Josephine Walton

CRISPR/Cas9-Mediated Trp53 and Brca2 Knockout to Generate Improved Murine Models of Ovarian High-Grade Serous Carcinoma

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.

CRISPR/Cas9-derived models of ovarian high grade serous carcinoma targeting Brca1 , Pten and Nf1 , and correlation with platinum sensitivity

Transplantable murine models of ovarian high grade serous carcinoma (HGSC) remain an important research tool. We previously showed that ID8, a widely-used syngeneic model of ovarian cancer, lacked any of the frequent mutations in HGSC, and used CRISPR/Cas9 gene editing to generate derivatives with deletions in Trp53 and Brca2. Here we have used one ID8 Trp53−/− clone to generate further mutants, with additional mutations in Brca1, Pten and Nf1, all of which are frequently mutated or deleted in HGSC. We have also generated clones with triple deletions in Trp53, Brca2 and Pten. We show that ID8 Trp53−/−;Brca1−/− and Trp53−/−;Brca2−/− cells have defective homologous recombination and increased sensitivity to both platinum and PARP inhibitor chemotherapy compared to Trp53−/−. By contrast, loss of Pten or Nf1 increases growth rate in vivo, and reduces survival following cisplatin chemotherapy in vivo. Finally, we have also targeted Trp53 in cells isolated from a previous transgenic murine fallopian tube carcinoma model, and confirmed that loss of p53 expression in this second model accelerates intraperitoneal growth. Together, these CRISPR-generated models represent a new and simple tool to investigate the biology of HGSC, and the ID8 cell lines are freely available to researchers.

Author Correction: CRISPR/Cas9-derived models of ovarian high grade serous carcinoma targeting Brca1 , Pten and Nf1 , and correlation with platinum sensitivity

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Role of innate immune responses in the effectiveness of oncolytic adenovirus as an anticancer agent

Abstract Background Ovarian cancer is the deadliest gynaecological cancer, with fewer than half of patients surviving for more than 5 years. Oncolytic viruses have been investigated as a new class of anticancer agent for this and other cancers. Oncolytic viruses infect and replicate selectively within malignant cells, while sparing normal cells. These viruses also induce profound immune responses, which might influence clinical efficacy. In this study we investigated the role of innate immune responses, in particular interleukin (IL) 17F, on the effectiveness of oncolytic adenovirus. Methods Changes in key human chemokines and cytokines in ovarian cancer lines (TOV21G, OVCAR4) after infection by the E1A CR2-deleted oncolytic adenovirus dl 922-947 were identified by PCR array (RT 2 Profiler, Qiagen, Hilden, Germany), a transcriptional screen. The observed changes were confirmed by real-time PCR and ELISA in six primary lines derived from malignant ovarian ascites ex vivo (primary lines) and by real-time PCR in human cancer xenografts in CD1 nu/nu mice (n=12). TOV21G IL17F −/– cells were generated with CRISPR–Cas9 technology (ThermoFisher, Waltham, MA, USA). Findings dl 922-947 infection led to consistent changes of chemokines and cytokines transcription ( r =0·56, p dl 922-947 in vivo (23, p=0·02). Moreover, upregulation of IL17F was confirmed by ELISA in TOV21G (n=2 in triplicates, p 6 vs 1381, p=0·03). IL17F has previously been implicated in neutrophil recruitment. Significant neutrophil infiltration was observed after dl 922-947 infection in HeLa subcutaneous xenografts by immunohistochemistry (median histoscores 2 for mock and 16·5 for tumours infected with dl 922-947, p=0·01). Interpretation Oncolytic adenovirus infection led to distinctive changes in chemokines and cytokines. In particular, IL17F, but not IL17A, was upregulated after adenovirus infection in vitro and in vivo. Moreover, dl 922-947 infection was associated with neutrophil infiltration in vivo. Ongoing experiments with TOV21G IL17F −/– cells generated using CRISPR–Cas9 technology will elucidate the influence of IL17F upregulation on tumour immune microenvironment and the effectiveness of dl 922-947. Funding Wellcome Trust.

