Key Points• Glutamine removal and knockdown of the glutamine transporter SLC1A5 have antileukemic activity in AML.• The glutaminase activity of L-asparaginase inhibits mTORC1 and protein synthesis and induces a strong autophagy in AML.Cancer cells require nutrients and energy to adapt to increased biosynthetic activity, and protein synthesis inhibition downstream of mammalian target of rapamycin complex 1 (mTORC1) has shown promise as a possible therapy for acute myeloid leukemia (AML). Glutamine contributes to leucine import into cells, which controls the amino acid/Rag/mTORC1 signaling pathway. We show in our current study that glutamine removal inhibits mTORC1 and induces apoptosis in AML cells. The knockdown of the SLC1A5 high-affinity transporter for glutamine induces apoptosis and inhibits tumor formation in a mouse AML xenotransplantation model. L-asparaginase (L-ase) is an anticancer agent also harboring glutaminase activity. We show that L-ases from both Escherichia coli and Erwinia chrysanthemi profoundly inhibit mTORC1 and protein synthesis and that this inhibition correlates with their glutaminase activity levels and produces a strong apoptotic response in primary AML cells. We further show that L-ases upregulate glutamine synthase (GS) expression in leukemic cells and that a GS knockdown enhances L-ase-induced apoptosis in some AML cells. Finally, we observe a strong autophagic process upon L-ase treatment. These results suggest that L-ase anticancer activity and glutamine uptake inhibition are promising new therapeutic strategies for AML. (Blood. 2013;122(20):3521-3532)

Key Points An increase in the classical monocyte subset to >94% of total monocytes discriminates CMML from other monocytoses with high specificity. This characteristic increase in classical monocytes disappears in CMML patients who respond to hypomethylating agents.

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...

The deregulation of translation markedly contributes to the malignant phenotype in cancers, and the assembly of the translation initiating complex eIF4F is the limiting step of this process. The mammalian Target of Rapamycin Complex 1 (mTORC1) is thought to positively regulate eIF4F assembly and subsequent oncogenic protein synthesis through 4E-BP1 phosphorylation. We showed here that the translation inhibitor 4EGI-1 decreased the clonogenic growth of leukemic progenitors and induced apoptosis of blast cells, with limited toxicity against normal hematopoiesis, which emphasize the importance of translation deregulation in acute myeloid leukemia (AML) biology. However, the mTORC1 inhibitor RAD001 (a rapamycin derivate) did not induce AML blast cell apoptosis. We herein demonstrated that mTORC1 disruption using raptor siRNA or RAD001 failed to inhibit 4E-BP1 phosphorylation in AML. Moreover, RAD001 failed to inhibit eIF4F assembly, to decrease the proportion of polysome-bound c-Myc mRNA, and to reduce the translation-dependent accumulation of oncogenic proteins. We identified the Pim-2 serine/threonine kinase as mainly responsible for 4E-BP1 phosphorylation on the S 65 residue and subsequent translation control in AML. Our results strongly implicate an mTORC1-independent deregulation of oncogenic proteins synthesis in human myeloid leukemogenesis. Di IntroductionAcute myeloid leukemia (AML) is a clonal hematologic disease characterized by differentiation arrest and by the proliferation of immature myeloid progenitors, both sustained by the deregulation of multiple signaling pathways. 1 Despite recent advances in the understanding of AML biology, the prognosis of this disease remains poor and new therapeutic perspectives are therefore under active investigation. 2 In most cancer models, the activity of mammalian Target of Rapamycin Complex 1 (mTORC1) is dependent on the activation of Akt, an oncoprotein that operates downstream of class IA phosphoinositide 3-kinase (PI3K). 3 In AML, mTORC1 is frequently activated, 4 but recent evidence has suggested that this activity does not rely on PI3K/Akt. These findings have shown that the p110␦ isoform of PI3K is principally responsible for PI3K activity 5 and that a specific p110␦ inhibitor, IC87114, fully inhibits Akt phosphorylation without affecting mTORC1 activity. 6 Moreover, the Src kinase Lyn has recently been shown to control mTORC1 activity but not Akt phosphorylation in primary AML cells. 7 The mTORC1 complex consists of mTOR, raptor, mLST8, and PRAS40. 8 It governs cell growth and regulates the cap-dependent translation of mRNAs. 9 Rapamycin and derivates, referred to as rapalogs (eg, RAD001), are closely related molecules demonstrating similar biologic activities by specifically repressing mTORC1 activity. 10,11 These compounds have recently been developed as anticancer therapeutics because of the frequent activation of mTORC1 in cancers and approved for clinical use. 12 Despite their seemingly clear mechanism of action and the rationale for their use as a canc...

