The hypoxia-activated prodrug TH-302 is reduced at its nitroimidazole group and under hypoxic conditions, releases Br-IPM, a bis-alkylating DNA crosslinker. We previously reported that Chk1 inhibitors are TH-302 sensitizers both in vitro and in vivo in the context of p53 checkpoint deficiency. To assess the impact of the Chk1 inhibitor AZD7762 on DNA damage in TH-302 treated cells directly, we used the single-cell electrophoresis-based ‘Comet’ assay. Cells treated with vehicle, TH-302, or AZD7762 exhibited no visible tail moment. In contrast, cells treated with both AZD7762 and TH-302 exhibited a tail moment, indicating the induction of double-strand breaks (DSBs). To determine the effect of AZD7762 on TH-302-induced DNA damage response, γH2AX was evaluated. We demonstrated in p53−/− HT29 cells that either TH-302 or AZD7762 induced γH2AX. However, treatment with both AZD7762 and TH-302 greatly increased γH2AX staining, demonstrating a greater DNA damage response with the combiniaton therapy. Induction of apoptosis was also assessed. TH-302 did not induce apoptosis in HT29, while AZD7762 caused a slight increase in apoptosis. However, treatment with both TH-302 and AZD7762 induced a dramatic increase in apoptosis, indicating that the Chk1 inhibitor AZD7762 sensitizes HT29 cells to apoptosis in response to the DNA damage induced by TH-302. To explore involved signaling pathways, we conducted immunoblot analysis and observed an increase in phospho-histone H3 at serine 10 in the combination treatment group, consistent with DNA damaged cells undergoing mitotic catastrophe. To investigate which DNA repair system is affected by Chk-1 inhibitor-mediated enhancement of TH-302 cytotoxicity, we utilized CHO cell-based DNA repair mutant cell line pairs. Potentiation of TH-302 cytotoxicity by AZD7762 was only observed in cell lines proficient in, and not deficient in, the specific type of DNA repair involved in the repair of TH-302 DNA damage (homology-dependent repair; HDR). To explore the role of HDR in AZD7762-mediated potentiation of TH-302 cytotoxicity further, we investigated the effects of AZD7762 on Rad51 levels. In response to TH-302, Rad51 expression levels were increased. However, combination treatment of cells with AZD7762 and TH-302 led to Rad51 expression levels lower than those observed in control cells. The results suggest that the down-regulation of Rad51-dependent HDR by AZD7762 results in the persistence of unrepaired DNA damage in response to TH-302 and underlies the mechanism of potentiation of TH-302 cytotoxicity by AZD7762. The preclinical data presented in this study support a new approach for the treatment of cancer by combining Chk1 inhibitors with the tumor-hypoxia targeted TH-302.
Citation Format: Fanying Meng, Deepthi Bhupathi, Charles P. Hart. Mechanistic studies of the potentiation of hypoxia-targeted drug TH-302 by co-administration of checkpoint kinase1 (Chk1) inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3289. doi:10.1158/1538-7445.AM2013-3289