mTOR and PI3 Kinase Inhibitor Synergy Figure 7. Mechanism of synergistic effect for combination treatment of temsirolimus and BEZ235. A, Phosphorylation of 4E-BP1 (P-4E- BP1 T37/T46) was assessed after incubating cells with the indicated treatments for 24 hrs. Total 4E-BP1 expression serves as a loading control. B, Cells were treated with 1 nM or 10 nM temsirolimus or BEZ235 for 72 hrs then phosphorylation of rS6 (P-rS6 S235/S236) determined by Western blotting. Total rS6 serves as a loading control. C, Proposed mechanism for synergistic effect. Temsirolimus blocks one arm of mTORC1 signaling as evidenced by lack of phosphorylation of rS6. As a dual inhibitor of PI3K and mTOR, BEZ235 acts both upstream as well as downstream of mTORC1 and mTORC2 through inhibition of Akt (upstream) and 4E-BP1 (downstream) phosphorylation. BEZ235 has minimal effects on rS6 phosphorylation, but combination with temsirolimus blocks both arms of mTORC1 signaling. As a specific PI3K inhibitor, ZSTK474 acts upstream of the PI3K/Akt/mTOR signaling pathway, and in combination with temsirolimus, can block rS6 but not 4E-BP1 activation. doi:10.1371/journal.pone.0026343.g007 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 abro- gated phosphorylation of rS6 at 1 nM. Thus, combining both agents (BEZ235 and temsirolimus) completely 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 (dual PI3K/mTOR inhibitor) [27], AZD2171 (dual VEGF2/PDGFR inhibitor); LBH589 (histone deacetylase inhib- itor) [23], LY294002 (PI3K inhibitor) [24], AZD6244 (MEK inhibitor) [25], and ZSTK474 (PI3K inhibitor) [34]. BEZ235 is a novel orally bioavailable inhibitor originally designed as a pan- PI3K family inhibitor based on the p110c (catalytic subunit of PI3K) kinase domain structure [6,27]. 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 [27]. Therefore, BEZ235 is classified as a dual inhibitor that is capable PLoS ONE | www.plosone.org 10 October 2011 | Volume 6 | Issue 10 | e26343