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

Gross S, Cairns RA, Minden MD, Driggers EM, Bittinger MA, Jang HG, Sasaki M, Jin S, Schenkein DP, Su SM, Dang L, Fantin VR, Mak TW - J. Exp. Med. (2010)

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agios Pharmaceuticals Incorporated, Cambridge, MA 02139, USA.

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.

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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.
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fig3: 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.

Mentions: To confirm that the R132C mutation of IDH1, and the R172K mutation of IDH2 confer a novel enzymatic activity that produces 2-HG, recombinant mutant enzymes were assayed for the NADPH-dependent reduction of α-KG. When samples were analyzed by liquid chromatography mass spectrometry (LC-MS) upon completion of the enzyme assay, 2-HG was identified as the end product for both the IDH1 R132C and IDH2 R172K mutant enzymes (Fig. 3 A). No isocitrate was detectable by LC-MS, indicating that 2-HG is the sole product of this reaction (Fig. 3 A). This observation held true even when the reductive reaction was performed in buffer containing NaHCO3 saturated with CO2 (unpublished data), which would be expected to favor the formation of isocitrate via the canonical reverse reaction.


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

Gross S, Cairns RA, Minden MD, Driggers EM, Bittinger MA, Jang HG, Sasaki M, Jin S, Schenkein DP, Su SM, Dang L, Fantin VR, Mak TW - J. Exp. Med. (2010)

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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2822606&req=5

fig3: 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.
Mentions: To confirm that the R132C mutation of IDH1, and the R172K mutation of IDH2 confer a novel enzymatic activity that produces 2-HG, recombinant mutant enzymes were assayed for the NADPH-dependent reduction of α-KG. When samples were analyzed by liquid chromatography mass spectrometry (LC-MS) upon completion of the enzyme assay, 2-HG was identified as the end product for both the IDH1 R132C and IDH2 R172K mutant enzymes (Fig. 3 A). No isocitrate was detectable by LC-MS, indicating that 2-HG is the sole product of this reaction (Fig. 3 A). This observation held true even when the reductive reaction was performed in buffer containing NaHCO3 saturated with CO2 (unpublished data), which would be expected to favor the formation of isocitrate via the canonical reverse reaction.

Bottom Line: 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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Agios Pharmaceuticals Incorporated, Cambridge, MA 02139, USA.

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.

Show MeSH
Related in: MedlinePlus