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) (PMCID:PMC5802386)
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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, Professor Karen and Athineos, Mr Dimitris and Vousden, Karen and Leung, Dr Elaine and Stevenson, Dr David and Strathdee, Mr Douglas 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 > School 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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