These findings are consistent with a recent study in melanoma cells in which dual treatment with the PI3K inhibitor PI-103 and rapamycin reversed compensatory Akt phosphorylation and induced cell cycle arrest, and xenograft studies demonstrated reduced tumor growth with this combination strategy. We extend these findings herein to define a potential mechanism by which the combination therapy promotes cell death. We found that BEZ235 alone blocked PI3K, mTORC1, and mTORC2 activity, in particular 4E-BP1 phosphorylation at a dose of 100 nM. However, BEZ235 was less effective in blocking rS6 phosphorylation. In comparison, temsirolimus completely abrogated phosphorylation of rS6 at 1 nM. Thus, combining both agentscompletely inhibited signaling throughout the pathway and synergistically induced cell death. Currently, combinatorial therapies are being applied to prevent resistance to single-agent treatments such as rapalogs. Examples of targeted small-molecule inhibitors under investigation include BEZ235, AZD2171; LBH589, LY294002, AZD6244, and ZSTK474. BEZ235 is a novel orally bioavailable inhibitor originally designed as a panPI3K family inhibitor based on the p110ckinase domain structure. Interestingly, when this compound was evaluated in preclinical studies, in vitro kinase assays revealed it also targets mTOR at a concentration of 20.7 nM. Therefore, BEZ235 is classified as a dual inhibitor that is capable of targeting both upstreamand downstreamof the PI3K/Akt/mTOR axis. BEZ235 has been reported to inhibit growth and proliferation and induce apoptosis in a variety of tumor cell lines, including breast cancer cells with mutant or amplified PIK3CA. BEZ235 showed antitumor activity in nude mice with few side effects. A recent report from a phase I study of BEZ235 in 59 patients with advanced solid tumors demonstrated antitumor effects and a favorable safety profile. ZSTK474, a pan-class I PI3K inhibitor, also demonstrated high potency against a panel of cancer cell lines and human tumor xenografts without toxicity to major organs. As discussed above, among all drugs tested, the agents which produced synergy with temsirolimus in our models were BEZ235 and ZSTK474. A main conclusion of our study is that combination treatment of ZSTK474 or BEZ235 with temsirolimus synergizes to decrease Y-27632 129830-38-2 viability in endometrial cancer cell lines. A potential mechanism of synergy from co-treatment with ZSTK474 and temsirolimus is the vertical NSC 136476 blockade of hyper-activated PI3K/Akt/mTOR signaling, specifically the simultaneous targeting of the upstream component PI3K by ZSTK474 and the downstream component mTORby temsirolimus. Temsirolimus alone only blocks rS6K activity downstream of mTORC1, whereas signaling through the other mTORC1 target 4E-BP1 is left intact. It has been documented in the literature that signaling through 4E-BP1 is required for Akt-mediated oncogenesis; therefore, inhibition of all components of this pathway is necessary to prevent tumor growth. Our data indicate that, in addition to inhibition of Akt activation, BEZ235 effectively blocks this residual signaling through 4E-BP1, which, when combined with temsirolimus inhibition of rS6K, synergistically blocks all arms of the PI3K/ Akt/mTOR pathway. Besides the observed inhibition of 4E-BP1 and rS6 with combined BEZ235and temsirolimus, another possibility might explain the observed synergy. Temsirolimus and BEZ235 target different structural domains of mTOR: temsirolimus is an allosteric inhibitor that targets the FKBP12-rapamycin-bindingdomain while BEZ235 is a catalytic inhibitor that targets the kinase domain.