Suzanne Dowson

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.

Paclitaxel resistance increases oncolytic adenovirus efficacy via upregulated CAR expression and dysfunctional cell cycle control

Resistance to paclitaxel chemotherapy frequently develops in ovarian cancer. Oncolytic adenoviruses are a novel therapy for human malignancies that are being evaluated in early phase trials. However, there are no reliable predictive biomarkers for oncolytic adenovirus activity in ovarian cancer. We investigated the link between paclitaxel resistance and oncolytic adenovirus activity using established ovarian cancer cell line models, xenografts with de novo paclitaxel resistance and tumour samples from two separate trials. The activity of multiple Ad5 vectors, including dl922‐947 (E1A CR2‐deleted), dl1520 (E1B‐55K deleted) and Ad5 WT, was significantly increased in paclitaxel resistant ovarian cancer in vitro and in vivo. This was associated with greater infectivity resulting from increased expression of the primary receptor for Ad5, CAR (coxsackie adenovirus receptor). This, in turn, resulted from increased CAR transcription secondary to histone modification in resistant cells. There was increased CAR expression in intraperitoneal tumours with de novo paclitaxel resistance and in tumours from patients with clinical resistance to paclitaxel. Increased CAR expression did not cause paclitaxel resistance, but did increase inflammatory cytokine expression. Finally, we identified dysregulated cell cycle control as a second mechanism of increased adenovirus efficacy in paclitaxel‐resistant ovarian cancer. Ad11 and Ad35, both group B adenoviruses that utilise non‐CAR receptors to infect cells, are also significantly more effective in paclitaxel‐resistant ovarian cell models. Inhibition of CDK4/6 using PD‐0332991 was able both to reverse paclitaxel resistance and reduce adenovirus efficacy. Thus, paclitaxel resistance increases oncolytic adenovirus efficacy via at least two separate mechanisms – if validated further, this information could have future clinical utility to aid patient selection for clinical trials.

RAD51 and BRCA2 Enhance Oncolytic Adenovirus Type 5 Activity in Ovarian Cancer.

Homologous recombination (HR) function is critically important in high-grade serous ovarian cancer (HGSOC). HGSOC with intact HR has a worse prognosis and is less likely to respond to platinum chemotherapy and PARP inhibitors. Oncolytic adenovirus, a novel therapy for human malignancies, stimulates a potent DNA damage response that influences overall antitumor activity. Here, the importance of HR was investigated by determining the efficacy of adenovirus type 5 (Ad5) vectors in ovarian cancer. Using matched BRCA2-mutant and wild-type HGSOC cells, it was demonstrated that intact HR function promotes viral DNA replication and augments overall efficacy, without influencing viral DNA processing. These data were confirmed in a wider panel of HR competent and defective ovarian cancer lines. Mechanistically, both BRCA2 and RAD51 localize to viral replication centers within the infected cell nucleus and that RAD51 localization occurs independently of BRCA2. In addition, a direct interaction was identified between RAD51 and adenovirus E2 DNA binding protein. Finally, using functional assays of HR competence, despite inducing degradation of MRE11, Ad5 infection does not alter cellular ability to repair DNA double-strand break damage via HR. These data reveal that Ad5 redistributes critical HR components to viral replication centers and enhances cytotoxicity. Implications: Oncolytic adenoviral therapy may be most clinically relevant in tumors with intact HR function. Mol Cancer Res; 14(1); 44–55. ©2015 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.

Copy number signatures and mutational processes in ovarian carcinoma

The genomic complexity of profound copy number aberrations has prevented effective molecular stratification of ovarian cancers. Here, to decode this complexity, we derived copy number signatures from shallow whole-genome sequencing of 117 high-grade serous ovarian cancer (HGSOC) cases, which were validated on 527 independent cases. We show that HGSOC comprises a continuum of genomes shaped by multiple mutational processes that result in known patterns of genomic aberration. Copy number signature exposures at diagnosis predict both overall survival and the probability of platinum-resistant relapse. Measurement of signature exposures provides a rational framework to choose combination treatments that target multiple mutational processes.The authors identify copy number signatures from shallow whole-genome sequencing of high-grade serous ovarian cancer (HGSOC) cases. HGSOC comprises a continuum of genomes shaped by multiple mutational processes that result in genomic aberration.

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

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

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.

The driver mutational landscape of ovarian squamous cell carcinomas arising in mature cystic teratoma.

Purpose: We sought to identify the genomic abnormalities in squamous cell carcinomas (SCC) arising in ovarian mature cystic teratoma (MCT), a rare gynecological malignancy of poor prognosis. Experimental design: We performed copy number, mutational state, and zygosity analysis of 151 genes in SCC arising in MCT (n = 25) using next-generation sequencing. The presence of high-/intermediate-risk HPV genotypes was assessed by quantitative PCR. Genomic events were correlated with clinical features and outcome. Results: MCT had a low mutation burden with a mean of only one mutation per case. Zygosity analyses of MCT indicated four separate patterns, suggesting that MCT can arise from errors at various stages of oogenesis. A total of 244 abnormalities were identified in 79 genes in MCT-associated SCC, and the overall mutational burden was high (mean 10.2 mutations per megabase). No SCC was positive for HPV. The most frequently altered genes in SCC were TP53 (20/25 cases, 80%), PIK3CA (13/25 cases, 52%), and CDKN2A (11/25 cases, 44%). Mutation in TP53 was associated with improved overall survival. In 8 of 20 cases with TP53 mutations, two or more variants were identified, which were bi-allelic. Conclusions: Ovarian SCC arising in MCT has a high mutational burden, with TP53 mutation the most common abnormality. The presence of TP53 mutation is a good prognostic factor. SCC arising in MCT share similar mutation profiles to other SCC. Given their rarity, they should be included in basket studies that recruit patients with SCC of other organs. Clin Cancer Res; 23(24); 7633–40. ©2017 AACR.

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.