Abstract B53: CRISPR/Cas9 mediated p53 and BRCA2 knockout to generate improved murine models of high grade serous ovarian cancer.
Authors: Walton Josephine , Dowson Suzanne , Ennis Darren , Leung Elaine , Farquharson Malcolm , Stevenson David , Blyth Karen , Strathdee Douglas , Balkwill Frances R. , Lockley Michelle , McNeish Iain A. 
Published: May 2016

Abstract
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&lt;0.0001 for all four lines). IHC characterization shows significantly higher Ki67 staining in p53 null tumors. In addition, F4/80+ macrophage staining is significantly greater in p53 null tumors. Whereas Trp53-/- sublines retain HR competence, Trp53-/-;Brca2-/- lines lose competence by Rad51 assay and show significantly increased sensitivity to rucaparib in vitro.
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.