A Mechanism for Synergy with Combined mTOR and PI3 Kinase Inhibitors
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.