Programmed Death Ligand 1 (PD - L1) Scoring Using the VENTANA PD - L1 (SP263) Assay in Non - Small - Cell - Lung Cancer (NSCLC)

Home , PAX9

PROGRAM ME

The 9 th International Conference Notch Targeting in Cancer

Grecian Park Hotel , Konnos Bay, Cyprus 2 6 th - 2 8 th June 201 9

WE ARE GRATEFUL TO OUR SPONSORS FOR THEIR SUPPORT AND SASKIA MULHEIM FOR HER INPUT AND ASSISTANCE

Anastasis Biotec Ltd

Wednesday 26 th June 2019

6. 30 - 6. 55 pm Registration

6. 55 – 7.00 pm Welcome: Agamemnon Epenetos

SESSION 1 - Chairperson Raj Lehal

7.00 - 7.30pm Freddy Radtke , Ecole Polytechnique Fédérale de Lausanne, Switzerland c - Myc and Notch

Abstract awaited

7.30 - 8. 00 pm Johanna K. Ahlskog, Emma Suhonen, and Cecilia Sahlgren , Faculty of Science and Engineering, Cell Biology, Åbo Akademi University, Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland

The Notch pathway counteracts replication stress to prevent cancer cell senescence

proliferatin g leading to tumorigenesis. The Notch pathway is critical in a multitude of fundamental cellular processes including cell - fate determination, differentiation and proliferation. Active Notch supports stem cell phenotype and is vital in the maintenance of ca ncer stem cells in a variety of different malignancies. In breast cancer, the Notch pathway is constitutively active and contributes to tumorigenesis. Our preliminary experiments show that Notch is essential for replication stress - induced DNA repair in bre ast cancer cells. We therefore hypothesize, that Notch is a key facilitator of the RSR to combat replication stress thereby evading senescence and maintaining proliferation. We aim at establishing the molecular mechanisms by which the Notch pathway regulat es the RSR and the biological consequences of this activity. Our approach is to explore the RSR in breast cancer cells exhibiting disturbed Notch activity. In addition to using classical methods for analyzing the RSR, senescence and DNA - damage repair mecha nisms upon replication stress, we will also identify new interaction partners of Notch by performing mass spectrometry on cells exposed to replication stress. Our results will bring unprecedented new knowledge about regulatory switches of cancer cell - fate, and define an unexplored new role for the Notch pathway in the replication stress response.

8.00 - 10.00pm Welcome Reception Cocktail and Canapes

Thursday 2 7 th June 201 9

SESSION 2 - Chairperson Freddy Radtke

9.00 - 9. 3 0 am Judith Hounjet, Daphne Peters, Arjan J. Groot and Marc Vooijs # , Department of Radiotherapy (MAASTRO)/GROW - School for Developmental Biology & Oncology, Maastricht University, Maastricht, The Netherlands # E - mail: [email protected]

Genome w ide shRNA - screening identifies novel regulators of the Notch pathway

NOTCH proteins are type I transmembrane receptors that interact with ligands expressed on adjacent cells and are activated by regulated intramembrane proteolysis. Ligand - binding to NOTCH receptors elicits consecutive S2 and S3 cleavage by Adam10 and the Presenilin - containing γ - secretase. Ligand - independent - oncogenic - activation of NOTCH also requires cleavage by Tace/Adam17. We found that in cells lacking both Adam1 0 and Adam17, oncogenic NOTCH1 proteins are fully active but still require γ - secretase for their activation suggesting heretofore unknown proteins that are sufficient to activate NOTCH1 signaling. To identify these proteins, we conducted a genome wide shRN A screen in Adam10/17 double KO MEF and identified a metal transporter as a novel essential regulator of Notch activity in these cells. Here we will present discuss the Notch - dependent phenotypes in cells lacking this transporter. Down - regulation of this transporter in Notch - dependent differentiation models for myoblasts (C12C12) and neuroblast (Neuro2A) caused premature and accelerated terminal differentiation. Mechanistically cells lacking this transporter are defective in physiological ligand - induced No tch cleavage and transcriptional activation. We will present data implicating endocytosis of Notch in these phenotypes.

9. 3 0 - 10.0 0 am Apostolos Klinakis , Investigator, Biomedical Research Foundation, Academy of Athens, Greece

Notch controls PTEN expression and PI3K output in urothelial carcinoma

Notch has been shown to act as a tumo u r suppressor in the mouse urothelium, in which loss of Notch activity leads to urothelial cell proliferation and urothelial carcinoma. Moreover, loss of function mutations and copy number loss of the NOTCH1 receptor have also been described in human bladde r cancer. Our group has shown that loss of Notch activity in mouse bladders leads to elevated

phosphorylated ERK1/2 levels and increased MAPK output. On the other hand, mouse models expressing mutant FGFR3 and Hras, the most commonly mutated oncogenes in h uman bladder cancer, hardly develop simple urothelial hyperplasias over a very long latency. This implies that MAPK activation is not sufficient to lead to urothelial tumorigenesis and other tumorigenic pathways are activated upon Notch loss, which in conc ert with the MAPK drive neoplastic transformation. We have generated novel data showing that the intracellular domain of the NOTCH1 receptor binds upon the promoter of the PTEN locus and controls its expression. Inactivation of the Notch pathway in vitro a nd in vivo in mice leads to PTEN downregulation and subsequent activation of the PI3K/PDK1/AKT axis. While this activation mediates neoplastic transformation, and possibly leads to a more aggressive tumo u r phenotype, at the same time it exposes novel vulne rabilities of the cancer lacking that could exploited therapeutically.

10.00 - 10.30 am Matteo Marchesini 1 , Andrea Gherli 1 , Anne - Marie Lund Winter 2 , Anna Montanaro 1 , Chiara Rompetti 3 , Claudia Sorrentino 1 , Luca Pagliaro 1 , Paolo Sportoletti 3 , William Dalby - Brown 2 , Giovanni Roti 1 1 University of Parma, Department of Medicine and Surgery, Parma, 43126, Italy 2 Cado Biotechnology IvS, Copenhagen, Denmark 3 University of Perugia, Department of Medicine, Hematology and Clinical Immunology, Perugia, 061 23, Italy

Targeting Oncogenic NOTCH1 in T - cell Acute Lymphoblastic Leukemia with SERCA Inhibitors.

The identification of SERCA (sarco - endoplasmic reticulum Ca 2+ ATPase) as a new target for modulating gain - of - function NOTCH1 mutations in relevant Notch - dependent disease models such as T - cell acute lymphoblastic leukemia (T - ALL) has spurred the development of chemical entities that modulate its activity. SERCA play s a critical role in Ca 2+ regulation particularly in myocytes, thus inhibiting it may increase the risk of heart failure, consequently limiting the development of this compound class for cancer therapeutics. Considering this toxicity challenge, urgent

disc overy and early optimization of chemical entities with better drug - like properties and reduced off - target toxicity is warranted. In this study, we describe a selective and potent SERCA inhibitor identified from a series of yeast plasma membrane H + - ATPase modulators. Similar to the SERCA inhibitor thapsigargin, CAD204520 inhibits Notch signaling in T - ALL leukemia cells causing an arrest on proliferation and apoptosis. CAD204520 preferentially targets mutated, including cells with isolated PEST mutations, ov er wild type NOTCH1 T - ALL proteins thereby overcoming the therapeutic limitation associated with the use of pan - Notch inhibitors. Remarkably however, CAD204520 resulted in an effective treatment in a NOTCH1 mutated T - ALL in vivo model without causing Ca 2+ - related cardiac toxicity. This study supports the development of tolerated SERCA inhibitors for Notch - dependent cancers and extends its application to cases with isolated PEST mutations such as mantle cell lymphoma (MCL) or chronic lymphocytic leukemia (CL L).

