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Inhibition of the DNA damage checkpoint kinase WEE1 potentiates genotoxic chemotherapies by abrogating cell-cycle arrest and proper DNA repair. However, WEE1 is also essential for unperturbed cell division in the absence of extrinsic insult. Here, we investigate the anticancer potential of a WEE1 inhibitor, independent of chemotherapy, and explore a possible cellular context underlying sensitivity to WEE1 inhibition. We show that MK-1775, a potent and selective ATP-competitive inhibitor of WEE1, is cytotoxic across a broad panel of tumor cell lines and induces DNA double-strand breaks. MK-1775–induced DNA damage occurs without added chemotherapy or radiation in S-phase cells and relies on active DNA replication. At tolerated doses, MK-1775 treatment leads to xenograft tumor growth inhibition or regression. To begin addressing potential response markers for MK-1775 monotherapy, we focused on PKMYT1, a kinase functionally related to WEE1. Knockdown of PKMYT1 lowers the EC50 of MK-1775 by five-fold but has no effect on the cell-based response to other cytotoxic drugs. In addition, knockdown of PKMYT1 increases markers of DNA damage, γH2AX and pCHK1S345, induced by MK-1775. In a post hoc analysis of 305 cell lines treated with MK-1775, we found that expression of PKMYT1 was below average in 73% of the 33 most sensitive cell lines. Our findings provide rationale for WEE1 inhibition as a potent anticancer therapy independent of a genotoxic partner and suggest that low PKMYT1 expression could serve as an enrichment biomarker for MK-1775 sensitivity. Mol Cancer Ther; 12(8); 1442–52. ©2013 AACR.

Inhibitors of the checkpoint kinases CHK1 and WEE1 are under development as sensitizers to chemotherapy or radiation therapy. Disruption of either CHK1 or WEE1 activity compromises the cell cycle arrest that normally accompanies the DNA damage response, forcing cells to prematurely divide before repairing damaged DNA. However, inhibition of either CHK1 or WEE1 alone also disrupts DNA replication, resulting in DNA double strand breaks (DSBs) primarily in S-phase cells. Here we describe the ability of inhibitors against CHK1 and WEE1 to synergize both in vitro and in vivo. MK-8776 and MK-1775 are advancing through early clinical trials as potent and selective inhibitors of CHK1 and WEE1, respectively. MK-8776 increases the cell-based potency of MK-1775 in multiple cancer cell lines by as much as a 10-fold EC50 shift in proliferation assays. Staining for the DSB marker γH2AX at low concentrations of each drug demonstrates that MK-8776 and MK-1775 in combination induce up to 50-fold more DNA damage than either MK-8776 or MK-1775 alone. The ability of the combination to drive premature mitosis, determined by pHH3 quantitation, was variable, suggesting that generation of DSBs rather than G2/M checkpoint abrogation is the mechanism underlying combination cytotoxicity. Induction of DSBs requires cyclin-dependent kinase activity and takes place largely in S-phase cells, implying deregulation of DNA replication. Furthermore, staining for DNA damage response markers γH2AX and pCHK1-ser345 was both more intense and more durable in a LoVo xenograft model of cancer when MK-8776 and MK-1775 were dosed together compared to either compound alone. At tolerated doses, the combination demonstrated significantly greater anti-tumor efficacy than either MK-8776 or MK-1775 alone in LoVo xenografts (91% tumor growth inhibition for the combination versus 28% and 41% for MK-8776 and MK-1775, respectively). Similar observations were made in additional xenograft models. These results demonstrate in vitro and in vivo synergy between MK-8776 and MK-1775, indicating unique and complimentary anti-tumor effects of CHK1 and WEE1 inhibition. Our studies provide preclinical rationale for combining WEE1 and CHK1 inhibitors as a novel cancer treatment regimen. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2051. doi:1538-7445.AM2012-2051

The Wee1 tyrosine kinase plays a key role in checkpoint arrest by phosphorylating and inhibiting cyclin-dependent kinases 1 and 2, two CDKs that drive S-phase and G2- to M-phase transitions, respectively. Interfering with Wee1 leads to elevated CDK1 and 2 activity, which in turn overcomes checkpoint arrest and causes cells to progress through the cell cycle in the presence of DNA damage. Wee1 inhibitors are therefore expected to sensitize cancer cells to DNA damaging chemotherapeutics. In particular, p53-deficient cells are postulated to be particularly sensitive to Wee1 inhibition because they lack a G1-checkpoint and rely more on S- and G2-phase checkpoints where Wee1 activity is critical. The p53 pathway is frequently inactivated in cervical carcinoma either through TP53 mutation or through functional inactivation by Human PapillomaVirus (HPV) gene products. We thus investigated the capability of MK-1775, a selective and potent inhibitor of Wee1, to sensitize cervical cancer cell lines to cisplatin and topotecan. We have evaluated the potential of the Wee1 inhibitor MK-1775 to sensitize cervical cancer cells to standard-of-care treatments both in vitro and in vivo. Using a panel of cervical cancer cell lines, we demonstrated that MK-1775 at clinically relevant concentrations potentiates the cytotoxic activity of cisplatin and topotecan in vitro as indicated by the average 3-fold and 2-fold shift in the CC50s for cisplatin and topotecan, respectively. Chemosenstization was observed in both HPV-negative/p53 mutant and HPV-positive p53/wild-type cervical cancer cell lines. Finally, we have demonstrated that MK-1775 significantly increases the in vivo efficacy of a cisplatin-topotecan combination therapy in xenograft models of HPV+ cervical carcinoma. Based on these data, MK-1775 is currently undergoing clinical evaluation in a phase 1b/2 trial in platinum combination for the treatment of cervical carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2969. doi:10.1158/1538-7445.AM2011-2969