Abstract 2852: Torin2 suppresses ionizing radiation induced DNA damage repair

Abstract

Abstract The phosphatidylinositol 3- kinase like kinase (PIKK) family of signaling proteins play a central role in stress response, DNA damage signaling, and sensing growth factor and nutrient levels to regulate cellular metabolism. In tumor cells, the PIKK signaling is often hyperactivated making it an attractive therapeutic target. In this study, we have focused our efforts on an ATP-competitive mTOR/PIKK inhibitor ‘Torin2′ which has been previously reported to exhibit potent activity against the DNA repair-associated proteins ATM, ATR and DNA-PK in addition to mTOR in biochemical profiling studies. Based on this profiling data, we hypothesized that Torin2 would have synergistic effects with agents or biological processes that cause DNA damage, especially in cases where high levels of replicative stress and non-oncogenic addiction to DNA repair factors are present. In order to understand the practical clinical implications, we evaluated how Torin2 modulates the effects of ionizing radiation (IR) induced DNA damage responses. With a defined set of biochemical and cytogenetic studies along with DNA repair reporter system, we established the role for Torin2 in double strand break repair responses in human tumor cell lines. Torin2 enhances ionizing radiation-induced cell killing in cancer cell lines independent of ATM status, suggesting that Torin2 simultaneously influences multiple PIKK targets. Moreover, Torin2 pre-treatment did not alter the appearance of γH2AX foci post irradiation but significantly delayed the disappearance of IR-induced γH2AX foci indicating a defective DNA repair processes. Pre-treatment of this drug decreased the number of IR-induced G2/S-phase specific chromosomal aberrations, and also reduces the frequency of the foci formation of IR-induced homologous recombination (HR) repair proteins, such as CtIP and Rad51, suggesting that the drug works by blocking HR, a repair process that predominantly works in S/G2 phase of the cell cycle. Consistent with these results, a HR reporter assay showed that Torin2 treatment reduces repair of I-SceI restriction enzyme induced DNA damage. Torin2 also influences DNA replicative stress through inhibition of ATR-Chk1 activation, and contributes to fork stalling during replication. With the above set of studies, we have identified key DNA damage signaling events by which Torin2 affects HR mediated double strand break repair after ionizing radiation treatment. This enhanced radiosensitizing effect of Torin2 merits further pre-clinical evaluation in combination with various radiotherapy modalities. Citation Format: Durga Udayakumar, Raj K. Pandita, Nobuo Horikoshi, Clayton R. Hunt, Qingsong Liu, Kwok-Kin Wong, Nathanael S. Gray, Tej K. Pandita, Kenneth D. Westover. Torin2 suppresses ionizing radiation induced DNA damage repair. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2852. doi:10.1158/1538-7445.AM2015-2852