10.30 - 11. 3 0 Coffee Break

SESSION 3 Chairperso n Kim Dale

11. 3 0 - 1 2.00 Francesca Anna Carrieri , Philip Murray, Paul Davies, Jacqueline Kim Dale * 1 Division of Cell and Developmental Biology, School of Life Sciences, University of Dundee, Dow Street, Dundee, DD15EH, Scotland, UK . 2 Department of Mathematics, University of Dundee, Nethergate, Dundee, DD14HN, Scotland, UK. 4 MRC Phosphorylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee, DD15EH, Scotland, UK .

Regulating NICD turnover

All vertebrates share a segmented body axis. Segments form from the rostral end of the presomitic mesoderm (PSM) with a periodicity that is regulated by the segmentation clock. The segmentation clock is a molecular oscillator that exhibits

dynamic clock gene expression across the PSM with a periodicity that matches somite formation. Notch signalling is crucial to this process. Altering Notch intracellular domain (NICD) stability affects both the clock period and somite size. Howeve r, the mechanism by which NICD stability is regulated in this context is unclear. We identified a highly conserved site crucial for NICD recognition by the SCF E3 ligase, which targets NICD for degradation. We demonstrate both CDK1 and CDK2 can phosphoryla te NICD in the domain where this crucial residue lies and that NICD levels vary in a cell cycle - dependent manner.

12.00 - 12.30 am Monticone G. 1 , Hossain F. 1 , Ucar A.D. 1 , Majumder S. 1 , Sorrentino C. 2 , Rodriguez P.C. 3 , Sierra R.A. 3 , Pannuti A. 1 , Osborne B.A. 4 , Minter L.M. 4 , Morello S. 2 and Miele L. 1

1 Department of Genetics and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA, United States, 2 Department of Pharmacy, University of Salerno, Fisciano, Italy, 3 H. L. Moffitt Comprehensive Cancer Center, Tam pa, FL, United States, 4 Department of Veterinary and Animal Sciences, University of Massachusetts, Amherst, MA, United States

Targeting adenosine A2A receptor in triple - negative breast cancer

One of the hallmarks of cancer is the ability to evade the host immune system and this is achieved through different mechanisms. Several studies have showed that the accumulation of adenosine in the tumor microenvironment suppresses T - cell function, thus helping the tumor to evade the immune system. To find a way to counteract adenosine - dependent immune suppression, we need to understand how adenosine regulates T - cell function. We examined the effect of adenosine in CD8+ T - cells, which are critical to cancer immunity. Notch1 is well known to be required for CD 8+ T - cell activation and function, therefore we tested whether adenosine regulates Notch1 signaling. Our results show that stimulation of adenosine A2A receptor (A2AR) reduces TCR - signaling and consequently Notch1 expression and activation in primary murin e CD8+ T - cells. This effect was accompanied by reduction of IFN - ϒ and granzyme B production. Conversely, the inhibition of A2AR restored TCR - signaling and Notch1 activation. Transgenic

expression of Notch1 intracellular domain under the Granzyme B promoter prevented the suppressive effects of A2AR stimulation. These findings suggest that A2AR stimulation suppresses CD8+ T - cell function through inhibition of TCR - induced Notch1 and this can be reversed by inhibition of A2AR and activation of Notch1. We hypoth esize that inhibition of A2AR may be able to protect CD8+ T - cells from the adenosine - dependent immune suppression in the tumor microenvironment. Consistent with this idea, our data show that treatment with a selective A2AR antagonist induces tumor cell dea th in immune stroma - containing spheroids from a mouse triple - negative breast cancer model. Further investigation is needed to establish whether A2AR inhibition can protect CD8+ T - cell function in tumors. Overall our current data provides a rationale for th e evaluation of A2AR antagonists as a Notch - modulating immunotherapy for triple - negative breast cancer.

12.30 - 1.00 pm Jelle Sleebom (1,2), Jaap den Toonder (1,3) and Cecilia Sahlgren (2,3,4) 1 Microsystems, Eindhoven University of Technology, Department of Mechanical Engineering, 5600MB, Eindhoven, the Netherlands. 2 Soft Tissue Engineering & Mechanobiology, Eindhoven University of Technology, Department of Biomedical Engineering, 5600MB, Eindho ven, the Netherlands. 3 Institute for Complex Molecular Systems, Eindhoven University of Technology, 5600MB, Eindhoven, the Netherlands. [email protected] . 4 Cell Fate Lab, Turku Bioscience, Åbo Akademi Unive rsity and University of Turku, FI - 20500 Turku, Finland

Breast Cancer Cells and Their CSC Population Migrate Towards Low Oxygen concentration

Most cancer deaths are caused by secondary tumo u rs formed through metastasis, yet due to our limited understanding of this process, prevention remains a major challenge. Recently, cancer stem cells (CSCs) have been proposed as the source of metastases, but only little is known about their migratory behavio u r. Oxygen gradients in the tumo u r have been linke d to directional migration of breast cancer cells. Here, we present a method to study the effect of oxygen gradients on the migratory behavio u r of breast CSCs, using a microfluidic device. Our chip contains

a chamber in which an oxygen gradient can be gene rated between hypoxic (<1%) and ambient (21%) conditions. We tracked the migration of CSCs obtained from MDA - MB - 231 breast cancer cells, and found that their migration patterns do not differ from the average MDA - MB - 231 population. Surprisingly, we found th at the cells migrate towards low oxygen levels, in contrast with earlier studies. We hypothesize that in our device, migration is exclusively due to the pure oxygen gradient, whereas the effects of oxygen in earlier work were modulated by additional cues f rom the tumo u r microenvironment (e.g. nutrients, metabolites). These results open new research directions into the role of oxygen in directing cancer and CSC migration.

