment which in turn suggests that LP226 induces cell death via UPR stimulation. Furthermore, the analysis of autophagy markers (LC3B-II and SQSTM1/p62) also demonstrated induction of this pathway in these cells although its role on cell death remains un-elucidated.

Innovative MLT-based drugs, being LP226 one of them, have shown to be a safe and efficacious therapeutic alternative that will bring dramatic improvements in the clinical outcome and in the quality of life for an important number of patients with cancer or other pathologies with unmet effective and safe treatment yet.

Poster abstracts


45

6

Bacteria-mediated cell death of colorectal tumors in mouse xenograft models

Madhura Bhave1, Patricia Conway1,2, Swee Hin Teoh1, Juliana Chan1

1 Nanyang Technological University, Singapore, SINGAPORE, 2 University of New South Wales, Sydney, AUSTRALIA

Bacterial cancer therapy has the potential to overcome the problems of tumor hypoxia and multi-drug resistance, using anaerobic spores of bacteria such as the proteolytic Clostridium sporogenes. However, the use of spores or live bacteria comes with the risk of toxicity and infection. To circumvent these issues, the anti-cancer effect of heat-inactivated C. sporogenes bacteria (IB) and its secreted bacterial proteins, known as conditioned media (CM) was investigated. After successfully inhibiting the growth of CT26 and HCT116 colorectal cancer cells in vitro, the efficacy of IB and CM was tested in mouse models of colorectal tumors using sub-cutaneous tumors of CT26 and HCT116 cells in Balb/C and athymic Balb/C nude mice respectively. These non-viable bacterial derivatives were administered through intra-tumoral injections over a period of 12 days, during which the tumor volume was measured. IB-mediated cell death inhibited the growth of the tumors with to a 60% tumor volume of the control by the end of the study in Balb/C mice. Notably, when the IB-treated mice were subsequently re-challenged with CT26 cells, the immune mediated-response prevented the establishment of a second tumor in 80% of the mice. Corresponding in vitro studies suggest that the cell death mechanism of IB and CM differs. IB-treated cells had higher numbers of necrotic cells while CM-treated cells had more apoptotic cells. secreted proteins present in CM were responsible for anti-cancer effect observed. Furthermore, it was found that physical interaction between the IB and the cancer cells is essential to affecting cell death in the latter. The study shows that bacterial derivatives inhibitive the growth on colorectal tumors by mediating cell death, indicating that there is a safer alternative to the use of spores and live bacteria. With further research, these non-viable derivatives could be developed as an alternative or adjunct to traditional cancer treatments.

Poster abstracts


46

7

Inhibitor of apoptosis proteins as targets for the treatment of malignant rhabdoid tumor

Rachel Coyle2,1, Maureen O’Sullivan1, Daniela Zisterer2

1 National Children’s Research Centre Crumlin, Dublin, EIRE, 2 Trinity Biomedical Sciences Institute, Dublin, EIRE

Malignant rhabdoid tumor (MRT) is an aggressive paediatric neoplasm, primarily diagnosed in those below the age of three. MRTs most commonly arise in the kidneys and central nervous system. Their diagnosis carries with it a poor prognosis, with survival rates of approximately 30 % despite intensive multimodal treatment. As such, new therapeutic strategies for the treatment of MRT are desperately needed. The inhibitor of apoptosis proteins (IAPs) have previously been shown to be overexpressed in numerous malignancies, with their overexpression commonly associated with a poorer prognosis. Therefore, they are currently being explored as clinical targets. Numerous small molecule IAP inhibitors have been shown to act as chemo- or radio- therapeutic sensitising agents in many cancer cell lines. However, the role of the IAPs in MRT has not yet been examined. In this study, the expression of a range of IAPs in a panel of MRT cell lines was demonstrated. Furthermore, the XIAP inhibitor embelin, was shown to sensitise TRAIL resistant MRT cells to cell death at a range of timepoints and concentrations. This synergistically enhanced cell death was dependent upon caspase 8 activation and correlated with caspase 8 and 3 cleavage suggesting apoptosis mediated by the extrinsic pathway. Furthermore, early downregulation of Mcl-1 upon combination treatment, and cleavage of Bid suggests engagement of the intrinsic or mitochondrial apoptotic pathway. In addition, FLIP and survivin downregulation, along with livin cleavage was observed upon co-treatment which may contribute to the enhanced cell death. Embelin treatment also enhanced cisplatin mediated cell death in MRT cells demonstrating its ability to synergise with intrinsic as well as extrinsic inducers of apoptosis. Thus, this study suggests that IAP inhibition via embelin in combination with traditional cancer therapeutics such as TRAIL or cisplatin has potential as a therapeutic strategy for the treatment of MRT.

Poster abstracts


47

8

Murine myeloma cells have a limited capacity to undergo immunogenic cell death in response to chemotherapy and epigenetic treatment

Lien De Beck1,2, Sarah Melhaoui2, Kim De Veirman1, Eline Menu1, Elke De Bruyne1, Karin Vanderkerken1, Karine Breckpot2, Ken Maes1

1 Laboratory of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel, Brussel, BELGIUM, 2 Laboratory of Molecular and Cellular Therapy, Vrije Universiteit Brussel, Brussel, BELGIUM

Dying cells can release danger-associated molecular patterns (DAMPs) and trigger an adaptive immune response, a process known as immunogenic cell death (ICD). Induction of ICD depends on the therapy and the capacity of cells to release the necessary DAMPs. Multiple myeloma (MM) is a plasma cell cancer which develops in the bone marrow. MM cells escape from the immune system via the PD1/PDL1 pathway and presence of inhibitory immune cells. Occurrence of ICD in MM cells has not been studied thoroughly. Better understanding of ICD in MM may result in strategies that exploit ICD to improve current therapies. Here, we investigated ICD in the 5T33 immunocompetent murine model for MM in response to chemotherapeutics (melphalan, bortezomib, mitoxanthrone), a DNA methyltransferase inhibitor decitabine and a histone deacetylase inhibitor quisinostat. Decitabine and quisinostat-treated 5T33vt cells and to a lesser extent melphalan and mitoxanthrone-treated cells increased the expression of maturation markers upon co-culture with bone marrow-derived dendritic cells. Vaccination of mice with 5T33vt cells treated with melphalan, mitoxanthrone or the combination of decitabine and quisinostat, delayed tumor progression and resulted in at most 30% tumor free mice indicating sub-optimal induction of ICD. We next investigated molecular hallmarks of ICD. Regardless of the treatment, an increased expression of calreticulin in 7AAD negative 5T33vt cells was observed which coincided with annexinV positivity. Moreover, the expression of the “don’t eat me” signal, CD47, was increased upon treatment. No increase in HMGB1 release above control conditions was observed. Melphalan, bortezomib, decitabine and quisinostat induced the expression of Ifnb and interferon stimulated genes (Mx1, Oasl2, Ifi27, Cxcl10). To conclude, dying murine myeloma cells have a limited ICD-inducing capacity, potentially due to the presence of CD47, absence of HMGB1 and co-incidence of calreticulin and phospatidylserine. These results warrant further validation in human MM cells.

Poster abstracts


48

9

MiR-205-5p promotes invasion and metastasis in breast cancer stem cells

Antonella De Cola2, Alessia Lamolinara3, Paola Lanuti3, Cosmo Rossi2, Manuela Iezzi3, Matilde Todaro1, Vincenzo De Laurenzi21 Cellular and Molecular Pathophysiology Laboratory, University of Palermo, DiBiMIS, Palermo, ITALY, 2 Department of Medical, Oral and Biotechnological Sciences, Center of Excellence on Aging and Translational Medicine (CeSi-Met), G. D’Annunzio University, Chieti-Pescara, ITALY, 3 Department of Medicine and Aging Science, Center of Excellence on Aging and Translational Medicine (CeSi-Met), G. D’Annunzio University, Chieti-Pescara, ITALY

Breast cancer stem cells, a subpopulation of tumor cells with stem-like properties, play a pivotal role in tumor growth and metastatic progression contributing to therapy resistance. Metastatic process is sustained by epithelial to mesenchymal transition (EMT) program by which epithelial cells convert into mesenchymal phenotype acquiring the ability to invade and disseminate from the primary tumor site to distant tissues. EMT is induced by several stimuli from the tumor microenvironment but also microRNAs can mediate it based on the activity on target genes. In this study we report that reduced level of miR-205-5p by Locked Nucleic Acids molecular approach (LNA), impairs metastatic potential and tumor progression of breast cancer stem cells, modulating the expression of EMT key transcription factors. Furthermore, miR-205 silencing attenuates breast cancer stem cells stemness phenotype, suggesting miR-205 as a novel target for breast cancer therapies.

Poster abstracts


49

10

Novel combination therapies targeting the PI3K/Akt/mTOR and MDM2/p53 pathways in testicular cancer

Gerda de Vries1, Ximena Rosas Plaza1, Albert Suurmeijer1, Marcel van Vugt1, Jourik Gietema1, Steven de Jong1

1 University Medical Center Groningen, Groningen, NETHERLANDS

Introduction: Metastatic testicular cancer (TC) is highly sensitive to cisplatin-based chemotherapy. However, patients with advanced disease in the poor prognosis group only have a 50% 5-year survival resulting from chemo-resistance. Previous data showed that the PI3K/Akt/mTOR pathway and the MDM2/p53 axis are involved in cisplatin resistance of TC cells. Here we investigated the inhibition of the PI3K/Akt/mTOR pathway in combination with cisplatin or Nutlin-3a as possible treatment options for TC.

Methods: TC cell lines sensitive or resistant to cisplatin were treated with Akt inhibitor MK2206, mTORC1 inhibitor everolimus or mTORC1/2 inhibitors AZD8055 and INK128 in combination with cisplatin or Nutlin-3a. DilC1(5)/PI stainings were used to determine the apoptosis inducing effect of different treatment combinations. Efficacy of drugs were tested with western blot. In vivo drug sensitivity was tested in TC PDX models. Mice were treated IP with cisplatin weekly for 3 weeks, or with AZD8055 daily for 3 weeks. Tumor growth and weight were measured.

Results: Apoptosis assays showed a ~2-3 fold increase in cisplatin induced apoptosis when combined with the mTORC1/2 inhibitors AZD8055 and INK128 in 2 resistant cell lines. Everolimus or MK2206 combined with cisplatin showed none to a small sensitizing effect. We also tested these inhibitors in combination with Nutlin-3a. A ~2-4-fold increase in apoptosis was observed in p53 wt cells at low Nutlin-3a concentrations when co-treated with MK2206, AZD8055 or INK128, associated with decreased p-MDM2 protein levels. The combination of everolimus and Nutlin-3a showed a lower apoptotic potential. Western blot confirmed protein inhibition. In vivo, chemo-sensitivity or resistance was reflected by the established PDX models in vivo. In addition, treatment with a high dose of AZD8055 stabilized tumor growth.

Conclusion: mTORC1/2 inhibition resulted in strong induction of apoptosis in combination with cisplatin or Nutlin-3a. These novel treatment combinations will be further investigated in vivo using TC PDX models.

Poster abstracts


50

11

New development of anticancer monoclonal antibodies targeting death receptor

Agathe Dubuisson3,1, Olivier Micheau3,2, Said El Alaoui1, Eric Fourmaux1, Sabrina Lareure1

1 CovalAb, Research Department, 11 Avenue Albert Einstein, Lyon, FRANCE, 2 INSERM, UMR1231, « Equipe labellisée Ligue contre le Cancer » and Laboratoire d’Excellence LipSTIC, Dijon, FRANCE, 3 Univ. Bourgogne Franche-Comté, INSERM, LNC UMR1231, Dijon, FRANCE

Restoring programmed-cell death of cancer cells is a real challenge in oncology. To date, several monoclonal antibodies targeting death receptors have been developed but they were either too toxic (anti-TNFα and anti-Fas) or failed to show significant clinical effects (anti-DR4 and anti-DR5). The effort should be continued to develop novel DR4 and DR5 antibodies with improved properties. In this regard, monoclonal antibodies targeting DR4 have been created here using DNA-immunisation. DNA-immunisation is a cost- and time-effective technique and provides antibodies that can recognise DR4 in their native forms. In this study, immunisations elicited significant humoral anti-DR4 responses and fusions resulted in numerous hybridomas that can specifically recognise DR4 in its native form. After an intensive screening, 17 clones were selected for production. They were then characterised and tested for their capacity to induce apoptosis in a wide range of cancer cell lines. These extensive tests allowed the identification of five antibodies of interest: one antibody agonistic to the ligand TRAIL, three others that can potentiate ligand action, and a last one showing antagonistic or competitive inhibition of TRAIL. Future in-vivo studies will be performed to confirm those results. This study shows efficiency of DNA-based immunization for generating efficient therapeutic monoclonal antibodies inducing cancer cell apoptosis.

Poster abstracts


51

12

The dark side of MET receptor: Impact on survival-apoptosis balance in vivo

Leslie Duplaquet1, Catherine Leroy1, Audrey Vinchent1, Fabien Vanden Abeele3, Alessandro Furlan1,2, David Tulasne1

1 University of Lille/CNRS UMR8161, Lille, FRANCE, 2 University of Lille/CNRS UMR8523, Villeneuve d’Ascq, FRANCE, 3 University of Lille/INSERM U1003, Villeneuve d’Ascq, FRANCE

MET is the receptor for HGF predominantly expressed by epithelial cells. MET signalling pathways play a major role in epithelial homeostasis, embryogenesis and tissue regeneration in adult. MET is also a powerful oncogene able to promote tumor invasion and metastasis in many types of cancer. However, in absence of ligand, MET is also able to promote apoptosis through its caspase cleavage leading to the release of a pro-apoptotic fragment named p40MET. This fragment is able to activate the intrinsic pathway of apoptosis by inducing mitochondrial membrane permeabilization. While survival pathways induced by the ligand-activated MET are well described, the physiological relevance and mechanisms allowing p40MET to promote apoptosis are still unknown.

To understand the in vivo involvement of MET in apoptosis, we developed knock-in mice in which the receptor is mutated on a caspase site. From primary hepatocytes derived from these mice, we showed that mutation of MET caspase site induces reduction of the caspase 3 activation and a loss of mitochondrial membrane permeabilization resulting in a decrease of the apoptotic response. Interestingly, p40MET is localized at the interface between the endoplasmic reticulum (ER) and mitochondria, a subcellular domain involved in calcium exchange between these organelles. Consistently, p40MET is able to induce calcium leakage from ER that ultimately induces mitochondria permeabilization and the inhibition of the calcium exchange abrogates p40MET apoptosis.

Thus, our work demonstrates that in addition to its ability to induce survival, MET is involved in the amplification of apoptosis evidenced in primary hepatocyte from knock-in mice, through an original mechanism involving calcium exchange between ER and mitochondria. This characteristic has important consequences on the role of MET during tumorigenesis. Indeed, in cancer the survival/apoptosis balance leans towards survival, the interest will be therefore to inhibit pro-survival signalling of the receptor while retaining its pro-apoptotic capacities.

Poster abstracts


52

13

Study of the effects of mimetic triglycerides on human pancreatic cancer

Javier Fernández-Díaz2,1, Roberto Beteta-Göbel2,1, Paula Fernández-García1, Victoria Lladó 2, Pablo V. Escribá2

1 Lipopharma Therapeutics S.L. , Balearic Islands, SPAIN, 2 University of the Balearic Islands, Balearic Islands, SPAIN

Pancreatic cancer ductal cells (PDAC) is the fourth leading cause of cancer death in the US and sixth in Europe. Its difficult early diagnosis accompanied by its high rate of metastasis makes this a fatal disease: the median survival time from diagnosis is 4-6 months. The only curative therapy is surgical resection. Pancreatic cancer requires the development of new therapies.

We have tested the antitumor effect of novel mimetic triglycerides (“TGMs”) on human pancreatic cancer cell lines. Two molecules, TGM4 and TGM5 were selected for in vivo studies using xenograft tumor models. Both demonstrated to be non-toxic and showed antitumor activity impairing the tumor growth and progression in comparison to control. TGM4 matched the efficacy of Gemcitabine, the standard of care for pancreatic cancer in humans.

TGM4 produced cell death and decreased the percentage of cells in G0/G1 phase, as determined by flow cytometry. This result was supported by the decrease in cell cycle regulatory protein cyclin D3. Furthermore, TGM4 seems to provoke cell death through a reticular stress (indicated by BIP overexpression) that triggers autophagy via CHOP. The autophagosomes formation was corroborated by the LC3B-II protein modification. Changes were also observed in signaling pathways involved in oncogenesis, such as the MAPK and PI3K / AKT pathways and the proto-oncogene c-jun. Finally, Thin-Layer Chromatography was use to study how TGM4 affected to the composition of the lipid membrane, inducing changes in the phospholipids cardiolipin and phosphatidylcholine.

These results indicate that the compounds included in the TGMs library have antiproliferative effect on pancreatic tumor cells. In fact, TGM4 and TGM5 have shown to be antitumor drugs on in vivo models. TGM4 is a molecule which due to its antiproliferative effects, apparent safety and antitumor effectiveness in vivo, could be an alternative future drug in the treatment of pancreatic cancer.

Poster abstracts


53

14

Investigating the activation of the non-canonical inflammasome in senescence

Irene Fernández-Duran1, Juan Carlos Acosta1

1 Edinburgh Cancer Research Centre, Edinburgh, UK

Senescent cells display an inflammatory phenotype which is controlled by the inflammasome. To better understand this process, we decided to study the role of inflammatory caspases in oncogene-induced senescence. We found that depletion of caspase-4 in a model of RASG12V oncogene-induced senescence in human primary fibroblasts results in bypass of the proliferation arrest as well as reduction of inflammatory markers.

Caspase-4, core protein of the non-canonical inflammasome, has been described to play an essential role in pyroptosis. In order to activate caspase-4, we transfected lipopolysaccharide (LPS) into IMR90 cells which resulted in cell death by pyroptosis as expected. However, the fraction of surviving cells displayed reduced cell proliferation and increased b-galactosidase staining in an LPS dose-dependent manner. ShRNA-mediated knockdown of caspase-4 followed by LPS transfection abrogated cell death as well as the senescence phenotype, confirming that both cell death and induction of senescence following LPS transfection are controlled by caspase-4. We are currently investigating the mechanism by which caspase-4 plays a role in senescence. We anticipate our findings may introduce a novel role for the non-canonical inflammasome by mediating senescence.

