Abstract PR14: CRISPR/Cas9-mediated Trp53, Brca1, Brca2, Pten, and Nf1 knockout to generate improved murine models of ovarian high-grade serous carcinoma
Authors: Walton Josephine , Farquharson Malcolm , Mason Susan , Blagih Julianna , Ennis Darren , Leung Elaine , Dowson Suzanne , Athineos Dimitris , Rami Damiano , Stevenson David , Coffelt Seth , Blyth Karen , Strathdee Douglas , Balkwill Frances , Vousden Karen , Lockley Michelle , McNeish Iain 
Published: August 2018

Abstract
Background: Transplantable murine models of ovarian high-grade serous carcinoma (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 a widely-used murine model of ovarian cancer. However, we previously showed that it is poorly characteristic of HGSC, with no functional alterations in key HGSC genes including Trp53, Brca1, Brca2, Pten, Nf1, and Rb1. We generated novel ID8 derivatives with single (Trp53-/-) and double (Trp53-/-;Brca2-/-) mutations using CRISPR/Cas9 gene editing.
Methods: We have now generated further ID8 derivatives: Trp53-/-;Brca1-/-, Trp53-/-;Pten-/- and Trp53-/-;Nf1-/-. In vitro, we assessed DNA double-strand break repair, and sensitivity to both platinum chemotherapy and PARP inhibition, as well as cytokine and chemokine production. In vivo, we investigated intraperitoneal growth, as well as immune cell infiltration into the tumor microenvironment.
Results: Assays of homologous recombination (HR) function confirm that loss of Brca1 function, but not Pten or Nf1, renders cells HR defective. This is accompanied by significant increases in sensitivity to both platinum and the PARP inhibitor rucaparib in Trp53-/-;Brca1-/- cells compared to Trp53-/-. Drug sensitivity in Trp53-/-;Pten-/- and Trp53-/-;Nf1-/- cells remains unchanged compared to Trp53-/- cells.
In vivo, loss of Pten and Nf1 significantly reduced time to reach humane endpoints following intraperiteonal injection (34 and 36.5 days respectively) compared to p53 loss alone (46 days, both p&lt;0.0001), while Brca1 loss had no effect on intraperitoneal growth (47 days).
There were significant differences in survival for the different genotypes following three doses of intraperitoneal cisplatin (5mg/kg on days 28, 35, and 42 only). Mice bearing control Trp53-/- tumors reached humane endpoint in a median of 81 days. Trp53-/-;Pten-/- and Trp53-/-;Nf1-/- tumors produced the worst survival (median 69 and 71 days, respectively; p&lt;0.01 for both compared to Trp53-/-). Survival was extended to 97 days for Trp53-/-;Brca1-/- (p=0.0003 compared to Trp53-/-), but even further to 113 days for Trp53-/-;Brca2-/- tumors (median 113 days), which was significantly longer than both Trp53-/- and Trp53-/-;Brca1-/- (p&lt;0.0001 for both).
In poor-prognosis Trp53-/-;Pten-/- and Trp53-/-;Nf1-/- tumors, whole-blood analysis of mice at endpoint shows a significant decrease in haemoglobin compared to single Trp53-/- tumors (p=0.0023). Furthermore, flow cytometry data show a significant increase in CD11b+;Ly6CG++ neutrophils in the ascites of mice compared to single Trp53-/-, suggesting an immunosuppressive microenvironment (p=0.0146). Analysis of whole blood at endpoint again shows a significant increase in neutrophils in mice bearing double Trp53-/-;Pten-/- tumors compared to single Trp53-/- (p=0.0203). Possible mechanisms suggested from cytokine array data include increased expression of CCL7 in Trp53-/-;Pten-/- cells, which is confirmed in tumors. Further work to characterize T-cell activation in these models is ongoing.
Conclusions: These novel ID8 models represent a new and simple tool to investigate the biology of HGSC. All cells will be made available to other researchers upon request.
This abstract is also being presented as Poster B61.
Citation Format: Josephine Walton, Malcolm Farquharson, Susan Mason, Julianna Blagih, Darren Ennis, Elaine Leung, Suzanne Dowson, Dimitris Athineos, Damiano Rami, David Stevenson, Seth Coffelt, Karen Blyth, Douglas Strathdee, Frances Balkwill, Karen Vousden, Michelle Lockley, Iain McNeish. CRISPR/Cas9-mediated Trp53, Brca1, Brca2, Pten, and Nf1 knockout to generate improved murine models of ovarian high-grade serous carcinoma. [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 PR14.