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There is a need for transplantable murine models of ovarian high-grade serous carcinoma (HGSC) with regard to mutations in the human disease to assist investigations of the relationships between tumor genotype, chemotherapy response, and immune microenvironment. In addressing this need, we performed whole-exome sequencing of ID8, the most widely used transplantable model of ovarian cancer, covering 194,000 exomes at a mean depth of 400× with 90% exons sequenced >50×. We found no functional mutations in genes characteristic of HGSC (Trp53, Brca1, Brca2, Nf1, and Rb1), and p53 remained transcriptionally active. Homologous recombination in ID8 remained intact in functional assays. Further, we found no mutations typical of clear cell carcinoma (Arid1a, Pik3ca), low-grade serous carcinoma (Braf), endometrioid (Ctnnb1), or mucinous (Kras) carcinomas. Using CRISPR/Cas9 gene editing, we modeled HGSC by generating novel ID8 derivatives that harbored single (Trp53–/–) or double (Trp53–/–;Brca2–/–) suppressor gene deletions. In these mutants, loss of p53 alone was sufficient to increase the growth rate of orthotopic tumors with significant effects observed on the immune microenvironment. Specifically, p53 loss increased expression of the myeloid attractant CCL2 and promoted the infiltration of immunosuppressive myeloid cell populations into primary tumors and their ascites. In Trp53–/–;Brca2–/– mutant cells, we documented a relative increase in sensitivity to the PARP inhibitor rucaparib and slower orthotopic tumor growth compared with Trp53–/– cells, with an appearance of intratumoral tertiary lymphoid structures rich in CD3+ T cells. This work validates new CRISPR-generated models of HGSC to investigate its biology and promote mechanism-based therapeutics discovery. Cancer Res; 76(20); 6118–29. ©2016 AACR.

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Background: Ovarian high-grade serous carcinoma (HGSC) remains a poor prognosis disease due to its late presentation and lack of “actionable” mutations to target pharmacologically. The host immune system, however, plays a pivotal role with the presence of cytotoxic T lymphocytes and chemokines that regulate T-cell trafficking both having positive effect on survival in preclinical and clinical studies. HGSC cells can achieve immune escape via epigenetic silencing of key immune-related genes, including via DNA methylation; when DNA methylation is pharmacologically reversed, genes involved in antigen presentation, receptor-dependent immune cell stimulation, and chemokine release are reactivated. One of these genes encodes the angiostatic chemokine Cxcl10, which is a T-lymphocyte attractant. In order to elucidate other potential epigenetic mechanisms directly or indirectly involved in Cxcl10 gene silencing, we will screen a library of novel epigenetic probes that target bromodomains, methyltransferases, demethylases, and others epigenetic pathways (Structural Genomic Consortium library). Aims: We will use Trp53-/- ID8 ovarian cancer cells to perform a medium-throughput screening of 40 novel epigenetic probes, measuring cxcl10 production, with the demethylating drug decitabine as positive control. Methods: Trp53-/- ID8 cells were treated with decitabine for up to 72 hours. Cell-cycle analysis was assessed via BrdU incorporation assay. Cytokine/chemokine transcription was assessed using RT2 profiler 84 gene chemo/cytokine PCR array (Qiagen), and verified using qRT-PCR. Acid-hydrolysed DNA was subjected to mass spectrometry/liquid chromatography to quantify the mean ratio of methyl-cytosine/cytosine. Western immunoblotting was performed to test DNMT1 protein levels in protein lysates after decitabine treatment. Cxcl10 levels in supernatant were quantified by ELISA. Results: Treatment of Trp53-/- ID8 cells with decitabine (DAC), at non-cytostatic/cytotoxic doses causes upregulation of multiple chemokines, specifically Cxcl10. 200nM DAC for 48 hours increased Cxcl10 transcription 22-fold (95% CI 20.6-21.1-fold, p=0.0001, values normalized to Gapdh control), and protein levels in supernatant three-fold (t test p Conclusions: Abrogation of DNA methylation with DAC in Trp53-/- ID8 cells induces an increase in the chemokine cxcl10, at both transcriptional and protein level. This provides a robust control for a medium-throughput screening of other epigenetic modifiers that are still at an early drug development stage, such as probes against bromodomains, methyltransferases, and demethylases. Such screening can unmask novel epigenetic mechanisms involved in immune-related gene transcription and offer new ways to potentiate immunotherapy in ovarian cancer. Citation Format: Pavlina Spiliopoulou, Josephine Walton, Suzanne Dowson, Alex Binks, Oliver Maddocks, Peter Adams, Iain McNeish. Epigenetic modification of ovarian cancer immunogenicity. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A37.

Introduction Oncolytic viruses are a promising new cancer therapy, since they can infect cancer cells, selectively multiply within them and induce direct cytotoxicity, leading to the release of mature viral particles that can infect other neighboring cancer cells. However, the mechanisms by which oncolytic adenoviruses induce cell death remains uncertain. It was long thought that DNA viruses induce apoptosis, but there is now evidence that cell death induced by adenovirus, vaccinia and HSV-1 displays features strongly resembling a form of programmed necrosis. Methods and Results In order to investigate the role of necrosis in cell death as a result of oncolytic adenovirus infection, cancer cells were infected with the E1A CR2-deleted adenoviral mutant dl922-947, which specifically replicates in cells that have abnormalities in the pRB-pathway. We specifically sought to investigate the role of the core necrotic proteins RIPK1, RIPK3 and MLKL in adenovirus cytotoxicity. Electron microscopy indicated that dl922-947 infection induces key morphological changes similar to necrotic death induced by TSZ (TNF-α, Smac-mimetic, Z.Vad.fmk) treatment. Using specific inhibitors of programmed necrosis (necrostatin-1, necrosulfonamide, GSK'840B and GSK'843A) as well as RNAi-mediated knockdown of RIPK1, RIPK3 and MLKL, we showed that adenovirus-infected cancer cells undergo RIPK3-dependent necrosis. We further found that, while TNF-α-induced programmed necrosis relies on the (RHIM)-dependent interaction of RIPK1 and RIPK3, dl922-947-induced cell death is independent of TNF-α signalling, does not involve RIPK1 and does not rely on the presence of MLKL. Caspase-8 inhibition, however, induces RIPK3-dependent necrosis that significantly enhances dl922-947 cytotoxicity. Using CRISPR/Cas9 gene editing, we have demonstrated that this increase in cytotoxicity during caspase inhibition was MLKL dependent. Using a RIPK3 overexpression model, we observed that the extent of adenovirus-induced cell death correlated with RIPK3 expression even in the absence of caspase inhibition. Using RIPK3 co-immunoprecipitation, we identified an interaction between RIPK3 and MLKL as well as an interaction between RIPK3 and adenoviral proteins. In vivo experiments using human xenografts showed that expression of RIPK3 significantly improved anti-tumor activity following intra-tumoral injection of dl922-947. Conclusions Our data suggest that adenovirus infection induces a novel form of programmed necrosis that differs from classical TSZ-induced necroptosis, but still relies on the kinase RIPK3. Unfortunately, many cancer cells do not express RIPK3 and can therefore not undergo programmed necrosis. The integration of human RIPK3 into an adenoviral vector offers a therapeutic window to eradicate cancer cells that are resistant to apoptosis. Citation Format: Melanie Weigert, Alex Binks, Suzanne Dowson, Elaine Leung, Dimitris Athineos, Xinzi Yu, Margaret Mullin, Josephine Walton, Clare Orange, Darren Ennis, Karen Blyth, Stephen Tait, Iain McNeish. Oncolytic adenovirus type 5 induces a novel form of programmed necrosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4379.