Poster abstracts


54

15

Development and pre-clinical assessment of a first-in-class small molecule inhibitor of FLIP

Jennifer P. Fox2, Catherine Higgins2, Joanna Majkut2, Greti Espona-Fiedler2, Emer Gates2, Nyree Crawford2, Christopher McCann2, Luke Humphreys2, Sandra Van Schaeybroeck2, Ray J. Boffey1, Trevor R. Perrior1, David Haigh2, Timothy Harrison2, Daniel B. Longley2

1 Domainex Ltd., Cambridge, UK, 2 Queens University Belfast, Belfast, UK

Evasion of cell death is a major cause of resistance to anti-cancer therapies. The anti-apoptotic protein FLIP is frequently overexpressed in a number of cancers and has been shown to be a major mediator of drug resistance. FLIP and procaspase-8 form complexes with the adaptor protein FADD in response to a variety of clinically relevant stimuli, including ligation of death receptors and chemotherapeutic agents. In these complexes, FLIP modulates the activation of procaspase-8, and thereby apoptosis and necroptosis - two major cell death mechanisms. We have found that there are important differences between FLIP and procaspase-8 in terms of their preferred modes of interaction with FADD that are therapeutically exploitable and subsequently identified a potential drug-binding pocket for selectively targeting FLIP. In silico followed by in vitro and cell-based screening identified small molecule inhibitors of the FLIP-FADD protein-protein interaction. Medicinal chemistry optimization of these hits identified a lead series that displays nanomolar, on-target (FADD and caspase-8-dependent) activity in cell-based assays in line with their binding affinity in an orthogonal biophysical assay. Moreover, the pro-apoptotic effects of these FLIP inhibitors were enhanced not only upon co-treatment with activators of the extrinsic apoptotic pathway such as TRAIL and IAP inhibitors (SMAC mimetics), but also in combination with standard-of-care chemotherapeutics (such as 5-Fluorouracil and platinum agents), targeted agents in relevant clinical subpopulations (such as MDM2 inhibitors in TP53 wild-type cancers and MEK inhibitors in BRAF mutant cancers) and ionizing radiation. Lead series compounds have also demonstrated in vivo activity alone and in combination with TRAIL and, importantly, are well-tolerated. Thus, we present first-in-class small molecules for targeting the cell death inhibitory protein FLIP that have numerous potential combination partners and therefore numerous potential applications for the treatment of therapy-resistant cancer.

This work is supported by the Wellcome Trust.

Poster abstracts


55

16

Acquired chemotherapeutic resistance to 5-FU is regulated by autophagy and senescence

Lahiru Gangoda1, Nidhi Abraham Mathew1, Selma Viden1, Shiva Keertikumar4, Ching-Seng Ang5, Amardeep Dillon3, John Mariadason3, Ramanuj Dasgupta2, Suresh Mathivanan1

1 Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Victoria, AUSTRALIA, 2 Genome Institute of Singapore, Agency for Science, Technology &Research (A*STAR), Singapore, SINGAPORE, 3 Olivia Newton-John Cancer Research Institute, Victoria, AUSTRALIA, 4 Peter MacCallum Cancer Centre, Victoria, AUSTRALIA, 5 The Bio21 Institute, University of Melbourne, Victoria, AUSTRALIA

Colorectal cancer is the third most common cancer in the world and the second leading cause of cancer-related death in the western world. Chemotherapy is the mainstay in the treatment of metastasized colorectal cancer. However, cancer cells acquire resistance to treatment by various mechanisms resulting in treatment failure. Even though the molecular mechanisms regulating acquired drug resistance is critical to overcome chemoresistance, it is poorly understood. We developed a panel of seven colorectal cancer (CRC) cells resistant to the chemotherapy drug, 5-Fluorouracil (5-FU). The parental and 5-FU resistant CRC cells were assayed for proteins known for their involvement in chemotherapeutic resistance in addition to unbiased quantitative proteomics and DNA methylation analysis. The integrated analysis revealed multiple mechanisms contributing to chemotherapeutic drug resistance including epithelial-to-mesenchymal transition, deregulation of apoptosis, dysregulated signaling pathways, dysregulation in mitochondrial energy metabolism, increased survival autophagy and senescence. We next attempted to sensitize the drug resistant cells by a combination of pharmacological inhibitors and CRISPR-based molecular biology techniques. While many other events were by-stander effects and did not confer to chemoresistance, inhibitors of late-autophagy and senescence sensitized the 5-FU resistant cells. As a follow up, patient derived xenograft (PDX) mouse models were established and made resistant to 5-FU. The mechanisms implicated in cell models were validated in PDX tissues. Overall, targeting autophagy and senescence was shown to overcome chemoresistance and hence could increase patient survival rates when used in combination with 5-FU.

Poster abstracts


56

17

Does Kremen-1 induce autophagic cell death?

Clara Geneste1, Ana Negulescu1, Shuheng Lin1, Baptiste Robin1, Elisabeth Errazuriz-Cerda1, Patrick Mehlen1, Olivier Meurette1

1 CRCL, Lyon, FRANCE

The transmembrane receptor Kremen1 (K1) has first been described as a receptor for Dickkopf ligands (DKK1, 2, 3 and 4) and its ability to block the WNT (wingless-related integration site) signalling. More recently, K1 was demonstrated to mediate cell death independently of its ability to inhibit Wnt signalling through a dependence receptor function. These receptors have a dual signalling ability depending on the availability of ligands. As such, K1 is inducing cell death unless bound to DKK1.

We showed that K1 is lost in many different cancers whereas DKK1 is up-regulated in cancers. In breast cancer carcinoma, K1 high expression is also a factor of good prognosis in patients in which DKK1 expression is low, showing a possible conditional tumour suppressive activity. We tested the ability of K1 to induced cell death in breast cancer cells. We showed that K1 overexpression or downregulation of DKK1 induces an atypical cell death mechanism, which is not inhibited by apoptosis or necroptosis inhibitors and that has autophagic phenotypic features. We confirmed autophagy induction by western blotting for the lipidated form of LC3B following expression of K1. Furthermore, this cell death is rescued by 3-methyladenine (an autophagy inhibitor) and enhanced by blocking the autophagic flux. We are currently validating putative partners obtained by a shRNA screen to decipher the precise mechanism of this pathway.

Poster abstracts


57

18

Cellular Senescence in Hodgkin’s Lymphoma

Siddharth Balachandran2, Jacob Gopas1,3, Estee Stern1, Udi Zurgil1, Janet Ozer1, Assaf Ben Ari1, Gregory Shubinsky1,3, Alex Braiman1, Rosa Sinay1, Rodda Ezratti1, Vladimir Dronov3, Daniel Benharroch1,3, Etta Livneh1

1 Ben Gurion University , Beer Sheva, ISRAEL, 2 Fox Chase Cancer Center, Philadelphia, USA, 3 Soroka University Medical Center, Beer Sheva, ISRAEL

Hodgkin’s lymphoma (HL), is a B cell malignancy. Although the rate of cure is high, about 20-35% of patients relapse, and about half of them eventually die of the disease or treatment-related late toxicities and secondary malignancies. Conceptually novel treatment strategies are thus needed, particularly for this category of patients. The malignant cells, called Hodgkin and Reed-Sternberg (HRS) cells, only make up 1-2% of the total tumor cellularity; the remaining mass comprising a mixed infiltrate population is thought to be recruited to the lymph node by HRS-driven pro-inflammatory signals. We propose that a sub-population of HRS cells, which we call herein large RS cells, have characteristics of senescent cells, and, thus produce large amounts of inflammatory mediators (the so-called “senescence-associated secretory phenotype”, or SASP). Senescence is characterized by permanent cell cycle arrest and loss of proliferative capacity, despite continued viability and metabolic activity. We show that the senescence markers the cell cycle inhibitors p16INK4a and p21Cip1 are expressed in large RS cells in all HL biopsies examined. Moreover, the large RS cells are negative for Ki-67 staining, demonstrating that these cells have ceased to proliferate. We also show that large, ‘RS-like’ cells in a HL-derived line, L428, stained for the characteristic senescence marker β-galactosidase (β-gal). Oxidative stress and chemotherapy increased the proportion of β-gal positive large RS cells. Furthermore, we show that the large RS cells secrete high levels of cytokines. We suggest that these senescent RS cells may be responsible, at least in part, for creating a pro-inflammatory microenvironment, promoting HL pathogenesis, and mediating chemo-resistance in relapsed disease. Understanding the pathways important for the establishment of senescence in HRS cells, as well gaining insight into targetable mechanisms for the eradication of these cells, will provide new therapeutic approaches for HL patients with recurrent or chemo-resistant disease.

Poster abstracts


58

19

Investigation of antitumor activity of LQB-223 compound in drug-resistant breast cancer cells

Lauana Greicy Tonon Lemos3,5, Gabriela Nestal-de-Moraes3, Gabriel Longo3, Flavia da Cunha Vasconcelos3, Paula Sabbo Bernardo3, Eric W-F Lam2, Camilla Djenne Buarque1, Paulo Roberto Ribeiro Costa4, Raquel Ciuvalschi Maia3

1 Departamento de Química, Pontifícia Universidade Católica do Rio de Janeiro, Rio de Janeiro, BRAZIL, 2 Department of Surgery and Cancer, Imperial Centre for Translational and Experimental Medicine (ICTEM), Imperial College London, London, UK, 3 Laboratório de Hemato-Oncologia Celular e Molecular, Programa de Hemato-Oncologia Molecular, Instituto Nacional do Câncer (INCA), Rio de Janeiro, BRAZIL, 4 Laboratório de Química Bioorgânica, Instituto de Pesquisas de Produtos Naturais, Universidade Federal do Rio de Janeiro, Rio de Janeiro, BRAZIL, 5 Programa de Pós-Graduação Strictu Sensu em Oncologia , Rio de Janeiro, BRAZIL

Breast is a leading cause of deaths among women worldwide. A major obstacle for successful chemotherapeutic treatment is multidrug resistance phenotype. Then, the development of novel anticancer compounds that might be able to surpass drug resistance mechanisms is crucial. The compound 11a-N-Tosyl-5-deoxi-pterocarpan, LQB-223, has been previously tested in vitro by our group and shown sensitize breast cancer cells, with little toxicity to non-neoplastic breast cells. The aim of this project was to assess LQB-223 effects in doxorubicin-resistant cells, MCF-7 DoxR. MCF-7 DoxR were exposed to LQB-223, and the toxic effects were evaluated by MTT and clonogenic assays. AnnexinV/PI labeling and cell cycle distribution were analyzed by flow cytometry. The cell migration was assessed by wound-healing assay. The effects of the combination of LQB-223 with drugs used in breast cancer treatment were evaluated by MTT assay. Protein expression was evaluated by Western blotting. LQB-223-mediated toxic effects in 3D conformation were analyzed by the APH assay, measurement of tumor size and cell migration. Our results show that LQB-223 treatment induced cytotoxicity and G2/M arrest in MCF-7 DoxR cells. Notably, we found a reduction in the migratory profile of MCF-7 and MCF-7 DoxR cells following LQB-223 treatment. Interestingly, LQB-223-mediated cytotoxic effects in the resistant cells were observed despite elevated Pgp expression and activity and overexpression of FOXM1, Survivin, XIAP, c-IAP1 and Mcl-1 chemoresistance-related proteins. Also, we found a synergistic effect for the combination of LQB-223 and docetaxel and doxorubicin in MCF-7 DoxR cells. In 3D conformation, LQB-223 treatment remarkably decreased cell viability, tumor size and cell migration. These results suggest the LQB-223 promotes cytotoxic effects and modulates the migratory profile in drug resistant breast cancer cells in 2D and 3D models, suggesting it acts against breast cancer cells, preferentially in tumors bearing the multidrug resistance phenotype.

Poster abstracts


59

20

Autophagy related markers (Beclin-1 and ATG4b) are strongly expressed in Wilms’ tumor and correlate with favourable histology

Maha Guimei3,2, Anu Ranade3, Mohamed Eladl3,1

1 Mansoura University, Mansoura, EGYPT, 2 University of Alexandria, Alexandria, EGYPT, 3 University of Sharjah, Sharjah, UNITED ARAB EMIRATES

Wilms’ tumor (WT) is the most common kidney malignancy in children. In spite of the great advances in treatment, unfavourable histology WTs and around 10% of favourable histology cases (FH) still fail to respond to current multimodality therapy. Of particular interest is the context-dependent role of autophagy in cancer. The characterization of potent, specific autophagy inhibitors with novel mechanisms of action is a very active area of research. To the best of our knowledge, the role of autophagy in WT has not been investigated. The current study was aimed to investigate the expression of autophagy related markers (ATG4 and Beclin1) in WT, its association with the different clinic-pathological parameters and its impact on patient survival. Twenty one formalin fixed paraffin embedded (FFPE) WT specimens were immunohistochemically stained using autophagy related markers; Beclin-1 and ATG-4b. All clinical, radiological and follow up data were retrieved from the patient records. All specimens showed positive expression of both Beclin-1 and AtG4b. The staining score (H-score) for Beclin1 varied between 50 and 300 and its expression was significantly associated with favourable histology (p=0.007). Similarly, ATG-4b expression was significantly higher in favourable histology tumours compared to unfavourable histology (p=0.046). A statistically significant positive correlation between Beclin-1 and ATG4 expression was observed. The cumulative disease free survival in patients with favourable histology was significantly higher compared to patients with unfavourable histology (p=0.0027). Beclin-1 and ATG4b expression were both found to be statistically significant discriminators of survival. Collectively these findings suggest that the expression of autophagy related markers is associated with a favourable histology and could predict a longer disease free survival as well as overall survival in these patients.

Poster abstracts


60

21

The selective cytotoxic effect of curcumin and doxorubicin combination on acute myeloid leukaemia cell line

Noor Hasan1

1 Middlesex University, London, UK

Frontline treatment for acute myeloid leukaemia (AML) involves chemotherapy drugs, however disease-free survival rarely goes beyond 5 years and clinical limitations include cardiotoxicity and early onset of drug resistance; therefore discovery of new therapeutic agents from natural products is increasing. Curcumin (Curcuma longa, turmeric) has shown to induce anti-oxidant and anti-inflammatory properties. The aim of this study was to investigate the effect of doxorubicin and curcumin individually and as combinations on healthy human monocyte cells and AML (MOLM13) cell line at 24 h and 48 h incubations. Cell viability was measured using CyQuant assay (binds to nucleic acids of live cells). From this, appropriate compound concentrations were selected for drug combination studies and the cell viability was measured. Neither doxorubicin (0.1-0.5 µM) nor curcumin (5-15 µM) incubation of normal monocyte cells showed any significant decrease in cell viability compared to negative control at 24 h and 48 h. AML cells incubated with doxorubicin or curcumin showed the drugs act in a dose and time-dependent manner. This shows curcumin to possess selective cytotoxic properties against the MOLM13 cell line and not the normal monocyte cells. The combination of doxorubicin (0.2 and 0.5 µM) with curcumin (10µM) also decreased the cell viability more pronouncedly in MOLM13 compared to normal monocytes. The effect of curcumin on the Bcl-2 family of proteins (involved in cell apoptosis) was also investigated using Western blot. It is thought that the combination of curcumin with doxorubicin has the potential to increase the therapeutic effect of chemotherapy while also reducing its cytotoxicity.

Poster abstracts


61

23

The Molecular Mechanisms Controlling Epithelial Cell Survival And Death In The Thymus During Cytoablative Treatment

Melanie Heinlein2,1, Reema Jain2,1, Andreas Strasser2,1, Daniel HD Gray2,1

1 Department of Medical Biology, The University of Melbourne, Parkville, Victoria, AUSTRALIA, 2 The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, AUSTRALIA

Thymic epithelial cells (TECs) direct the process of T cell differentiation and are essential for cellular immunity. Atrophy of the thymus following cyto-ablative therapy impairs immune recovery in patients. This is a significant cause of morbidity and mortality. Despite their importance for immunity, there is little understanding of the mechanisms that control TEC survival and death, especially in the context of treatments that damage the immune system (e.g. chemotherapeutic agents or γ-irradiation). The aim of this project is to elucidate: 1) the mechanisms of TEC death in response to different cyto-ablative therapies, and; 2) which pro-survival BCL-2 family proteins are necessary for TEC survival and thymus regeneration.

To assess whether TEC death is induced by cyto-ablative treatments directly or secondary to the loss of thymocytes, the intrinsic apoptotic pathway was genetically blocked by deleting BAX and BAK exclusively in TECs (using Foxn1Cre Baxfl/fl Bak-

/- mice) or thymocytes (using CD4Cre Baxfl/fl Bak-/- mice). We found that γ-irradiation-induced TEC loss was rescued in CD4Cre Baxfl/fl Bak-/- mice, indicating that TEC death during thymic injury is caused by the loss of thymocytes.

Our recent studies found that BCL-2 and BCL-XL are not critical for TEC homeostasis under steady-state conditions. However, we now show that thymic recovery in Foxn1Cre Bcl2fl/fl and Foxn1Cre BclxLfl/fl mice following γ-irradiation is crippled. We found that TEC residing in the medulla, which are critical for thymic tolerance, were particularly effected by the loss of these pro-survival proteins. These data reveal a differential role for these proteins in TEC survival under steady-state homeostasis versus injury. Together, these studies reveal the molecular mechanisms that govern thymic dysfunction and recovery following cyto-ablative therapies. This insight will be useful for the design of strategies that target TEC survival to preserve or recover thymic function in patients.

