Rapamycin derivatives reduce mTORC2 signaling and inhibit AKT activation in AML

Abstract

The mTOR complex 2 (mTORC2) containing mTOR and rictor is thought to be rapamycin insensitive and was recently shown to regulate the prosurvival kinase AKT by phosphorylation on Ser473. We investigated the molecular effects of mTOR inhibition by the rapamycin derivatives (RDs) temsirolimus (CCI-779) and everolimus (RAD001) in acute myeloid leukemia (AML) cells. Unexpectedly, RDs not only inhibited the mTOR complex 1 (mTORC1) containing mTOR and raptor with decreased p70S6K, 4EPB1 phosphorylation, and GLUT1 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 cells. Similar observations were made in samples from patients with hematologic malignancies who received RDs in clinical studies. Our study provides the first evidence that rapamycin derivatives inhibit AKT signaling in primary AML cells both in vitro and in vivo, and supports the therapeutic potential of mTOR inhibition strategies in leukemias.

Figures

Figure 1

Rapamycin derivatives inhibit mTORC1 and mTORC2 signaling in AML cell lines and in primary AML samples. (A-B) U937 cells (A) or cell lysates (B) were treated with different concentrations of CCI-779 for 24 hours. Cell lysates and mTOR immunoprecipitates prepared from the lysates were analyzed by Western blot for the levels of mTOR, rictor, and raptor. (C) U937 cells were treated with indicated concentrations of CCI-779 for 24 hours, and cell lysates were analyzed by immunoblotting for the indicated proteins and phosphorylation states. (D) The effects of mTOR inhibition on transcriptional level of CCND1/CCND2 and GLUT1 were assessed via real-time PCR. Error bars denote half the difference between the maximum and minimum values that arose on substituting ΔCt − SD or ΔCt + SD, respectively, for ΔCt in the formula RE = 100 × 2 exp [−ΔCt]. (E) OCI-AML3 cells were treated with indicated concentrations of CCI-779 and RAD001 for 24 hours. The level of mTOR, rictor, and raptor from cell lysates and mTOR immunoprecipitates was evaluated by Western blot. (F-G) The effect of mTOR inhibition on mTOR upstream regulators (Akt) and downstream targets was detected by Western blot (F) and real-time PCR (G). (H) Primary AML blasts or cell lysates from 2 patients' samples were treated with CCI-779 for 24 hours, and immunoprecipitation of mTOR was carried out as described in panel A. Representative results of 2 of the 8 primary samples yielded similar results. (I-J) Effects of mTOR inhibition on downstream mTOR and AKT substrates were examined by immunoblotting of cell lysates from patient no. 1 (I), and on the transcriptional levels of CCND1/CCND2 and glut-1 by real-time PCR (J).

Figure 2

Rapamycin derivatives inhibit AKT signaling in leukemic cells in vivo. (A) Peripheral blood mononuclear cells from patients treated with either everolimus or temsirolimus were subjected to immunoblotting analyses of pAKT, total AKT, and GAPDH, and (B) quantitative real-time PCR analysis of CCND1/CCND2 and GLUT1 transcription. The data shown are derived from TaqMan PCR analyses of these genes. (C) Changes in white blood cell count (WBC, 109/L) and absolute blast count (ABC, 109/L) during temsirolimus treatment. (i) Patient with relapsed refractory pre-B-cell acute lymphoblastic leukemia (pre-B-ALL) received 3 doses of temsirolimus at a dose of 25 mg intravenously every week (indicated by arrows). (ii) Patient with primary refractory AML has completed 2 courses of temsirolimus (4 weekly injections each, at a dose of 25 mg intravenously every week) and received 2 doses of temsirolimus in course 3.