While CHK1 1 siRNA, CHK1 2 siRNA, and G?6976 each likely possess activities unrelated to CHK1 function, the recapitulation of the same description phenotype using these three independent agents in multiple cell lines suggests CHK1 to be the most likely target. FA pathway deficient tumor cells are hypersensitive to CHK1 inhibition To test our hypothesis that FA deficient tumor lines are hyper dependent on CHK1 function, we tested a pair of isogenic FA proficient and deficient tumor cell lines with regard to sensitivity to CHK1 siRNA and G?6976. The 2008 ovarian carcinoma line is deficient in FA pathway function due to methylation of the FANCF promoter region. This cell line can be functionally corrected with an exogenously expressed FANCF gene to create the 2008F line.

Indeed, the FA deficient 2008 cell Inhibitors,Modulators,Libraries line was found to be more sensitive to G?6976 than the FANCF comple mented 2008F at all doses tested. Since G?6976 inhibits kinases unrelated to CHK1, we wished to confirm our Inhibitors,Modulators,Libraries results with another inhibitor that has a relatively high specificity for CHK1 but exhibits a different specificity profile with regard to non CHK1 kinases. Such an inhibitor would unlikely recapitulate the effect of G?6976 if the underlying mech anism was independent of CHK1. We selected UCN 01 for this purpose. Comparable to that observed for G?6976, the FA deficient 2008 cell line was hyper sensi tive to UCN 01 relative to the FA restored 2008F cell line. This finding supported our hypothesis that FA deficient tumor cells are hyper dependent on CHK1 for cell viability.

While G?6976 and UCN 01 exhibited differential specifi city for non CHK1 related kinases for the most part, both inhibited Protein Kinase C alpha and Protein Kinase C beta 1 in addition to CHK1 at the con centrations tested in this study. To exclude PRKC and PRKC 1 inhibition as the cause underlying the FA specific Inhibitors,Modulators,Libraries tumor killing of G?6976 and UCN Inhibitors,Modulators,Libraries 01, we tested the effect of independent siRNAs directed against PRKC and PRKC 1. The specificity of both siRNAs was validated in a previous study. At 80% silencing efficiency, neither siRNAs caused preferential killing of the 2008 line relative to the 2008F line. In contrast, an siRNA directed against CHK1 caused preferential killing of the 2008 line relative to the 2008F. While the G?6976, UCN 01, CHK1 siRNA, PRKC , and PRKC 1 data sets were each individually imperfect, com bined they offer strong support for the hypothesis that FA deficient tumors are hyper dependent Inhibitors,Modulators,Libraries on CHK1 function. Overall, PCI-34051 the FA specific tumoricidal effect of CHK1 silenc ing inhibition was comparable to those that we previ ously reported for ATM silencing inhibition.