Rapamycin Analogs Reduce mTORC2 Signaling and Inhibit AKT Activation in AML.

The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin-insensitive, and was recently shown to regulate the pro-survival kinase AKT by phosphorylation on Ser473 (Sarbassov Science 2005;307 and Mol Cell 2006;22). We investigated the molecular effects of mTOR inhibition by rapamycin analog CCI-779 in AML cells. Unexpectedly, CCI-779 not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased phosphorylation of p70S6K, 4EPB1 and reduction in Glut-1 mRNA, but also blocked AKT activation via inhibition of mTORC2 formation. This resulted in suppression of phosphorylation of the direct AKT substrate FKHR and decreased transcription of D-Cyclins in AML cell line and 5 of 8 primary AML samples in vitro. Similar observations were made in samples from patients with hematological malignancies who were treated with the rapamycin analogs temsirolimus or everolimus: the levels of Ser473 phosphorylated AKT decreased in 3/5 patient samples at 1 or 24 hour(s) of temsirolimus treatment, and in 6/8 patient samples treated with everolimus. In the 9 samples in which AKT was inhibited, ≥2-fold decrease in Cyclin D1 mRNA was observed in 5, Cyclin D2 in 3, both, Cyclin D1 and D2 in 1 sample, and Glut-1 in 4 patient samples. In 7 of the 9 patients in whom AKT was inhibited, a >50% decrease in peripheral blood absolute blast count (3 AML, 1 ALL) or absolute lymphocyte count (1 CLL) for >1 week duration was documented, and two patients with RAEB-1 had improvements in platelet counts, one fulfilling the criteria for clinical response (hematological improvement). No change in peripheral blood counts or progression of leukemia was seen in 6 patients. Of these, decrease in pAKT was observed in 2, no change in 3 and increase in 1 (Fisher exact two-tailed p= 0.021). Altogether, our study provides first evidence that rapamycin analogs inhibit AKT signaling in primary AML cells both in vitro and in vivo, and support the therapeutic potential of mTOR inhibition strategies in leukemias.