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.

The phenotypic change characteristic of Aurora B inhibition is the induction of polyploidy. Utilizing specific siRNA duplexes and a selective small molecule inhibitor (AZD1152) to inhibit Aurora B activity in tumor cells, we sought to elucidate the mechanism by which Aurora B inhibition results in polyploidy. Cells treated with AZD1152 progressed through mitosis with misaligned chromosomes and exited without cytokinesis and subsequently underwent endoreduplication of DNA despite activation of a p53-dependent pseudo G1 checkpoint. Concomitant with polyploid cell formation, we observed the appearance of Rb hypophosphorylation, an event that occurred independently of cyclindependent kinase inhibition. We went on to discover that Aurora B directly phosphorylates Rb at serine 780 both in vitro and in vivo. This novel interaction plays a critical role in regulating the postmitotic checkpoint to prevent endoreduplication after an aberrant mitosis. Thus, we propose for the first time that Aurora B determines cellular fate after an aberrant mitosis by directly regulating the Rb tumor suppressor protein.

Malignant peripheral nerve sheath tumors (MPNST) are soft-tissue tumors with a very poor prognosis and largely resistant to chemotherapy. MPNSTs are characterized by activation of the Ras pathway by loss of tumor suppressor neurofibromatosis type 1. In view of this, MPNST may be susceptible to inhibition of the activated Ras/Raf/mitogenactivated protein kinase pathway by the B-Raf inhibitor sorafenib. MPNST (MPNST and ST8814) and dedifferentiated liposarcoma (LS141 and DDLS) human tumor cell lines were characterized for Ras activation and B-Raf expression. Tumor cells were treated with sorafenib and examined for growth inhibition, inhibition of phospho-MEK, phospho-ERK, cell cycle arrest, and changes in cyclin D1 and pRb expression. MPNSTs were sensitive to sorafenib at nanomolar concentrations. This appeared to be due to inhibition of phospho-MEK, phospho-ERK, suppression of cyclin D1, and hypophosphorylation of pRb at the CDK4-specific sites, resulting in a G 1 cell cycle arrest. These effects were not seen in the liposarcoma cells, which either did not express B-Raf or showed decreased Ras activation. Small interfering RNA -mediated depletion of B-Raf in MPNSTs also induced a G 1 cell cycle arrest in these cells, with a marked inhibition of cyclin D1 expression and Rb phosphorylation, whereas depletion of C-Raf did not affect either. With growth inhibition at the low nanomolar range, sorafenib, by inhibiting the mitogenactivated protein kinase pathway, may prove to be a novel therapy for patients with MPNST. [Mol Cancer Ther 2008; 7(4):890 -6]

Supplementary Material 3 from CHIR-124, a Novel Potent Inhibitor of Chk1, Potentiates the Cytotoxicity of Topoisomerase I Poisons <i>In vitro</i> and <i>In vivo</i>

Supplementary Material 1 from CHIR-124, a Novel Potent Inhibitor of Chk1, Potentiates the Cytotoxicity of Topoisomerase I Poisons <i>In vitro</i> and <i>In vivo</i>