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 AML cells. Glutaminase C (GAC) is the GA isoform that is most abundantly expressed in AML. Both knockdown of GLS1 expression and pharmacological 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⁺ progenitors. Strikingly, a GLS1-knockdown dramatically inhibited AML development in NSG mice. The anti-leukemic activity of CB-839 was abrogated by both the expression of a hyperactive GAC^K320A allele and the addition of the TCA cycle product α-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.

The 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 p110delta 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 anti-leukemic 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).

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.

The phosphatidylinositol 3-kinase (PI3K)/Akt and mTORC1 pathways are frequently activated, representing potential therapeutic targets in acute myeloid leukemia (AML). In 19 AML samples with constitutive PI3K/Akt activation, the rapamycin derivative inhibitor everolimus (RAD001) increased Akt phosphorylation. This mTOR C1-mediated Akt up-regulation was explained by an insulin-like growth factor-1 (IGF-1)/IGF-1 receptor autocrine loop: (1) blast cells expressed functional IGF-1 receptors, and IGF-1-induced Akt activation was increased by RAD001, (2) a neutralizing anti-IGF-1R α-IR3 monoclonal antibody reversed the RAD001-induced Akt phosphorylation, and (3) autocrine production of IGF-1 was detected in purified blast cells by quantitative reverse transcription-polymerase chain reaction and immunofluorescence. This RAD001-induced PI3K/Akt up-regulation was due to an up-regulated expression of the IRS2 adaptor. Finally, we observed that concomitant inhibition of mTORC1 and PI3K/Akt by RAD001 and IC87114 induced additive antiproliferative effects. Our results suggest that dual inhibition of the mTORC1 complex and the IGF-1/IGF-1R/PI3K/Akt pathway in AML may enhance the efficacy of mTOR inhibitors in treatment of this disease.

Acute myeloid leukemias arise from a rare population of leukemic cells, known as leukemic stem cells, which initiate the disease and contribute to frequent relapses. Although the phenotype of these cells remains unclear in most patients, these cells are enriched within the CD34(+)CD38(low/-) compartment expressing the interleukin-3 alpha chain receptor, CD123. The aim of this study was to determine the prognostic value of the percentage of blasts with the CD34(+)CD38(low/-)CD123(+) phenotype. The percentage of CD34(+)CD38(low/-)CD123(+) cells in the blast population was determined at diagnosis using flow cytometry. One hundred and eleven patients under 65 years of age with de novo acute myeloid leukemia and treated with intensive chemotherapy were retrospectively included in the study. Correlations with complete response, disease-free survival and overall survival were evaluated with univariate and multivariate analyses. A proportion of CD34(+)CD38(low/-)CD123(+) cells greater than 15% at diagnosis and an unfavorable karyotype were significantly correlated with a lack of complete response. By logistic regression analysis, a percentage of CD34(+)CD38(low/-)CD123(+) higher than 15% retained significance with an odds ratio of 0.33 (0.1-0.97; P=0.044). A greater than 1% population of CD34(+)CD38(low/-)CD123(+) cells negatively affected disease-free survival (0.9 versus 4.7 years; P<0.0001) and overall survival (1.25 years versus median not reached; P<0.0001). A greater than 1% population of CD34(+)CD38(low/-)CD123(+) cells retained prognostic significance for both parameters after multivariate analysis. The percentage of CD34(+)CD38(low/-)CD123(+) leukemic cells at diagnosis was significantly correlated with response to treatment and survival. This prognostic marker might be easily adopted in clinical practice to rapidly identify patients at risk of treatment failure.