Cancer-associated IDH1 mutations produce 2-hydroxyglutarate - PubMed (original) (raw)

. 2009 Dec 10;462(7274):739-44.

doi: 10.1038/nature08617.

David W White, Stefan Gross, Bryson D Bennett, Mark A Bittinger, Edward M Driggers, Valeria R Fantin, Hyun Gyung Jang, Shengfang Jin, Marie C Keenan, Kevin M Marks, Robert M Prins, Patrick S Ward, Katharine E Yen, Linda M Liau, Joshua D Rabinowitz, Lewis C Cantley, Craig B Thompson, Matthew G Vander Heiden, Shinsan M Su

Affiliations

Cancer-associated IDH1 mutations produce 2-hydroxyglutarate

Lenny Dang et al. Nature. 2009.

Abstract

Mutations in the enzyme cytosolic isocitrate dehydrogenase 1 (IDH1) are a common feature of a major subset of primary human brain cancers. These mutations occur at a single amino acid residue of the IDH1 active site, resulting in loss of the enzyme's ability to catalyse conversion of isocitrate to alpha-ketoglutarate. However, only a single copy of the gene is mutated in tumours, raising the possibility that the mutations do not result in a simple loss of function. Here we show that cancer-associated IDH1 mutations result in a new ability of the enzyme to catalyse the NADPH-dependent reduction of alpha-ketoglutarate to R(-)-2-hydroxyglutarate (2HG). Structural studies demonstrate that when arginine 132 is mutated to histidine, residues in the active site are shifted to produce structural changes consistent with reduced oxidative decarboxylation of isocitrate and acquisition of the ability to convert alpha-ketoglutarate to 2HG. Excess accumulation of 2HG has been shown to lead to an elevated risk of malignant brain tumours in patients with inborn errors of 2HG metabolism. Similarly, in human malignant gliomas harbouring IDH1 mutations, we find markedly elevated levels of 2HG. These data demonstrate that the IDH1 mutations result in production of the onco-metabolite 2HG, and indicate that the excess 2HG which accumulates in vivo contributes to the formation and malignant progression of gliomas.

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Figures

Figure 1

Figure 1. Cells expressing human R132H IDH1 contain dramatically elevated levels of 2HG

(a) Western blots for myc-tagged human isocitrate dehydrogenase 1 (IDH1-myc) or R132H mutant (R132H-myc) in stably transfected U87MG human glioblastoma cells, (b) Metabolite profiles from cells expressing R132H IDH1 or WT IDH1 detected by LC-MS scanning for species between 110–1000 m/z (M-H+). Red spots labeled A, B, and C represent species assigned to 2HG, dehydro-2HG, and 2HG-sodium adduct respectively. Spectrometric details supporting the identification of species “A” as 2HG are shown in the right panel, (c) Cells expressing R132H IDH1 contain elevated levels of 2HG. Data were normalized by cell number and expressed as fold difference relative to parental values. Error bars depict one standard deviation (SD) from the mean of 3 independent experiments, (d) Cells expressing R132H IDH1 display time-dependent accumulation of 2HG in cell culture media, normalization was as described in (c). Errors bars depict one SD from the mean of four independent experiments.

Figure 2

Figure 2. R132H mutation alters the enzymatic properties of IDH1

Kinetic parameters of oxidative and reductive reactions as measured for WT and R132H IDH1 enzymes are shown. KM and kcat values for the reductive activity of the WT enzyme were unable to be determined as no measurable enzyme activity was detectable at any substrate concentration.

Figure 3

Figure 3. R132H mutation in IDH1 results in production of R(−)-2HG

(a) 2HG was identified as the reductive reaction product of recombinant human R132H mutant IDH1 using LC-MS as shown, (b) The chirality of 2HG produced by R132H mutant IDH1 was assessed as by diacetyl-L-tartaric anhydride derivatization and LC-MS analysis. Normalized LC-MS signal for the reductive reaction (rxn) product alone, an R(−)-2HG standard alone, and the two together (Rxn + R(−)-2HG) are shown as is the signal for a racemic mixture of R(−) and S(+) forms (2HG Racemate) alone or with the reaction products (Rxn + Racemate).

Figure 4

Figure 4. Structural analysis of R132H mutant IDH1

(a) On the left is shown an overlay of R132H mutant IDH1 (green) and WT IDH1 (gray) structures in the ‘closed’ conformation. On the right is shown an overlay of WT IDH1 (blue) structure in the ‘open’ conformation with mutant IDH1 (green) for comparison, (b) Close-up comparison of the R132H IDH1 active site (left) with αKG (yellow) and NADPH (gray) and the WT IDH active-site (right) with isocitrate (yellow) and NADP (gray). Residues coming from the other monomer are denoted with a prime (‘) symbol. In addition to the mutation at residue 132, the major changes are the positions of the catalytic residues Tyr 139 and Lys 212′. (c) Walleyed stereo image showing the composite omit map for αKG, NADPH, calcium ion, His 132 and other key catalytic residues in the R132H mutant active site contoured at 1σ level.

Figure 4

Figure 4. Structural analysis of R132H mutant IDH1

(a) On the left is shown an overlay of R132H mutant IDH1 (green) and WT IDH1 (gray) structures in the ‘closed’ conformation. On the right is shown an overlay of WT IDH1 (blue) structure in the ‘open’ conformation with mutant IDH1 (green) for comparison, (b) Close-up comparison of the R132H IDH1 active site (left) with αKG (yellow) and NADPH (gray) and the WT IDH active-site (right) with isocitrate (yellow) and NADP (gray). Residues coming from the other monomer are denoted with a prime (‘) symbol. In addition to the mutation at residue 132, the major changes are the positions of the catalytic residues Tyr 139 and Lys 212′. (c) Walleyed stereo image showing the composite omit map for αKG, NADPH, calcium ion, His 132 and other key catalytic residues in the R132H mutant active site contoured at 1σ level.

Figure 5

Figure 5. Human malignant gliomas containing R132 mutations in IDH1 contain increased concentrations of 2HG

Human glioma samples obtained by surgical resection were snap frozen, genotyped to stratify as wild-type (WT) (N=10) or carrying an R132 mutant allele (Mutant) (n=12) and metabolites extracted for LC-MS analysis. Among the 12 mutant tumors, 10 carried a R132H mutation, one an R132S mutation, and one an R132G mutation. Each symbol represents the amount of the listed metabolite found in each tumor sample. Red lines indicate the group sample means. The difference in 2HG observed between WT and R132 mutant IDH1 mutant tumors was statistically significant by Student’s t-test (p<0.0001). There were no statistically significant differences in αKG, malate, fumarate, succinate, or isocitrate levels between the WT and R132 mutant IDH1 tumors.

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