Cancer-associated metabolite 2-hydroxyglutarate accumulates in acute myelogenous leukemia with isocitrate dehydrogenase 1 and 2 mutations

Abstract

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2), are present in most gliomas and secondary glioblastomas, but are rare in other neoplasms. IDH1/2 mutations are heterozygous, and affect a single arginine residue. Recently, IDH1 mutations were identified in 8% of acute myelogenous leukemia (AML) patients. A glioma study revealed that IDH1 mutations cause a gain-of-function, resulting in the production and accumulation of 2-hydroxyglutarate (2-HG). Genotyping of 145 AML biopsies identified 11 IDH1 R132 mutant samples. Liquid chromatography-mass spectrometry metabolite screening revealed increased 2-HG levels in IDH1 R132 mutant cells and sera, and uncovered two IDH2 R172K mutations. IDH1/2 mutations were associated with normal karyotypes. Recombinant IDH1 R132C and IDH2 R172K proteins catalyze the novel nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of alpha-ketoglutarate (alpha-KG) to 2-HG. The IDH1 R132C mutation commonly found in AML reduces the affinity for isocitrate, and increases the affinity for NADPH and alpha-KG. This prevents the oxidative decarboxylation of isocitrate to alpha-KG, and facilitates the conversion of alpha-KG to 2-HG. IDH1/2 mutations confer an enzymatic gain of function that dramatically increases 2-HG in AML. This provides an explanation for the heterozygous acquisition of these mutations during tumorigenesis. 2-HG is a tractable metabolic biomarker of mutant IDH1/2 enzyme activity.

Figure 1.

IDH1/2 mutant AML cells and sera have increased levels of 2-HG. (A) Extracts from IDH1/2 WT (n = 10) and IDH1/2 mutant (n = 16) patient leukemia cells obtained at presentation and relapse, and IDH1 R132 mutant leukemia cells grown in culture for 14 d (n = 14) were analyzed by LC-MS to measure levels of 2-HG. (B) 2-HG was measured in sera of patients with IDH1 WT or IDH1 R132 mutant leukemia. In A and B, each point represents an individual patient sample. Diamonds represent WT, circles represent IDH1 mutants, and triangles represent IDH2 mutants. Horizontal bars indicate the mean. * indicates a statistically significant difference relative to WT patient cells (P < 0.05).

Figure 2.

IDH1/2 mutant AML cells do not display altered levels of central carbon metabolites. Extracts from leukemia cells of AML patients carrying an IDH1/2 mutant allele (mutant; n = 16) or WT (n = 10) obtained at initial presentation and relapse were assayed by LC-MS for levels of α-KG, succinate, malate, and fumarate. Each point represents an individual patient sample. Open circles represent WT, closed circles represent IDH1 mutants, and triangles represent IDH2 mutants. Horizontal bars represent the mean. There were no statistically significant differences between the WT and IDH1/2 mutant AML samples.

Figure 3.

Recombinant IDH1 R132C and IDH2 R172K produce 2-HG. (A) LC-MS analysis of in vitro reactions using recombinant IDH1 R132C and IDH2 R172K confirms that 2-HG and not isocitrate is the end product of the mutant enzyme reactions. Reactions were performed in triplicate in each of two independent experiments; typical chromatograms are presented. (B) The WT IDH1 enzyme catalyzes the oxidative decarboxylation of isocitrate to α-KG, with the concomitant reduction of NADP to NADPH. The IDH1 R132C and IDH2 R172K mutants reduce α-KG to 2-HG while oxidizing NADPH to NADP. These are referred to in the text as the “forward” and “partial reverse” reactions, respectively.