Purpose: Chk1kinase is a critical regulator of both S and G 2 -M phase cell cycle checkpoints in response to DNA damage. This study aimed to evaluate the biochemical, cellular, and antitumor effects of a novel Chk1inhibitor, CHIR124. Experimental Design: CHIR-124 was evaluated for its ability to abrogate cell cycle checkpoints, to potentiate cytotoxicity, and to inhibit Chk1-mediated signaling induced by topoisomerase I poisons in human tumor cell line and xenograft models. Results: CHIR-124 is a quinolone-based small molecule that is structurally unrelated to other known inhibitors of Chk1. It potently and selectively inhibits Chk1 in vitro (IC 50 = 0.0003 Amol/L). CHIR-124 interacts synergistically with topoisomerase poisons (e.g., camptothecin or SN-38) in causing growth inhibition in several p53-mutant solid tumor cell lines as determined by isobologram or response surface analysis. CHIR-124 abrogates the SN-38^induced S and G 2 -M checkpoints and potentiates apoptosis in MDA-MD-435 breast cancer cells. The abrogation of the G 2 -M checkpoint and induction of apoptosis by CHIR-124 are enhanced by the loss of p53.We have also shown that CHIR-124 treatment can restore the level of cdc25A protein, which is normally targeted by Chk1for degradation following DNA damage, indicating that Chk1 signaling is suppressed in the presence of CHIR-124. Finally, in an orthotopic breast cancer xenograft model, CHIR-124 potentiates the growth inhibitory effects of irinotecan by abrogating the G 2 -M checkpoint and increasing tumor apoptosis. Conclusions: CHIR-124 is a novel and potent Chk1inhibitor with promising antitumor activities when used in combination with topoisomerase I poisons.

Dedifferentiated liposarcoma (DDLS) is a rare but aggressive cancer with high recurrence and low response rates to targeted therapies. Increasing treatment efficacy may require combinations of targeted agents that counteract the effects of multiple abnormalities. To identify a possible multicomponent therapy, we performed a combinatorial drug screen in a DDLS-derived cell line and identified cyclin-dependent kinase 4 (CDK4) and insulin-like growth factor 1 receptor (IGF1R) as synergistic drug targets. We measured the phosphorylation of multiple proteins and cell viability in response to systematic drug combinations and derived computational models of the signaling network. These models predict that the observed synergy in reducing cell viability with CDK4 and IGF1R inhibitors depend on activity of the AKT pathway. Experiments confirmed that combined inhibition of CDK4 and IGF1R cooperatively suppresses the activation of proteins within the AKT pathway. Consistent with these findings, synergistic reductions in cell viability were also found when combining CDK4 inhibition with inhibition of either AKT or epidermal growth factor receptor (EGFR), another receptor similar to IGF1R that activates AKT. Thus, network models derived from context-specific proteomic measurements of systematically perturbed cancer cells may reveal cancer-specific signaling mechanisms and aid in the design of effective combination therapies.

(2010) Chk1 inhibition after replicative stress activates a double strand break response mediated by ATM and DNA-dependent protein kinase, Cell Cycle, 9:5, 995-1004,

The G 2 /M cell cycle checkpoint is regulated by a multitude of signaling pathways after genotoxic stress. Herein, we report that treatment with the 90-kDa heat shock protein (Hsp90) molecular chaperone inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) selectively abrogates the G 2 /M checkpoint induced by 7-ethyl-10-hydroxycamptothecin (SN-38), an active metabolite of irinotecan, in p53-null compared with p53-intact HCT116 colon cancer cells. The basis for this selectivity can be explained in part by the lack of p21 induction in p53-null cells. In accord with published results, we could show that treatment with 17AAG resulted in depletion of Chk1, a known Hsp90 client protein. In addition, we observed a time-and dose-dependent decrease in Wee1 kinase level, a negative regulator of mitosis, after 17AAG treatment in gastrointestinal cancer cells. Depletion of Wee1 protein preceded mitotic entry induced by 17AAG, and this decrease could be partially rescued by cotreatment with a proteasome inhibitor. Coimmunoprecipitation experiments showed that Hsp90 and Wee1 interacted in whole cells, and 17AAG treatment decreased the degradative half-life of Wee1, indicating that Wee1 is another Hsp90 client in mammalian cells. Knockdown of Chk1 and Wee1 by short interfering RNA each resulted in abrogation of the G 2 /M checkpoint induced by SN-38. The combination of SN-38 and 17AAG was shown to be synergistic in p53-null but not in parental HCT116 cells by median effect/combination index analysis. Taken together, 17AAG specifically inhibits the G 2 /M checkpoint in p53-defective cells by down-regulation of two critical checkpoint kinases, Chk1 and Wee1.

Purpose Both Hsp90 and checkpoint kinase 1 (Chk1) have emerged as novel therapeutic targets. We conducted a phase I study of irinotecan and the Hsp90 inhibitor 17AAG, which can also down-regulate Chk1, in patients with solid tumors. Experimental Design During the dose-escalation phase, patients received intravenous irinotecan followed by 17AAG once weekly for 2 weeks in a 21-day cycle. At the maximum tolerated dose (MTD), additional patients were enrolled to undergo pre- and post-17AAG tumor biopsies for pharmacodynamic evaluation. The pharmacokinetics of irinotecan, 17AAG, and their metabolites were characterized. Tumor p53 status as determined by immunohistochemistry was correlated with antitumor activity. Results Twenty-seven patients with a variety of solid tumors were enrolled. Four patients developed dose-limiting toxicity (DLT) at dose level 4 (100 mg/m2 irinotecan and 375 mg/m2 17AAG) including nausea, vomiting, diarrhea, and pulmonary embolism. The pharmacokinetics of 17AAG and its metabolite were not significantly affected by coadministration of irinotecan, and vice versa. There was no partial response although tumor shrinkage was observed in 6 patients. Five of 10 patients with p53-mutant tumor had stable disease as the best response compared with 2 of 6 patients with p53-wildtype tumor (P=0.63). Evidence for Hsp90 inhibition by 17AAG, resulting in phospho-Chk1 loss, abrogation of the G2/M cell cycle checkpoint, and cell death could be demonstrated in tumor biopsy samples obtained at the MTD. Conclusions The combination of irinotecan and 17AAG can be given to patients with acceptable toxicity. The recommended phase II dose of the combination is 100 mg/m2 irinotecan and 300 mg/m2 17AAG.