Abstract 1784: Enhancement of hypoxia-activated prodrug TH-302 activity by Chk1 inhibition
Authors: Meng Fanying , Bhupathi Deepthi , Matteucci Mark D. , Hart Charles P. 
Published: August 2012

Abstract
TH-302 is a hypoxia-activated prodrug currently in clinical trials for the treatment of cancer. TH-302 releases the DNA cross-linking bromo-isophosphoramide mustard under hypoxic conditions. When DNA damage occurs, a signal transduction cascade is activated in response to the damage and transmits signals to the downstream effectors that connect with the cell cycle machinery. Checkpoint kinase 1 (Chk1) is a vital link between the upstream sensors of the DNA damage checkpoints and the cell cycle effectors. Generally, cell cycle progression is interrupted at the stage where the cell was when injured to give the cell time to repair the damage by activating DNA damage response and repair pathways. Due to its integral role in maintaining genomic integrity, Chk1 has been considered an attractive molecular target for inhibition to treat cancer. In recent years, Chk1 inhibitors have been studied for use in combination with DNA damaging anticancer agents that cause S and G2/M arrest in attempts to increase the efficacy of cancer treatment while sparing normal cells. We have shown that TH-302 induces G2/M arrest at low concentrations and a pan-cell cycle arrest at high concentrations. We hypothesized that the pharmacological inhibition of Chk1 kinase activity could potentiate the efficacy of TH-302. To investigate this possibility, we tested Chk1 inhibitors PF477736, AZD7762 and LY2603618 in combination with TH-302 in HeLa cervical, HT-29 colon, and H460 NSCLC cell lines. Employing an in vitro proliferation assay, we show that TH-302 activity is greatly enhanced (15 to 50 fold) by the addition of the Chk1 inhibitors in the two p53-deficient HeLa and HT29. In contrast, TH-302 activity is not affected by the presence of the Chk1 inhibitors in the p53 wild-type H460. The differential effects on TH-302 activity in combination with the Chk1 inhibitors were confirmed using clonogenic survival assays. These results are consistent with published studies showing p53 status playing a critical role in the activity of Chk1 inhibitors in combination with other DNA damaging agents. We hypothesized that TH-302 induced cell-cycle arrest at the G2/M phase may be mediated by activation of Chk1 and prevention of the activation of downstream Cdc2 kinase activity. We show that Chk1 inhibitors can abrogate TH-302-induced G2 checkpoint arrest in HeLa cells. Since Chk1 affects Cdc2 phosphorylation, we also evaluated the status of Cdc2 phosphorylation in response to the Chk1 inhibitor alone, TH-302 alone, or the combination of Chk1 inhibitor and TH-302. The results demonstrate that TH-302 alone, but not Chk1 inhibitor alone, increases Cdc2-Y15 phosphorylation due to the induced G2/M arrest. In the combination study, the addition of the Chk1 inhibitor blocks the TH-302-induced increase of Cdc-Y15 phosphorylation. Taken together, the results suggest a novel approach for the treatment of cancer combining Chk1 inhibitors with the tumor-hypoxia targeted TH-302.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1784. doi:1538-7445.AM2012-1784