Abstract AP24: CRISPR/CAS9–MEDIATED TRP53, BRCA1, BRCA2 AND PTEN KNOCKOUT TO GENERATE IMPROVED MURINE MODELS OF OVARIAN HIGH GRADE SEROUS CARCINOMA

BACKGROUND: Ovarian high grade serous carcinoma (HGSC) is the commonest subtype of human ovarian cancer, and prognosis remains poor. Transplantable murine models of HGSC that recreate key mutations seen in the human disease are greatly needed. These models would assist investigation of the relationships between tumor genotype, chemotherapy response and immune microenvironment. ID8 is the most widely-used mouse model of ovarian cancer, but it has not been characterized in light of current understanding of HGSC biology. METHODS: We performed whole exome sequencing of ID8, covering 194,000 exons at a mean depth of 400x with 90% exons sequenced >50x. Using CRISPR/Cas9 gene editing, we have generated novel ID8 derivatives with single (Trp53-/-) and double (Trp53-/-;Brca2-/-, Trp53-/-;Brca1-/- and Trp53-/-;Pten-/-) deletions. We have characterized intra-peritoneal growth of these novel mutants, and investigated platinum and PARP inhibitor sensitivity and immune cell infiltration into the tumor microenvironment. RESULTS: Whole exome sequencing showed no functional mutations in genes characteristic of HGSC (Trp53, Brca1, Brca2, Nf1, Rb1). In addition, mutations typical of clear cell (Arid1A, Pik3ca), low grade serous (Braf), endometrioid (Ctnnb1) and mucinous (Kras) carcinomas were notably absent. ID8 also demonstrated intact p53 function and homologous recombination in functional assays. We show that loss of p53 alone significantly increases tumor growth rate within the peritoneal cavity of C57Bl/6 mice, and has marked effects upon immune microenvironment. In particular, loss of p53 increases CCL2 expression and induces immunosuppressive myeloid cell infiltration into tumor and ascites. Trp53-/-;Brca2-/- and Trp53-/-;Brca1-/- cells show significantly increased sensitivity to the PARP inhibitor rucaparib in vitro compared to parental and Trp53-/- cells. In vivo, Trp53- /-;Brca2-/- tumors are more sensitive to platinum chemotherapy than Trp53-/-. They also exhibit slower intraperitoneal growth, with appearance of intra-tumoral CD3+ cell-rich tertiary lymphoid structures, a phenotype observed in human tumors with high mutational burden. Mice bearing Trp53-/-;Pten-/- tumors have significantly reduced survival compared to Trp53-/-. Full in vivo assessment of the Trp53-/-;Brca1-/- and new tripleTrp53-/-;Brca2-/- ;Pten-/- lines is on-going. CONCLUSIONS: These models represent a new and simple tool to investigate the biology of HGSC. All cells will be made available to other researchers upon request. Citation Format: Josephine Walton, Julianna Blagih, Malcolm Farquharson, Darren Ennis, Elaine Leung, Suzanne Dowson, Laura A. Tookman, Clare Orange, Dimitris Athineos, Susan Mason, David Stevenson, Karen Blyth, Douglas Strathdee, Frances R. Balkwill, Karen Vousden, Michelle Lockley, Iain A. McNeish. CRISPR/CAS9–MEDIATED TRP53, BRCA1, BRCA2 AND PTEN KNOCKOUT TO GENERATE IMPROVED MURINE MODELS OF OVARIAN HIGH GRADE SEROUS CARCINOMA [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP24.

RIPK3 promotes adenovirus type 5 activity

Oncolytic adenoviral mutants infect human malignant cells and replicate selectively within them. This induces direct cytotoxicity that can also trigger profound innate and adaptive immune responses. However, the mechanism by which adenoviruses produce cell death remains uncertain. We previously suggested that type 5 adenoviruses, including the E1A CR2 deletion mutant dl922-947, might induce a novel form of programmed death resembling necroptosis. Here we have investigated the roles of core necrosis proteins RIPK1, RIPK3 and MLKL in the cytotoxicity of dl922-947 and other adenovirus serotypes. By electron microscopy, we show that dl922-947 induces similar necrotic morphology as TSZ treatment (TNF-α, Smac mimetic, zVAD.fmk). However, dl922-947-mediated death is independent of TNF-α signalling, does not require RIPK1 and does not rely upon the presence of MLKL. However, inhibition of caspases, specifically caspase-8, induces necroptosis that is RIPK3 dependent and significantly enhances dl922-947 cytotoxicity. Moreover, using CRISPR/Cas9 gene editing, we demonstrate that the increase in cytotoxicity seen upon caspase inhibition is also MLKL dependent. Even in the absence of caspase inhibition, RIPK3 expression promotes dl922-947 and wild-type adenovirus type 5 efficacy both in vitro and in vivo. Together, these results suggest that adenovirus induces a form of programmed necrosis that differs from classical TSZ necroptosis.

