The mTOR Inhibitor Rapamycin Demonstrates Activity Against AML in Combination with Imatinib Mesylate and with 5-Azacytidine.

The serine/threonine kinase mTOR (mammalian target of rapamycin) integrates pathways involved in cell cycle regulation and apoptosis and has been shown to be aberrantly activated in AML. mTOR (mammalian target of rapamycin) serine threonine kinase is involved in cell cycle regulation and apoptosis. mTOR inhibitors are a class of signal transduction inhibitors, originally developed as immunosuppressive agents, with antineoplastic activity in solid tumor models. Acute myelogenous leukemia blasts and stem cells have been reported previously to be inhibited by rapamycin, an mTOR inhibitor. As a single agent, rapamycin has minimal activity, however, suggesting that for meaningful clinical efficacy, it would require combination with other agents. We have therefore begun to examine its effects in combination with other signal transduction inhibitors and demethylating agents. Rapamycin by itself had minimal inhibitory effect on AML cell lines or primary AML samples. However, in combination with the c-kit inhibitor, imatinib mesylate, 2.5–25 uM, rapamycin demonstrated synergistic inhibition of AML cell lines such as MV411 as measured in a standard MTT assay (combination index 0.32 to 0.576 as determined with the Calcusyn program),, indicating strong synergism. In this cell line, rapamycin by itself demonstrated no significant inhibitory activity. Synergism with imatinib mesylate was also noted in the U937 and HL60 cell lines but not in the KG1a cell line. The combination of rapamycin and imatinib inhibited AKT phosphorylation to a greater extent than either agent alone in both the MV411 and U937 cell lines, but the combination did not effectively inhibit ERK phosphorylation. Inhibition of phosphorylation of p70S6 kinase by the combination of 20 uM imatinib mesylate and 80uM rapamycin did not occur to a greater extent than that seen with rapamycin alone, although phosphorylation of 4EBP1 was inhibited to a greater extent when both agents were added concurrently. When AML cell lines were exposed to 5-azacytidine alone, 1 to 16 uM, the IC50 was 4–8uM. 5-azacytidine increased apoptosis above control whereas rapamycin by itself had minimal effect on apoptosis as assessed in an Annexin V flow cytometric assay. In the U937 cell line, the combination index for the U937 cell line was 0.465 (10 uM 5-azacytidine and 100 nM rapamycin), indicating synergism. For MV411, the combination index was 0.736, for KG1a, 0.118, and for HL60, no synergism was noted. Exposure to the combination of 5-azacytidine and rapamycin resulted in greater suppression of AKT and 4EBP1 phosphorylation than did either agent singly. These data suggest that simultaneous targeting of more than one signaling pathway may improve inhibition of leukemic cell growth. Furthermore, combining signal transduction inhibitors with hypomethylating agents may also result in effective suppression of AML proliferation.