MTOR Inhibitor Rapamycin Interacts Synergistically with Farnesyltransferase Inhibitor FTI-277 To Induce Growth Inhibition in Human Leukemia Cells.

Abstract

Proteins regulating the mammalian target of rapamycin (mTOR), as well as some of the targets of the mTOR kinase, are overexpressed or mutated in cancer. Rapamycin inhibits the growth of cell lines derived from multiple tumor types in vitro, and tumor models in vivo. However, it has been suggested that substantial proportion of acute myeloid leukemia (AML) cells showed resistance to rapamycin-induced growth inhibition.Aim: We aim to investigate the effects of the farnesyltransferase inhibitor (FTI)-277 on the rapamycin-induced growth inhibition of human leukemia cells.Patients and methods: Flow cytometric evaluation and Western blot analysis for mTOR and Ras-like GTPase Rheb expression in the leukemia cell lines (HL60,NB4,THP1,KG1,U937) and primary leukemia cells obtained from AML patients were performed. We also observed the inhibition of cell growth and the changes in expression of mTOR and up- or down-streams of mTOR after mTOR inhibitor rapamycin treatment with or without FTI-277.Results: Both flow cytometric evaluation and Western blot analysis demonstrated that mTOR expression in the leukemia cell lines (HL60, NB4, THP1, KG1, U937) and primary leukemia cells obtained from AML patients were significantly higher compared to normal bone marrow mononuclear cells (p<0.001). Expression of Ras-like GTPase Rheb, a mTOR upstream, was also significantly increased in the leukemia cell lines and primary AML cells compared to normal bone marrow mononuclear (p<0.001 and p<0.005, respectively). We observed the inhibition rate of leukemia cell growth after treatment of cells with mTOR inhibitor rapamycin (100mM) in the absence or presence of farnesyltransferase inhibitor FTI-277 (10mM). Clonogenic cell growth in the leukemia cell lines was 69.3 ± 5.3% in the rapamycin group and 78.7 ± 4.4% in the FTI-277 group compared to that of the control group. Cotreatment of THP1 and HL-60 leukemia cells with rapamycin and FTI-277 exerted synergistic decrease in the clonogenic cell growth, as well as arrest at the G2/M phase of cell cycle, in a dose-and time-dependent manner (p<0.01). This was associated with marked attenuation of protein levels of Rheb, phospho-mTOR, and mTOR downstreams phospho-p70S6 kinase, phospho-4E-BP1. Interestingly decreased expression of mTOR upstreams Akt/PKB activity, Akt/PKB phosphorylation and PTEN phosphorylation was also observed in these leukemia cells after cotreatment with FTI-277 and rapamycin. These findings were also observed in the primary leukemia cells obtained from untreated patients with AML.Conclusions: Taken together, these findings indicate that farnesyltransferase inhibitor FTI-277 potentially enhance the growth-inhibitory property of rapamycin, with inducing multiple perturbations in PI3K - Akt/PKB - mTOR signaling pathway in human leukemia cells. Combined rapamycin and FTI blockade can exert powerful anti-leukemia effects and could be developed into a novel therapeutic strategy for AML.