1 2.3 0 – 2.30 pm Lunch Break

2.30 – 3.30 pm Open Air workshop : Co - ordinators Cecilia Sahlgren and Martin , Baron Topic : What do we want: Notch Inhibition or Activation

SESSION 4 Chairperso n : Gareth Williams

4.00 - 4. 30 Marco Loddo and Gareth Williams Oncologica UK Ltd, Suite 15 - 16, The Science Village, Chesterford Research Park, Cambridge, UK

The Oncofocus® precision oncology test: a comprehensive companion diagnostic for NOTCH directed therapies

The Notch pathway is an evolutionarily conserved, intercellular signalling cascade which plays a critical role in cell fate decision, tissue patterning, and morphogenesis. There is mounting evidence that this pathway is dysregulated in a

variety of maligna ncies and can behave as either an oncogene or a tumour suppressor depending upon cell context. Aberration of the Notch signalling pathway has been identified in a wide range of tumours from haematological cancers, such as leukaemia and lymphoma, through to lung, skin, breast, pancreas, colon, prostate, ovarian, brain, and liver. The Notch signalling pathway has become of major interest as a novel target for cancer therapeutic intervention. The efficacy of a targeted agent and its potential benefit to cancer patients is critically dependent on the performance of the accompanying companion diagnostic. The companion diagnostic must also be applicable to the routine paraffin wax embedded biopsy sample for clinical relevance and this is challenging in relation to somatic cancer genetics due to the fragmented nature of the DNA and RNA templates following formalin fixation. The Oncofocus® precision oncology platform provides comprehensive analysis of the NOTCH target genes and has been optimised for routine formalin fixed biopsy samples. Oncofocus includes analysis of the full length coding sequence of NOTCH1, NOTCH2 and NOTCH3 and also detection of the NOTCH fusion genes. The Oncofocus platform provides linkage to NOTCH directed therapies for all solid tumour types and is updated on a 3 monthly basis using an integrated bioinformatics compendium of genomic information linked to efficacy data. Here we present examples how the Oncofocus test enables patients with a wide variety of different tumour types to be linked t o NOTCH directed targeted therapies. Secondly, our screening of tumours in a routine clinical setting provides information about the frequency of this target across a range of tumour types in routine clinical practice and therefore the potential impact of these targeted agents in the future treatment of cancer.

4.30 - 5.00 pm Laurence Cohen , Partner, Longacre Funding Partners LLP, Birchwood, The Avenue, Radlett WD7 7DQ ,UK

From Lab to Market - Getting an invention to market

Getting a new invention off the ground requires persistence, perseverance, patience, some luck, and some skill. But it is possible and as the inventor you will need to learn fast about many things that were outside your experience. Most new developments come from academic institutions and charitable foundations. They generally provide the earliest stage funding, which is sufficient to ascertain if the project is worth continuing with and to provide the initial patent advice, and probably the initial pate nt application. To turn an idea into a commercial product takes a lot of persistence. Here are some of the steps. In order to get started, the team leader needs to get a motivated team round him and to identify what product or product class or platfor m is the ultimate goal. This includes identifying what is to be provided to the market and for what purpose. The market purpose needs to be identified, together with the market potential, whether the market is crowded or is there an untapped need, and whe ther it is a new drug or a re - purposing. The latter have an even more uphill struggle. The competition needs to be sized up. There is a world of difference between a cancer drug for an indication for which there is an unmet need and an anti - infective com peting against low cost and generally effective products in a crowded market. To bring a project to fruition, there needs to be management. Funders want to know that the managers of the project will be able to bring it to fruition, noting that much of the development work, at least in the early stages and clinical trials will be out sourced in many cases. The experience of a successful entrepreneur in the biotech/pharma space is invaluable. Other skills include lawyers (at least on tap), accountants (at least on tap), clinical trials experts and patent experts (normally external). Risk mitigation is an important element of any request for funding. It is necessary to identify the technical and commercial risks and how they will be dealt with. For example, and already synthesised product is less risk that one in which the synthesis path has to be determined, because only very small amount of the active ingredient have been inefficiently synthesised in the lab. Synthesis to GMP adds a

further risk. The tox risk must be factored in. Products with in vivo results, even in animals are less risky than those without. And it is necessary to address efficacy, as many drugs fail not because they are no good, but because they are not as good as the standard of care . When all that has been reviewed and put into executive summaries (2 - 3 pages of 4 — 5 slides) and the business plan written up, funders have to be located and approached. Leveraging personal relationships is a very good way of getting started. Finally look for funders who operate in the market for the immediate needs; seed, up to £10m, and beyond £10m. Charities like CRUK need to be included and can validate the science. Fundraising takes as much effort as the research, and with less reward and high degre es of dilution of the founders’ financial interests.

5.00 - 6.0 0 pm Brief Oral presentations of Posters :

 Samira Sadat Hosseini Alghaderi, Tao Wang, Keith Brennan, Martin Baron , Faculty of Biology, Medicine and Health , The University of Manchester , Level3 Michael Smith Building , Oxford Road , Manchester M13 9PL

Investigation of Notch3 trafficking and processing in the endosomal pathway

Notch3 misregulation has been linked to a number of human diseases including Cancer and vascular dem entia. The latter causes misaccumulation of the extracellular domain of Notch3 related to mis - trafficking. Endocytosis has been previously shown to be involved in both misactivation and down regulation of Notch, dependent on the compartment to which Notch is trafficked. We are interested to understand how Notch3 is trafficked in the endosomal pathway and what part endocytosis plays in its regulation and in the consequence of pathogenic mutations. Here we show, in a RPE model cell line, that expressed Notch3 undergoes processing and separation of extracellular and intracellular domains,

which then traffic separately in the endosomal pathway. We found that a cysteine mutant that disrupts the ligand binding site N3 traffics substantially as wild type and shows the similar separation of extracellular and intracellular epitopes, suggesting the processing of Notch3 can be independent of ligand interactions. We further compared several cysteine mutations in different regions of the Notch extracellular domain and obs erved mis - accumulation of mutant Notch3 occurs around and above the nucleus and at cell boundaries, including clumps of Notch3 ECD - only staining which may explain its CADASIL disease phenotype. Non - permeabilised cell immunostaining indicates that such acc umulations are, in part, surface - exposed for the ECD epitope.

 Marika Sjöqvist, 1 , Ronald Österbacka 2 , Luisa Torsi 2,3 & Cecilia Sahlgren 1,4

1 Faculty of Science and Engineering, Biosciences, Åbo Akademi University, Turku, Finland 2 Faculty of Science and Engineering, Physics, Åbo Akademi University, Turku, Finland 3 Dipartimento di Chimica, Università degli Studi di Bari “Aldo Moro”, 70125 Bari, Italy 4 Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands

Bioelectronic activation of Notch – possibilities for Notch targeted regenerative therapies

biofun ctionalized scaffolds provide intriguing possibilities for biomedical applications in the field of tissue engineering and regenerative therapies. Myocardial infarction (MI) is the leading cause of death worldwide. The infarcted area suffers from cardiomyocyte apoptosis, fibrosis, ischemia etc. Activation of Notch promotes the healing process after MI. Our goal is to utilize the bioelectronic Jagged1 platform as a cardiac patch, in order to control Notch activation after MI and thereby enhance cardiomyocyte survival, promote reperfusion of the infarcted area and reduce fibrotic scarring.

 Chabanais Julien 1 , Labrousse François 2 , Chaunavel Alain 2 , Maftah Abderrahman 1 , Germot Agnès 1

1 Glycosylation and Cell Differentiation, Limoges University, PEIRENE, EA 7500, F - 87060 Limoges cedex, France. 1 Department of Pathology, Limoges University Hospital, 87042 Limoges cedex, France.

POFUT1 overexpression in colorectal cancer: a novel biomarker for early diagnosis?

Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the se cond in females with 1.65 million new cases and almost 835,000 deaths worldwide in 2015 (Fitzmaurice et al. , 2017). Comprehension of mechanisms which initiate tumor development is crucial since an early diagnosis of cancer can trigger treatment and increas e the patient chances of recovery. Therefore, research of new potential diagnostic markers of cancer occupies a substantial part in the scientific field. R ecently, studies focused on glycosylation, especially in malignant tumor development (Munkley et al. , 2016). In that context, O - fucosylation, linked to expression of Protein O - fucosyltransferase 1 (POFUT1) and its activity on EGF - like domains, appears promising. POFUT1 adds O - fucose on a S or T residue within 2 3 the consensus sequence C X 4 (S/T)C of EGF - like domains (Takeuchi et al. , 2010) present in some cell surface and secreted proteins (Wang et al. , 1996). In humans, 87 putative POFUT1 targets had been referenced (Schneider et al. , 2017). Among those, NOTCH receptors are the most described in literatu re and their O -

fucosylation was shown to be essential for their interaction with ligands and therefore for NOTCH signaling (Stahl et al. , 2008), this pathway being implicated in tumor development ( Weng , 2004; Göthert et al. , 2007). While POFUT1 overexpress ion has been recently proposed as a potential biomarker for oral, hepatocellular and breast cancers (Yokota et al. , 2013; Ma et al. , 2016; Wan et al. , 2017), no study was carried out on POFUT1 implication in CRC. We analyzed data from 626 tumors and 51 non - tumor adjacent colorectal tissues available in FireBrowse. We found that POFUT1 is significantly overexpressed in CRC tissues from the stage I and 76.02% of tumors have a 20q11.21 amplification (where POFUT1 gene is located), associated in 90.13% of cases with a POFUT1 overexpression, compared to non - tumor adjacent tissues. Its overexpression had been confirmed with immunolabelling analysis performed on six cancer tissues and four colorectal cell lines. Furthermore, POFUT1 expression was correlated with th ose of NOTCH receptor family genes, particularly NOTCH1 . Their link was consolidated by the higher expression levels of NOTCH target genes implicated in biological processes such as proliferation, apoptosis and epithelial - mesenchymal transition. Lastly, co mbined POFUT1/NOTCH1 overexpressions were significantly associated with several clinical parameters. We conclude that POFUT1 is overexpressed in CRC from stage I, and its high expression is associated with metastatic process, probably through NOTCH pathway activation. Then, POFUT1 could represent a potential novel biomarker for CRC diagnosis.

8. 0 0 pm until late Conference Dinner

Friday 2 8 th June 201 9

SESS ION 5 - Chairperso n : Cecilia Sahlgren

9.00 - 9. 3 0 am Valeriy M Paramonov 1,2,3 , Veronika Mamaeva 2,3† , Iida Laiho 2,3 , Pauliina Cronqvist 4 , Marcin Chrusciel 1 , Adolfo Rivero - Müller 1,2,3,5 , Christian W Siebel 6 and Cecilia Sahlgren 2,3,7 1 Institute of Biomedicine, Research Centre for Integrative Physiology and Pharmacology , University of Turku, Finland; 2 Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Finland; 3 Faculty of Science and Engineering, Cell Biology, Åbo Akademi University, Finland; 4 Department of Pathology and Forensic Medicine, University of Turku, Finland; 5 Department of Biochemistry and Molecular Biology, Medical University of Lublin, Poland; 6 Genentech Inc., Department of Discovery Oncology, San Francisco, CA, USA; 7 Institute for Complex Molecular Systems, Eindhoven Univers ity of Technology, Eindhoven, the Netherlands.

† Deceased 06 January 2018

Dissecting Somatostatin Receptor – Notch signaling crosstalk in digestive neuroendocrine neoplasias.

Distinctive contributions of individual Notch family members to the pathological changes of the endocrine pancreas remain controversial, despite a relatively well - understood role of Notch pathway in lineage commitment and checkpoints in pancreatic developm ent. We constructed and subjected to immunohistochemical analysis tissue microarrays from a large set of archival surgical samples from patients with neuroendocrine neoplasias (NENs) of pancreas, small - intestine and colon, aiming to profile all Notch recep tors and ligands together with a number of Notch targets, markers of endocrine development and somatostatin receptors (SSTRs). The analysis revealed a consistent, paralog - specific Notch expression pattern in the studied NENs, allowing to reconsider “oncoprotective” role of Notch in these tumours. Another intriguing finding was a prominent downregulation of selected SSTR subtypes, specifically observed in a cohort of poorly - differentiated NENs. These insights urged us to seek for possible c onnection of SSTR and Notch signalling in advanced NENs. Subsequent in vitro studies hinted to dysregulated Jagged1 in pancreatic NENs cell lines with differential expression of SSTRs, providing extra support for our hypothesis. Further proof of SSTR - Notch connection came from in vivo experiments in mice, where xenografts of SSTR - null pancreatic

NEN cells not only exhibited more aggressive growth, but were also selectively responsive to the systemic treatment with the investigational Jagged1 - blocking antibo dy. These results depict SSTR - Notch crosstalk as a candidate « molecular switch » controlling progression of pancreatic NENs and introduce jagged1 blockade as a novel treatment modality for advanced NENs.

9. 3 0 - 10.00 am Martin Baron , School of Biological Sciences, University of Manchester, UK

Shaping Notch signalling responses through an endocytic regulatory network

Understanding the connections between genotype and phenotype remains an important and challenging research problem in biology and medicine today. The Notch gene in Drosophila, encodes a highly modular developmental signalling receptor with widespread roles in development and adult tissue homeostasis. In Drosophila, historical studies have revealed the complex genetic architectu re of Notch, comprising a diverse allelic series with intriguing tissue specificities and varying degrees of Notch loss or gain of function. More recently this genetic complexity is being recapitulated by 100's of human Notch mutations, uncovered through c ancer genome sequencing. This diverse collection of mutations now provides an important resource to probe Notch structure/function and regulation. However much of this diversity is not easily interpretable at a structure/function level in terms of straight forward alterations to the core canonical Notch activation mechanism. Work in my group, combining genetic, cell biological and computational approaches, has revealed how a network of endocytic trafficking routes, including novel ligand - independent signal a ctivation mechanisms, acts to tune Notch signalling levels in response to environmental and other signalling inputs by directing Notch to different endocytic destinations associated with its activation or down regulation. We have further found that differe nt Notch missense mutants "postcode" Notch to different endosomal subdomains, helping

to provide some new mechanistic understanding of their different phenotypic outcomes.

10.00 - 10.30 am Gareth H. Williams 1 , Andrew G. Nicholson 2 , David R. J. Snead 3 , Erik Thunnissen 4 , Sylvie Lantuejoul 5 , Paul Cane 6 , Keith M. Kerr 7 , Marco Loddo 1 , Marietta L. Scott 8 , Paul W. Scorer 8 , Craig Barker 3

1 Oncologica UK Ltd., Cambridge, UK; 2 Royal Brompton and Harefield NHS Foundation Trust and National Heart and Lung Institute, Imperial College, London, UK; 3 University Hospitals Coventry and Warwickshire NHS Trust, Coventry, UK; 4 VU University Medical Center, Amsterdam, The Netherlands; 5 Centre Léon Bérard UNICANCER and Grenoble Alpes University, Lyon, France; 6 Guy’s and St Thomas’ NHS Foundation Trust, London, UK; 7 Aberdeen University Medical School and Aberdeen Royal Infirmary, Aberdeen, UK; 8 Precision Medicine Laboratories, Pre cision Medicine and Genomics, IMED Biotech Unit, AstraZeneca, Cambridge, UK

Inter - rater reliability of programmed death ligand 1 (PD - L1) scoring using the VENTANA PD - L1 (SP263) assay in Non - Small - Cell - Lung Cancer (NSCLC)

Background Monoclonal antibodies directed against PD - L1 and PD - 1 is a major focus of immunotherapies being developed by a number of major Biopharma. The VENTANA PD - L1 (SP263) assay has been developed as a companion diagnostic for anti - PD - L1 immune checkpoint inhibito rs. Here we investigate assay inter - rater reliability, applied to PD - L1 scoring of tumour cells (TCs) and immune cell (IC) infiltrates in NSCLC.