Abstract A28: Adenoviruses up-regulate IL17F, but not IL17A, and activate NK cells, with potential impact on oncolytic adenovirus efficacy

Oncolytic viruses (OVs) are a new class of anti-cancer agent. They infect and replicate selectively within malignant cells, whilst sparing normal cells. OVs disrupt chemokine and cytokine networks, which may influence clinical efficacy. This study investigates the role of innate immune responses, in particular IL17F and Natural Killer cells (NK), on the efficacy of oncolytic adenoviruses. A transcriptional array (Qiagen RT2 Profiler) of 84 human chemokines and cytokines revealed highly consistent changes (r=0.623, p 1. Alvarez-Breckenridge CA, et al. Nat Med. 2012 Dec;18(12):1827-34. Citation Format: Elaine YL Leung, Melanie Weigert, Josephine Walton, Darren Ennis, Dimitris Athineos, Suzanne Dowson, Chris Hansell, Karen Blyth, Gerard Graham, Iain McNeish. Adenoviruses up-regulate IL17F, but not IL17A, and activate NK cells, with potential impact on oncolytic adenovirus efficacy. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A28.

Abstract B53: CRISPR/Cas9 mediated p53 and BRCA2 knockout to generate improved murine models of high grade serous ovarian cancer.

Background: There has been great progress in recent years in unfolding the molecular biology of high grade serous ovarian cancer (HGSOC). HGSOCs are universally TP53 mutated with largescale genetic instability and chromosomal rearrangements. In addition, c.20% have mutations in BRCA1 or BRCA2, and a further 30% may also have defective homologous recombination (HR) through other mechanisms. HGSOCs with defective HR are more likely to respond to platinum chemotherapy and have increased sensitivity to Poly (ADP ribose) polymerase (PARP) inhibitors. There is great need for transplantable murine models that accurately reflect human disease. The most commonly used is ID8, first published in 2000: ovarian surface epithelial cells were isolated from C57Bl/6 mice, and with EGF stimulation, underwent spontaneous transformation in vitro. Single cell clones were derived, and following intra-peritoneal injection of the ID8 clone in syngeneic mice, diffuse peritoneal carcinomatosis developed, with extensive blood-stained ascites at a median of 110 days. Over 100 papers have been published using ID8, but none has assessed its suitability as a model of HGSOC. Methods and Results: We performed whole-exome next generation (NGS) on ID8 cells and subsequent Sanger sequencing of key genes. ID8 cells are Trp53 wild-type by both techniques. This is confirmed in intra-peritoneal ID8 tumors. Using RT-qPCR we show that p53 protein is transcriptionally active in ID8 cells, with robust upregulation of Cdkn1a (p21) transcription following DNA damage with cisplatin. In addition, by NGS both Brca1 and Brca2 are wild-type in ID8 cells. Using a functional assay of homologous recombination, which measures the ability of cells to form Rad51 foci in response to PARP inhibition, we show that ID8 cells are HR competent. Together, these results suggest that ID8 poorly recapitulates HGSOC and lacks two defining features of the disease. We used CRISPR/Cas9 technology to create four Trp53-/- ID8 sublines with bi-allelic deletions in Trp53 exon 5. We have also created double mutant Trp53-/-;Brca2-/- lines, which have additional bi-allelic deletions in Brca2 exon 3. Three different guide RNAs were used both to target Trp53 and Brca2. In vivo growth of Trp53-/- and Trp53-/-;Brca2-/- tumors was assessed following intraperitoneal injection into female C57Bl/6 mice. Mice were monitored daily and killed when they reached UK Home Office limits. Immunohistochemistry (IHC) was carried out on resected tumors. All four Trp53-/- ID8 sublines lines have significantly reduced Trp53, Cdkn1a and Bax transcription at baseline, and no p53 protein on immunoblot. Following intra-peritoneal injection of Trp53-/- cells, mice develop characteristic tumor nodules and ascites, and have a median survival of 42-57 days, which is highly significantly shorter than mice injected with parental ID8 cells (p Conclusion: Parental ID8 is unrepresentative of human HGSOC. Using CRISPR/Cas9 technology, we have created ID8 sublines that recapitulate key mutations in HGSOC. Loss of wild-type p53 accelerates intra-peritoneal growth and also alters the tumor microenvironment, with increased intra-tumoral F4/80+ macrophages. We aim to characterize the immune infiltrate in tumors and ascites. Trp53-/-;Brca2-/- mutant cells provide suitable models for HR defective HGSOC. In vivo characterization of these tumors is ongoing. Thus, we have used CRISPR/Cas9 technology to generate more realistic transplantable models of human HGSOC. Citation Format: Josephine Walton, Suzanne Dowson, Darren Ennis, Elaine Leung, Malcolm Farquharson, David Stevenson, Karen Blyth, Douglas Strathdee, Frances R. Balkwill, Michelle Lockley, Iain A. McNeish. CRISPR/Cas9 mediated p53 and BRCA2 knockout to generate improved murine models of high grade serous ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B53.