Metabolic reprogramming has been described as a hallmark of transformed cancer cells. In this study, we examined the role of the glutamine (Gln) utilization pathway in acute myeloid leukemia (AML) cell lines and primary AML samples. Our results indicate that a subset of AML cell lines is sensitive to Gln deprivation. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes the conversion of Gln to glutamate. One of the two GLS isoenzymes, GLS1 is highly expressed in cancer and encodes two different isoforms: kidney (KGA) and glutaminase C (GAC). We analyzed mRNA expression of GLS1 splicing variants, GAC and KGA, in several large AML datasets and identified increased levels of expression in AML patients with complex cytogenetics and within specific molecular subsets. Inhibition of glutaminase by allosteric GLS inhibitor bis-2-(5-phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide or by novel, potent, orally bioavailable GLS inhibitor CB-839 reduced intracellular glutamate levels and inhibited growth of AML cells. In cell lines and patient samples harboring IDH1/IDH2 (Isocitrate dehydrogenase 1 and 2) mutations, CB-839 reduced production of oncometabolite 2-hydroxyglutarate, inducing differentiation. These findings indicate potential utility of glutaminase inhibitors in AML therapy, which can inhibit cell growth, induce apoptosis and/or differentiation in specific leukemia subtypes.

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Despite high initial response rates, acute myeloid leukemia (AML) treated with the BCL-2–selective inhibitor venetoclax (VEN) alone or in combinations commonly acquires resistance. We performed gene/protein expression, metabolomic and methylation analyses of isogenic AML cell lines sensitive or resistant to VEN, and identified the activation of RAS/MAPK pathway, leading to increased stability and higher levels of MCL-1 protein, as a major acquired mechanism of VEN resistance. MCL-1 sustained survival and maintained mitochondrial respiration in VEN-RE cells, which had impaired electron transport chain (ETC) complex II activity, and MCL-1 silencing or pharmacologic inhibition restored VEN sensitivity. In support of the importance of RAS/MAPK activation, we found by single-cell DNA sequencing rapid clonal selection of RAS-mutated clones in AML patients treated with VEN-containing regimens. In summary, these findings establish RAS/MAPK/MCL-1 and mitochondrial fitness as key survival mechanisms of VEN-RE AML and provide the rationale for combinatorial strategies effectively targeting these pathways.

BackgroundCalorie restriction (CR) prevents obesity and exerts anticancer effects in many preclinical models. CR is also increasingly being used in cancer patients as a sensitizing strategy prior to chemotherapy regimens. While the beneficial effects of CR are widely accepted, the mechanisms through which CR affects tumor growth are incompletely understood. In many cell types, CR and other nutrient stressors can induce autophagy, which provides energy and metabolic substrates critical for cancer cell survival. We hypothesized that limiting extracellular and intracellular substrate availability by combining CR with autophagy inhibition would reduce tumor growth more effectively than either treatment alone.ResultsA 30 % CR diet, relative to control diet, in nude mice resulted in significant decreases in body fat, blood glucose, and serum insulin, insulin-like growth factor-1, and leptin levels concurrent with increased adiponectin levels. In a xenograft model in nude mice involving H-RasG12V-transformed immortal baby mouse kidney epithelial cells with (Atg5+/+) and without (Atg5−/−) autophagic capacity, the CR diet (relative to control diet) genetically induced autophagy inhibition and their combination, each reduced tumor development and growth. Final tumor volume was greatest for Atg5+/+ tumors in control-fed mice, intermediate for Atg5+/+ tumors in CR-fed mice and Atg5−/− tumors in control-fed mice, and lowest for Atg5−/− tumors in CR mice. In Atg5+/+ tumors, autophagic flux was increased in CR-fed relative to control-fed mice, suggesting that the prosurvival effects of autophagy induction may mitigate the tumor suppressive effects of CR. Metabolomic analyses of CR-fed, relative to control-fed, nude mice showed significant decreases in circulating glucose and amino acids and significant increases in ketones, indicating CR induced negative energy balance. Combining glucose deprivation with autophagy deficiency in Atg5−/− cells resulted in significantly reduced in vitro colony formation relative to glucose deprivation or autophagy deficiency alone.ConclusionsCombined restriction of extracellular (via CR in vivo or glucose deprivation in vitro) and intracellular (via autophagy inhibition) sources of energy and nutrients suppresses Ras-driven tumor growth more effectively than either CR or autophagy deficiency alone. Interventions targeting both systemic energy balance and tumor-cell intrinsic autophagy may represent a novel and effective anticancer strategy.

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