A Mechanism for Synergy with Combined mTOR and PI3 Kinase Inhibitors Shujie Yang1,2, Xue Xiao1,2, Xiangbing Meng1,2, Kimberly K. Leslie1,2* 1 Department of Obstetrics & Gynecology, The University of Iowa, Iowa City, Iowa, United States of America, 2 Holden Comprehensive Cancer Center, The University of Iowa, Iowa City, Iowa, United States of America Abstract Dysregulation of the mammalian target of rapamycin (mTOR) signaling has been found in many human cancers, particularly those with loss of the tumor suppressor PTEN. However, mTORC1 inhibitors such as temsirolimus have only modest activity when used alone and may induce acquired resistance by activating upstream mTORC2 and Akt. Other tumors that do not depend upon PI3K/Akt/mTOR signaling for survival are primarily resistant. This study tested the hypothesis that the limited clinical efficacy of temsirolimus is due to a compensatory increase in survival signaling pathways downstream of Akt as well as an incomplete block of 4E-BP1-controlled proliferative processes downstream of mTOR. We explored the addition of a PI3K inhibitor to temsirolimus and identified the mechanism of combinatorial synergy. Proliferation assays revealed that BEZ235 (dual PI3K/mTOR inhibitor) or ZSTK474 (pan PI3K inhibitor) combined with temsirolimus synergistically inhibited cell growth compared to cells treated with any of the agents alone. Co-treatment resulted in G0/G1 cell cycle arrest and up- regulation of p27. Cell death occurred through massive autophagy and subsequent apoptosis. While molecular profiling revealed that, in most cases, sensitivity to temsirolimus alone was most marked in cells with high basal phospho-Akt resulting from PTEN inactivation, combining a PI3K inhibitor with temsirolimus prevented compensatory Akt phosphorylation and synergistically enhanced cell death regardless of PTEN status. Another molecular correlate of synergy was the finding that temsirolimus treatment alone blocks downstream S6 kinase signaling, but not 4E-BP1. Adding BEZ235 completely abrogated 4E-BP1 phosphorylation. We conclude that the addition of a PI3K inhibitor overcomes cellular resistance to mTORC1 inhibitors regardless of PTEN status, and thus substantially expands the molecular phenotype of tumors likely to respond. Citation: Yang S, Xiao X, Meng X, Leslie KK (2011) A Mechanism for Synergy with Combined mTOR and PI3 Kinase Inhibitors. PLoS ONE 6(10): e26343. doi:10.1371/journal.pone.0026343 Editor: Eric J. Bernhard, National Cancer Institute, United States of America Received June 6, 2011; Accepted September 25, 2011; Published October 19, 2011 Copyright: 2011 Yang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was supported in part by NIH R01CA99908 to KKL and Department of Obstetrics and Gynecology Research Development Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: kimberly-leslie@uiowa.edu Introduction Alterations in the phosphoinositide-3-kinase (PI3K)/Akt/mam- malian target of rapamycin (mTOR) signaling pathway have been found in many human tumors. In particular, amplification and mutation of PIK3CA, mutation of PIK3R and Akt, and loss of tumor suppressor PTEN (phosphatase and tensin homolog deleted from chromosome 10) contribute to constitutive activation of this signaling pathway [1,2,3,4]. Understanding the interplay among signaling molecules in the PI3K/Akt/mTOR pathway is of utmost importance. Two distinct mTOR complexes, mTORC1 and mTORC2, have been identified and have differential sensitivity to rapamycin. mTORC1 is downstream of Akt, sensitive to rapamycin inhibition, and controls cap-dependent protein trans- lation [5]. The two best-studied mTORC1 substrates are 40S ribosomal S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), which mediate efficient protein translation. In contrast, mTORC2 is directly upstream of Akt and is resistant to rapamycin. Akt can be activated by phosphorylation at two different sites, S473 by mTORC2 and T308 by phosphoinositide-dependent kinase 1 (PDK1). Constitu- tive activation of the PI3K/Akt/mTOR signaling axis leads to uncontrolled tumor cell proliferation and survival [1]. Given the importance of the mTOR pathway in cancer cell growth, significant efforts have attempted to identify targeted inhibitors. Rapamycin and its analogs (rapalogs), such as RAD001 (everolimus), AP23573 (ridaforolimus) and CCI-779 (temsirolimus) are allosteric inhibitors of mTOR [6]. However, single agent rapalogs have only achieved modest antitumor activity in the clinic [7]. The limited anticancer efficacy of the rapalogs can be explained by two possible mechanisms: (1) rapalogs inhibit only mTORC1 (not mTORC2), thereby inducing feedback activation of survival signaling pathways such as Akt phosphorylation [7,8,9]; or (2) rapalogs incompletely block mTORC1 downstream signaling. For example, in some cells mTOR inhibitors prevent phosphorylation of S6K1 but not 4E-BP1, thus allowing the cells to escape growth inhibition [10,11,12]. Previous studies indicate that PTEN inactivation, PIK3CA mutation, and mTOR dysregulation are common molecular signatures for endometrial carcinoma [1,13]. Furthermore, PI3K activation is a hallmark for aggressive tumors at this site [14]. mTOR inhibitors (temsirolimus, everolimus, and ridaforolimus) have been tested in phase I and II clinical trials for advanced and recurrent endometrial carcinomas with some promising clinical outcomes; however, response rates are not robust. In general, responses are partial and vary from 8%–26% with an additional PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26343