Abstract 3377: Integrated molecular analysis of ovarian cancer cells identifies the ATR signaling axis central to cisplatin resistance.
Authors: Teng Pang-Ning , Wang Guisong , Litzi Tracy J. , Hood Brian L. , Blanton Brian , Conrads Kelly A. , Darcy Kathleen M. , Hamilton Chad A. , Mcguire William P. , Maxwell G. Larry , Bakkenist Chris J. , Conrads Thomas P. 
Published: August 2014

Ovarian cancer is the most lethal gynecological malignancy due primarily to the high percentage of women diagnosed at advanced stage. Although most patients initially respond to treatment, recurrence is common and second-line treatment is less effective, particularly in platinum-resistant disease. An integrated molecular analysis of an ovarian cancer cell line, A2780, and its isogenic cisplatin resistant line, A2780-CP20, was conducted utilizing next generation sequencing of the exome and the transcriptome (RNA-seq), along with a comprehensive analysis of the proteome and the phosphoproteome. Integration of these data revealed a number of differentially expressed candidates at the level of the proteome, the mechanistic nature of which could be explained by mutational status or differential splicing. We focused our attention on the ataxia telangiectasia mutated and Rad3-related (ATR) kinase. ATR activity is induced by DNA damage when single strand DNA results from persistently stalled replication forks. The gene and transcript for ATR were observed as mutated, alternatively spliced or altered at the transcript level in A2780-CP20 versus A2780 cells. The gene product was identified and validated to be ∼50-fold elevated in A2780-CP20 relative to A2780 cells. We hypothesized that inhibition of ATR would sensitize ovarian cancer cells to cisplatin-induced DNA lesions. To test this hypothesis undifferentiated (A2780, A2780-CP20) and papillary serous ovarian cancer cells (OV90 and OVCAR3) were challenged with cisplatin in the presence and absence of a selective ATR small molecule inhibitor (5 μM ATRi, ETP46464). The results demonstrate significantly increased cisplatin sensitivity in all cell lines tested by 54-99%. We further evaluated the ATR signaling axis, and repeated the cisplatin challenge experiments with a selective small molecule inhibitor against the serine/threonine kinase Chk1 (LY2603618), a downstream target that is phosphorylated by activated ATR. Similarly to what we observed with inhibition of ATR, these data reveal significant sensitization of ovarian cancer cells to cisplatin when co-cultured with the Chk1 inhibitor. To evaluate the selectivity of this sensitization response, the ovarian cancer cells were co-cultured with a selective inhibitor to the serine/threonine kinase Chk2 (2-(4-(4-Chlorophenoxy)phenyl)-1H-benzimidazole-5-carboxamide), which revealed no change in cisplatin sensitivity. We conclude that selective inhibition of ATR or Chk1, but not Chk2, significantly sensitizes ovarian carcinoma cell lines to cisplatin. Furthermore, we suggest that ATR and Chk1 represent key signaling nodes that are centrally involved in cisplatin resistance in ovarian cancer, and therefore represent attractive candidates for molecularly targeted therapy in the setting of resistant/recurrent ovarian cancer.

Citation Format: Pang-Ning Teng, Guisong Wang, Tracy J. Litzi, Brian L. Hood, Brian Blanton, Kelly A. Conrads, Kathleen M. Darcy, Chad A. Hamilton, William P. Mcguire, G. Larry Maxwell, Chris J. Bakkenist, Thomas P. Conrads. Integrated molecular analysis of ovarian cancer cells identifies the ATR signaling axis central to cisplatin resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3377. doi:10.1158/1538-7445.AM2013-3377