The Dual PI 3 K / mTOR Inhibitor NVP-BEZ 235 Is a Potent Inhibitor of ATM-and DNA-PKCs-Mediated DNA Damage Responses 1 , 2

Inhibitors of PI3K/Akt signaling are being actively developed for tumor therapy owing to the frequent mutational activation of the PI3K-Akt-mTORC1 pathway inmany cancers, including glioblastomas (GBMs). NVP-BEZ235 is a novel and potent dual PI3K/mTOR inhibitor that is currently in phase 1/2 clinical trials for advanced solid tumors. Here, we show that NVP-BEZ235 also potently inhibits ATM and DNA-PKcs, the two major kinases responding to ionizing radiation (IR)–induced DNA double-strand breaks (DSBs). Consequently, NVP-BEZ235 blocks both nonhomologous end joining and homologous recombination DNA repair pathways resulting in significant attenuation of DSB repair. In addition, phosphorylation of ATM targets and implementation of the G2/M cell cycle checkpoint are also attenuated by this drug. As a result, NVP-BEZ235 confers an extreme degree of radiosensitization and impairs DSB repair in a panel of GBM cell lines irrespective of their Akt activation status. NVP-BEZ235 also significantly impairs DSB repair in a mouse tumor model thereby validating the efficacy of this drug as a DNA repair inhibitor in vivo. Our results, showing that NVP-BEZ235 is a potent and novel inhibitor of ATM and DNA-PKcs, have important implications for the informed and rational design of clinical trials involving this drug and also reveal the potential utility of NVP-BEZ235 as an effective radiosensitizer for GBMs in the clinic. Neoplasia (2012) 14, 34–43 Introduction The phosphatidylinositol 3-kinase (PI3K)–Akt-mTORC1 pathway is frequently activated in a variety of human cancers, including glioblastomas (GBMs). Therefore, inhibitors of PI3K/Akt signaling are being actively developed for tumor therapy (reviewed in Liu et al. [1] and Garcia-Echeverria and Sellers [2]). Dual PI3K-mTOR inhibitors are particularly effective in blocking Akt activation because they prevent the feedback activation of PI3K signaling normally observed with mTORC1 inhibitors, such as rapamycin. NVP-BEZ235 is a potent dual PI3K-mTOR inhibitor [3] that has shown great efficacy in inhibiting tumor growth in preclinical mouse models [4–11] and is currently being evaluated in phase 1/2 clinical trials for advanced solid tumors (colorectal, breast, non–small cell lung carcinoma, renal, and sarcoma; reviewed in Garcia-Echeverria and Sellers [2]). The design of these trials is based on the premise that this compound specifically inhibits PI3Ks [half-maximal inhibitory concentration (IC50) = 4-75 nM] and mTOR (IC50 = 20 nM) with little cross-reactivity toward other kinases, as previously reported [3]. However, mTOR belongs to the PI3K-like kinase (PI3KK) family, which includes the two major kinases that respond directly to DNA double-strand breaks (DSBs), ATM and DNA-PKcs, both of which have catalytic domains highly homologous to that of the PI3Ks [1,12]. Because DSBs are induced during radiotherapy and commonly generated during chemotherapy, it is particularly important to understand the effects of NVP-BEZ235 on these two kinases. A number of recent reports identify an important role for the PI3KAkt pathway in radioresistance (reviewed in Mukherjee et al. [13] and Address all correspondence to: Sandeep Burma, PhD, Division of Molecular Radiation Biology, Department of Radiation Oncology, University of Texas Southwestern Medical Center, 2201 Inwood Rd, NC7-214E, Dallas, TX 75390. E-mail: sandeep. burma@utsouthwestern.edu S.B. is supported by grants from the National Institutes of Health (RO1 CA149461), National Aeronautics and Space Administration (NNX10AE08G), and the Cancer Prevention and Research Institute of Texas (RP100644). This article refers to supplementary materials, which are designated by Tables W1 and W2 and Figures W1 to W6 and are available online at www.neoplasia. com. These authors equally contributed to this study. Received 26 October 2011; Revised 23 December 2011; Accepted 3 January 2012 Copyright © 2012 Neoplasia Press, Inc. All rights reserved 1522-8002/12/$25.00 DOI 10.1593/neo.111512 www.neoplasia. com Volume 14 Number 1 January 2012 pp. 34–43 34