TCs expressing PD - L1 (TM score), tumour - associated IC population as a percentage of total tumour area (PIC value), and percentage of ICs expressing PD - L1 (IC score). Scores were analysed using intra - c lass correlation coefficient (ICC) and patient classification using Fleiss’ Kappa.

Results Interim results were available for 3 pathologists and 180 cases. TM scoring between pathologists showed strong pair - wise correlations between individuals (R2>0.90) with an ICC >0.95. Pair - wise and overall agreement was ≥85% for TC ≥ 1% and >93% for TC ≥ 20%, TC ≥ 25%, and TC ≥ 50%. Fleiss’ Kappa showed substantial agreement for TC ≥ 1% and excellent agreement for TC ≥ 20%, TC ≥ 25%, and TC ≥ 50%. There were systemati c and substantial differences in PIC values and IC scores between pathologists with poor pair - wise correlations. ICC indicated poor reliability for both IC score (0.36) and PIC values (0.044). Fleiss’ Kappa showed poor agreement for IC ≥ 25% (0.183).

Conc lusions Assessment of TM score in NSCLC was highly reproducible using VENTANA PD - L1 (SP263) assay, building confidence in the accuracy of this assay in patient selection for anti - PD - L1 therapy. However, expert pathologists were unable to reproducibly asses s IC score in NSCLC suggesting assessment methodology is unreliable for this tumour type and assay. This contrasts with urothelial cancer (UC) in which pathologist agreement for PIC values and IC scores was generated as part of UC VENTANA PD - L1 (SP263) IHC assay CE marking and FDA approval. This difference in pathology of the different tumour types requires further investigation.

10. 3 0 - 11. 3 0 am Coffee Break

SESSION 6 Chair person : Lucio Miele

11. 3 0 - 12.00 Zhaohui Xiong 1,2 , Menghan Shi 1,2 , Shuang Ren 1,2 , Ning Gu 2 , Hao Chen 2 , Jing Li 3 , Yuning Han 3 , Jianwen Que 4 , Zheng Sun 1,* , and Xiaoxin Luke Chen 2,5,*

1 Department of Oral Medicine, Beijing Hospital for Stomatology, Capital Medical University, 4 Tian - Tan - Xi - Li, Beijing 100050, China. 2 Cancer Research Program, Julius L. Chambers Biomedical Biotechnology Research Institute, North Carolina Central University , 700 George Street, Durham, NC 27707, USA. 3 Department of Thoracic Surgery, Ningxia Medical University General Hospital, Yinchuan, Ningxia 750004, China. 4 Center for Human Development & Division of Digestive and Liver Diseases, Columbia University Medic al Center, NY 10032, USA. 5 Center for Gastrointestinal Biology and Disease, Division of Gastroenterology and Hepatology, Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

Suppression of the NOTCH - PAX9 signaling in esophageal squamous epithelial cells by alcohol drinking

Introduction: Esophageal squamous cell carcinoma (ESCC) is the 8 th most common cancer in the world. Many studies have shown alcohol drinking as a leading risk factor for the development of the disease, however, the molecular mechanisms of ethanol - associated ESCC remain largely unclear. Our previous study has shown that ethanol exposure downregulated PAX9 expression in human esophageal epithelial cells in vi tro and mouse forestomach and tongue in vivo . PAX9 was downregulated in human ESCC, and its downregulation was associated with alcohol drinking and promoter hypermethylation. Both alcohol drinking and Pax9 deficiency promoted carcinogen - induced squamous ce ll carcinogenesis in mouse tongue, esophagus, and forestomach. Here we aimed to determine whether NOTCH regulates PAX9 and whether ethanol inhibits NOTCH - PAX9 signaling in esophageal squamous epithelial cells in vitro and in vivo . Results: Using the Cignal Finder 45 - Pathway Reporter Array, we found inhibition of the NOTCH signaling in KYSE510 cells by ethanol exposure. Western blotting of NICD1 and NOTCH effectors (STAT3 and c - Myc) validated NOTCH inhibition by

ethanol. NOTCH activa tion (by JAG1 treatment or NICD1 transfection) or NOTCH inhibition (by gene knockdown and a chemical inhibitor) up - regulated or down - regulated PAX9 expression respectively in KYSE510 and KYSE410 cells. RBPJ ChIP - PCR confirmed the binding of RBPJ to the pro moter of Pax9 gene. Furthermore, ethanol exposure inhibited NOTCH - PAX9 signaling in time - and dose - dependent manners and NOTCH activation counteracted the effect of ethanol on PAX9. In vivo , alcohol drinking inhibited NOTCH expression and activity, and con ditional NOTCH activation up - regulated PAX9 expression, in mouse esophagus. Finally, PAX9 expression was positively associated with HES1 expression in the human esophagus, and NICD1 expression in the esophageal epithelium of heavy drinkers was lower than t hat in non - drinkers. Conclusion: NOTCH regulates PAX9 expression in esophageal squamous epithelial cells. Ethanol exposure suppresses the NOTCH - PAX9 signaling and may thus promote ESCC.

12.00 - 12.30 am Fokhrul Hossain 1 , Deniz A. Ucar 1 , Samarpan Majumder 1 , Margarite Matossian 2 , Keli Xu 3 , Yong Ran 4 , Lisa Minter 5 , Yaguang Xi 1 , Matthew Burow 2 , Todd Golde 4 , Barbara Osborne 5 and Lucio Miele 1 . 1 Department of Genetics and Stanley S. Scott Cancer Center, LSUHSC, New Orleans; 2 Tulane University Cancer Center; 3 University of Mississippi Cancer Institute; 4 University of Florida at Gainesville; 5 University of Massachusetts at Amherst.

Sulindac Sulfide as a Gamma Secretase Modifier to Target Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is heterogeneous group of clinically aggressive breast cancers with high risk of recurrence and metastasis. There is strong evidence support ing the involvement of Notch signaling in TNBC progression. Expression of Notch1 and Jagged1 correlate with poor prognosis and mediate cross - talk between TNBC cells and the immune microenvironment. Notch inhibitors, Including Gamma Secretase Inhibitors (GS Is) are quite effective in preclinical models of TNBC.