Poster abstracts


62

24

Epigallocatechin gallate-dependent regulation of mTOR-AMPK pathways upon endoplasmic reticulum stress

Marianna Holczer1, Boglárka Besze1, Gábor Bánhegyi1, Orsolya Kapuy1

1 Semmelweis University, Department of Medical Chemistry, Molecular Biology and Pathobiochemistry , Budapest, HUNGARY

Keywords: EGCG, AMPK, mTOR, GADD34, autophagy, endoplasmic reticulum stress

The maintenance of cellular homeostasis is mainly dependent on the ability of cells to take precise actions with respect to various stimuli (such as nutrient availability, growth factors etc.). We have recently proposed a regulatory network, where the life-and-death decision of ER stress response mechanism is defined by the positive and negative feedback loops of autophagy, apoptosis and mTOR-AMPK pathways. The transient switch from autophagy-induced survival to apoptotic cell death is controlled by GADD34 with respect to endoplasmic reticulum (ER) stress. Recently it has been suggested that the major polyphenol of green tea (called epigallocatechin gallate or EGCG for short) enhances autophagy at various diseases, however the details of the regulatory features of this natural compound on the self-eating process are lacking.

The aim of the present study is to investigate the role of EGCG in promoting autophagy-dependent survival and to verify the key role of GADD34 in the connection with mTOR-AMPK pathways upon prolonged ER stress.

EGCG treatment is able to extend cell viability via inducing cytoprotective autophagy, meanwhile mTOR gets down-regulated and AMPK gets up-regulated. EGCG treatment induces the self-eating process through ULK1-AMPK-mTOR regulatory network. We show that inhibition of GADD34 by guanabenz or siGADD34 results in down-regulation of autophagy-dependent survival and a quick activation of mTOR/inactivation of AMPK, followed by apoptotic cell death. The negative effect of GADD34 inhibition is diminished by combined treatment with EGCG. Our data suggest a novel role for EGCG by promoting cell survival via imbalancing mTOR-AMPK pathways with respect to ER stress.

Since many studies have proved a link between ER stress and cancer long-term significance of this work shows medical purposes.

This work was supported by the COST ACTION BM1307 PROTEOSTASIS and ÚNKP-17-4-III-SE-75 New National Excellence Program of the Ministry of Human Capacities.

Poster abstracts


63

25

Caspase-independent cell death (CICD) does not elicit the compensatory proliferation of melanoma cancer cells

Gabriel Ichim1

1 Center for Research on Cancer of Lyon (CRCL), Lyon, FRANCE

Apoptosis, a form of programmed cell death, gives the shape of all multicellular organisms, while keeping them healthy. During development, apoptosis removes unwanted cells and corrects growth-related errors. In cancer, it is the preferential killing weapon employed by tumor suppressor genes and most chemotherapeutic drugs. Although efficient in killing cancer cells, apoptosis has been recently shown to trigger the proliferation of neighboring cells while also having an immune silencing effect.

Therefore, the aim of this study is to examine whether triggering alternative types of cell death would also impact on the proliferation of surviving cancer cells. For this purpose, we set up an in vitro model to trigger either apoptosis or caspase-independent cell death (or CICD) in melanoma cells and compare the kinetics of compensatory proliferation engaged by both types of cell death. More specifically, apoptosis is triggered by a doxycycline-inducible BAX expression while CICD is efficiently induced using the same system in a CRISPR/Cas9-generated APAF-1 KO background. We then co-cultured apoptotic or CICD cells with melanoma cells stably expressing H2B-mCherry and we followed their proliferation using an Incucyte Imager. As expected, apoptosis elicits an increase in the proliferation of surviving neighboring cells. Interestingly, CICD did not produce the same effect, although it initiated a complex cytokine storm. This effect seems to be independent of the MITF-ZEB1 axis of transcription factors, which is important for melanoma proliferation. Moreover, melanoma cells grown in the presence of CICD conditioned media have impaired migration capacities.

To conclude, this study enforces the idea that engaging caspase-independent cell death for cancer therapy might be preferable over apoptosis since it does not have a compensatory proliferation effect on surviving cancer cells.

Poster abstracts


64

26

Aurora A is essential for survival of head and neck cancer cells expressing high-risk human papilloma virus E7 oncogene

Adi Idris1, Nigel McMillan1

1 Menzies Health Institute Queensland and School of Medical Science, Griffith University, Southport, Queensland, AUSTRALIA

Human papilloma virus (HPV) is the main culprit in cancers of the cervix, penis, anus, skin, eye and head and neck and is responsible for ~5% of the total cancer burden worldwide. Although the HPV vaccine is now available, the slow and gradual process for HPV cancers to form means little will change, even for vaccinated individuals, for the next 25 years. This warrants the development of new therapeutic strategies in both the newly diagnosed and recurrent patients. Importantly, a curative therapeutic regime to treat HPV cancer types have not yet been discovered, let alone for head and neck cancers. We have previously shown that Aurora kinase A is critical for survival of high-risk HPV type 16-transformed cervical cancer cells expressing the major HPV E7 oncogene. Using an Aurora kinase A-specific inhibitor, ALISERTIB, E7-expressing, not HPV negative, cervical cancer cells died by apoptosis through reduction of a pro-survival protein, MCL-1. Our findings highlight the requirement of E7 to sensitise cells to Alisertib-mediated cell death. In this study, we wanted to investigate whether these observations could be replicated in HPV positive head and neck cancer cells. Furthermore, we wanted to explore the range of HPV types sensitive to ALISERTIB. We have engineered non-HPV head and neck cancer cells, SCC25, to express a range of HPV E7 genes from low- and high-risk types with varying oncogenic potential. Out of all the high-risk HPV types tested, high-risk HPV type 52 E7 confers the highest sensitivity to MLN8237-mediated toxicity leading to PARP cleavage. MCL-1 was reduced in ALISERTIB-treated HPV52 E7-expressing cells, but not in SCC25 wild type cells, at least partially via a ubiquitin-proteosomal degradation mechanism. This study demonstrates that HPV 52 E7 oncogene hyper-sensitises head and neck cancer cells to ALISERTIB-mediated cell death. We are currently elucidating the mechanism MCL-1 ubiquitination.

Poster abstracts


65

27

Application of a Single-Cell Assay to Identify Drugs Targeting the Human Nucleotide Excision Repair Pathway

Feridoun Karimi-Busheri1, Xiaoyan Yang1, Razmik Mirzayans1, Michael Weinfeld1

1 University of Alberta, Department of Oncology, Edmonton, Alberta,, CANADA

Nucleotide excision repair (NER) is a multistep process and one of the critical DNA repair mechanisms uniquely capable of removing a broad spectrum of bulky DNA lesions that cause a major distortion to the DNA helix, thereby blocking replication and transcription. Such lesions include cyclobutane pyrimidine dimers (CPDs) induced by ultraviolet light (UV) as well as interstrand and intrastrand crosslinks induced by platinum-based cancer chemotherapeutic drugs. We are interested in identifying small molecule inhibitors of key players of NER that are capable of increasing the susceptibility of colon cancer cells to killing by bulky DNA-damaging agents. Here we report a proof-of-principle study in which we determined the feasibility of immunofluorescence microscopy to identify drugs capable of inhibiting UV-induced CPDs in individual cells. Employing an improved immunofluorescence protocol, we report a remarkable variation in the sensitivity of CPD-specific antibodies acquired from different sources to detect DNA lesions. The antibody supplied by Kamiya Biomedical (Seattle, WA, USA) proved to be highly sensitive to detect CPDs induced by UV (254-nm) under our conditions. Using this antibody coupled with immunofluorescence microscopy, we show that UV-induced CPDs are efficiently repaired by normal human fibroblasts and human colon carcinoma cells, but such lesions persist in the genome of NER-deficient (xeroderma pigmentosum complementation group A) fibroblasts. In addition, we report that treatment of colon carcinoma cells with small molecule inhibitors of XPA or ERCC1/XPF proteins markedly inhibited their ability to remove UV-induced CPDs.

Poster abstracts


66

28

Protein and chemotherapy profiling of extracellular vesicles harvested from therapeutic induced senescent triple negative breast cancer cells

Emma Kavanagh6, Sinéad Lindsay6, Melinda Halasz5,7, Luke Gubbins6, Karolina Weiner-Gorzel6, Matthew Ho Zhi Guang6, Aloysius McGolrick6, Eoin Collins6, Michael Henry3, Alfonso Blanco-Fernández6, Peter O’Gorman2, Patricia Fitzpatrick8, Michaela Higgins4, Paul Dowling1, Amanda McCann6,7

1 Biology Department, National University of Ireland Maynooth, Kildare, EIRE, 2 Haematology Department, Mater Misericordiae University Hospital, Dublin, EIRE, 3 National Institute for Cellular Biotechnology, Dublin City University, EIRE, 4 Oncology Department, Mater Misericordiae University Hospital, Dublin, EIRE, 5 Systems Biology Ireland (SBI), University College Dublin (UCD), EIRE, 6 UCD Conway Institute of Biomolecular and Biomedical Research, School of Medicine, University College Dublin (UCD), EIRE, 7 UCD School of Medicine, College of Health and Agricultural Science, University College Dublin (UCD), EIRE, 8 UCD School of Public Health, Physiotherapy and Sports Science, University College Dublin, EIRE

Triple negative breast cancer (TNBC) is an aggressive subtype with relatively poor clinical outcomes and limited treatment options. Chemotherapy, while killing cancer cells, can result in the generation of highly chemoresistant therapeutic induced senescent (TIS) cells that potentially form stem cell niches resulting in metastases. Intriguingly, senescent cells release significantly more extracellular vesicles (EVs) than non-senescent cells. Our aim was to profile EVs harvested from TIS TNBC cells compared with control cells to identify a potential mechanism by which TIS TNBC cells maintain survival in the face of chemotherapy. TIS was induced and confirmed in Cal51 TNBC cells using the chemotherapeutic paclitaxel (PTX) (Taxol). Mass spectrometry (MS) analysis of EVs harvested from TIS compared with control Cal51 cells was performed using Ingenuity Pathway Analysis and InnateDB programs. We demonstrate that TIS Cal51 cells treated with 75 nM PTX for 7 days became senescent (senescence-associated β-galactosidase (SA-β-Gal) positive, Ki67-negative, increased p21 and p16, G2/M cell cycle arrest) and released significantly more EVs (P=0.0002) and exosomes (P=0.0007) than non-senescent control cells. Moreover, TIS cells displayed an increased expression of the multidrug resistance protein 1/p-glycoprotein. MS analysis demonstrated that EVs derived from senescent Cal51 cells contained 142 proteins with a significant increased fold change compared with control EVs. Key proteins included ATPases, annexins, tubulins, integrins, Rabs and insoluble senescence-associated secretory phenotype (SASP) factors. A fluorescent analogue of PTX (Flutax-2) allowed appreciation of the removal of chemotherapy in EVs from senescent cells. Treatment of TIS cells with the exosome biogenesis inhibitor GW4869 resulted in reduced SA-β-Gal staining (P=0.04). In summary, this study demonstrates that TIS cells release significantly more EVs compared with control cells, containing chemotherapy and key proteins involved in cell proliferation, ATP depletion, apoptosis and the SASP. These findings may partially explain why cancer senescent cells remain viable despite chemotherapeutic challenge.

Poster abstracts


67

29

Malignant gliomas can be converted to benign glial cells through treatment with a combination of small molecules

Jinsoo Oh2, Yongbo Kim2,1, Yoon Ha2,1

1 Brain Korea 21 PLUS Project for Medical Science, College of Medicine, Yonsei University, Seoul, SOUTH KOREA, 2 Department of Neurosurgery, Spine & Spinal Cord Institute, College of Medicine, Yonsei University, Seoul, SOUTH KOREA

Gliomas, the most malignant central nervous system tumors, are known to have a very poor survival rate. Given that gliomas are derived from mutations of glial precursor cells, a considerable number of them still strongly react with glial-precursor-cell-specific markers. Thus, we investigated whether malignant gliomas can be converted to glial cells through the regulation of endogenous gene expression implicated in glial precursor cells. In this study, we used three small molecule compounds (cyclic adenosine monophosphate (cAMP) enhancer, mechanistic target of rapamycin (mTOR) inhibitor and Bromodomain and Extra-Terminal motif (BET) inhibitor) to convert malignant glioma cells to benign glial cells. Small-molecule-induced gliomas (SMiGs) were not only transformed into the glial-specific morphology but also showed positive reactions with glial-specific markers such as GFAP, CNP, and RIP. A microarray analysis showed that SMiGs exhibited a marked increase of glial-enriched genes, whereas the malignant-cancer-cell-specific gene was greatly decreased. Moreover, proliferation of malignant glioma cells could be greatly suppressed after being converted to benign glial cells. Our findings show the proof of concept that malignant gliomas can be reprogrammed to benign glial cells, using a combination treatment of small molecules, and their proliferation can be regulated by the differentiation. We suggest that our small molecule composition (i.e., forskolin, rapamycin, and I-BET151) may be the next generation of anticancer agent, which acts by reprogramming malignant gliomas to differentiate into glial cells.

Poster abstracts


68

30

Dexamethasone-mediated downregulation of cFLIP protein promotes Dexamethasone/BV6-induced cell death

Lara Kleinesudeik3,2, Katharina Rohde3, Simone Fulda3,2

1 German Cancer Consortium (DKTK), Frankfurt, GERMANY, 2 German Cancer Research Center (DKFZ), Heidelberg, GERMANY, 3 Institute for Experimental Cancer Research in Pediatrics, Goethe University, Frankfurt, GERMANY

We recently reported that the Smac mimetic BV6 and glucocorticoids, e.g. Dexamethasone (Dexa), synergize to induce cell death in vitro and in vivo. Here, we discover that this synergism involves Dexa-stimulated downregulation of cellular FLICE-like inhibitory (cFLIP) protein in acute lymphoblastic leukemia (ALL) and diffuse large B-cell lymphoma (DLBCL) cell lines. Dexa rapidly decreases cFLIP protein levels in ALL cells, which is further enhanced by addition of BV6. In DLBCL cells which constitutively harbor high cFLIP levels, Dexa prevents BV6-stimulated increase of cFLIP. While Dexa attenuates the BV6-stimulated activation of non-canonical NF-κB signaling, it suppresses cFLIPL protein but not mRNA levels pointing to a transcription-independent downregulation of cFLIP by Dexa. Analysis of protein degradation pathways indicates that Dexa causes cFLIP depletion independently of proteasomal, lysosomal or caspase pathways, as inhibitors of the proteasome, lysosomal enzymes or caspases all failed to protect from Dexa-mediated loss of cFLIP protein. Also, Dexa alone or in combination with BV6 does not affect overall activity of the proteasome. Overexpression of cFLIP to an extent that is no longer subject to Dexa-imposed downregulation rescues Dexa/BV6-mediated cell death. Vice versa, knockdown of cFLIP significantly increases BV6-mediated cell death in both ALL and DLBCL cell lines, thus mimicking the effect of Dexa. Altogether, these data demonstrate that Dexa-mediated downregulation of cFLIP protein promotes Dexa/BV6-mediated cell death, thereby providing novel insights into the synergistic antitumor activity of this combination treatment.

Poster abstracts


69

31

Interplay between POPX2 and TAK1 in the regulation of apoptosis

Cheng-Gee Koh2,1, Ting Weng2,1

1 Mechanobiology Institute, Singapore, SINGAPORE, 2 Nanyang Technological University, Singapore, SINGAPORE

POPX2 is a serine/threonine phosphatase belonging to the PP2C family. Our earlier work has shown that cells with high POPX2 levels exhibit increased motility and invasiveness. This is consistent with our findings that invasive breast cancer cells such as MDA-MB-231 have higher levels of POPX2 compared to the low invasive cell types such as MCF7. Knocking down POPX2 in MDA-MB-231 cells resulted in lower motility and invasiveness suggesting links between POPX2 levels and invasiveness. Indeed, a survey of genomic data available in databases found that triple negative breast cancer cells have higher levels of POPX2. These observations might suggest that POPX2 could be a possible target for therapeutic intervention.

However, we found that cancer cells with POXP2-knockdown may confer better survival when the cells are challenged with etoposide. In this study, we found that POPX2 interacts with TGF-beta Activated Kinase (TAK1). TAK1 plays essential roles in the regulation of innate and adaptive immunity and has been found to be a powerful pro-survival protein. It is well documented that TAK1 activates the IKK-NF-kB pathway to elicit its anti-apoptotic activities. We found that POPX2 dephosphorylates TAK1 and down-regulates TAK1 activity, suggesting that POPX2 negatively regulates TAK1-IKK-NF-κB signaling. When we treated cells with VP16, a topoisomerase inhibitor, to induce DNA double stranded breaks and apoptosis, we found elevated nuclear translocation of NF-kB and increased levels of NF-kB mediated genes in POPX2-knockdown cells. Our data suggest that activation of the TAK1-IKK-NF-kB axis in POPX2-knockdown cells enhances cell survival when the cells suffer DNA damage. Hence, the levels of POPX2 in the cells can influence the therapeutic outcome of chemotherapy. Modulation of POPX2 levels could be an avenue to fine-tune therapeutic responses.

Poster abstracts


70

32

The impact of low endogenous Caspase 3 level on apoptosis and chemo resistance in the AB thymoma cell line TAB1

Stefan Küffer1, Denise Müller1, Vivek Venkataramani1, Alexander Marx2, Philipp Ströbel11 Institute of Pathology / University Medical Center Göttingen, GERMANY, 2 Institute of Pathology / University Medical Center Mannheim, GERMANY

Thymomas (TH) and thymic carcinomas (TC) are rare but the most common primary mediastinal tumours. They tend to be aggressive and have a high frequency for local therapy failures. Chemotherapy is used in patients with non-resectable or recurrent disease, however an optimal treatment has not yet been established and tumours often become resistant. One of the early events in resistance is the inactivation of the apoptosis- or activation of anti-apoptotic-factors. We observed that the AB thymoma cell line TAB1 was not able to induce apoptosis due to the lack of Caspase 3 (Casp3) expression. This correlated well with immunohistochemical stainings of TH and TC tissue samples which revealed a decreased expression in the majority of type A and AB thymomas. Even though A and AB thymomas have favourable prognosis, low levels of Casp3 might be an initial events in avoiding cell death and in cancer onset. The elucidation of the regulation of Casp3 expression and the underlining mechanism might help in the decision for different treatment options.

