CRISPR/Cas9-mediated Trp53 and Brca2 knockout to generate improved murine models of ovarian high grade serous carcinoma

Walton, J. et al. (2016) CRISPR/Cas9-mediated Trp53 and Brca2 knockout to generate improved murine models of ovarian high grade serous carcinoma. Cancer Research, 76(20), pp. 6118-6129. (doi:10.1158/0008-5472.CAN-16-1272) (PMID:27530326)

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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 400x with 90% exons sequenced >50x. We found no functional mutations in genes characteristic of HGSC (Trp53, Brca1, Brca2, Nf1, 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 CCL 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 to Trp53-/- cells, with an appearance of intra-tumoral 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.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Orange, Miss Clare and Walton, Miss Josephine and Mcneish, Professor Iain and Blyth, Dr Karen and Athineos, Mr Dimitris and Vousden, Karen and Leung, Dr Elaine and Strathdee, Dr Douglas and Stevenson, Dr David and Ennis, Dr Darren and Mason, Miss Susan and Dowson, Miss Suzanne and Farquharson, Dr Malcolm
Authors: Walton, J., Blagih, J., Ennis, D., Leung, E., Dowson, S., Farquharson, M., Tookman, L. A., Orange, C., Athineos, D., Mason, S., Stevenson, D., Blyth, K., Strathdee, D., Balkwill, F. R., Vousden, K. H., Lockley, M., and McNeish, I. A.
College/School:College of Medical Veterinary and Life Sciences > Institute of Cancer Sciences
College of Medical Veterinary and Life Sciences > School of Medicine, Dentistry & Nursing
Journal Name:Cancer Research
Publisher:American Association for Cancer Research
ISSN:0008-5472
ISSN (Online):1538-7445
Published Online:16 August 2016
Copyright Holders:Copyright © 2016 American Association for Cancer Research
First Published:First published in Cancer Research 76(20):6118-6129
Publisher Policy:Reproduced in accordance with the copyright policy of the publisher

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
647981CR-UK Centre renewalKaren VousdenCancer Research UK (CAN-RES-UK)18076RI CANCER SCIENCES
643981Personalised biomarkers of response in high-grade serious ovarian cancerIain McNeishCancer Research UK (CAN-RES-UK)C608/A15973RI CANCER SCIENCES
712701Clinical Training Award Cycle 2Andrew BiankinCancer Research UK (CAN-RES-UK)20921ICS - TRANSLATIONAL RESEARCH CENTRE