However, the success of GSIs in clinical trials has been limited by their intestinal toxicity and potential for adverse immunological effects. The prognostic value of tumor - infiltrating lymphocytes (TI L) and the recent approval of atezolizumab for TNBC underscore the need to target Notch without suppressing tumor immunity. Recent work by the Fuchs lab shows that cancer stem - like cells (CSC), which in TNBC can be Notch - dependent, promote resistance to im munotherapy. Our overarching goal is to replace GSIs with agents that lack their systemic toxicity and adverse immunological effects to target CSC while stimulating tumor immunity. We identified Sulindac Sulfide (SS), the active metabolite of FDA - approved NSAID Sulindac, as a potential candidate to replace GSIs. SS has documented Gamma Secretase Modifier (GSM) activity, in addition to cyclo - oxygenase (COX) inhibition, cGMP phosphodiesterase inhibition (which results in decreased Wnt signaling), PPARδ, RXR and SERCA1 inhibition. We confirmed that SS inhibits Notch1 cleavage in TNBC cells, but - importantly - not in murine T - cells where endosomal Notch activation is triggered by TCR engagement. SS significantly inhibited mammosphere growth in all human and muri ne TNBC models we tested: 1) human MDA - MB - 231 cells; 2) murine TNBC model C0321, from targeted conditional knockout of Lunatic Fringe ( LFng - / - ); and 3) Two TNBC patient - derived xenograft models, 2K1 and 4IC. As we observed for GSIs, SS is synergistic with AKT inhibitors in these mammospheres. In C0321 tumors, which recapitulate human mesenchymal TNBC, we found that SS had remarkable single - agent in vivo activity and virtually eliminated Notch1 expression in tumors without intestinal toxicity. SS caused an increase in intra - tumoral CD11c+ dendritic cells, but decreased CD4 cells, which in this model are largely PD - 1 positive (exhausted). CD8 cells were modestly increased. SS did not affect the numbers of tumor infiltrating macrophages or myeloid - derived supp ressor cells (MDSC). However, SS blocked the maturation and immunosuppressive function of bone marrow - derived MDSC. In tumor - derived spheroids containing immune cells, SS strongly potentiated the cytotoxic effect of anti - PD1. RNA - Sequencing of SS - treated t umors confirmed inhibition of HES1 expression and revealed significant reduction of CXCL14, EGR1, HOXC6, MAGI2, NCAM1, APOE, CLU (a Wnt target) , DTX4 (an E3 - ligase positive regulator of Notch activation) , and TGFB3 genes and upregulation of CCL17, EPCAM, F ABP4, C4A, LTF, ZBTB16, INADLFGFR2,MME, TIAM1 genes. We are investigating the mechanisms

of this anti - tumor activity and the role of Notch inhibition in it. Our data support further investigation of SS for the treatment of TNBC, with standard of care or with immunotherapy.

1 2 . 3 0 – 2.3 0 pm Lunch break

2.30 - 3.3 0pm Open Air workshop: Co - ordinators : Rajwinder Lehal and Lucio Miele - Topic: Notch Therapeutics

SESSION 7 : Chairperson : Agamemnon Epenetos

4.00 - 4.30 pm Judith Hounjet , Roger Habets, Marco B. Schaaf, Tessa C. Hendrickx, Lydie M.O. Barbeau, Sanaz Yahyanejad, Kasper M. Rouschop, Arjan J. Groot and Marc Vooijs # Department of Radiotherapy/GROW - School for Developmental Biology & Oncology, Maastricht University, Maastricht, the Netherlands # Corresponding author: Marc Vooijs, Department of Radiotherapy (MAASTRO)/GROW – School for Developmental Biology & Oncology, Maastricht University, Universiteitssingel 50, 6229 ER Maastricht, The Netherlands, Tel: +31 (0)433882912; E - mail: [email protected]

Chloroquine sensitizes oncogenic NOTCH1 driv en human T cell leukemia to γ - secretase inhibition

T - cell acute lymphoblastic leukemia (T - ALL) is an aggressive cancer arising from T - cell progenitors in the bone marrow. Although current treatments, including chemotherapy and glucocorticoids, have significantly improved survival, T - ALL still remains a fat al disease and new treatment options are needed. Since more than 60% of T - ALL cases bear oncogenic NOTCH1 mutations, inhibitors of NOTCH1 signaling are being actively investigated for the treatment of T - ALL, however, they have limited efficacy in tumors du e to dose - limiting toxicities. Here, we show that the clinically used drug chloroquine (CQ), an inhibitor of lysosomal function and autophagy, decreases T - ALL cell viability and proliferation. Mechanistically, CQ impairs the redox balance and induces DNA d amage with a subsequent DNA

damage response. In addition, we observed that CQ interferes with the intracellular trafficking and processing of oncogenic NOTCH1. Interestingly, we show for the first time that the addition of CQ to γ - secretase inhibition (GSI ) has a synergistic therapeutic effect on T - ALL cells and reduces the concentration of GSI required to block proliferation in vitro . This effect of CQ was not observed in GSI - resistant T - ALL cell lines. Overall, our results suggest that CQ may be a promisi ng alternative as a repurposed drug in the treatment of T - ALL, either as a single treatment or in combination with NOTCH1 inhibition, increasing the therapeutic ratio.

4. 30 - 5.00 pm Rajwinder Lehal 1 , Michele Vigolo 2, Jelena Zaric 2 Charlotte Urech 1 , Maximilien Murone 1 ,Freddy Radtke 2 . 1 Cellestia Biotech AG, Basel, Switzerland 2 Ecole Polytechnique Fédérale de Lausanne, Switzerland

Direct targeting of NOTCH Transcription Complex by a novel small molecule CB - 103 circumvents dose limiting toxicities assoc iated with pan - NOTCH inhibitors.

NOTCH signalling is a key development pathway whose aberrant activation plays an onco - driver role in human cancers. In human tumo u rs the NOTCH pathway can be activated by various genetic lesions such as over expression of ligands/receptors, GOF mutations in NOTCH receptors, chromosomal translocations, or loss - of - function mutations in the negative regulators of the pathway. Activation of NOTCH due to above mentioned mechanisms can be addressed in part using blo cking antibodies against NOTCH ligands/receptors or small molecule inhibitors of the gamma secretase enzyme (GSIs). In addition to their limitations in targeting tumo u rs harbouring chromosomal translocations in NOTCH receptors, the use of specific blocking antibodies against NOTCH receptors/ligands allows targeting of only a narrow spectrum of NOTCH positive tumo u rs. On the other hand, GSIs act as pan - NOTCH inhibitors and thereby are able to target human tumo u rs positive for NOTCH1 - NOTCH4 receptors. Howeve r, clinical development of these pan - NOTCH inhibitors is hindered due to dose limiting toxicities (DLTs) associated with GSIs. The main DLTs associated with GSIs have been diarrhoea and vomiting due induction of goblet cell metaplasia in

intestine, where N OTCH is known to regulate proliferation and differentiation of intestinal stem cells. A sustained blockage of NOTCH signalling leads to an inhibition of stem cell proliferation and differentiation of stem cells into mucous producing goblet cells. The effec t on stem cell proliferation and differentiation is mediated via a subset of specific NOTCH target genes. Effect on proliferation is mostly driven by downstream target genes such as HES1, 3, and 5, c - MYC and BMI1. The effect on stem cell differentiation is predominantly mediated by repression of MATH1. Therefore, MATH1 upregulation upon NOTCH inhibition is a pre - requisite to induce goblet cell metaplasia. Given the role of NOTCH signalling in human cancers and DLTs associated with 1 st and 2 nd generation NOT CH targeting agents (GSIs and blocking Abs), there is a need to develop novel pan - NOTCH inhibitors able to circumvent these DLTs. We have previously reported discovery and development of a novel class of pan - NOTCH inhibitors that blocks signalling by direc tly targeting the NOTCH transcription complex.