Casp3 was transiently reintroduced in TAB1 and apoptosis was induced with staurosporine Annexin V and the corresponding apoptotic and anti-apoptotic signalling factors (Casp3, 7, 8, 9, XIAP, PARP, gH2AX, BCL2, MCL1, AIF, Calpain and DFF45) were monitored by flow cytometry and western blot. Tissue microarrays (TMAs) including 150 TH and TC samples were stained for Casp3.

When re-expressing Casp3 in TAB1 apoptosis was induced without any death stimulus. And although antiapoptotic factors were strongly presented in TAB1 Casp3 expression reduced protein levels of BCL2 and MCL1 dramatically.

The reduction of Casp3 expression inhibits apoptosis in cancer cells. However, when Casp3 is re-expressed in this state apoptosis is induced without an upstream activation and cells die. Therefore approaches to reactivate or to surround Casp3 expression in certain cancer types may support treatment response.

Poster abstracts


71

33

Synergistic activity of sorafenib and betulinic acid against colony-forming ability of non-small cell lung cancer cells

Justyna Kutkowska1, Andrzej Rapak1, Leon Strzadala1

1 Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, POLAND

Sorafenib is a multi-targeted kinase inhibitor that has shown efficacy against a wide variety of tumors in preclinical models, including human non-small cell lung cancer (NSCLC). Previously reported in vitro and in vivo studies have demonstrated that betulinic acid has anti-tumor and anti-proliferative properties, and induce apoptosis in tumor cells. Combination of drugs with different targets is a logical approach to overcome multilevel cross-stimulation among key signaling pathways in NSCLC progression.

NSCLC cell lines: A549, H358 and A427, with different KRAS mutations, and normal human peripheral blood lymphocytes cells (PBL), were treated with sorafenib and betulinic acid alone and in combination. We examined the effect of different combined treatments on viability (MTS test), proliferation and apoptotic susceptibility analyzed by flow cytometry, alterations in signaling pathways by Western blotting and colony-forming ability.

The combination of sorafenib with betulinic acid had a strong effect on the induction of apoptosis of different NSCLC cell lines. Also, this combination was not toxic for human PBL cells. Combination treatment changed the expression of proteins involved in the mitochondrial apoptosis pathway and induced apoptotic death by caspase activation. Also, the combination treatment completely inhibited expression or phosphorylation of ERK1/2, AKT and mTOR in NSCLC cell lines. Importantly, combination treatment with low drug concentrations tremendously reduced colony-forming ability of A549, H358 and A427 cells, as compared to both compounds alone.

Our study suggests that combination therapy with low concentrations of sorafenib and betulinic acid had the capacity to induce high levels of cell death and abolish clonogenic activity in some NSCLC cell lines.

Poster abstracts


72

34

Activation of the Siglec-7 inhibitory receptor on human mast cell leukemia cells induces their death in vitro and in vivo

Nadine Landolina1, Francesca Levi-Schaffer1

1 Hebrew University of Jerusalem, Jerusalem, ISRAEL

Introduction: Blocking antibodies against inhibitory receptors (IRs, also known as “immune checkpoints”), are revolutionizing cancer treatment. IRs, however, can be expressed, not only on immune cells, but also on immune-cell derived neoplasms. Whether activation of these IRs through activating monoclonal antibodies (mAb) can lead to tumor growth inhibition is, so far, understudied. The IR Siglec-7 is expressed on various immune cells, including mast cells (MCs) and eosinophils. The aim of this work was to investigate the effects of anti-Siglec-7 mAb (Anti-S7) on MC leukemia cell line (HMC-1) growth in vitro and in vivo.

Method: Expression of Siglec-7 on bone marrow aspirates and stability of the receptor up to 72h on HMC-1 were evaluated by FACS. Anti-S7 mediated effects on HMC-1 and on transfected RPMI8866 replication and survival were tested using trypan blue exclusion test, MTT assay and Annexin V PI FACS staining. Evaluation of phosphorylation states of signaling molecules were performed using Western Blot and intracellular FACS. In vivo effects of Anti-S7 were checked on HMC-1 grafted SCID/Beige mice ip treated with Anti-S7.

Results: Here we show that neoplastic MCs of mastocytosis patients express Siglec-7 and its activation inhibits HMC-1 replication and survival. Siglec-7 activation causes phosphorylation of Src-homology region 2 domain-containing phosphatase-1 (SHP-1), reduced phosphorylation of c-Kit and its downstream signalling effectors. Furthermore, using Siglec-7 transfectants we found that activation of Siglec-7 leads to apoptosis and inhibits cell growth via its cytoplasmic domain. Importantly, we showed that Anti-S7 treatment leads to reduced tumor growth in vivo in SCID beige mice, even when tumors were already established.

Conclusion: Collectively, we demonstrated that activation of Siglec-7 by specific mAbs is an effective anti-MC leukemia treatment placing Siglec-7 as a new specific target possibly also of other cancers expressing this IR.

Poster abstracts


73

35

New platforms to screen for Hsp70 chaperone inhibitors and the discovery of a colchicine derivative that sensitizes melanoma and glioblastoma tumors to doxorubicin

Vladimir Lazarev2, Dmitry Sverchinsky2, Elena Mikhaylova2, Pavel Semenyuk1, Elena Komarova2, Sergey Niskanen2, Anton Burakov1, Viktor Kartsev3, Irina Guzhova2, Boris Margulis2

1 A. N. Belozersky Research Institute of Physico-Chemical Biology, Moscow State University, Moscow, RUSSIAN FEDERATION, 2 Institute of Cytology RAS, St. Petersburg, RUSSIAN FEDERATION, 3 InterBioscreen, Chernogolovka, Moscow, RUSSIAN FEDERATION

Tumor progression requires the maintenance of chaperone machinery to provide the appropriate control over protein synthesis, transport and utilization in rapidly and continuously renewing cancer cells. Since Hsp70 chaperone performs the above functions and therefore becomes an important target for therapeutic compounds. The aim of this study was to discover low-toxic factor using which we can inhibit Hsp70-based protective mechanism and by this to sensitize cells to an efficiently acting anti-tumor drug. We developed two novel assays for compounds able to affect substrate-binding and refolding activity of Hsp70 and performed screening of small molecules from the collection of InterBioScreen LLC. One of the positive compounds was N-amino-ethyl amino derivative of colchicine (AEAC) whose toxicity was two orders lower than that of original compound. Unlike colchicine, AEAC did not demonstrate microtubule-depolymerizing activity, whereas its Hsp70 inhibitory capacity was two orders higher than that of colchicine. According to the data of Drug affinity Responsive Target Stability assay and thermophoresis technique AEAC efficiently binds Hsp70; this fact was proved by the data of molecular docking. Importantly, in C6 rat glioma and B16 mouse melanoma cells AEAC was found to inhibit the efficacy of Hsp70-based refolding system although its effect on cell growth was negligible. In combination with doxorubicin AEAC increases the anti-tumor efficiency of the latter in both cell models. When both tumors were inoculated in animals AEAC administered together with doxorubicin was found to exert therapeutic effect by prolonging animal survival and reducing tumor growth rate. In conclusion, two novel assays were created for the high-throughput analysis of chaperone inhibitor and one of compounds discovered in the chemical library screening, derivative of colchicines demonstrated pronounced anti-tumor activity in combination with the established drug, doxorubicin, and may become novel medicines with the broad spectrum of effects.

Poster abstracts


74

36

Depletion of CTCF in breast cancer cells selectively induces cancer cell death via p53

Ji-Yeon Lee1, Mustafa Muhammad1, Clara Yuri Kim1, Myoung Hee Kim1

1 Yonsei University College of Medicine, Seoul, SOUTH KOREA

CCCTC-binding factor (CTCF), a ubiquitous 11-zinc finger multifunctional protein, has distinct molecular functions, such as transcriptional activation, repression, and chromatin barrier activity, in a locus-specific manner. Elevated CTCF levels in breast cancer cells are known to contribute to tumorigenesis; however, the underlying mechanism remains elusive. We investigated the effect of CTCF expression on breast cancer cell survival and elucidated its mechanism. CTCF depletion in MCF-7 cells led to a decreased cell growth and proliferation, surpassing the growth of normal cells under co-culture system of MCF-7-GFP and MCF10A. Here we propose that the phenotypes observed in CTCF-depleted MCF-7 cancer cells, such as reduced cell proliferation, increased apoptosis, and cell cycle arrest, are closely linked with the activation of p53. The consensus CTCF-binding site, located approximately 800 bp upstream of the first exon of TP53, was marked by H3K27me3, but not by the active mark H3K4me3, although CTCF is expressed. Knockdown of CTCF conversely led to the recruitment of H3K4me3 instead of H3K27me3, accompanying with the higher enrichment of PolII in the proximal promoter region of TP53. With the activation of p53, increased p21 and Bax expressions were observed in CTCF knockdown MCF-7 cells. Elucidating functional roles of CTCF and regulation mechanisms may help to guide CTCF and/or its related molecules as a therapeutic target to prevent cancer cell growth.

Poster abstracts


75

37

Synergetic effect of Coptis chinensis with gefitinib in gefitinib resistant non-small cell lung cancer cells by inducing apoptosis through suppression of Mcl-1 and Bcl-2

Jae Hwan Kim4, Seong-Hee Park4, Eun- Jung kim1, Jinkyung Rho5, Myung-Jin Park3, Changil Kim2, Ji-Yun Lee4

1 Dongguk University, Ilsan, SOUTH KOREA, 2 Konkuk University , Chungju , SOUTH KOREA, 3 Korea Institute of Radiological and Medical Sciences, Seoul, SOUTH KOREA, 4 Korea University, Seoul, SOUTH KOREA, 5 University of Ulsan, Seoul, SOUTH KOREA

The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) gefitinib is an effective therapeutic drug against non-small cell lung cancers (NSCLCs) harboring EGFR mutations. However, both adaptive and acquired resistance significantly limits the efficacy of EGFR-TKI and thus the current chemotherapeutic strategies for NSCLCs. There is therefore a need to overcome this resistance. In this study, we investigated the anticancer potential of natural extracts of Coptis chinensis (ECC) against the gefitinib-resistant (GR) lung cancer cells PC9GR and A549GR. ECC inhibited cell viability, migration, and invasion, and induced apoptosis effectively in GR cells. These effects were associated with the suppression of EGFR-AKT signaling and anti-apoptotic proteins Mcl-1 and Bcl-2, which were overexpressed in GR cells. Moreover, combination treatment with ECC and gefitinib enhanced the sensitivity of GR cells to gefitinib. These results indicate the potential of ECC in the treatment of NSCLC, particularly in combination with EGFR-TKI therapy in EGFR-TKI resistance.

Poster abstracts


76

38

Inhibition of Nrf2 activity suppressed cell proliferation and induced cell death pathway in the keap1 mutant gefitinib resistant lung cancer cell.

Seong-Hee Park2, Jae Hwan Kim2, Jeong-Yub Kim2, Myung-Jin Park1, Min Jung Kim4, Hyemin Seo4, Shibo Li3, Ji-Yun Lee2

1 KIRMS, Seoul, SOUTH KOREA, 2 Korea University, Seoul, SOUTH KOREA, 3 OUHSC, OK city, USA, 4 Sookmyung Women’s University, Seoul, SOUTH KOREA

Lung cancer is the leading cause of cancer-related mortality worldwide. The most generalized form of lung cancer is non-small cell lung cancer (NSCLC), comprising 80% of all lung cancer. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), gefitinib, is effective therapy against NSCLCs which with specific EGFR-activation mutation. However, such cancer eventually develops acquired resistance by various mechanisms such as a secondary EGFR mutation, T790M, in spite of their initial response. Therefore, an understanding of the mechanisms behind TKI resistance is essential for improving EGFR-TKI therapeutic efficacy in non-small cell lung cancer (NSCLC) patients. In this study, we discovered that overexpression of ARE-containing Nrf 2 target genes by increased transactivation of Nrf2 due to the acquired Keap1 mutation in gefitinib resistant (GR) NSCLC cell we established. This GR cells also acquired cross-resistant to irreversible EGFR-TKIs, afatinib and osimertinib, and showed increased viability, invasiveness, proliferation, and tumorigenicity both in vitro and in vivo. This result was confirmed by the fact that inhibition of Nrf2 activity by treatment with brusatol or exogenously expressing wildtype Keap1 suppressed tumor cell proliferation and induced cell death pathway. These data suggest that the Keap1-Nrf2 pathway needs to be considered in EGFR-TKI treatment lung cancer as well as other cancers.

Poster abstracts


77

39

CDK9-Inhibition and TRAIL overcomes Sorafenib-resistance of hepatocellular carcinoma

Johannes Lemke2, Ella Kitzig2, Anna-Laura Kretz2, Uwe Knippschild2, Henning Walczak1, Doris Henne-Bruns2

1 Centre for Cell Death, Cancer and Inflammation, UCL Cancer Institute, University College London, London, UK, 2 University of Ulm, Clinic of General and Visceral Surgery, Ulm, GERMANY

Sorafenib remains the standard therapy for patients with advanced HCC despite considerable side-effects and the fact that systemic treatment only prolongs survival by a few months. One major reason for its limited efficiency is the fact that that HCC cells commonly exhibit or acquire resistance to Sorafenib. Therefore, novel effective and cancer-selective therapeutic strategies are urgently needed. Recently, we identified the combination of the death ligand TRAIL and CDK9 inhibition as an exceptional potent strategy to selectively kill tumor cells. Here, we evaluated the combination of the clinical tested CDK9-inhibitor Dinaciclib and TRAIL for HCC and investigated underlying molecular mechanism.

The combination of Dinaciclib and TRAIL synergistically and effectively reduced cell viability in HCC cells and, importantly, almost completely abolished clonogenic survival of these cells. The combination induced cell cycle arrest and caspase-8-dependent apoptosis in these cells. Moreover, this combination displayed a superior cytotoxic effect in HCC cells compared to Sorafenib treatment alone. Interestingly, this combination overcame acquired Sorafenib-resistance in HCC cells by the shift of the ratio of pro- and anti-apoptotic proteins on the transcriptional level.

In conclusion, due to its potency, CDK9-inhibition in combination with TRAIL provides a novel and promising therapeutic approach for Sorafenib-resistant HCC.

Poster abstracts


78

40

Activators of the SHIP1 lipid phosphatase; a novel therapeutic strategy to suppress B-cell receptor signalling and induce apoptosis in human B-cell cancers

Elizabeth Lemm2, Beatriz Valle-Argos2, Lindsay Smith2, Nicky Weston-Bell2, Yohannes Gebreselassie2, Andrew J Steele2, Freda Stevenson2, Matthew J Carter2, Mark Cragg2, Francesco Forconi3,2, Jennifer Cross1, Curtis Harwig1, Lloyd Mackenzie1, Graham Packham2

1 7Aquinox Pharmaceuticals (Canada), Inc., Vancouver, CANADA, 2 Cancer Sciences Unit, University of Southampton, Southampton, UK, 3 Haematological Oncology Group, University of Southampton, Southampton, UK

Signalling via the B-cell receptor (BCR) is a major driver of malignant B-cell proliferation and survival in B-cell malignancies including chronic lymphocytic leukaemia (CLL). The role of kinases in BCR signalling is well understood and kinase inhibitors are effective therapies for B-cell cancers. However, resistance is increasingly common and new drugs are required. In this study we investigated responses to a novel chemical activator of SHIP1, a PI(3,4,5)P3 (PIP3)-specific phosphatase which suppresses PI(3,4,5)P3-dependent signalling downstream of PI3 kinase (PI3K) using primary CLL cells.

We focused on the exemplar compound, AQX-C5, which is structurally related to the natural product pelorol. AQX-C5 interfered with the ability of anti-IgM to activate PIP3-dependent signalling, including downstream phosphorylation of AKT and p70-S6K, and induction of MYC and the BCL2-related survival protein MCL1. AQX-C5 also triggered downmodulation of the chemokine receptor CXCR4 which is a target for SHIP1-mediated modulation in other systems. Consistent with the known survival function of PIP3- signalling, SHIP1 activation increased caspase-dependent apoptosis of CLL cells when cultured alone or in the presence of survival-inducing conditions such as anti-IgM or treatment with IL-4 and CD40L. CLL cells were more sensitive to killing compared to B cells from healthy donors and non-malignant T cells present in CLL samples.

When investigated in the absence of anti-IgM stimulation, we found that AQX-C5 caused a rapid, but transient increase in expression of MCL1 and a prolonged increase in NOXA (proapoptotic BCL2 family protein), via post-transcriptional mechanisms. Despite modulation of MCL1/NOXA, expression of BCL2 itself was not altered.

These findings suggest that pelorol analogues such as AQX-C5 may be interesting therapeutic agents for B-cell cancers.

Poster abstracts


79

41

Progestin-induced senescence-like phenotype is associated with autophagy in breast cancer cells

Natasa Bajalovic1, Amanda RE Woo1, Valerie Lin1

1 Nanyang Technological University, Singapore, SINGAPORE

Progesterone is an ovarian steroid hormone essential for the development of the mammary gland. It has also been implicated in the development of breast cancer. Large clinical trials of hormone replacement therapy using progestin medroxyprogesterone acetate in post-menopausal women concluded that the progestin significantly increase risks of breast cancer. On the other, the same compound had been used as a second-line endocrine therapy for metastatic breast cancer for nearly two decades prior to the publication of the trial results. A better understanding of the mechanisms of the action of progesterone will help explain these conflicting observations. In this report, we used progesterone receptor-transfected MCF-7 cells as the cell model to clarify the function of progestin. Although progestin R5020 induced cell cycle progression initially (in the first 24h), a single dose of R5020 induced marked cell cycle arrest from 48h onward and this inhibition became irreversible despite of the removal of R5020. At 96h after treatment, the cells exhibited extensive spreading and hardly any sign of further cell proliferation based on BrdU labelling. This growth arrest is associated with a secretory phenotype with increased expression of cytokines such as IL-1α and IL-1β. It is also associated with increased autophagic flux that is likely mediated by the down-regulation of PI3K-AKT signaling. The study suggests that progestin exerts powerful anti-tumoral effect in breast cancer cells that express high levels of progesterone receptor. Nonetheless, future study should address if these effects have any undesirable influence on the tumor micorenvironment.

