Therefore, we determined the effects of simultaneous inhibition of WEE1 and CHK1 on the cell selleck chemicals cycle profile of asynchron ously growing cell populations. We selected two cell lines where synergy was observed, NCI H2009 NSCLC and Su. 86. 86 pancreatic cancer cells, and used concentrations of the two inhibitors that had little effect on cell prolifera tion when dosed alone, but had a profound effect in combination. We also included normal human renal epi thelial cells and normal human mammary epithelial cells, which did not score high in the combin ation synergy screen and were unresponsive to the concentrations of inhibitors used. In all the cells tested, MK 1775 had no dis cernible effect on the cell cycle profile, whereas MK 8776 caused an increase in the number of G1 S phase cells in the cancer lines.

Notably, the combination of MK 1775 and MK 8776 led to a dramatic accumulation of cells with G1 S phase DNA content in both tumor lines. Again, this effect was not seen in either of the normal cell populations tested, consistent with published observations that WEE1 knockdown is more deleterious in transformed cells than in non transformed cells. Inhibition of WEE1 and CHK1 leads to synergistic accumulation of DNA damage Inactivating mutations of p53 can impair the G1 check point arrest that typically follows DNA damage. Due to a compromised G1 checkpoint, it is suggested that p53 deficient tumor cells are more dependent on the G2 checkpoint and therefore more likely to be sensitized to DNA damaging agents by G2 checkpoint modulators, i. e. WEE1 or CHK1 inhibitors.

Mutational status of p53 was found for 31 of the 39 cancer cell lines screened and these lines were analyzed for synergy of the MK 1775 and MK 8776 combination. Synergy scores ranged from 0 to 0. 4 among both p53 wild type and p53 mutant cell lines, but we failed to observed any difference in synergy among the two groups. The same cell panel did demonstrate a trend toward greater overall sensitivity to the MK 1775 plus MK 8776 combination among p53 mutant lines. Two p53 wild type or null isogenic cell line pairs demonstrated similar synergy and overall response to the MK 1775 and MK 8776 combination. Loss of either WEE1 or CHK1 function through siRNA depletion or small molecule inhibition results in an accumu lation of DNA damage. Therefore, we considered the likeli hood that combining MK 1775 with MK 8776 might result in increased DNA damage. To differentiate effects of the combination from effects of either single agent, we selected concentrations of MK 1775 and Carfilzomib MK 8776 that alone had limited effect in a cell proliferation assay, but when com bined led to 80% growth inhibition.