Abstract 943: The critical role of CDK2 activation in determining the differential sensitivity of cell lines to Chk1 and Wee1 inhibitors

Abstract

Abstract Anticancer DNA damaging agents activate checkpoints that halt the cell cycle to prevent further DNA replication and mitosis until the damage has been repaired. One of the important regulatory proteins activated in this pathway is Checkpoint kinase 1 (Chk1). Hence, Chk1 inhibitors have emerged as promising novel therapeutics. In clinical trials, Chk1 inhibitors are primarily being evaluated in combination with various DNA damaging agents. However, Chk1 inhibitors also show single agent activity in a subset of cell lines, and this provides a unique therapeutic opportunity to target these tumors. The goal of this study is to define the mechanism of this sensitivity, and identify biomarkers that would permit selection of appropriate patients for clinical trials. From screening a large panel of cancer cell lines, we observed that 15% were sensitive to the Chk1 inhibitor MK-8776, with rapid (<6 h) induction of DNA double-strand breaks (DSB) in S phase cells. Most of the resistant cell lines were sensitive to the Wee1 inhibitor, MK-1775 (AZD1775), while all the cell lines were sensitive to the Wee1/Myt1 dual inhibitor, PD-166285. Wee1 suppresses activation of CDK1 and CDK2, suggesting that MK-1775-mediated activation of one or other (or both) of these kinases leads to DSB. MK-8776 did not activate CDK1, so we hypothesized that activation of CDK2 was critical. To differentiate between the role of CDK1 and CDK2 in cell lines sensitive or resistant to MK-8776, we used inhibitors to CDK1/2. We found that CVT-313 was a selective inhibitor of CDK2 at low concentrations, but that Ro-3306 failed to discriminate between these two kinases in cells. CVT-313 prevented DSB and the associated phosphorylation of histone-H2AX induced by MK-8776 and MK-1775. Cyclin E is a direct target of CDK2; phosphorylation of cyclin E leads to its degradation which was only observed in sensitive cells and prior to the formation of DSB. The acute cytotoxicity was dependent on cyclin A rather than cyclin E, as siRNA-mediated knockdown of cyclin A but not cyclin E, prevented DSB. We conclude that activation of cyclin A/CDK2 determines the sensitivity of cells to MK-8776. Resistance to MK-8776 results from failure to activate CDK2, presumably through failure to activate the upstream CDC25A phosphatase. MK-1775 can circumvent this resistance by directly activating CDK2. The untimely and abnormal activation of CDK2 is then cytotoxic to cancer cells. Citation Format: Nandini Sakurikar, Ruth Thompson, Ryan Montano, Alan Eastman. The critical role of CDK2 activation in determining the differential sensitivity of cell lines to Chk1 and Wee1 inhibitors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 943. doi:10.1158/1538-7445.AM2015-943