Poster abstracts


80

42

Promotion of senescence by oxidative stress and chemotherapy: role of protein kinase C

Assaf Ben Ari1, Udi Zurgil1, Etta Livneh1

1 Ben-Gurion University of the Negev, Beer Sheva, ISRAEL

Senescence is characterized by permanent cell cycle arrest and loss of proliferative capacity, despite continued viability and metabolic activity. Senescent cells undergo massive genome modulation including induction of the senescence-associated secretory phenotype (SAPS). Components of SASP can implement cell cycle arrest or recruit the immune system to clear senescent cells, thus contributing to tumor suppression. However, some secreted pro-inflammation molecules, act as pro-tumorigenic agents contributing to tumor progression, suggesting that elimination of senescent cells could be beneficial. Very few studies showed a role for PKC in senescence. Here we show that PKCη, an epithelial specific and anti-apoptotic kinase (epithelia is the origin of about 90% of human tumors), promotes senescence induced by oxidative stress and DNA damage. Using PKCη-knockdown breast adenocarcinoma MCF-7 cells we show that PKCη promotes senescence induced by oxidative stress and DNA damage via its ability to upregulate the expression of the cell cycle inhibitors p21Cip1 and p27Kip1 and to modulate transcription of major components of SASP such as IL-6 and IL-8. Moreover, we demonstrate that PKCη creates a positive loop for reinforcing senescence by increasing the transcription of both IL-6 and IL-6 receptor. Thus, the presence/absence of PKCη modulates major components of SASP. Furthermore, our studies demonstrate that PKCη interferes with gH2AX phosphorylation, which mark DNA double-strand breaks for repair. The phosphorylation of gH2AX may reflect the extent of DNA breaks but also the cellular response to DNA damage. Our experiments suggest that the response to DNA damage (repair processes) was more efficient in PKCη-knockdown cells. The phosphorylation on ATM and Chk2 was also lower in PKCη expressing cells. Our studies provide clues for the underlying mechanisms that promote senescence as some chemotherapy drugs function also through senescence induction. This will point for therapeutic targets for intervention in cancer and for the elimination of senescent cells.

Poster abstracts


81

43

Apoptosis-mediated cell death via Human antigen R silencing in colon cancer cells

Yu-Li Lo1, Vivian Juang1, Chen-Shen Wang1, Guan-Liang Lin2, Huei-Ju Ting2

1 Department and Institute of Pharmacology, National Yang-Ming University, Taipai, TAIWAN ROC, 2 Department of Biological Sciences and Technology, National University of Tainan, Tainan, TAIWAN ROC

Human antigen R (HuR), a RNA-stabilizing protein, is important in posttranscriptional regulation of oncogenes and antiapoptosis-related genes, including galectin-3, β-catenin and Bcl-2. In this study, we aim to verify if siRNA against HuR (siHuR) may suppress antiapoptosis signaling pathway via inhibition of β-catenin and Bcl-2 in colon cancer cells. Our results showed that siHuR decreased the mRNA expressions of galectin-3, β-catenin and Bcl-2 in chemotherapy-treated colon cancer cells. Accordingly, the co-treatment of chemotherapy and siHuR decreased the protein expressions of galectin-3, β-catenin and Bcl-2. HuR silencing enhanced the intracellular accumulation of chemotherapy in colon cancer cells. Furthermore, siHuR significantly enhanced chemotherapy-mediated apoptosis and thus intensified the cytotoxicity of chemotherapy. The combinatorial therapy of siHuR and chemotherapy significantly reduced the expression of Bcl-2, but remarkably increased the expression of Bax, as well as the activity levels of caspase-3 and -9. Collectively, this is a novel study associating the post-transcriptional control by HuR silencing with survival signaling repression and apoptosis induction in colon cancer cells.

Poster abstracts


82

44

Pre-clinical evaluation of targeting autophagy for the treatment of Oral Squamous Cell Carcinoma

Stefania Magnano1, Jeffrey O’Sullivan1, Daniela M. Zisterer1

1 Trinity College Dublin, Dublin, EIRE

Background: Oral Squamous Cell Carcinoma (OSCC) is the sixth most common form of cancer worldwide. The 5-year survival rate in oral cancer is only 50% mostly due to chemoresistance and late diagnosis (1). There is a compelling demand for improved therapeutic options for OSCC. It has been demonstrated that a cell survival pathway known as autophagy is frequently activated in tumour cells treated with chemotherapeutics (2). The role of autophagy in OSCC remains poorly understood and further study is required to clarify whether it plays a role in chemoresistance and in tumour progression.

Objectives: To investigate cell death mechanisms induced in oral cancer cells in response to standard OSCC chemotherapeutics (e.g. cisplatin) and to examine the role of autophagy in chemoresistance. The long term goal is to develop selective treatment strategies to bypass drug resistance and improve clinical outcome in OSCC patients.

Methods: The effect of cisplatin on the viability of the SCC4 cell line was evaluated by the Alamar Blue assay and IC50 values were determined using GraphPad Prism software. Apoptosis was examined through flow cytometric analysis of Annexin V/PI stained cells and by immunoblotting of caspase3 cleavage products. Autophagy was investigated by the Cyto-ID Autophagy Detection Kit and immunoblotting of LC3II and p62 proteins.

Results: Cisplatin reduced the viability of SCC4 cells and induced apoptosis (resulting in caspase3 activation) and autophagy in a dose- and time-dependent manner. Additionally, preliminary data suggest that targeting autophagy enhances cisplatin induced apoptosis in SCC4 cells.

Conclusions: Cisplatin induces both apoptosis and autophagy in OSCC cells. Cisplatin-induced autophagy may play an important role in chemoresistance. Combining cisplatin with autophagy inhibitors may represent a valuable treatment strategy to promote OSCC cytotoxicity and diminish resistance in OSCC patients.

1. Bagan J et al., Oral Oncol. 2010 Jun;46(6):414-7.

2. Ahn MY et al., Oral Oncol. 2011 Nov;47(11):1032-8.

Poster abstracts


83

45

The NRF2-regulated oxidative stress influences cell cycle regulatory network

Margita Márton1, Nikolett Tihanyi1, Gábor Bánhegyi1, Orsolya Kapuy1

1 Semmelweis University, Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Budapest, HUNGARY

Keywords: oxidative stress, NRF2, Cyclin D1

Oxidative stress is considered as a redox imbalance between oxidative free radicals formation and antioxidant compounds by making cellular damages and even leading to cell death. Treatment with oxidative agents results in activation of several signal transduction pathways; meanwhile the ongoing cell division cycle has to be blocked. The PERK-substrate NRF2 (nuclear factor erythroid 2-related factor 2) has key role in empowering cell adaptation to oxidative stress through transcriptionally controlling more than 2000, mainly cytoprotective, genes. However, details about its effect on cell cycle regulation have not been explored yet. Cell cycle is a precisely regulated process driven by cyclin dependent kinase (CDK)/cyclin complexes. It was shown that Cyclin D1 got immediately down-regulated via PERK pathway in response to oxidative stress resulting in cell cycle arrest. Using molecular biological techniques, our goal was to reveal a crosstalk between NRF2 and the key elements of cell cycle regulatory network upon oxidative stress.

Treatments with oxidative stressor were combined with silencing PERK or NRF2 in HEK293T cells. Both protein’s silencing increased cell viability during oxidative stress, meanwhile autophagy had an activation peak. Apoptosis got activated only upon oxidative treatment. Cyclin D1 level was almost constant in siPERK and siNrf2 transfected and non-transfected cells too, but certain CDK inhibitors were down-regulated in silenced treatments. We suggest that PERK kinase inhibits the cyclin molecule throughout NRF2 induction by various methods. We also assume that the indirect regulatory connection between NRF2 and Cyclin D1 is generated by CDK inhibitors.

The high amount of oxidative free radicals promotes carcinogenesis, and the increased activation of NRF2 has been noticed in several cancers therefore our study might have medical importance.

This work was supported by the COST ACTION BM1307 PROTEOSTASIS and ÚNKP-17-3-I-SE-30 New National Excellence Program of the Ministry of Human Capacities.

Poster abstracts


84

46

Discovery of therapies for lymphomas and leukaemia: synthesis and antiproliferative action of novel ethanoanthracenes

James Patrick Mc Keown3, Andrew Byrne3, Clara Charleton2, Keith Ferris1, Mary Jane Meegan3

1 School of Biotechnology, Dublin City University, Glasnevin, Dublin, EIRE, 2 School of Chemistry, NUI Maynooth, Kildare, EIRE, 3 School of Pharmacy & Pharmaceutical Sciences, Trinity College Dublin, EIRE

CLL (Chronic Lymphocytic Leukaemia) is the most common leukaemia in developed countries globally, primarily affecting the elderly. CLL is classed as a clonal disorder of mature B-lymphocytes and its clinical patient prognoses being affected mainly by the mutational status of the Immunoglobulin G Heavy Chain Variable region (IGHV) (with unmutated IGHV holding a better patient prognosis than the wild type variant)1.

Structures related to tricyclic and tetracyclic anti-depressants (fluoxetine and maprotiline respectively) have been previously shown to express potent, selective antiproliferative and pro-apoptotic effects in vitro and were met with marked success in related B cell malignancy cell lines, namely Burkitt’s Lymphoma (BL) cell lines DG-75 and MUTU-12.

Based on these preliminary studies, libraries of structurally related compounds were designed, based on the proven effectiveness of nitrostyrene core moiety derivatives and evidence of effectiveness of chalcone moieties in leukemic cell lines3. These compounds were synthesised using Henry-Knoevenagel condensation, Claisen-Schmidt condensation and Diels Alder cycloaddition reactions. Each compound was subsequently characterised by 1H NMR, 13C NMR, IR spectroscopy and high resolution mass spectrometry (HRMS).

The antiproliferative activity of each compound was determined using both the Alamar Blue assay on the two types of CLL cell lines: HG-3 and PGA-1, representative of bad and good prognosis respectively. The antiproliferative effects and structure activity compound relationships, together with preliminary effects on the induction of apoptosis will be discussed.

1. Scarfò, L.; Ferreri, A. J. M.; Ghia, P., Chronic lymphocytic leukaemia, Critical Reviews in Oncology/Hematology, 2016, 104, 169-182.

2. Mc Namara, Y. M., Bright, S.A., Byrne, A.J., Williams, D.C., Meegan, M.J., Synthesis and biochemical evaluation of a novel series of maprotiline analogues, European Journal of Medicinal Chemistry 2014, 71, 333.

3. Zhuang, C.; Zhang, W.; Sheng, C.; Zhang, W.; Xing, C.; Miao, Z., Chalcone: A Privileged Structure in Medicinal Chemistry, Chemical Reviews, 2017.

Poster abstracts


85

47

5FU-based Chemotherapy and TNFα sensitized Ex Vivo Colorectal Cancer Explant Cultures to IAP Antagonist Birinapant Treatment

Mariangela Meyer4,2, Joanna Fay3, Rebecca Monaghan4,2, Orna Bacon4,2, Elaine W. Kay3, Deborah A. McNamara5, Daniel B. Longley1, Markus Rehm4,6, Jochen H.M. Prehn4,2

1 Centre for Cancer Research Biology, Queen’s University, Belfast, UK, 2 Centre for Systems Medicine, Royal College of Surgeons in Ireland, Dublin, EIRE, 3 Department of Pathology, Beaumont Hospital, Dublin, EIRE, 4 Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland, Dublin, EIRE, 5 Department of Surgery, Beaumont Hospital, Dublin, EIRE, 6 Institute for Cell Biology and Immunology, Stuttgart University, Stuttgart, GERMANY

The Inhibitor of Apoptosis Protein (IAP) family encodes a group of proteins with diverse roles in apoptosis regulation, acting as inhibitors of caspases activation and by mediating cell survival through the NF-kB signalling. Upregulation of IAPs has been correlated with disease progression and poor prognosis in many types of cancer whence, synthetic inhibitors of IAPs known as Second Mitochondrial-derived Activator of Caspases (SMAC) mimetics have been developed in recent years. These target IAPs by mimicking the N-terminal IAP-binding motif of SMAC, the natural IAPs antagonist. The principal aim of this study is to assess the efficacy of SMAC mimetic treatment in colorectal cancer (CRC) with emphasis on the role of its pro-inflammatory tumour microenvironment (TME) with high levels of Tumour Necrosis Factor alpha (TNFα).

An ex vivo model has been established using dental sponges as a semi-solid support for CRC patient-derived explants. Tissues were treated with a bivalent SMAC mimetic known as Birinapant, alone and in combination with 5Fluorouracil (5FU)/Oxaliplatin or TNFα. Caspases activation was analysed by immunohistochemistry and western blotting and compared to expression of IAPs and proliferation markers. Levels of cFLIP, RIPK1 and FADD were also determined. Apoptosis was evaluated using Terminal Deoxynucleotidyl Transferase (TdT) assay.

Treatment of explants with 5FU/Oxaliplatin or TNFα, sensitised tumours to SMAC mimetic-mediated cell death via caspase-dependent apoptotic mechanisms, triggering activation of caspase-8, -9, and -3. Birinapant induced downregulation of IAPs and this effect was enhanced by co-therapies. Birinapant in combination with the selected compounds also reduced the expression of Ki-67, Zeb1 and cFLIP and promotes the processing of FADD and RIPK1.

This 3D culture system retains the in vivo tissue architecture and overall molecular expression, promising to serve as a powerful experimental platform to identify key biomarkers which could serve as predictors of treatment response to IAP antagonist-based therapy.

Poster abstracts


86

48

Targeting multiple myeloma cells by inhibition of BCL-2 family proteins in a personalized manner

Laura Moesbergen1, Anne Slomp1, Reinier Raaijmakers1, Monique Minnema1, Victor Peperzak1

1 UMC Utrecht, Utrecht, NETHERLANDS

Despite improvements in patient survival rates and the increase in number of anti-cancer drugs, multiple myeloma (MM) remains an incurable disease. Therefore, a better, more personalized strategy is needed to treat this highly heterogeneous malignancy. Pro-survival BCL-2 family proteins BCL-2, BCL-XL and MCL-1 are potent inhibitors of apoptosis and are often overexpressed in B cell malignancies. In MM, MCL-1 was shown to be consistently overexpressed and identified as one of the most important genes for MM cell survival. Increased expression of BCL-2 and BCL-XL was also observed, but varies greatly between patients. At the moment it is unclear whether expression of MCL-1, BCL-2 or BCL-XL correlates to the cytogenetic profile, disease progression or sensitivity to single use, or combination of, BH3 mimetic inhibitors targeting these molecules. We addressed these questions using a cohort of primary MM samples from the Dutch Parelsnoer Institute.

We used in vitro cultures with the MS-5 bone marrow (BM) stromal cell line to keep primary MM cells viable throughout culture. Expression of BCL-2, BCL-XL and MCL-1 was measured using intracellular staining followed by flow cytometric analysis. Apoptosis induction by BCL-2 family inhibitors ABT-199 (BCL-2), A-1155463 (BCL-XL) and S63845 (MCL-1) was determined using DiOC6 and TO-PRO-3 staining.

Expression of BCL-XL, and to a lesser extend of BCL-2, correlated with the percentage of MM cells in the BM aspirate. The level of MCL-1 expression correlated with sensitivity to MCL-1 inhibitor S63845, while this was not clear for BCL-2 or BCL-XL. Although sensitivity to the single inhibitors varied, we observed clear synergistic effects when combining 2 different inhibitors. We therefore propose to treat MM patients with (combinations of) BCL-2 family inhibitors, where the choice of inhibitors is based on disease stage and expression of the BCL-2 family proteins.

Poster abstracts


87

49

Yarrow Supercritical Extract inhibits pancreatic cancer cell growth by targeting lipogenic genes

Lamia Mouhid1, Marta Gómez de Cedrón1, Guillermo Reglero1,2, Tiziana Fornari2, Ana Ramírez de Molina1

1 Precision Nutrition and Cancer Program, Molecular Oncology Group, Madrid Institute for Advanced Studies on Food (IMDEA-Food), SPAIN, 2 Production and Characterization of Novel Foods Department, Institute of Food Science Research (CIAL) CSIC-UAM, Madrid, SPAIN

Pancreatic cancer is one of the most aggressive cancers, being the absence of effective treatments the main cause of death. Metabolic reprogramming is well recognized as a hallmark of cancer1 and thus, there is an increased interest on targeting the altered metabolism in cancer treatment. Particularly, an exacerbated lipid metabolism is considered a stimulus to tumor malignancy2. On the other hand, plants extracts constitute a relevant source of bioactive components which might efficiently inhibit tumor cell progression. Herein, we proposed an extract of Achillea Millefolium (commonly known as Yarrow), obtained by supercritical fluid extraction3 as well as it has antitumoral properties in pancreatic cancer derived cell lines -diminishes cell viability, induces apoptosis, inhibits invasion, and inhibits 3D growth and colony formation-. Moreover, Yarrow synergizes with 5-fluororacil (5-Fu), which is currently used in the clinics, to inhibit pancreatic cancer cell proliferation. Importantly, Yarrow specifically targets SREBF1 (downregulation), a master regulator of lipid metabolism, and the lipogenic genes FASN (fatty acid synthase) and SCD (stearoyl-CoA desaturase), regulated by SREBF1 at the transcriptional level, are also diminished. Finally, Yarrow decreases tumor growth in an in vivo xenograft mouse model.

In summary, our results indicate that Yarrow supercritical extract is a promising antitumoral co-adjuvant in pancreatic cancer treatment.

1. Hanahan D, Weinberg RA. Hallmarks of cancer: The next generation. Cell. 2011;144(5):646-674.

2. Luo X, Cheng C, Tan Z, et al. Emerging roles of lipid metabolism in cancer metastasis. Molecular Cancer. 2017;16(1):76.

3. García-Risco MR, Mouhid L, Salas-Pérez L, et al. Biological activities of asteraceae (achillea millefolium and calendula officinalis) and lamiaceae (melissa officinalis and origanum majorana) plant extracts. Plant Foods for Human Nutrition. 2017;72(1):96-102.