Here we present further in vivo characterization of the development candidate CB - 103. A comprehensive in vitro study demonstrated that CB - 103 acts as a pan - NOTCH inhibitor. Furthermore, in vitro and in vivo pharmacological studies show that CB - 103 circumvents DLTs, namely goblet cell metaplasia, associated with GSIs, due to the novel mode of action. We present molecular evidence that due to differential regulation of downstream target genes, CB - 103 fully enga ges the NOTCH pathway in intestine and spares MATH1 gene repression and thereby does not cause goblet cell metaplasia.

5.00 - 5. 3 0 PM M arianna P rokopi 1 , 2 , C ostas P itsillides 2 , M ahendra D eonarain 3 , K onstantinos K apnisis 1 , S pyros S tylianou 4 , G eorge K ousparos 5 , C hristina K ousparou 6 , A ndreas A nayiotos 1 , A gamemnon E penetos 7

1 . D epartment of M echanical and M aterials S cience and En gineering, C yprus U niversity of T echnology, L imassol, C yprus 2 Theramir ltd, L imassol, C yprus 3 Antikor ltd, S tevenage, H erts, UK 4 Nipd genetics plc, N icosia, C yprus 5 Frimley P ark H ospital, NHS foundation trust, S urrey, UK

6 Novartis C yprus, N icosia, C yprus 7 Imperial C ollege L ondon, UK

Monitoring tumour response to therapeutic tr4 fusion protein via in vivo imaging

Introduction The Notch signaling pathway has been associated with breast, cervical and lung cancer, neuroblastomas and acute T - cell lymphoblastic leukemia and is strongly implicated in tumori genesis. [1] Notch genes encode large single pass transmembrane proteins that regulate cell fate. Upon activation, Notch undergoes cleavage, releasing its intracellular domain NICD and translocates into the nucleus for transcriptional activation of its dow nstream target genes. [2] Discovery of this pathway has prompted many pharmaceutical companies to develop novel anti - Notch therapeutics. We have generated a novel fusion protein TR4 that inhibits the Notch pathway at the transcriptional level and can poten tially reduce tumour growth in vivo . TR4 consists of the truncated version Mastermind - like (MAML) that behaves in a dominant negative fashion and inhibits Notch activation and the cell penetrating peptide Antennapedia (Antp). The fusion protein is able to translocate into the cell nucleus and suppress Notch activation thus reverting the transformed phenotype, inhibiting the anchorage dependent growth and inducing apoptosis in highly metastatic epithelial human breast cancer cells. Utilizing a state - of - the - a rt in vitro pharmacokinetics and toxicology program, in combination with innovative imaging techniques such as in vivo flow cytometry and whole - body fluorescence reflectance imaging, we have investigated the pharmacokinetic and pharmacodynamic behaviour of the TR4 protein in order to define its mode of action and establish safety and efficacy in treating tumours in animal models of breast adenocarcinoma[3].

M ethods A comprehensive in vitro and in vivo pharmacokinetics study that included apoptotic, toxicology and immunogenicity assays was undertaken through administration of various concentrations of TR4 in immuno - competent mice in order to determine the effects of TR4 and define the mechanism(s) of ac tion. Blood

samples were collected for toxicological assays and to monitor the immune response by ELISA whereas major organs were excised for immunohistochemistry to determine tissue toxicity and induction of apoptosis. The dynamics of the TR4 fusion prot ein were investigated in animal cancer models, through the successful conjugation of the TR4 to proprietary fluorescent markers (Cy5.5). Molecular modelling studies were performed to identify appropriate conjugation sites on the TR4 molecule to ensure no l oss of the membrane transfer capabilities of the fusion protein. A group of fluorescent dyes was prepared, with maleide groups appropriate for bioconjugation to thiols on the TR4 molecule. TR4 - Cy5.5 conjugates were chosen based on their optical properties (absorption 600 - 650 nm, emission 650 - 700 nm) and suitability for the in vivo imaging study. Conjugated TR4 was purified using proprietary methods to ensure protein solubility and avoid excessive aggregation and was further tested to ensure that the protein retained the ability to translocate in breast cancer cells. The in vivo pharmacodynamic behaviour of the fluorescent TR4 - Cy5.5 was investigated using fluorescence - based in vivo flow cytometry and whole - body imaging. These molecular imaging techniques hav e the ability to monitor and quantify fluorescently labelled vehicles in circulation, to detect and image the biodistribution and incorporation of therapeutic agents in cells and organs of both healthy and tumour - bearing mice, as well as to provide a quant ifiable assessment of tumour growth over time. Following intravenous administration of the TR4 - Cy5.5 in non - tumour bearing mice the non - specific uptake of the protein by circulating blood cells was assessed via in vivo flow cytometry, while the biodistribu tion of the protein accumulating in different organs/tissues was imaged using whole body reflectance imaging. The accumulation of TR4 in various major organs (liver, kidney, lung and brain) was further verified by Western blot analysis. An orthotopic mous e model for breast cancer disease was developed via injection of MDA - MB - 231 GFP labeled cells in SCID mice, in order to enable the imaging and assessment of tumo u r growth in vivo . Animals that expressed a detectable fluorescent signal from established orth otopic tumors were selected and divided into two groups: untreated (control) and treated with TR4 (day 0). Mice were monitored for 2 weeks via in vivo imaging and were then euthanized at the end of the experiment (day 14).

Results The pharmacokinetics stu dies indicated that TR4 did not induce any overt toxicity, notable death of cells, organ dysfunction, or any toxic manifestations that were predicted materially to reduce the life span of the animals. The immunogenicity tests indicated that animals do not raise an antibody response against the injected TR4 protein. Finally, no murine antibodies against full - length TR4 were detected by ELISA in mice serum. TR4 - Cy5.5 conjugates prepared via molecular mode l ling studies and selected based on their optical characteristics were first validated in vitro for their membrane translocation ability prior to their use for the in vivo part of the study. In vivo flow cytometry in mice, injected i.v. with the TR4 - Cy5.5 c onjugates, showed that free, non - internalized protein was rapidly cleared from circulation, as the background (noise) signal collected from the blood vessels did not substantially increase. Furthermore, fluorescently label l ed circulating cells that may hav e internalized the TR4 protein were not detected at any time point. Whole body imaging showed that TR4 - Cy5.5 accumulated in some of the major organs/tissues imaged and was cleared within 24 hrs. Assessment of tumo u r burden via in vivo imaging demonstrated a reduction in tumo u r growth over time in mice treated with TR4 - Cy5.5 compared to untreated mice. The figure shows an overall increase of less than 50% in the relative intensity of fluorescence signal in the treated mice compared to a 3 - fold increase in s ignal and thus tumo u r size in untreated mice by day 14.

Discussion In this report we have performed preclinical studies to validate TR4 as a promising therapeutic strategy for breast adenocarcinoma. The dynamics of the drug molecule have been investigat ed in an in vivo setting in order characterize its therapeutic efficacy in an animal tumo u r model. Mechanisms of action have been investigated via the application of innovative imaging modalities with the aim of guiding the technology closer to the market by completing the necessary preclinical studies in order to optimize the TR4 fusion protein as a novel therapy for invasive cancer disease.

