[Abstract unavailable

• Genetic-or compound CB-839-induced GAC inhibition reduces OXPHOS and has antileukemic activity in AML.• GAC inhibition synergizes with BCL-2 inhibition by compound ABT-199.Cancer cells require glutamine to adapt to increased biosynthetic activity. The limiting step in intracellular glutamine catabolism involves its conversion to glutamate by glutaminase (GA). Different GA isoforms are encoded by the genes GLS1 and GLS2 in humans. Herein, we show that glutamine levels control mitochondrial oxidative phosphorylation (OXPHOS) in acute myeloid leukemia (AML) cells. Glutaminase C (GAC) is the GA isoform that is most abundantly expressed in AML. Both knockdown of GLS1 expression and pharmacologic GLS1 inhibition by the drug CB-839 can reduce OXPHOS, leading to leukemic cell proliferation arrest and apoptosis without causing cytotoxic activity against normal human CD34 1 progenitors. Strikingly, GLS1 knockdown dramatically inhibited AML development in NSG mice. The antileukemic activity of CB-839 was abrogated by both the expression of a hyperactive GAC K320A allele and the addition of the tricarboxyclic acid cycle product a-ketoglutarate, indicating the critical function of GLS1 in AML cell survival. Finally, glutaminolysis inhibition activated mitochondrial apoptosis and synergistically sensitized leukemic cells to priming with the BCL-2 inhibitor ABT-199. These findings show that targeting glutamine addiction via GLS1 inhibition offers a potential novel therapeutic strategy for AML. (Blood. 2015;126(11):1346-1356

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)

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).

Tyrosine kinase inhibitors have revolutionized the treatment of chronic myeloid leukemia and are increasingly used for other indications. Fluid retention, however, including pleural effusions, are a significant side effect of imatinib, the first-line treatment for chronic myeloid leukemia. We investigated pleural and pulmonary complications in patients treated with dasatinib, a novel multitargeted tyrosine kinase inhibitor, as part of clinical trial protocols. Of 40 patients who received dasatinib (70 mg twice daily) for imatinib resistance or intolerance, 9 (22.5%) developed dyspnea, cough, and chest pain. Of these nine patients, six had pleural effusions (all were exudates) and seven had lung parenchyma changes with either ground-glass or alveolar opacities and septal thickening (four patients had both pleural effusions and lung parenchyma changes). Lymphocytic accumulations were detected in pleural and bronchoalveolar lavage fluids in all patients except for one who presented with neutrophilic alveolitis. Pleural biopsies revealed lymphocytic infiltration in one patient and myeloid infiltration in another. After dasatinib interruption, lung manifestations resolved in all cases and did not recur in three of four patients when dasatinib was reintroduced at a lower dose (40 mg twice daily). Thus, lung physicians should be aware that lung manifestations, presumably related to an immune-mediated mechanism rather than fluid retention, may occur with dasatinib treatment.

The mammalian target of rapamycin (mTOR) and the phosphoinositide 3-kinase (PI3K) signaling pathways are commonly deregulated in cancers and promote cellular growth, proliferation, and survival. mTOR is part of two complexes, mTORC1 and mTORC2, with different biochemical structures and substrates specificity. PI3K/AKT activation may result from genetic hits affecting different components of the pathway, whereas the mechanisms leading to constitutive mTORC1 activation remain globally unknown. The connections between the PI3K and mTOR kinases are multiple and complex, including common substrates, negative feedback loops, or direct activation mechanisms. First-generation allosteric mTOR inhibitors (eg, rapamycin) are mainly active on mTORC1 and mostly display cytostatic anti-tumor activity. Recently, second-generation catalytic mTOR inhibitors targeting both mTOR complexes 1 and 2 have been developed. Some of them also inhibit class IA PI3K. Here, we highlight recent data generated with these new inhibitors against cancer cells and their potential as anti-cancer drugs.