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Background Chk1 inhibitors are currently under clinical evaluation as single agents and in combination with cytotoxic chemotherapy. Understanding determinants of sensitivity and novel combinations is critical for further clinical development. Methods Potentiation of mTOR inhibitor cytotoxicity by the Chk1 inhibitor V158411 was determined in p53 mutant colon cancer cells. DNA damage response, expression levels of repair proteins, cell cycle effects and the contribution of alternative DSB repair pathways were further evaluated by western blotting and high content analysis. Results mTOR inhibitors AZD8055, RAD-001, rapamycin and BEZ235 induced synergistic cytotoxicity with the Chk1 inhibitor V158411 in p53 mutant colon cancer cells. Reduced FANCD2, RAD51 and RPA70, core proteins in homologous recombination repair (HRR) and interstrand crosslink repair (ICLR), following inhibition of mTOR was associated with increased V158411 induced DSBs and caspase 3-independent cell death. Dual mTOR and Chk1 inhibition activated DNA-PKcs. Cells defective in DNA-PKcs exhibited increased resistance to V158411 with Chk1 expression closely correlated to DNA-PKcs expression in various types of cancer. Conclusions Down regulation of proteins involved in HRR or ICLR by mTOR inhibitors is associated with increased sensitivity of human tumours to Chk1 inhibitors such as V158411. High levels of DNA-PKcs may be a potential biomarker to stratify patients to Chk1 inhibitor therapy alone or in combination with mTOR inhibitors.

Background Chk1 inhibitors are currently in clinical trials as putative potentiators of cytotoxic chemotherapy drugs. Chk1 inhibitors may exhibit single agent anti-tumor activity in cancers with underlying DNA repair, DNA damage response or DNA replication defects. Methods Here we describe the cellular effects of the pharmacological inhibition of the checkpoint kinase Chk1 by the novel inhibitor V158411 in triple-negative breast cancer and ovarian cancer. Cytotoxicity, the effect on DNA damage response and cell cycle along with the ability to potentiate gemcitabine and cisplatin cytotoxicity in cultured cells was investigated. Western blotting of proteins involved in DNA repair, checkpoint activation, cell cycle and apoptosis was used to identify potential predictive biomarkers of Chk1 inhibitor sensitivity. Results The Chk1 inhibitors V158411, PF-477736 and AZD7762 potently inhibited the proliferation of triple-negative breast cancer cells as well as ovarian cancer cells, and these cell lines were sensitive compared to ER positive breast and other solid cancer cells lines. Inhibition of Chk1 in these sensitive cell lines induced DNA damage and caspase-3/7 dependent apoptosis. Western blot profiling identified pChk1 (S296) as a predictive biomarker of Chk1 inhibitor sensitivity in ovarian and triple-negative breast cancer and pH2AX (S139) in luminal breast cancer. Conclusions This finding suggests that Chk1 inhibitors either as single agents or in combination chemotherapy represents a viable therapeutic option for the treatment of triple-negative breast cancer. pChk1 (S296) tumor expression levels could serve as a useful biomarker to stratify patients who might benefit from Chk1 inhibitor therapy.

Background Chk1 inhibitors are currently in clinical trials in combination with a range of cytotoxic agents and have the potential to potentiate the clinical activity of a large number of standard of care chemotherapeutic agents. Utilizing pharmacodynamic biomarkers to optimize drug dose and scheduling in these trials could greatly enhance the likelihood of clinical success. Methods In this study, we evaluated the in vitro potentiation of the cytotoxicity of a range of cytotoxic chemotherapeutic drugs by the novel Chk1 inhibitor V158411 in p53 mutant colon cancer cells. Pharmacodynamic biomarkers were evaluated in vitro. Results V158411 potentiated the cytotoxicity of a range of chemotherapeutic agents with distinct mechanisms of action in p53 mutant colon cancer cell lines grown in anchorage dependent or independent culture conditions. Analysis of pharmacodynamic biomarker changes identified dependencies on the chemotherapeutic agent, the concentration of the chemotherapeutic and the duration of time between combination treatment and biomarker analysis. A reduction in total Chk1 and S296/S317/S345 phosphorylation occurred consistently with all cytotoxics in combination with V158411 but did not predict cell line potentiation. Induction of γH2AX levels was chemotherapeutic dependent and correlated closely with potentiation of gemcitabine and camptothecin in p53 mutant colon cancer cells. Conclusions Our results suggest that Chk1 phosphorylation could be a useful biomarker for monitoring inhibition of Chk1 activity in clinical trials involving a range of V158411-chemotherapy combinations and γH2AX induction as a predictor of potentiation in combinations containing gemcitabine or camptothecin.