Poster abstracts


88

50

Cyclin dependent kinase inhibitors: a novel treatment strategy to sensitize glioblastoma to TRAIL-induced apoptosis

Janis Noonan3, Monika Jarzabek3, Frank Lincoln3, Arhona Pariag3, Leonie Young4, Keith Ligon2, Hanne Jahns7, Avi Ashkenazi5, Daniella Zheleva1, Annette Byrne3, Jochen Prehn3, Markus Rehm6, Brona Murphy3

1 Cyclacel Ltd, Dundee, UK, 2 Department of Pathology, Harvard Medical School, Boston, Massachusetts, USA, 3 Department of Physiology & Medical Physics, Royal College of Surgeons in Ireland, Dublin 2, EIRE, 4 Endocrine Oncology Research Group, Department of Surgery, Royal College of Surgeons in Ireland, Dublin 2, EIRE, 5 Genentech, San Francisco, California , USA, 6 Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, GERMANY, 7 Pathobiology Section, School of Veterinary Medicine, University College Dublin, Dublin 4, EIRE

Glioblastoma (GBM) is the most common primary brain tumour and no cure presently exists. The average survival rate for patients is only 15 months. The clinical management of these tumours is hampered by the fact that these tumours are highly resistant to apoptotic death which the treatment strategies of radiation and chemotherapy attempt to induce.

Recent efforts to sensitize GBM to apoptosis have focused on ligands to death receptors, such as tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), to activate the extrinsic pathway of apoptosis. Numerous studies have demonstrated that many malignancies, including GBM, are completely resistant to monotherapy with TRAIL however, due in part to their overexpression of anti-apoptotic proteins, such as Mcl-1. Our group has published that R-roscovitine, a first generation CDK inhibitor, down-regulates Mcl-1 in GBM. Additionally, we have demonstrated that combining R-roscovitine with TRAIL re-establishes apoptotic sensitivity in GBM cells grown as monolayers.

We have now assessed the effectiveness of our novel combination strategy in superior preclinical models of GBM – patient-derived neurospheres and in a murine orthotopic patient-derived xenograft (PDX) model. Significant levels of apoptosis were observed in patient-derived neurospheres upon treatment with R-roscovitine and TRAIL, highlighting that 3-D cultures of patient-derived neurospheres could also be sensitised to the apoptotic-inducing effects of TRAIL upon co-administration of R-roscovitine. Following the successful establishment of our murine orthotopic PDX model, the co-treatment strategy displayed similar efficacy and critically, substantially extended the survival times of the animals. These findings were not evident in the 3-D cultures or animals treated with single administration of either agent. Furthermore, we also provide evidence that the CDK inhibitor successfully crossed the blood brain barrier of the tumour bearing animals.

These robust preclinical findings highlight that cyclin-dependent kinase inhibition in conjunction with death receptor ligand ligation represents an exciting novel approach for anti-GBM cancer therapy.

Poster abstracts


89

51

D-Α-Tocopheryl polyethylene glycol 1000 succinate and a small molecule inhibitor of Survivin synergistically induce apoptosis in breast cancer cells

Christiana Neophytou1, Avgoustinos Mesaritis1, Andreas Constantinou1

1 University of Cyprus, Nicosia, CYPRUS

Breast cancer is the most frequently diagnosed and second in mortality rate malignancy among women. Despite the many advances in breast cancer treatment in recent years, there is still need for the discovery of novel approaches to improve drug effectiveness and reduce negative side effects. D-alpha-tocopheryl polyethylene glycol succinate (TPGS) is a vitamin E synthetic derivative frequently used in the development of drug delivery systems to improve the pharmacokinetics of anti-cancer drugs and reduce multi-drug resistance. We have previously shown that TPGS not only acts as a carrier molecule but also exerts anti-cancer effects by inducing apoptosis and cell cycle arrest in breast cancer cells that overexpress the anti-apoptotic protein Survivin. As part of this study, we investigated the effect of TPGS with a small molecule inhibitor of Survivin (SMI), in various breast cancer cell lines representing different subtypes of the disease. Our aim was to evaluate the presumed synergistic effect of the TPGS-SMI combination and reveal its mechanism of action. Our results show that the TPGS-SMI combination acts synergistically to reduce specifically the viability of Her2Neu-overexpressing SKBR3 cells. The combination of agents decreased Survivin mRNA levels, increased the subG1 phase of the cell cycle, and induced PARP cleavage. In addition, the TPGS-SMI treatment led to the cleavage of Caspases 9 and 7 and reduced the levels of the anti-apoptotic protein Bcl-2 indicating that the intrinsic pathway of apoptosis is induced. Importantly, the TPGS-SMI combination did not affect the viability of MCF-10A normal immortalized cells. Since SKBR3 cells overexpress the Her2neu protein, we could potentially synthesize a TPGS-based micelle, loaded with the SMI and conjugated with the antibody Herceptin for targeted delivery to the cancer site. Further studies will examine the therapeutic potential of the TPGS-SMI conjugate in Her2/neu positive breast cancer.

Poster abstracts


90

52

RING-dependent cell growth and drug resistance induced by nuclear X-linked inhibitor of apoptosis protein (XIAP) in breast cancer

Deborah Delbue2, Pedro I. Lucena2, Marcela C. Robaina2, João P.B. Viola2, Eric W.F Lam1, Raquel C. Maia2, Gabriela Nestal de Moraes2

1 Imperial College London, London, UK, 2 Instituto Nacional de Câncer (INCA), Rio de Janeiro, BRAZIL

Breast cancer is the most common malignancy within women worldwide. Apoptosis is deregulated in most cancers, including breast cancer. XIAP belongs to the family of inhibitor of apoptosis proteins and acts by binding to and inhibiting caspases as well as ubiquitinating target proteins. XIAP is mainly found at the cytoplasm, but it can be detected at the nucleus in normal and tumor cells types. However, little is known on the role of XIAP subcellular localization in breast cancer drug resistance. Here, we show that XIAP expression is detected in breast-derived cell lines but it can be found more abundantly at the nuclear fraction in the doxorubicin resistant cells, MCF-7 DoxR, as assessed by subcellular fractionation and Western blotting. Accordingly, paclitaxel-resistant MCF-7 TaxR cells showed XIAP nuclear expression, confirming an association between nuclear XIAP and chemoresistance. Cell transfections revealed that XIAPΔRING (RING deletion) and XIAPNLS C-term (Nuclear localization signal insertion at C-terminus) mutants were localized at the nucleus, differently from wild-type XIAP. Results from cell counting, flow cytometry, clonogenic, cell viability and crystal violet assays show that overexpression of XIAPNLS C-term, but not XIAPΔRING, induced doxorubicin resistance and increased proliferative capacity, suggesting that XIAP functions at the nucleus depend on its RING domain. Analysis of Kaplan-Meyer curves revealed that XIAP nuclear localization conferred poor prognosis in hormone receptor-negative patients, differently from cytoplasmic XIAP. Accordingly, cytoplasmic XIAP expression was associated with age ≥ 50 years and T1 tumor size, known favorable prognostic factors in breast cancer. In multivariate analyses, we found that nuclear XIAP was an independent prognostic factor in hormone receptor-negative patients. Altogether, our findings show that nuclear XIAP associated with poor clinical outcome in hormone receptor-negative breast cancer patients as well as promoted cell growth and drug resistance in vitro in a RING-dependent fashion.

Poster abstracts


91

53

Losing mitochondrial membrane potential: the role of essential oils in mediating unhealthy mitochondrial cells to undergo autophagic and apoptotic death of human brain cancer cells in vitro

Nik Nursyazni Nik Mohd Kamal1, Bebe Syakinah Hashim1, Fatin Athirah Abdul Aziz1, Wen-Nee Tan2

1 Cluster of Integrative Medicine, Advanced Medical and Dental Institute, Universiti Sains Malaysia, Kepala Batas, Pulau Pinang, MALAYSIA, 2 School of Distance Education, Universiti Sains Malaysia, Minden, Pulau Pinang, MALAYSIA

The aim of this study was to investigate the cell death effects of the essential oils extracted from the leaf (EO-L) and stem bark (EO-SB) of Garcinia atroviridis on DBTRG-05MG and U87 human brain cancer cells. Based on the MTT cell proliferation assay result, EO-L potentially inhibited both DBTRG-05MG and U87 cells in comparison to EO-SB. The growth inhibition induced by EO-L was significantly observed in U87 when compared to DBTRG-05MG cells. EO-L has induced the loss of mitochondrial membrane potential in U87 cells, which is an early indicator for the mechanism of apoptotic cell death. This study also found that co-treatment of EO-L and BEZ235 inhibitor potently induced the autophagy activity in U87 cells. This result also suggests that EO-L as an agent to mediate the unhealthy mitochondrial cells to undergo autophagosome degradation. Flow cytometric analysis of double staining with Annexin V-FITC and propidium iodide further confirm the role of EO-L in activating apoptotic cell death in U87 cells. At 24 h post-treatment, EO-L significantly has induced apoptosis when compared to control. Taken together, EO-L treatment has induced cytotoxicity in U87 human brain cancer cells, synergistically promoted the effect of BEZ235 inhibitor in eliminating the unhealthy mitochondrial cells through autophagosome process, which ultimately lead to apoptotic cell death. Our data epitomize basic cell death mechanism induced by naturally-derived essential oils as potential future translatable brain cancer therapy.

Poster abstracts


92

54

Neutrophil aggregates in human colorectal cancers are associated with apoptosis rather than necroptosis

Katharina Strasser 1,2, Victoria Brandel 2, Peter Stoll 2, Hanna Birnleitner 2, Thomas Bachleitner-Hofmann2, Michael Bergmann 2, Rudolf Oehler 2

1 CBmed GmbH – Center for Biomarker Research in Medicine, Graz, AUSTRIA, 2 Dept. of Surgery, Medical University of Vienna, Vienna, AUSTRIA

Local accumulation of dead cell debris is a common histological feature in colorectal cancer. We analyzed which type of cell death is going on and which type of phagocytes is attracted for dead cell clearance. Surgical resections of colorectal cancer (CRC) and distant normal mucosa were collected from 15 patients. The samples were either shock frozen for histological analysis or disintegrated to single cell suspensions for analysis by flow cytometry. Immunohistochemistry showed that areas of massive cell death in CRC are highly infiltrated with neutrophils. Macrophages, in contrast, were only found outside these areas. Flow cytometric analysis confirmed an immense accumulation of CD66b+ neutrophils in cancerous colon tissue as compared to distant normal mucosa. In vitro assays revealed that neutrophils are highly efficient in efferocytosis. It is well known that neutrophils are primarily attracted by IL-8. In vitro induction of different types of cell death showed that necroptosis induces the highest release of IL-8 when co-cultured with PBMCs. However, pMLKL+ necroptotic cells were rarely detectable in the in CRC samples. In contrast, we found a strong activation of caspase 3 in areas of neutrophil infiltration. These data suggest that the dead cell debris in CRC are the result of apoptotic cell death and attract neutrophils for efficient clearance by efferocytosis.

Poster abstracts


93

55

The role of histone deacetylase 8 (HDAC8) in neuroblastoma pathobiology

Jing Shen1, Sara Najafi1,5, Sina Stäble1, Johannes Fabian1, Emily Koeneke1,5, Fiona R. Kolbinger1,5, Tino Heimburg7, Wolfgang Sippl7, Manfred Jung6, Heike Peterziel1,5, Dominique Kranz4,2, Michael Boutros4,2, Frank Westermann8, Olaf Witt1,5, Ina Oehme1,5

1 CCU Pediatric Oncology, DKFZ, Heidelberg, GERMANY, 2 Department for Cell and Molecular Biology, Medical Faculty Mannheim, Mannheim, GERMANY, 3 Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg Medical Center, GERMANY, 4 Division of Signaling and Functional Genomics, DKFZ, Heidelberg, GERMANY, 5 Hopp Children’s Cancer Center at NCT Heidelberg (KiTZ), GERMANY, 6 Institute of Pharmaceutical Sciences, University of Freiburg, GERMANY, 7 Institute of Pharmacy, Martin-Luther University of Halle-Wittenberg, Halle/Saale, GERMANY, 8 Research Group Neuroblastoma Genomics, DKFZ, Heidelberg, GERMANY

The prognosis of advanced stage neuroblastoma patients remains poor and, despite intensive therapy, the five-year survival rate remains less than 50%. We identified histone deacetylase (HDAC) 8 as an indicator of poor clinical outcome and a selective drug target for differentiation therapy in vitro and in vivo. Selective inhibition of HDAC8 slows neuroblastoma growth, induces a more differentiated phenotype and serves as a potent enhancer of retinoic acid-mediated anti-neuroblastoma activity. RNA interference (RNAi) screens are commonly applied to identify novel limiting factors for drug responsiveness and to unravel targeted combinations of specific drugs to overcome these limitations. We used a kinome-wide RNAi screen to identify new combinations that enhance the sensitivity of neuroblastoma to HDAC8 inhibitors. We identified receptor tyrosine kinases (RTKs) as druggable neuroblastoma cell survival activators that can be targeted by treatment with small molecule inhibitors. Treatment of neuroblastoma cell lines with the combination of an HDAC8 inhibitor with RTK inhibitors efficiently blocked the activation of growth receptor survival signaling and shifted the cell cycle arrest and differentiation phenotype toward effective cell death.

Poster abstracts


94

56

Dual role of HDAC10 in lysosomal exocytosis and DNA repair promotes neuroblastoma chemoresistance

Johannes Ridinger2,1, Emily Koeneke2,1, Fiona Kolbinger2,1, Katharina Koerholz2,1, Siavosh Mahboobi3, Nikolas Gunkel1, Aubry Miller1, Heike Peterziel2,1, Anne Hamacher-Brady4, Olaf Witt2,1, Ina Oehme2,1

1 German Cancer Research Center, Heidelberg, GERMANY, 2 Hopp Children’s Cancer Center at NCT Heidelberg (KiTZ), Heidelberg, GERMANY, 3 Institute of Pharmacy, University of Regensburg, GERMANY, 4 Johns Hopkins University, Bloomberg School of Public Health, Baltimore. Maryland, USA

Drug resistance is a leading cause for treatment failure in many cancers, including neuroblastoma, the most common solid extracranial childhood malignancy. Previous studies from our lab indicate that histone deacetylase 10 (HDAC10) is important for the homeostasis of lysosomes, i.e. acidic vesicular organelles involved in the degradation of various biomolecules. Here, we show that depleting or inhibiting HDAC10 results in accumulation of lysosomes in chemotherapy-resistant neuroblastoma cell lines, as well as in the intracellular accumulation of the weakly basic chemotherapeutic doxorubicin within lysosomes. Interference with HDAC10 does not block doxorubicin efflux from cells via P-glycoprotein inhibition, but rather via inhibition of lysosomal exocytosis. In particular, intracellular doxorubicin does not remain trapped in lysosomes but also accumulates in the nucleus, where it promotes neuroblastoma cell death. Inhibition of HDAC10 further interferes with DNA double strand break (DSB) repair, providing an additional mechanism of sensitizing neuroblastoma cells to doxorubicin. Taken together, we demonstrate that HDAC10 inhibition in combination with doxorubicin kills neuroblastoma, but not non-malignant cells, both by impeding drug efflux and enhancing DNA damage, providing a novel opportunity to target chemotherapy resistance.

Poster abstracts


95

57

α-mangostin, singly and in combination with doxorubicin, as pro-apoptotic agents and protein kinase inhibitors in the AML cell line, MOLM13

Cynthia U. Osemeke1


Acute myeloid leukaemia is the most common form of adult leukaemia with several kinase mutations. The FLT3-ITD mutation is a common kinase mutation (20-30%) observed in AML patients and the most involved in the prognosis of AML. Kinases are crucial in controlling different cellular activities, and mutations are well known to signal cancer development and progression. A tyrosine kinase inhibitor, imatinib (Gleevec ST1571), has been successfully used in treating CML by selectively targeting genetically altered tyrosine kinase. However, due to the complexity and the high frequency of the mutations identified in AML, the success rates from commonly used tyrosine kinase inhibitors are low in comparison to CML. The aim of this study is to investigate in vitro, the effect of the phytochemical, α-mangostin, singly and in combination with doxorubicin (DOX) as pro-apoptotic agents and protein kinase inhibitors in AML cell line MOLM13. The phytochemical α-mangostin (1- 50 µM) was used to treat MOLM13 for 72 h. CyQUANT proliferation assay was used to determine its effect on cell viability. Apoptosis and cell cycle analysis were conducted using a flow cytometer. Expression of proteins was determined using western blot technique. Statistical analysis including ANOVA, Student T-test were chosen for comparing the effects of different treatments. Results showed that α-mangostin at the concentration (≥20 µM) inhibited cell growth in a dose-dependent manner. More apoptotic effects were observed when α-mangostin (20 µM) was combined with Dox (1 µM), with more TUNEL positive cells and increased expression of the pro-apoptotic protein BAK. The G2/M phase cell cycle arrest was induced by combination with increased expression of p21 protein and reduced expression of the phosphorylated CDC25 proteins. Further studies to explore the mechanism of potential kinase inhibition of FLT3-ITD mutation is currently carried out. Our findings may provide relevant information in identifying potential kinase inhibitors in treating AML.

