Whilst this cell necessary line had low levels of Chk1 phosphorylation at serine 296, it was the only cell line with high expression of pH2AX potentially indicative of a reasonable level of DNA breakage in prolif erating cells. Shibata et al, identified elevated expres sion levels of pChk1 and to a lesser extent pH2AX as being predictive of the sensitivity of breast can cer cell lines to the Chk1 inhibitor PF 477736. Sensitivity to V158411 appeared independent of both p53 and kRas mutational status, both of which have previously been im plicated in Chk1s mechanism of action. The outlier in this analysis was the ovarian cancer cell line ES 2. This cell line had high expression levels of pChk1 but was relatively resistant to growth inhibition by all three Chk1 inhibitors.

Further work is needed to understand the relative resistance of this cell line to Chk1 inhibition. The underlying mechanism for the sensitivity of these cancer cell types to single Chk1 inhibitor therapy is not yet clear and the phosphorylation events identified as potential predictive markers of sensitivity, and pH2AX are most likely symptomatic rather than the cause of the underlying sensitivity. This observation suggests that the molecular defects in these cell lines occur in pathways for which Chk1 can mutu ally compensate to protect genomic integrity and there fore Chk1 inhibition is lethal. An example of this so far discovered is the Fanconis Anemia DNA repair path way. Cells defective in FA were sensitive to Chk1 siRNA and the small molecule Go6976 due to an accumulation of unrepairable DNA double strand breaks.

The basal like breast cancer cell line HCC9137 harbors a homozy gous truncation mutation in the DNA repair gene BRCA1 and this reduced capacity to repair DNA breaks may underlie this cell lines sensitivity. Underlying defects in DNA repair would be predicted to confer increased sensitivity to DNA damaging cytotoxic drugs such as cisplatin. The correlation between sensitivity to cispla tin and V158411 was cell line dependent and not con sistent across the panel of breast and ovarian lines studied. For example, the BRCA defective cell line HCC1937 was highly sensitive to cisplatin and V158411 whilst the ovarian cell line SKOV 3 was equally sensitive to V158411 but 8 fold more resistant to cisplatin than the HCC1937 cell line.

This suggests that different mecha nisms may account for the V158411 sensitivity in different Carfilzomib cell lines. Cells under replicative stress due to oncogene amplifi cation or activation of oncogenic signaling pathways are addicted to Chk1 kinase activity for the completion of a normal S phase. The sensitivity of neuroblast oma and melanoma cell lines has been suggested to be likely related to oncogenic replicative stress. In the case of neuroblastoma, this has been linked to members of the Myc family of oncogenes.