Abstract 5727: Competitive inhibition of mTOR results in tumor specific radiosensitization

Abstract

Abstract Translational control of gene expression has been implicated as a component of the cellular response to ionizing radiation (IR). The mammalian target of rapamycin (mTOR) is a critical kinase in the regulation of translation. mTOR exists in two distinct complexes: mTORC1 and mTORC2. mTORC1 regulates 4E-BP1 and p70-s6k, whereas mTORC2 regulates several AGC kinases including AKT. Most studies of mTOR have focused on the use of the allosteric inhibitor rapamycin and its analogs, which incompletely inhibit mTORC1 and do not inhibit mTORC2. In contrast, newly developed competitive mTOR inhibitors (e.g. PP242) inhibit mTORC1 output more completely and inhibit mTORC2. In the study described here, we compared the effects of rapamycin and PP242 on cellular radiosensitivity using a clonogenic assay. In two tumor cell lines originating from different histologies, U251 and MDA-MB-231, PP242 treatment 1h before irradiation increased radiosensitivity with DEFs (dose enhancement factors at 0.1 surviving fraction) of 1.52 and 1.34 respectively, whereas rapamycin had no effect. However, PP242 had no effect on the radiosensitivity of the normal human fibroblast cell line, MRC9. PP242 treatment of U251 cells resulted in a dose-dependent increase in radiosensitivity. Furthermore, addition of PP242 immediately, 1, or 6h after IR resulted in increased radiosensitivity with DEFs of 1.60, 1.50, and 1.26, respectively. To investigate the mechanism of this radiosensitization U251 cells were treated with PP242, irradiated (2Gy) and stained for γH2AX, a marker of DNA double strand breaks (DSBs). PP242 exposure significantly delayed the dispersal of radiation-induced γH2AX foci, consistent with an inhibition of DSB repair. Under conditions of clonogenic survival, exposure of U251 or MDA-MB-231 cells to 2Gy resulted in no detectable increase in mTOR output. PP242 treatment of both tumor lines resulted in complete inhibition of mTORC1/2 output whereas rapamycin only partially inhibited mTORC1 and did not inhibit mTORC2. m7-GTP batch chromatography showed PP242 induced a decrease in cap-bound eIF4G and an increase in cap-bound 4E-BP1, while rapamycin slightly increased 4E-BP1 binding and had no effect on eIF4G binding. Consistent with the effects on cap-complex formation, PP242 treatment caused a considerably greater decrease in polysome abundance than rapamycin. These results suggest that competitive inhibitors of mTOR, in contrast to rapalogs, induce tumor selective radiosensitization. Moreover, the mechanism appears to involve the inhibition of DSB repair and the suppression of translation. 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 5727. doi:1538-7445.AM2012-5727