A cknowledgements This work was co - funded by Eureka Eurostars Program and the Cyprus Research Promotion Foundation (E!8520 - TROJANDRUG).

REFERENCES [1] Prokopi M, Kousparou CA, Epenetos AA. The Secret Role of microRNAs in Cancer Stem Cell Development and Potential Therapy: A Notch - Pathway Approach. Front Oncol. 2014; 4: 389. [2] Kopan R, Ilagan MX. The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 2000 137:216 – 33. [3] Takebe, N. et al. Targeting Notch, Hedgehog, and Wnt pathways in cancer stem cells: clinical update . Nat. Rev. Clin. Oncol. 2015 12, 445 – 464

5.30 pm A gam emnon Epenetos Farewell

POSTERS

 M.Sjöqvist 1 , Ronald Österbacka 2 , Luisa Torsi 2,3 & Cecilia Sahlgren 1,4

Bioelectronic activation of Notch – possibilities for Notch targeted regenerative therapies

During development, strictly organised spatial and te mporal expression of Notch receptors and ligands is essential for proper development of different organs, with each consecutive activation of Notch driving a specific morphogenic process . We want to mimic the precise control of Notch signaling, by creating a bioelectronic platform for Notch activation. With this device, we want to achieve spatiotemporal control of Notch signaling. Jagged1 peptides will be introduced into a gated organic field effect transistor (OFET) by coupling them to the gate ele ctrode through a self - assembled monolayer (SAM). Due to the properties of the biofunctionalized OFET (BIOFET) Jagged1 peptide conformation can be changed (from inactive to active) in response to electrical stimuli, enabling Notch activation on demand. This type of biofunctionalized scaffolds provide intriguing possibilities for biomedical applications in the field of tissue engineering and regenerative therapies. Myocardial infarction (MI) is the leading cause of death worldwide. The infarcted area suffers from cardiomyocyte apoptosis, fibrosis, ischemia etc. Activation of Notch promotes the healing process after MI. Our goal is to utilize the bioelectronic Jagged1 platform as a cardiac patch, in order to control Notch activation after MI and thereby enhance cardiomyocyte survival, promote reperfusion of the infarcted area and reduce fibrotic scarring.

 Gareth H. Williams 1 , Andrew G. Nicholson 2 , David R. J. Snead 3 , Erik Thunnissen 4 , Sylvie Lantuejoul 5 , Paul Cane 6 , Keith M. Kerr 7 , Marco Loddo 1 , Marietta L. S cott 8 , Paul W. Scorer 8 , Craig Barker 3

1 Oncologica UK Ltd., Cambridge, UK; 2 Royal Brompton and Harefield NHS Foundation Trust and National Heart and Lung Institute, Imperial College, London, UK; 3 University Hospitals Coventry and Warwickshire NHS Trust, Coventry, UK; 4 VU University Medical Center, Amsterdam, The Netherlands; 5 Centre Léon Bérard UNICANCER and Grenoble Alpes University, Lyon, France; 6 Guy’s and St Thomas’ NHS Foundation Trust, London, UK; 7 Aberdeen University Medical School and Aberdeen Royal Infirmary, Aberdeen, UK; 8 Precision Medicine Laboratories, Precision Medicine and Genomics, IMED Biotech Unit, AstraZeneca, Cambridge, UK

Inter - rater reliability of programmed death ligand 1 (PD - L1) scoring using the VENTANA PD - L1 (SP263) assay in Non - Small - Cell - Lung Cancer (NSCLC)

Background Monoclonal antibodies direct ed against PD - L1 and PD - 1 is a major focus of immunotherapies being developed by a number of major Biopharma. The VENTANA PD - L1 (SP263) assay has been developed as a companion diagnostic for anti - PD - L1 immune checkpoint inhibitors. Here we investigate assa y inter - rater reliability, applied to PD - L1 scoring of tumour cells (TCs) and immune cell (IC) infiltrates in NSCLC.

Methods Six expert European pulmonary pathologists independently scored 200 NSCLC samples stained using the VENTANA PD - L1 (SP263) assay. A rchival, commercially - sourced formalin - fixed paraffin - embedded resections were selected to represent the dynamic range of PD - L1 expression. Each pathologist scored the proportion of TCs expressing PD - L1 (TM score), tumour - associated IC population as a perc entage of total tumour area (PIC value), and percentage of ICs expressing PD - L1 (IC score). Scores were analysed using intra - class correlation coefficient (ICC) and patient classification using Fleiss’ Kappa.

Results Interim results were available for 3 pathologists and 180 cases. TM scoring between pathologists showed strong pair - wise correlations between individuals (R2>0.90) with an ICC >0.95. Pair - wise and overall agreement was ≥85% for TC ≥ 1% and >93% for TC ≥ 20 %, TC ≥ 25%, and TC ≥ 50%. Fleiss’ Kappa showed substantial agreement for TC ≥ 1% and excellent agreement for TC ≥ 20%, TC ≥ 25%, and TC ≥ 50%. There were systematic and substantial differences in PIC values and IC scores between pathologists with poor pai r - wise correlations. ICC indicated poor reliability for both IC score (0.36) and PIC values (0.044). Fleiss’ Kappa showed poor agreement for IC ≥ 25% (0.183).

Conclusions Assessment of TM score in NSCLC was highly reproducible using VENTANA PD - L1 (SP263) assay, building confidence in the accuracy of this assay in patient selection

for anti - PD - L1 therapy. However, expert pathologists were unable to reproducibly assess IC score in NSCLC suggesting assessment methodology is unreliable for this tumour type and assay. This contrasts with urothelial cancer (UC) in which pathologist agreement for PIC values and IC scores was generated as part of UC VENTANA PD - L1 (SP263) IHC assay CE marking and FDA approval. This difference in pathology of the different tumour typ es requires further investigation.

 Johanna K. Ahlskog, Emma Suhonen, and Cecilia Sahlgren Faculty of Science and Engineering, Cell Biology, Åbo Akademi University, Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland

The Notch pathway counteracts replication stress to prevent cancer cell senescence

In normal cells, the replication stress response (RSR) induc es cell differentiation, apoptosis, or senescence in response to excessive replication stress. However, in cancer cells, defects in RSR enables cells to escape senescence and continue proliferating leading to tumorigenesis. The Notch pathway is critical in a multitude of fundamental cellular processes including cell - fate determination, differentiation and proliferation. Active Notch supports stem cell phenotype and is vital in the maintenance of cancer stem cells in a variety of different malignancies. In b reast cancer, the Notch pathway is constitutively active and contributes to tumorigenesis. Our preliminary experiments show that Notch is essential for replication stress - induced DNA repair in breast cancer cells. We therefore hypothesize, that Notch is a key facilitator of the RSR to combat replication stress thereby evading senescence and maintaining proliferation. We aim at establishing the molecular mechanisms by which the Notch pathway regulates the RSR and the biological consequences of this activity. Our approach is to explore the RSR in breast cancer cells exhibiting disturbed Notch activity. In addition to using classical methods for analyzing the RSR, senescence and DNA - damage repair mechanisms upon replication stress, we will also identify new int eraction partners of Notch by

performing mass spectrometry on cells exposed to replication stress. Our results will bring unprecedented new knowledge about regulatory switches of cancer cell - fate, and define an unexplored new role for the Notch pathway in the replication stress response.