Poster abstracts


96

58

A new and essential function for the mitochondrial pro-apoptotic protein SMAC/Diablo in cancer

Avijit Paul1, Yakov Krelin1, Tasleem Arif1, Varda Shoshan-Barmatz1

1 Ben-Gurion University of the Negev, Beersheba, ISRAEL

The mitochondrial pro-apoptotic protein SMAC/Diablo participates in apoptosis by negatively regulating inhibitor of apoptosis proteins (IAPs), activating the caspase cascade, and thus, apoptosis. Unexpectedly, we found that SMAC/Diablo is over-expressed in cancer. This paradox was addressed here by silencing SMAC/Diablo expression using specific siRNA (si-hSMAC). In cancer cell lines and sub-cutaneous lung cancer xenografts in mice, such silencing reduced cell and tumor growth. Immunohistochemistry and electron microscopy of the residual tumor following si-hSMAC treatment demonstrated morphological changes, including cell differentiation and reorganization into glandular/alveoli-like structures and elimination of surfactant-producing organs, the lamellar bodies. Next-generation sequencing of tumors treated with non-targeted- or si-hSMAC revealed altered expression of proteins associated with the cellular membrane, extracellular matrix, proteins found in the lumen of the ER and Golgi, and in exosomal networks, proteins involved in lipid metabolism and transporters of lipids, metabolites and ions. SMAC/Diablo silencing decreased phospholipid and phosphatidylcholine levels, as well as the expression of enzymes associated with their synthesis. These findings suggest that SMAC/Diablo possesses additional non-apoptotic functions associated with regulating lipid synthesis essential for cancer growth and development and may explain SMAC/Diablo over-expression in cancer. The new lipid synthesis-related function of the pro-apoptotic protein SMAC/Diablo in cancer cells makes SMAC/Diablo a promising therapeutic target.

Poster abstracts


97

59

Increasing telomerase expression by novel compounds in senescent human dermal fibroblast cultures partially restores their proliferative capacity

Snir Yehuda1, Vadim, E. Fraifeld1, Esther Priel11 Ben-Gurion University of the Negev, Beer-Sheva, ISRAEL

Telomerase, a ribonucleoprotein that is responsible for the re-elongation of telomeres, is not expressed or only slightly expressed in most of the human somatic cells, which results in telomere shortening following each cycle of DNA replication and cell division. Telomere shortening is known to be the major factor for induction of replicative cellular senescence (CS). In our previous study, we showed that human dermal fibroblasts (HDFs) expressed the catalytic subunit of telomerase – TERT, and exhibited telomerase activity. Both TERT and telomerase activity were markedly decreased in the course of replicative CS in primary cultures of HDFs. We synthesized novel compounds (AGS) that activate the expression of TERT, increase the activity of telomerase and increase telomere length in various human and animal cells in vitro and in several in vivo animal models. Here, we examined the ability of these compounds to increase TERT expression and telomerase activity in senescent HDFs and their effect on proliferative capacity of HDF cultures. We found that the AGS compounds can (i) increase TERT expression and telomerase activity in senescent cells, and (ii) partially restore their proliferative capacity. The results suggest that re-expression of telomerase in senescent cells by pharmaceutical compounds may influence their senescent state.

Poster abstracts


98

60

Modulation of HES1 expression as a candidate for 2OHOA’s antiproliferative effect in glioma cells

Raquel Rodríguez Lorca1, Laura Arbona González1, Victoria Lladó1, Pablo V. Escribá1,2, Paula Fernández García1,2

1 Laboratory of Molecular Cell Biomedicine, University of the Balearic Islands, Palma de Mallorca, SPAIN, 2 Lipopharma Therapeutics SL, Palma de Mallorca, SPAIN

Glioblastoma (GBM) is the most common malignant brain tumor with a poor prognosis of survival. 2-hydroxyoleic acid, 2OHOA, is an antitumor drug, currently entering a multicenter phase IIb clinical trial for the treatment of gliomas and other solid tumors. Its mechanism of action involves important changes in membrane lipid composition and organization, as well as modulation of several signaling pathways. Previous studies have demonstrated 2OHOA induces cell cycle arrest in cancer cells, driving glioma-to-glial cell differentiation and, finally, leading to a selective autophagic cell death through the inhibition of the Ras-MAPK, Cyclin/CDK-DHFR, and PI3K-Akt pathways in human glioma cells.

One of the transcriptional regulators modulated by 2OHOA is Hes1, involved in the control of differentiation and proliferation. We hypothesized that the possible mechanism of action could be related with a down expression of this gene. In this context, we focus on the modulation of HES1 expression in U118 GBM cells under activation or inhibition of different signaling pathways.

In the light of our results, HES1 expression is up-regulated by activation of Notch, EGFR/MAPK, JNK and Wnt/β-catenin signaling pathways and the inhibition of Wnt and Hedgehog pathway. Moreover, 2OHOA decreased HES1 expression through several signaling pathways related to changes in plasma membrane composition and organization. In this scenario, pro-survival (EGFR/MAPK) and stress induced (JNK) pathways balance can be altered, leading to cell cycle arrest. Even more, 2OHOA could possibly modulate the NICD (Notch IntraCellular Domain) production and trafficking to the nucleus, and regulate the β-catenin pathway by targeting to the destruction complex (GSK-3β/AXIN/APC), its recruitment to the cytoplasm or Wnt receptor blockade. In summary, 2OHOA is a promising therapeutic drug against GBM treatment and its final autophagic event could be associated with regulation of HES1 expression.

Poster abstracts


99

61

Checkpoint kinase 1 (CHK1) controls normal B cell development, lymphomagenesis and cancer cell survival

Fabian Schuler1, Johannes Weiss1, Silke Lindner1, Michael Lohmüller1, Sebastian Herzog1, Simon Spiegl2, Philipp Menke2, Stephan Geley2, Verena Labi1, Andreas Villunger1,3

1 Division of Developmental Immunology, Biocenter, Medical University of Innsbruck, Tyrol, AUSTRIA, 2 Division of Molecular Pathophysiology, Biocenter, Medical University of Innsbruck, Tyrol, AUSTRIA, 3 Tyrolean Cancer Research Institute, Innsbruck, Tyrol, AUSTRIA

Checkpoint kinase 1 (CHK1) is critical for intrinsic cell cycle control and coordination of cell cycle progression in response to DNA damage. Despite its essential function, CHK1 has been identified as a target to kill cancer cells, especially those lacking functional p53. Studies using Chk1 haploinsufficient mice initially suggested a role as tumor suppressor in line with its function to halt cell cycle progression and orchestrate DNA repair in response to replication stress or when cells experience exogenous DNA damage. Yet, in cancer cells Chk1 appears to display pro-survival properties, suggesting context dependent activities. Here, we report on the key-role of CHK1 in normal B-cell development, lymphomagenesis and cell survival. Chemical CHK1-inhibition induces BCL2-regulated apoptosis in primary as well as malignant B-cells and CHK1 expression levels control the timing of lymphomagenesis in mice. Furthermore, loss of Chk1 precludes the outgrowth of lymphomas in Eµ-MYC transgenic mice, while a reduction in gene-dose delays tumor onset. This effect was associated with an obvious increase in replication stress and hallmarks of DNA damage in premalignant MYC transgenic B cells. Moreover, total ablation of Chk1 in B-cells arrests their development at the pro-B cell stage, a block that, surprisingly, cannot be overcome by inhibition of mitochondrial apoptosis, as cell cycle arrest is initiated as an alternative fate to limit the spread of damaged DNA.

Together, these findings suggest that targeting CHK1 is a valid strategy to hit hard-to-treat blood-cancer with high apoptotic threshold or low apoptotic priming. A limitation of this strategy, however, might be that, in the absence of functional p53, or reduced cell cycle check-point proficiency, BCL2-controlled apoptosis resistance can trigger aneuploidy tolerance, increasing the chances for treatment failure.

Poster abstracts


100

62

Cytotoxic and apoptotic effects of resveratrol, cisplatin and their combinations on MDA-MB-231 breast adenocarcinoma cells

Arda Sever2, Filiz Özdemir1

1 Anadolu University, Faculty of Pharmacy, Department of Biochemistry, Eskişehir, TURKEY, 2 Anadolu University, Graduate School of Health Sciences, Department of Biochemistry, Eskişehir, TURKEY

Breast cancer is the most common type of cancer among women and is an important disease causing high mortality. Chemotherapy is one of the most widely used treatment regimens in cancer. Resveratrol is a non-flavonoid polyphenol with potent antioxidant and anticancer effects and it has been shown that it interacts with several cellular processes including angiogenesis, inflammation, cell cycle progression and apoptosis. Cisplatin is a chemotherapeutic agent used in a variety of malignancies. Its antineoplastic activity occurs from DNA cross-links and adducts, in addition to the generation of superoxide radicals. In this study, we aimed to investigate the combinative effects of cisplatin and resveratrol on MDA-MB-231 human breast adenocarcinoma cell line. Cytotoxic activity of the compounds were determined by using standard 3-(4,5-dimetilltiyazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) colorimetric assay. IC50 values for cisplatin and resveratrol were determined as 46 µM and 144 µM respectively at 24 h. Combination doses were prepared according to IC50 doses of cisplatin and resveratrol. [23 µM cisplatin + 72 µM resveratrol] combination, which is 50% of their IC50 values and [18.5 µM cisplatin + 57.5 µM resveratrol], which is 40% of their IC50 values, inhibited cell proliferation by 50.28% and 34.93% respectively. Analysis of apoptosis was conducted by using BD Pharmingen FITC Annexin V Apoptosis Detection Kit. Apoptotic cell percentage for IC50 doses of cisplatin and resveratrol were 16.9% and 26.2% respectively, whereas 13.7% and 41.4% with their %40 and %50 combinations. DNA fragmentation is one of the hallmarks of apoptosis and it occurs in the late phase of the mechanism. DNA double-strand breaks in the cells were imaged with a fluorescence microscope by using Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method. These results showed that combination of cisplatin and resveratrol causes cytotoxicity and induces apoptosis on MDA-MB-231 cell line.

Poster abstracts


101

63

Targeting PP2A to manipulate MCL-1 expression in malignant B cells

Anne Slomp2, Laura Moesbergen2, Monique Minnema1, Victor Peperzak2

1 Department of Hematology, University Medical Center Utrecht, NETHERLANDS, 2 Laboratory of Translational Immunology, University Medical Center Utrecht, NETHERLANDS

Pro-survival B Cell Lymphoma (BCL-2) family proteins BCL-2, BCL-XL, and MCL-1 are potent inhibitors of apoptosis and are often overexpressed in lymphoid malignancies. MCL-1 contributes to survival of malignant B cells and intervening in MCL-1 expression may therefore improve treatment of B cell malignancies. Direct inhibition of MCL-1, as is currently being done for BCL-2 in the treatment of chronic lymphocytic leukemia (CLL), may result in significant side effects, since MCL-1 expression is crucial for survival of many healthy cells and tissues. Therefore, we investigate strategies for indirect targeting of MCL-1. MCL-1 protein stability is increased in post-germinal center (GC) B cell malignancies, such as multiple myeloma (MM). This may be the result of increased activity of specific phosphatases that prevent MCL-1 phosphorylation, ubiquitination, and subsequent proteasomal degradation. We aim to identify phosphatases that control MCL-1 half-life in a cancer- or tissue-specific manner in order to find new therapeutic targets for treatment of B cell malignancies.

Using a phosphatase siRNA screen, we sought for phosphatases that stabilize MCL-1 in MM cells. The results were validated by comparing the effects of phosphatase inhibitors on MCL-1 phosphorylation and protein half-life. These experiments suggest that the PP2A phosphatase complex is directly responsible for dephosphorylation and stabilization of MCL-1. One regulatory PP2A subunit that was identified in the screen is currently being examined using CRISPR/Cas9-mediated knockout. Since it may be a tissue-specific component of the PP2A complex, it may be an interesting target for therapy.

Indirect targeting of MCL-1 through inhibition of a specific regulatory PP2A subunit may circumvent tissue toxicity and may therefore be a new strategy for therapy, possibly in combination with inhibitors of other pro-survival BCL-2 family proteins.

Poster abstracts


102

64

The role of cell competition in tumor growth

Saskia Suijkerbuijk3,1, Golnar Kolahgar1, Iwo Kucinski1, Eugenia Piddini1, Jacco van Rheenen3,2

1 Gurdon Institute - University of Cambridge, Cambridge, UK, 2 Hubrecht Institute (KNAW), Utrecht, NETHERLANDS, 3 Netherlands Cancer Institute (NKI-AvL), Amsterdam, NETHERLANDS

Interactions between tumor cells and their environment play an important role in the modulation of tumor growth. Comprehension of the types and consequences of this communication is therefore essential to successfully understand cancer and develop targeted therapies. Here we use the adult fly intestine and mouse organoids to characterize the role of competitive cell interactions to tumor growth. We show that, in the fly intestine, APC-/- driven adenomas cause attrition of surrounding host tissue through cell competition.

Furthermore, inhibition of cell competition, through blocking apoptosis, can prevent loss of host tissue and overgrowth of adenomas. Interestingly, both JNK signaling and an imbalance in YAP activity are required for cell competition and thereby tumor growth. Importantly, competitive cell interactions between tumor and healthy cells are not restricted to fly epithelia, but are also observed in mammalian intestinal organoids. We show that wild-type small intestine cells are gradually lost from organoids through competition with carcinoma cells. Tumor cells use these competitive interactions to boost their own proliferation. Together these data indicate that, in Drosophila, the intrinsic over-proliferative capacity of tumor cells is not uncontrolled and can be constrained by host tissues if cell competition is inhibited. The presence of similar interactions in mammalian models indicates the potential for new therapeutics.

Poster abstracts


103

65

Agonist-mediated activation of STING induces apoptosis in B cell cancer

Chih-Hang Tang2, Joseph Zundell2, Sujeewa Ranatunga1, Cindy Lin2, Yulia Nefedova2, Juan Del Valle1, Chih-Chi Hu2

1 The University of South Florida, Tampa, USA, 2 The Wistar Institute, Philadelphia, USA

STING (stimulator of interferon genes) is an endoplasmic reticulum (ER)-resident protein critical for cytoplasmic DNA sensing and production of type I interferons to help boost our immune response to combat infections or cancer. We showed that the IRE-1/XBP-1 pathway of the ER stress response is required for the function of STING, as evidenced by that both IRE-1-deficient and XBP-1-deficient mouse embryonic fibroblasts failed to respond to STING agonists by producing interferons. While various Toll-like receptor ligands induce B cells to undergo rapid proliferation and differentiation, we discovered that STING agonists potently induced mitochondria-mediated apoptosis in normal and malignant B cells while triggering production of interferons in many other cell types, including fibroblasts, melanoma, hepatoma and Lewis lung cancer cells. STING agonists clearly induced apoptosis in malignant B cells through STING because no cytotoxicity was observed in B cell lymphoma and multiple myeloma cells in which the STING gene was deleted with zinc finger nucleases. Different from fibroblasts, melanoma, hepatoma and Lewis lung cancer cells, normal and malignant B cells were not capable of degrading STING after stimulations with STING agonists, suggesting that prolonged activation of STING in B cells could engage apoptotic machineries. Injections of the STING agonist, 3’3’-cGAMP, induced apoptosis and regression of cancer in Eμ-TCL1 mice carrying spontaneously developed chronic lymphocytic leukemia. Injections of 3’3’-cGAMP also resulted in the prolonged survival of syngeneic mice grafted with multiple myeloma, and suppressed myeloma growth in immunodeficient NSG mice. Thus, other than the potential use of STING agonists in boosting anti-tumor immune response, these agonists can directly target B cell malignancies. In addition, while STING agonists have been recently proposed to be used as adjuvants, our data showing that STING agonists are cytotoxic to B cells suggest that their use as adjuvants to boost antibody production may need to be re-evaluated.

Poster abstracts


104

66

Cell death enhancement and chemosensitisation effect of 5-fluorouracil in colorectal cancer cells via the combination with delta-tocotrienol

Shiau-Ying Tham3, Chun-Wai Mai1, Ju-Yen Fu2, Hwei-San Loh3

1 International Medical University, Bukit Jalil, Wilayah Perseketuan Kuala Lumpur, MALAYSIA, 2 Malaysian Palm Oil Board , Kajang, Selangor, MALAYSIA, 3 University of Nottingham Malaysia Campus, Semenyih, Selangor, MALAYSIA

5-Fluorouracil (5-FU) is a popular chemotherapeutic drug for treating colorectal cancers. However, its therapeutic outcome has often been limited by high-dose toxicities and drug-resistance development. Hence, combined treatment with a chemosensitising agent may offer a solution. The current research focuses a combinatorial application of the natural chemosensitiser, delta-tocotrienol (δ-T3) and 5-FU as a novel treatment approach for colorectal cancers. This study aimed to investigate the combinatorial effects of δ-T3 and 5-FU on cell death and chemosensitivity enhancement. A cell viability assay was conducted to investigate the individual and combined treatments of δ-T3 (0.1-100μM) and 5-FU (0.1-100μM) on HCT116, HCC2998, Caco-2 and SW48 colorectal cancer cells. Subsequently, the synergistic combination was evaluated for the clonogenicity, cell cycle and apoptotic effects. The morphologies of induced cell death were characterised by bright-field and fluorescence microscopies. The protein expression profiles of several cell death and survival markers were determined by Western blotting. The sub-effective concentrations of δ-T3 in the combined treatment significantly lowered the IC50 of 5-FU on Caco-2 and SW48 cells for 16 and 4 folds respectively, signifying a chemosensitising effect. Clonogenic survival assay showed that the combined treatment profoundly hampered the cell survival more than the individual treatments. The presence of δ-T3 did not interfere with the primary action of 5-FU in causing an S-phase arrest. However, cellular apoptosis induced by the combined treatment was confirmed using the flow cytometry. Featured cell death morphologies including nuclear condensation, cell shrinkage, cytoplasmic vacuolation and extension were observed. The combined treatment was also found to upregulate the pro-apoptotic markers, namely p53 and Bid and downregulate the pro-survival markers such as XIAP and survivin. Collectively, these findings suggest that δ-T3 has enhanced the cell death and chemosensitisation effect in 5-FU-treated colorectal cancer cells and thus prove the potential synergy in this combined treatment.

