Interestingly, synergy between MK 1775 and MK 8776 did not correlate with the p53 status of the cell line, though overall sensitivity to the drugs might favor p53 mutant lines. Furthermore, three of the seven lines described in Figure 1 are wild type for p53. Further examination of other putative markers such as expression of WEE1, CHK1, or cyclin B1, will be important future questions to Cabozantinib XL184 address in understanding the cellular context of WEE1 and CHK1 inhibitor activity. Mechanistic studies suggest that WEE1 and CHK1 inhi bitors combine synergistically due to, at least in part, alterations of the cell cycle and compounded DNA dam age. Though both MK 1775 and MK 8776 are chemosensitizers that potentiate the anti proliferative effects of DNA damaging chemotherapeutics, it is also known that knockdown or inhibition of either WEE1 or CHK1 alone leads to DNA damage.

Therefore, it is likely that MK 1775 and MK 8776 work together in an analogous fashion as they do in combination with gen otoxic agents to prevent proper checkpoint response and damage control. Importantly, DNA damage incurred by WEE1 and CHK1 inhibition occurs primarily in S phase and requires CDK activity, consistent with findings that disruption of either WEE1 or CHK1 individually leads to S phase arrest, slowed DNA replication, and induced DNA damage. Increased accumulation and duration of DNA damage by MK 1775 and MK 8776 was observed in vivo, and accordingly the combination led to inhibition of tumor growth in xenograft models.

WEE1 and CHK1 inhibition was unable to prevent tumor regrowth, how ever, suggesting either that not all cells are affected or that following drug treatment cells are able to sufficiently re pair damaged DNA. Along these lines, we were unable to find robust evidence of apoptosis both in vitro and in vivo. The WEE1 inhibitor MK 1775 is known to reduce phos phorylation on tyrosine 15 of CDK1 2, resulting in increased CDK1 2 activity. Inhibition of CHK1 increases the activity of the protein phosphatases CDC25A B C, thereby reducing phosphorylation of tyrosine 15 and indirectly increasing CDK1 2 activity. We hypothesized, therefore, that combined inhibition of WEE1 and CHK1 could result in an additive inhibition of phospho CDK1 2Y15. However, we were unable to observe a substantial decrease in phospho CDK1 2Y15 beyond the effect of MK 1775 alone, suggesting that CHK1 inhibition by MK 8776 compliments inhibition of WEE1 through mechanism and target distinct from CDK1 2.

The synergistic antiproliferative effect of combined WEE1 and CHK1 inhibition was also noted by Davies et al. and Carrassa L et al. Each of these studies identified the WEE1 gene as an siRNA target that could sensitize to either a CHK1 inhibitor or a CHK1 siRNA in solid tumor cell lines. Davies et al. reported synergy Carfilzomib between WEE1 and CHK1 inhibitors in four cell lines, three of which are reported p53 wild type. Similarly, Carrassa et al.