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Differential Sensitivity to CDK2 Inhibition Discriminates the Molecular Mechanisms of CHK1 Inhibitors as Monotherapy or in Combination with the Topoisomerase I Inhibitor SN38.

DNA damage activates checkpoints to arrest cell cycle progression in S and G2 phases, thereby providing time for repair and recovery. The combination of DNA-damaging agents and inhibitors of CHK1 (CHK1i) is an emerging strategy for sensitizing cancer cells. CHK1i induce replication on damaged DNA and mitosis before repair is complete, and this occurs in a majority of cell lines. However, ∼15% of cancer cell lines are hypersensitive to single-agent CHK1i. As both abrogation of S phase arrest and single-agent activity depend on CDK2, this study resolved how activation of CDK2 can be essential for both replication and cytotoxicity. S phase arrest was induced with the topoisomerase I inhibitor SN38; the addition of CHK1i rapidly activated CDK2, inducing S phase progression that was inhibited by the CDK2 inhibitor CVT-313. In contrast, DNA damage and cytotoxicity induced by single-agent CHK1i in hypersensitive cell lines were also inhibited by CVT-313 but at 20-fold lower concentrations. The differential sensitivity to CVT-313 is explained by different activity thresholds required for phosphorylation of CDK2 substrates. While the critical CDK2 substrates are not yet defined, we conclude that hypersensitivity to single-agent CHK1i depends on phosphorylation of substrates that require high CDK2 activity levels. Surprisingly, CHK1i did not increase SN38-mediated cytotoxicity. In contrast, while inhibition of WEE1 also abrogated S phase arrest, it more directly activated CDK1, induced premature mitosis, and enhanced cytotoxicity. Hence, while high activity of CDK2 is critical for cytotoxicity of single-agent CHK1i, CDK1 is additionally required for sensitivity to the drug combination.