Poster abstracts


105

67

HMGB1 protein as a key regulator of the cell fate under stress conditions in cellular models of lung cancer

Jordana Todorova1, Maria Schröder1, Maria Petrova1, Ivan Iliev1

1 Roumen Tsanev Institute of Molecular Biology, Bulgarian Academy of Sciences, Sofia, BULGARIA

The balance between autophagy and apoptosis is regulated by various cell signals and crosstalk between these pathways determines cell fate under stress conditions. High mobility group box 1 (HMGB1), a chromatin-associated nuclear protein and extracellular damage associated molecular pattern molecule (DAMP), can bind to different receptors and causes huge variety of cellular responses. Recently HMGB1 was described as one of the main players in the crosstalk between autophagy and apoptosis pathways. However, whether HMGB1plays a role in autophagy in lung cancer is elusive. In this study, we used two lung cancer cell lines A549 and H1299 as a model. We observed that autophagic stimuli such as starvation or rapamycin treatment induced HMGB1-GFP translocation from the nucleus to the cytoplasm where the protein colocalized with Beclin1 in both cell lines. To assess the role of HMGB1 in autophagy we monitored autophagic flux by LC3-GFP puncta formation and LC3-II expression in cells after treatment with rapamycin or starvation. It is known that both starvation and rapamycin treatment enhance autophagy and induce apoptosis in some cell lines. When we blocked autophagy by 3-methyladenine (3-MA), an inhibitor that blocks the formation of autophagosomes, we observed increased annexin V staining, which is an indicator of enhanced apoptosis, in A549 cells. In p53-null H1299 cells, such increase of apoptotic cells was not observed at the same experimental conditions. In these experiments, we also monitored the cytosolic fraction of HMGB1 and we found that in A549 cells the level of the protein was decreased, but surprisingly in H1299 cells it remained increased. More experiments should be done for clarifying the last observations.

This work was supported by grant DN01/10 16.12.2016 of the Bulgarian National Science Fund.

Poster abstracts


106

68

CD31 expression determines redox status and chemoresistance in human angiosarcomas

Vivek Venkataramani3,1, Stefan Küffer4, Kenneth Cheung2, Xuejun Jiang1, Lorenz Trümper3, Gerald Wulf3, Philipp Ströbel41 Memorial Sloan Kettering Cancer Center, Cell Biology Program, New York, USA, 2 Queen Mary University of London, William Harvey Research Institute, London, UK, 3 University Medical Center Göttingen (UMG), Department of Hematology and Oncology, Göttingen, GERMANY, 4 University Medical Center Göttingen (UMG), Institute of Pathology, Göttingen, GERMANY

Angiosarcomas (AS) are soft tissue sarcomas with endothelial differentiation and vasoformative capacity. Most AS show strong constitutive expression of the endothelial adhesion receptor CD31/PECAM-1 pointing to an important role of this molecule. However, the biological function of CD31 in AS is unknown. The expression levels of CD31 in AS cells and its effects on cell viability, colony formation and chemoresistance was evaluated in human AS clinical samples and in cell lines through isolation of CD31high and CD31low cell subsets. The redox-regulatory CD31 function linked to YAP signaling was determined using a CD31 blocking antibody and siRNA approach and was further validated in CD31-knockout endothelial cells. We found that most AS contain a small CD31low cell population. CD31low cells had lost part of their endothelial properties, were more tumorigenic and chemoresistant than CD31high cells due to more efficient reactive oxygen species (ROS) detoxification. Active downregulation of CD31 resulted in loss of endothelial tube formation, nuclear accumulation of YAP, and YAP-dependent induction of antioxidative enzymes. Addition of pazopanib, a known enhancer of proteasomal YAP degradation re-sensitized CD31low cells for doxorubicin resulting in growth suppression and induction of apoptosis. Human AS contain a small aggressive CD31low population that have lost part of their endothelial differentiation programs and are more resistant against oxidative stress and DNA damage due to intensified YAP signaling. Our finding that the addition of YAP inhibitors can re-sensitize CD31low cells towards doxorubicin may aid in the rational development of novel combination therapies to treat AS.

Poster abstracts


107

69

Protein arginine methyltransferase PRMT5 in high-risk multiple myeloma: a possible new treatment strategy?

Philip Vlummens2,1, Kim De Veirman2, Eline Menu2, Karin Vanderkerken2, Elke De Bruyne2, Ken Maes2

1 Department of Clinical Hematology, Ghent University Hospital, Ghent, BELGIUM, 2 Department of Hematology and Immunology, Myeloma Center Brussels, Vrije Universiteit Brussel, BELGIUM

Multiple myeloma (MM), even in the age of novel agents, remains an incurable B-cell malignancy. It is known that a certain subset of patients have high-risk features linked with dismal outcome. Therefore, the need for additional therapeutic options remains high. The past years, a role for epigenetic dysregulations and DNA repair pathway alterations have been identified in MM pathogenesis. We analyzed the prognostic significance of different epigenetic and DNA repair target genes using publically available gene-expression data of MM patients. We identified Protein Arginine Methyltransferase 5 (PRMT5), known to play a role in epigenetic regulation of tumor suppressor genes and protein methylation, as a potential target gene in high-risk patients. We validated the clinical and prognostic significance of PRMT5 in the CoMMpass Study, showing a significant decrease in progression-free survival in patients with high PRMT5 expression (112.7 vs 189.9 weeks, p=0.003). We evaluated the role of PRMT5 as a druggable target in different human myeloma cell lines with the use of a PRMT5 inhibitor, EPZ015666. EPZ015666 treatment of OMP2, U266 and JJN3 cells showed a significant decrease in cell growth over the course of treatment, without striking effects on the cell cycle profile. Increased amount of AnnexinV positivity was observed starting from 6 days of treatment. Furthermore, a combinatory effect on MM cell viability was seen when EPZ015666 was combined with melphalan, an alkylating agent. On a protein level, a decrease of global symmetric arginine di-methylation was seen alongside decreased levels of E2F1, a PRMT5 target involved in the Rb1/E2F cell cycle regulatory pathway. In conclusion, we have identified that PRMT5 is important for MM cell growth and survival. The role of PRMT5 in MM pathogenesis and treatment strategies warrants further investigation.

Poster abstracts


108

70

Targeting mitochondrial priming with BH3-mimetics in diffuse large B-cell lymphoma

Kristina Henz3, Victoria Smith4, Martin JS Dyer4, Simone Fulda3,1, Meike Vogler3,4

1 German Cancer Consortium (DKTK), Heidelberg, GERMANY, 2 German Cancer Research Centre (DKFZ) , Heidelberg, GERMANY, 3 Institute for Experimental Cancer Research in Pediatrics, Goethe University, Frankfurt, GERMANY, 4 University of Leicester, Leicester, UK

The induction of apoptosis at the mitochondria is critically regulated by BCL2 protein family. In cancer cells with high expression of pro-apoptotic BH3-only proteins these are sequestered by the anti-apoptotic BCL2 proteins and cells are primed to undergo mitochondrial apoptosis. Primed cells are particularly sensitive to treatment with BH3-mimetics, which target and inhibit the anti-apoptotic BCL2 proteins.

Here, we hypothesize that besides BCL2, also the related anti-apoptotic BCL2 proteins MCL1 and BCL-XL may be promising therapeutic targets in DLBCL. To investigate the importance of the different anti-apoptotic BCL2 proteins we directly compared the efficacy to BH3-mimetics selectively targeting BCL2, BCL-XL or MCL1. Data derived from a range of well-characterised DLBCL cell lines as well as primary patient-derived cells indicate a very heterogeneous response to these selective BH3-mimetics. In line with the results obtained in clinical trials, some DLBCL cells are highly sensitive to Venetoclax, while a majority displays resistance. Notably, some DLBCL cell lines are clearly more dependent on MCL1 or BCL-XL than on BCL2, and are highly susceptible to the selective BCL-XL inhibitor A1331852 or the MCL1 inhibitor S63845.

To investigate the importance of MCL1 or BCL-XL independently of small molecule inhibitors, we performed siRNA mediated knockdown of MCL1 or BCL-XL that resulted in increased cell death in those cell lines also displaying sensitivity to the selective BH3-mimetics. Interestingly, some cell lines that are more resistant to BCL2, BCL-XL or MCL1 inhibition exhibited high expression of BCL2A1 indicating that besides BCL2, BCL-XL and MCL1 also BCL2A1 may be an important therapeutic target in DLBCL.

Taken together, the response of DLBCL cells to BH3-mimetics is highly diverse and complex, highlighting the need to understand the mechanisms underlying sensitivity to BH3-mimetics and the development of biomarkers that can be used to stratify patients most likely to respond to selective BH3-mimetics.

Poster abstracts


109

71

Betulinic acid enhanced doxorubicin-induced apoptotic cell death in acute myeloid leukaemia cell lines

Milan Vu1


Inbalance of the B-cell lymphoma 2 (Bcl-2) family of proteins in acute myeloid leukaemia (AML) contributes to its chemoresistance. The Bcl-2 family of proteins regulate cell death by apoptosis and it has been suggested that targeting the Bcl-2 proteins family may increase patients’ response to chemotherapy. Betulinic acid (BetA) has shown potential to selectively sensitise cancer cells to apoptotic cell death in cancers of epithelial origin and it may have potential to stimulate cell death in blood cancers. The aim of this study was to investigate the effect of BetA, either as a monotherapy or in combination with established leukaemic drug doxorubicin in inducing apoptotic cell death in AML cell lines. MOLM13, acute myeloid leukaemia cell line, was treated with BetA (5-40 μM) alone or in combination with doxorubicin (0.5 and 1 μM). Cell viability was measured by the CyQuant Direct assay (which estimates cell numbers via nuclear content of viable cells). 5-(and -6) carboxyfluorescein diacetate succinimidyl ester (CFSE) cell labelling was used to assess the ant-proliferative effect of the compounds on MOLM13. To investigate reactive oxygen species (ROS) activity, cells were stained with 2’,7’–dichlorofluorescin diacetate (DCFDA). Cell death population was measured via flow cytometry using Annexin V and PI dye after 48 h cell treatments. The regulation of Bcl-2 family of proteins was examined by western blot analysis. The study demonstrated that BetA is cytotoxic and inhibits proliferation in AML, which was selective in MOLM13 cell lines but not in normal monocyte cells. Combination treatments elevated ROS levels compared to individual treatments. The potential synergism of BetA 20 μM and doxorubicin at 1 μM has shown enhanced anti-cancer properties in this study, compared to doxorubicin alone. Further studies are underway to investigate the effect of BetA and combination treatment to induce other cell death pathways such as autophagy and necroptosis, and overcome leukaemia cancer resistance.

Poster abstracts


110

72

Exploring genomic biomarkers of Vitamin C induced cell death in pancreatic ductal adenocarcinoma

Cassia Warren6,7, Joanna Karasinska7, Andrew Metcalfe7, Wieslawa Dragowska5, Tiaxiang Wang7, Donald Yapp5, Janel Kopp2, Ling Huang1, Senthil Muthuswamy1, Daniel Renouf7,3, David Schaeffer7,4

1 Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA, 2 Department of Cellular & Physiological Sciences, University of British Columbia, Vancouver, BC, CANADA, 3 Department of Medicine, University of British Columbia,, Vancouver, BC, CANADA, 4 Division of Anatomic Pathology, Vancouver General Hospital, Vancouver, BC, CANADA, 5 Experimental Therapeutics, BC Cancer Agency, Vancouver, BC, CANADA, 6 Interdisciplinary Oncology Program, University of British Columbia, Vancouver, BC, CANADA, 7 Pancreas Centre British Columbia, Vancouver, BC, CANADA

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest forms of cancer, with nearly 5000 newly-diagnosed individuals per year in Canada and a 5-year survival of less than 10%. These challenges are exacerbated by late-stage diagnosis, limited therapeutic options and a lack of well-established predictors of treatment response. Treatment with high-dose Vitamin C (VitC) has been reported to be cytotoxic in KRAS mutant colorectal cancer cell lines. Over 90% of PDAC cases have oncogenic KRAS mutations, suggesting that treatment with VitC may be a promising option, but whether PDAC response to VitC is dependent on KRAS status or other molecular subtypes is yet to be established.

To determine cytotoxicity effects of VitC, a panel of 10 cell lines (7 PDAC and 3 non-cancerous) and patient-derived organoids (PDOs) harbouring known mutant or wildtype KRAS were treated with VitC alone (0-20mM), or in combination with gemcitabine (gem) for 24 or 72 hours. Cell viability was assessed and compared to see differences in VitC sensitivity.

We identified two highly sensitive cell lines, MIA Paca2 (EC50=0.96mM) and Panc1 (EC50=1.78mM) while other cell lines showed reduced or no response. Furthermore, MIA Paca2 and Panc1 cells were responsive at VitC doses that were not toxic in non-cancerous cells. Notably, not all KRAS mutant cells were sensitive to VitC suggesting that other factors in addition to KRAS mutation may in part influence response. Further work is focused on determining VitC sensitivity by optimizing drug screening in PDOs with matched genomic, transcriptomic, and proteomic data, to identify predictive biomarkers. Knowledge of molecular signatures associated with VitC sensitivity will allow us to identify a subpopulation of tumours that may benefit from this potential new therapeutic strategy for PDAC.

Poster abstracts


111

73

Shedding light on the new form of targeted therapy in acute and chronic myeloid leukemia

Narjes Yazdi4, Mohammad Houshmand2, Alireza Kazemi3, Amir Atashi5, Mahin Nikougoftar Zarif1

1 Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, IRAN, 2 Department of Clinical and Biological Sciences, University of Turin, Turin, ITALY, 3 Department of Hematology and Blood Banking, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences,, Tehran, IRAN, 4 Department of Molecular Genetics, Tehran Medical Branch, Islamic Azad University, Tehran, IRAN, 5 Stem cell and Tissue Engineering Research Center, Shahroud University of Medical Sciences, Shahroud, IRAN

Background: hematologic malignancies account for about 10% of all cancers in developed countries. Due to the limitation and drawback of conventional treatments such as chemotherapy, different targeted therapies have introduced. As cancerous cells utilize compensatory pathways to circumvent these agents, renewing and finding up to the minute therapy is of the essence. Long non-coding RNA PVT1, which located in chromosomal region 8q24, a tried and tested oncogene in solid tumors, by the preservation of c-Myc, Nop2, FOXM1 and sponging many tumor suppressor microRNAs, play a crucial role in regulating cell cycle, apoptosis, proliferation, and invasion of cancerous cells. In this study, we determined to shed light on the effect of PVT1 knockdown on the suppression of acute and chronic myeloid leukemia cells development.

Materials and Methods: after measurement of the normal expression of the lncRNA PVT1 in TF-1 (acute myeloid leukemia) and k562 (chronic myeloid leukemia) cells, PVT1 knock-down was performed. The expression level of c-Myc, hTERT, Bcl2, Caspase-3, p15, and p16 was evaluated by qRT-PCR before and after siRNA treatment. The proliferation rate by CFSE, cell cycle analysis by Propidium Iodide and apoptosis assay by utilizing Annexin-PI were performed. Moreover, to verify the protein expression of c-Myc and h-TERT, flow cytometry analysis and western blot were carried out.

Results: Our results demonstrated that PVT1 knock-down results in c-Myc and h-TERT mRNA down-regulation and c-Myc and h-TERT protein degradation. Moreover, it induced apoptosis, G0/G1 arrest, and reduction of proliferation rate. All the results confirmed in molecular levels. Conclusion: due to the role of lncRNA PVT1 in participating in the different cellular process, considering this potent oncogene for targeted therapy may be a suitable approach for the treatment of both acute and chronic myeloid leukemia.

Keyword: Long non-coding RNA, PVT1, c-Myc, h-TERT, Leukemia

Poster abstracts


112

74

Autophagy inhibition reinforces the effect of 5-azacytidine in induction of apoptosis and suppression of telomerase activity in acute myeloid leukemia

Narjes Yazdi4, Alireza Kazemi3, Mohammad Houshmand2, Mahin Nikougoftar Zarif1

1 Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, IRAN, 2 Department of Clinical and Biological Sciences, University of Turin, Turin, ITALY, 3 Department of Hematology and Blood Banking, School of Allied Medical Sciences, Shahid Beheshti University of Medical Sciences,, Tehran, IRAN, 4 Department of Molecular Genetics, Tehran Medical Branch, Islamic Azad University, Tehran, IRAN, 5 Stem cell and Tissue Engineering Research Center, Shahroud University of Medical Sciences, Shahroud, IRAN

Background: Malignant cells utilize various mechanisms to ameliorate the process of senescence and programmed cell death. One of the most potent mechanisms that resulted in the development of neoplasms is autophagy. Autophagy is a cytoprotective process that removes misfolded proteins and damaged organelles under stressful conditions. Moreover, extended telomere length is associated with infinite proliferation and viability in cancer cells. Telomeres are nucleoprotein complexes at the end of human chromosomes and have a crucial role in genome integrity and stability. Lots studies indicated that telomere shortening contributes to induction of aging and apoptosis and have a close bond with autophagy process.

Materials and methods: In this study, for the first time, we resolved to utilize 5-azacytidine (5-aza), as a DNA hypomethylating agent which has therapeutic efficacy in acute myeloid leukemia, along with Chloroquine (CQ), as an autophagy inhibitor, on apoptosis, cell cycle, autophagic flux and molecular features (including expression levels of hTERT, c-Myc, LC3-B, p62, Beclin-1, Bax, Bcl-2, p21 and p27) in TF-1 and HL-60 cell lines.

Results: Our results showed that inhibition of autophagy via CQ is more efficient in the induction of apoptosis, cell cycle arrest and elevated Sub-G1 compartment in 5-aza treated cells. On the other hand, higher down-regulation of mRNA and protein expression levels of hTERT, Bcl-2 and also up-regulation of Bax, p21, p27 in combined modality were seen.

Conclusion: On balance, our findings suggest that autophagy inhibition via CQ seems to be most effective in the promotion of anti-cancer effects 5-aza on acute myeloid leukemia cells in vitro.

Keywords: autophagy, hTERT, Chloroquine, 5-Azacytidine, leukemia

Poster abstracts


113

75

Caspase-10: a molecular switch from cell-autonomous apoptosis to communal cell death in response to chemotherapeutic drug treatment

Andrea Mohr1, Sylwia Jencz1, Yasamin Mehrabadi1, 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 a novel apoptosis-inducing complex, we termed FADDosome, which is driven by ATR-dependent caspase-10 upregulation. During FADDosome-induced apoptosis, cFLIPL is ubiquitinated by TRAF2, leading to its degradation and subsequent FADD-dependent 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. It leads to processing of cFLIPL to cFLIPp43, TNF-alpha 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

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

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