Groupe Ouest Est des Leucémies et Autres Maladies du Sang (GOELAMS), FranceThe PI3K/AKT and mTOR signaling pathways are activated in acute myeloid leukemia, including in the more immature leukemic populations. Constitutive PI3K activation is detectable in 50% of acute myeloid leukemia samples whereas mTORC1 is activated in all cases of this disease. In leukemic cells, the PI3K activity relates to the expression of the p110d isoform of class IA PI3K. Constitutive PI3K activation is the result of autocrine IGF-1/IGF-1R signaling in 70% of acute myeloid leukemia samples but specific inhibition of this pathway does not induce apoptosis. Specific inhibition of PI3K/AKT or mTORC1 alone in vitro has antileukemic effects which are essentially exerted via the suppression of proliferation. However, as mTORC1 activation is independent of PI3K/AKT in acute myeloid leukemia, dual PI3K and mTOR inhibitors may induce apoptosis in blast cells. Moreover, mTORC1 inhibition using sirolimus overactivates PI3K/AKT via the upregulation of IRS2 expression and by favoring IGF-1/IGF-1R autocrine signaling. Recent data also indicate that mTORC1 does not control protein translation in acute myeloid leukemia. These results open the way for the design of direct inhibitors of protein synthesis as novel acute myeloid leukemia therapies and also for the development of second generation mTOR inhibitors (the TORKinhibs).

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Finding an effective treatment for acute myeloid leukemia (AML) remains a challenge, and all cellular processes that are deregulated in AML cells should be considered in the design of targeted therapies. We show in our current study that the LKB1/AMPK/TSC tumor suppressor axis is functional in AML and can be activated by the biguanide molecule metformin, resulting in a specific inhibition of mammalian target of rapamycin (mTOR) catalytic activity. This induces a multisite dephosphorylation of the key translation regulator, 4E-BP1, which markedly inhibits the initiation step of mRNA translation. Consequently, metformin reduces the recruitment of mRNA molecules encoding oncogenic proteins to the polysomes, IntroductionIn acute myeloid leukemia (AML), the oncogenic deregulation of mRNA translation markedly contributes to the malignant phenotype. However, the efficacy of clinically available therapies that target this process, such as rapamycin, remains moderate due to the induction of multiple resistance mechanisms, 1 and new approach should be considered to bypass these processes.The liver kinase B1 (LKB1) serine/threonine kinase is encoded by the tumor-suppressor gene, STK11, which harbors germ-line mutations in the inherited cancer predisposition, Peutz-Jeghers syndrome, and somatic mutations in sporadic cancers. 2 The search for substrates of LKB1 that mediate its tumor-suppressor function led to the identification of the LKB1/AMPK/TSC adenosine monophosphate-activated protein kinase (AMPK) as a direct LKB1 substrate. 3 AMPK is a heterotrimeric complex comprising a catalytic ␣-and 2 regulatory ␤-and ␥-subunits, and LKB1 enhances AMPK activity through the phosphorylation of the ␣-subunit at T 172 . 4 AMPK is allosterically activated by the accumulation of AMP molecules, due to metabolic stresses that inhibit adenosine triphosphate (ATP) production (eg, hypoxia, glucose deprivation) or stimulate ATP consumption 5 and is also activated by drugs used to treat type 2 diabetes, including metformin. 6 AMPK is therefore the main cellular energy sensor, acting as a central negative regulator of metabolic pathways, such as fatty acid oxidation and glucose consumption. 7 The LKB1/AMPK pathway also regulates the protein synthesis rate through the control of the serine/threonine kinase mammalian target of rapamycin (mTOR), 8 a process that is consistently deregulated in AML cells. 9 When activated, AMPK stimulates tuberous sclerosis complex 1/2 (TSC 1/2 ), which comprises the TSC1/hamartin and TSC2/tuberin proteins, through AMPK-mediated TSC2 phosphorylation at the T 1227 and S 1345 residues. 10 This stimulates the GTPase-activating protein (GAP) function of TSC2 toward the small G-protein Rheb (Ras homolog enriched in brain), and increases the pool of guanosine diphosphate (GDP)-bound Rheb molecules. Although the molecular mechanism underlying mTOR activation by Rheb-guanosine triphosphate (GTP) is still under debate, [11][12][13] it is well established that TSC2 activation switches off Rheb, resulting in the inhibition o...