Using small molecule inhibitors currently under early clinical development, we have shown that simultaneous inhibition of the WEE1 and CHK1 kinases results in synergistic potentiation of each drug for a variety of cell types in proliferation assays. Knockout of WEE1 results in embryonic lethality before day 3.5 , and knockdown of WEE1 is known to inhibit proliferation of several cancer cell lines in vitro [27, 28]. Similarly, anti-proliferative effects of CHK1 inhibition via siRNA or pharmacologic inhibition have been described . The increased potency of MK-1775 and MK-8776 when combined supports the notion that WEE1 and CHK1 have non-overlapping activity. Potentially predictive biomarkers for each class of drug have been described for their chemosensitizing effects, including p53 status for both WEE1 and CHK1 [2, 31], WEE1 expression levels for WEE1 , and cyclin B levels for CHK1 . 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 (A2780, LoVo, and A427). Further examination of other putative markers such as expression of WEE1, CHK1, or cyclin B1, will be important future questions to address in understanding the cellular context of WEE1 and CHK1 inhibitor activity.
Mechanistic studies suggest that WEE1 and CHK1 inhibitors combine synergistically due to, at least in part, alterations of the cell cycle and compounded DNA damage (Figures 2, 3, and 4). 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 genotoxic 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, however, suggesting either that not all cells are affected or that following drug treatment cells are able to sufficiently repair damaged DNA. Along these lines, we were unable to find robust evidence of apoptosis both in vitro and in vivo (data not shown).
The WEE1 inhibitor MK-1775 is known to reduce phosphorylation 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(s) and target(s) 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 (Davies et al.) or a CHK1 siRNA (Carrassa et al.) in solid tumor cell lines. Davies et al. reported synergy between WEE1 and CHK1 inhibitors in four cell lines, three of which are reported p53 wild type . Similarly, Carrassa et al. reported synergy in seven cell lines regardless of p53 status . This manuscript extends earlier findings into 37 cancer cell lines using compounds that are currently under early stage clinical development. Our findings align with those reported demonstrating that the mechanism underlying synergy between WEE1 and CHK1 inhibition is ubiquitous as well as with the finding that p53 status does not affect this synergy.
Davies et al. reported an absence of premature mitosis in the HEL92.1.7 cell line, though this experiment was conducted with an excess of WEE1 and CHK1 inhibitors required for inhibition of cell proliferation (compare Figures 2C and 5 in ). Carrassa et al. conducted mechanistic studies in one cell line, OVCAR-5, and concluded that premature mitosis accompanied the simultaneous inhibition of WEE1 and CHK1 inhibition . It was unclear in that study whether concentrations of inhibitors used to study biochemical correlates coincided with the concentrations required to inhibit proliferation. By examining the effects of MK-1775 and MK-8776 at the lowest concentrations needed to achieve antiproliferative activity, individualized for multiple cell lines, we are able to demonstrate that DNA damage rather than premature mitosis seems to be the primary cause of synergistic cytoxicity (Figure 4), though we do find that select cell lines, i.e. HT-29, may undergo premature mitosis as well. Importantly, these findings were corroborated in vivo where LoVo xenograft tumor samples demonstrated synergistic increases in the DNA damage markers γH2AX and pCHK1S345 but not in the mitosis marker pHH3 (Figure 7 and data not shown). Collectively these data argue that nonoverlapping functions of the WEE1 and CHK1 kinases during S- phase are responsible for the widespread and strong synergy observed following their inhibition.
Our studies describe synergy achieved by simultaneous inhibition of the WEE1 and CHK1 kinases and, together with the work of Davies et al.  and Carrassa et al. , provide pharmacologic evidence that the two kinases have unique and nonoverlapping activities. Combined treatment with MK-1775 and MK-8776 demonstrates synergistic DNA damage and anti-tumor efficacy at tolerated doses, suggesting possible clinical use of the drugs in combination. The robust and ubiquitous nature of the synergy may suggest potential toxicity in normal tissue and therefore identification of mechanisms underlying sensitivity will be important in understanding the potential clinical application of this combination.