We analyzed prognostic factors of response, response duration, and possible impact on survival of epoetin ␣, epoetin ␤, or darbepoetin ␣ (DAR) with or without granulocyte colony-stimulating factor in 403 myelodysplastic syndrome (MDS) patients. Sixty-two percent (40% major and 22% minor) and 50% erythroid responses were seen, and median response dura-

IntroductionAcute myeloid leukemia (AML) is associated with a low survival rate. Therefore, new therapeutic strategies may prove effective in addition to chemotherapy.The deregulation of several signal transduction pathways is a common feature in AML. The phosphatidylinositol 3-kinase (PI3K)/ Akt pathway is activated in AML blast cells. [1][2][3][4][5] We showed previously that the p110 ␦ isoform of PI3K is a potential therapeutic target and that the p110 ␦-selective inhibitor IC87114 blocks AML cell proliferation. 6,7 The mammalian target of rapamycin (mTOR) is activated in response to stimuli activating the PI3K pathway, 8 and mTORC1 inhibitors may have therapeutic value in the treatment of patients with AML 9,10 ; however, rapamycin alone led to modest antileukemic activity. 10 We investigated the effect of the mTORC1 inhibitor RAD001 (Everolimus; Novartis Pharmaceuticals, Basel, Switzerland) in 19 bone marrow samples from patients with newly diagnosed AML. We show that mTORC1 inhibition with RAD001 increased Akt activating phosphorylation, as a result of up-regulated expression of the IRS-2 protein adaptor that promoted insulin-like growth factor-1 (IGF-1) /IGF-1R signaling. Moreover, we show that the enhanced activation of Akt was dependent on the IGF-1 autocrine production by leukemic cells. Our results provide a rationale for combined inhibition of both mTORC1 and PI3K/Akt pathways in AML, and we observed an additive effect of both RAD001 and IC87114 on blast cell proliferation. MethodsBone marrow (BM) samples were obtained from 19 patients with newly diagnosed AML, all treated in the AML2001 chemotherapy trial, initiated by the French Multicenter Group, Groupe Ouest Est des Leucémies et Autres Maladies du Sang (GOELAMS). All biologic studies were approved by the GOELAMS Institutional Review Board, and signed informed consent was obtained in accordance with the Declaration of Helsinki. The clinical characteristics of patients are summarized in Table S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). All patients presented a constitutive activation of Akt at diagnosis, as reported previously. 5 Cells were incubated with the following inhibitors: RAD001 (Everolimus) kindly provided by Novartis, IC87114 provided by ICOS (Bothell, WA), LY294002 from Sigma (St Louis, MO), and ␣IR3 from Calbiochem (La Jolla, CA). IGF-1 was from Sigma. Expression of total and phosphorylated proteins was detected by Western blot (WB) analysis as reported previously. 7 The references for the antibodies are summarized in Table S2. Immunofluorescence staining for IGF-1 expression and quantitative reverse transcription-polymerase chain reaction (RT-PCR) were performed on blast cells, sorted according to their CD45 low expression and side scatter (see Document S1 for details). Blast cell BM blast cells from patients G192 and G194 were collected after Ficoll-Hypaque density gradient separation, then washed once in PBS buffer. Blast cells (5 ϫ 10 5 /mL) from patient G192 were starv...