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).