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

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Title: CRISPR/Cas9-Mediated Trp53 and Brca2 Knockout to Generate Improved Murine Models of Ovarian High-Grade Serous Carcinoma
Authors: Walton, J
Blagih, J
Ennis, D
Leung, E
Dowson, S
Farquharson, M
Tookman, LA
Orange, C
Athineos, D
Mason, S
Stevenson, D
Blyth, K
Strathdee, D
Balkwill, FR
Vousden, K
Lockley, M
McNeish, IA
Item Type: Journal Article
Abstract: 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.
Issue Date: 16-Aug-2016
Date of Acceptance: 2-Aug-2016
URI: http://hdl.handle.net/10044/1/55480
DOI: https://dx.doi.org/10.1158/0008-5472.CAN-16-1272
ISSN: 1538-7445
Publisher: American Association for Cancer Research
Start Page: 6118
End Page: 6129
Journal / Book Title: Cancer Research
Volume: 76
Issue: 20
Copyright Statement: © 2016 American Association for Cancer Research.
Keywords: Science & Technology
Life Sciences & Biomedicine
Oncology
REGULATORY T-CELLS
FALLOPIAN-TUBE
INTRAEPITHELIAL-CARCINOMA
MOUSE MODEL
CANCER
TUMOR
SITE
P53
TRANSFORMATION
EXPRESSION
Animals
BRCA2 Protein
CRISPR-Cas Systems
Cell Line, Tumor
Cystadenocarcinoma, Serous
Disease Models, Animal
Exome
Female
Gene Editing
Mice
Mice, Inbred C57BL
Mice, Knockout
Ovarian Neoplasms
Poly(ADP-ribose) Polymerase Inhibitors
Tumor Microenvironment
Tumor Suppressor Protein p53
1112 Oncology And Carcinogenesis
Oncology & Carcinogenesis
Publication Status: Published
Appears in Collections:Division of Surgery
Division of Cancer
Faculty of Medicine



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