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

• Published in: Cancer research (Chicago, Ill.) Ill.), 2016-10, Vol.76 (20), p.6118-6129
• Main Authors: Walton, Josephine, Blagih, Julianna, Ennis, Darren, Leung, Elaine, Dowson, Suzanne, Farquharson, Malcolm, Tookman, Laura A, Orange, Clare, Athineos, Dimitris, Mason, Susan, Stevenson, David, Blyth, Karen, Strathdee, Douglas, Balkwill, Frances R, Vousden, Karen, Lockley, Michelle, McNeish, Iain A
• Format: Article
• Language: English
• Subjects: Animals; BRCA2 Protein - genetics; BRCA2 Protein - physiology; Cell Line, Tumor; CRISPR-Cas Systems - physiology; Cystadenocarcinoma, Serous - drug therapy; Cystadenocarcinoma, Serous - etiology; Disease Models, Animal; Exome; Female; Gene Editing; Mice; Mice, Inbred C57BL; Mice, Knockout; Ovarian Neoplasms - drug therapy; Ovarian Neoplasms - etiology; Ovarian Neoplasms - genetics; Poly(ADP-ribose) Polymerase Inhibitors - therapeutic use; Tumor Microenvironment; Tumor Suppressor Protein p53 - genetics; Tumor Suppressor Protein p53 - physiology
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/0008-5472.can-16-1272

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.