mTOR Activation Is Necessary for Primary AML Cells to Survive Genotoxic Stress.

The signaling pathways that regulate the survival of AML cells and whether those pathways can be targeted therapeutically are still poorly defined. We have previously demonstrated that AML cells require PI3 kinase activation for survival but clinical targeting of PI3 kinase itself is not currently feasible. Therefore, we have chosen to study the role of the mammalian target of rapamycin or mTOR protein in the survival of leukemic cells. mTOR mediates the effects of PI3 kinase and can be inhibited by the immunosuppressant, rapamycin. For these studies, patient samples were collected from patients seen at the Hospital of the University of Pennsylvania and we selected samples with greater than 75% blast cells for analysis. Here we show that mTOR is activated in over 90% of patient samples examined as shown by constitutive phosphorylation of the mTOR target protein, p70 S6 kinase (p70S6K). A second substrate, 4EBP1, is phosphorylated in the majority of AML samples but less consistently than p70S6K. Inhibition of mTOR with rapamycin caused only a modest 20% decrease in cell survival based on a 48 hour in vitro assay. Strikingly, however, when AML blasts were incubated with a combination of a topoisomerase II inhibitor, etoposide, and rapamycin, the dose response to etoposide was shifted to the left with the ED50 decreased by at least one log in each of 5 tested samples. Rapamycin inhibited mTOR as shown by inhibition of p70S6K phosphorylation and this inhibition was not altered in the presence of etoposide. To determine if combinations of rapamycin and etoposide would have combined effects on leukemic stem cells, AML cells were incubated in etoposide alone or etoposide with rapamycin for 16 hours and then transplanted into NOD/SCID mice for analysis of stem cell survival. Engraftment of AML cells into NOD/SCID animals was decreased by etoposide and this inhibition was increased by the combination of etoposide and rapamycin. These studies suggest that mTOR regulates a critical cell survival pathway in AML stem cells and this pathway is necessary for survival in the presence of a distinct apoptotic signal. Furthermore, these studies suggest that combinations of rapamycin with chemotherapy may enhance the efficacy of chemotherapeutic agents in the treatment of AML and, therefore, we have recently initiated a Phase I trial of rapamycin in combination with MEC chemotherapy for the treatment of relapsed AML.