glutaminase and alpha-hydroxyglutarate

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.

Topics: Antineoplastic Agents; Apoptosis; Benzeneacetamides; Cell Differentiation; Cell Proliferation; Energy Metabolism; Enzyme Inhibitors; Glutamate Dehydrogenase; Glutamic Acid; Glutaminase; Glutamine; Glutarates; Humans; Isocitrate Dehydrogenase; Leukemia, Myeloid, Acute; Mutation; Protein Isoforms; Sulfides; Thiadiazoles

Since polycystic kidney disease (PKD) was first noted over 30 years ago to have neoplastic parallels, there has been a resurgent interest in elucidating neoplasia-relevant pathways in PKD. Taking a nontargeted metabolomics approach in the B6(Cg)-Cys1(cpk/)J (cpk) mouse model of recessive PKD, we have now characterized metabolic reprogramming in these tissues, leading to a glutamine-dependent TCA cycle shunt toward total 2-hydroxyglutarate (2-HG) production in cpk compared with B6 wild-type kidney tissue. After confirmation of increased 2-HG expression in immortalized collecting duct cpk cells as well as in human autosomal recessive PKD tissue using targeted analysis, we show that the increase in 2-HG is likely due to glutamine-sourced α-ketoglutarate. In addition, cpk cells require exogenous glutamine for growth such that inhibition of glutaminase-1 decreases cell viability as well as proliferation. This study is a demonstration of the striking parallels between recessive PKD and cancer metabolism. Our data, once confirmed in other PKD models, suggest that future therapeutic approaches targeting this pathway, such as using glutaminase inhibitors, have the potential to open novel treatment options for renal cystic disease.

Topics: Animals; Cells, Cultured; Creatine Kinase; Disease Models, Animal; DNA Mutational Analysis; Enzyme Inhibitors; Female; Glutaminase; Glutamine; Glutarates; Humans; Infant; Infant, Newborn; Male; Metabolomics; Mice; Models, Genetic; Polycystic Kidney Diseases

Isocitrate dehydrogenase 1 (IDH1), which localizes to the cytosol and peroxisomes, catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and in parallel converts NADP(+) to NADPH. IDH1 mutations are frequently detected in grades 2-4 gliomas and in acute myeloid leukemias (AML). Mutations of IDH1 have been identified at codon 132, with arginine being replaced with histidine in most cases. Mutant IDH1 gains novel enzyme activity converting α-KG to D-2-hydroxyglutarate (2-HG) which acts as a competitive inhibitor of α-KG. As a result, the activity of α-KG-dependent enzyme is reduced. Based on these findings, 2-HG has been proposed to be an oncometabolite. In this study, we established HEK293 and U87 cells that stably expressed IDH1-WT and IDH1-R132H and investigated the effect of glutaminase inhibition on cell proliferation with 6-diazo-5-oxo-L-norleucine (DON). We found that cell proliferation was suppressed in IDH1-R132H cells. The addition of α-KG restored cell proliferation. The metabolic features of 33 gliomas with wild type IDH1 (IDH1-WT) and with IDH1-R132H mutation were examined by global metabolome analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We showed that the 2-HG levels were highly elevated in gliomas with IDH1-R132H mutation. Intriguingly, in gliomas with IDH1-R132H, glutamine and glutamate levels were significantly reduced which implies replenishment of α-KG by glutaminolysis. Based on these results, we concluded that glutaminolysis is activated in gliomas with IDH1-R132H mutation and that development of novel therapeutic approaches targeting activated glutaminolysis is warranted.

Topics: Brain Neoplasms; Cell Line, Tumor; Dacarbazine; Glioma; Glutaminase; Glutamine; Glutarates; HEK293 Cells; Humans; Isocitrate Dehydrogenase; Ketoglutaric Acids; Metabolome; Mutation; Temozolomide

Recently identified isocitrate dehydrogenase (IDH) mutations lead to the production of 2-hydroxyglutarate (2HG), an oncometabolite aberrantly elevated in selected cancers. We developed a facile and inexpensive fluorimetric microplate assay for the quantitation of 2HG and performed an unbiased small-molecule screen in live cells to identify compounds capable of perturbing 2HG production. Zaprinast, a phosphodiesterase 5 inhibitor, was identified as an efficacious modulator of 2HG production and confirmed to lower 2HG levels in vivo. The mechanism of action was not due to cGMP stabilization, but rather, profiling of metabolites upstream of mutant IDH1 pointed to targeted inhibition of the enzyme glutaminase (GLS). Zaprinast treatment reversed histone hypermethylation and soft-agar growth of IDH1-mutant cells, and treatment of glutamine-addicted pancreatic cancer cells reduced growth and sensitized cells to oxidative damage. Thus, Zaprinast is efficacious against glutamine metabolism and further establishes the therapeutic linkages between GLS and 2HG-mediated oncogenesis.. Gain-of-function IDH mutations are common events in glioma, acute myelogenous leukemia, and other cancer types, which lead to the accumulation of the oncometabolite 2HG. We show that the drug Zaprinast is capable of reducing cellular 2HG levels by inhibiting the upstream enzyme GLS, thus identifying a new strategy to target 2HG production in selected IDH-mutant cancers.

Topics: Animals; Cell Line, Tumor; Enzyme Inhibitors; Fibrosarcoma; Glutaminase; Glutarates; HEK293 Cells; High-Throughput Screening Assays; Histones; Humans; Isocitrate Dehydrogenase; Methylation; Mice; Mice, Nude; Neoplasms, Experimental; Purinones

Mutation at the R132 residue of isocitrate dehydrogenase 1 (IDH1), frequently found in gliomas and acute myelogenous leukemia, creates a neoenzyme that produces 2-hydroxyglutarate (2-HG) from α-ketoglutarate (α-KG). We sought to therapeutically exploit this neoreaction in mutant IDH1 cells that require α-KG derived from glutamine. Glutamine is converted to glutamate by glutaminase and further metabolized to α-KG. Therefore, we inhibited glutaminase with siRNA or the small molecule inhibitor bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) and found slowed growth of glioblastoma cells expressing mutant IDH1 compared with those expressing wild-type IDH1. Growth suppression of mutant IDH1 cells by BPTES was rescued by adding exogenous α-KG. BPTES inhibited glutaminase activity, lowered glutamate and α-KG levels, and increased glycolytic intermediates while leaving total 2-HG levels unaffected. The ability to selectively slow growth in cells with IDH1 mutations by inhibiting glutaminase suggests a unique reprogramming of intermediary metabolism and a potential therapeutic strategy.

Topics: Blotting, Western; Cell Line, Transformed; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Glioma; Glutamic Acid; Glutaminase; Glutarates; Humans; Isocitrate Dehydrogenase; Ketoglutaric Acids; Mutation; RNA Interference; Sulfides; Thiadiazoles; Time Factors