Profiling the effects of isocitrate dehydrogenase 1 and 2 mutations on the cellular metabolome

Abstract

Point mutations of the NADP(+)-dependent isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2) occur early in the pathogenesis of gliomas. When mutated, IDH1 and IDH2 gain the ability to produce the metabolite (R)-2-hydroxyglutarate (2HG), but the downstream effects of mutant IDH1 and IDH2 proteins or of 2HG on cellular metabolism are unknown. We profiled >200 metabolites in human oligodendroglioma (HOG) cells to determine the effects of expression of IDH1 and IDH2 mutants. Levels of amino acids, glutathione metabolites, choline derivatives, and tricarboxylic acid (TCA) cycle intermediates were altered in mutant IDH1- and IDH2-expressing cells. These changes were similar to those identified after treatment of the cells with 2HG. Remarkably, N-acetyl-aspartyl-glutamate (NAAG), a common dipeptide in brain, was 50-fold reduced in cells expressing IDH1 mutants and 8.3-fold reduced in cells expressing IDH2 mutants. NAAG also was significantly lower in human glioma tissues containing IDH mutations than in gliomas without such mutations. These metabolic changes provide clues to the pathogenesis of tumors associated with IDH gene mutations.

Fig 1.

Metabolite profile of a glioma cell line expressing IDH1-R132H or IDH2-R172K. (A) Heat map showing 314 biochemicals in lysates from six replicates each of HOG cells expressing IDH1-WT, IDH1-R132H, IDH2-WT, IDH2-R172K, or vector alone, arranged by unsupervised hierarchical clustering. The level of each biochemical in each sample is represented as the number of SDs above or below the mean level of that biochemical (z score). (B) Venn diagrams indicating the number of biochemicals with mean levels that are significantly (P < 0.05) higher or lower in cells expressing each transgene compared with the vector. (C) PCA of metabolite profile dataset. The percentage of variance in the dataset reflected by the first six PCs is shown in the histogram, and PC1 and PC2 for each sample are plotted.

Fig 2.

Metabolite profile of a glioma cell line expressing IDH1-R132H, treated with 2HG, or with knocked-down IDH1. (A) Heat map showing z scores for 202 biochemicals in HOG cell lysates arranged by unsupervised hierarchical clustering. Six replicates each of cells stably expressing IDH1-R132H, IDH1 shRNA, or scrambled shRNA and cells treated with medium containing 0 (vector), 7.5 mM, or 30 mM 2HG for 72 h before analysis are shown. (B) Venn diagrams indicating the number of biochemicals with mean levels that are significantly (P < 0.05) higher or lower in each group of cells compared with vector and the number of these changes shared by cells in the indicated groups. (C) PCA of this metabolite profile dataset. The percentage of variance in the dataset reflected by the first six PCs is shown in a histogram, and PC1 and PC2 for each sample are plotted.

Fig 3.

Metabolites altered twofold or more by IDH1-R132H expression. Biochemicals that were on average more than twofold higher or lower in HOG IDH1-R132H cells relative to vector cells are displayed. The fold-change of these biochemicals in cells expressing IDH1-WT, IDH2-R172K, IDH2-WT, treated with 2HG, or expressing IDH1 shRNA is shown also. All changes shown here that were greater than twofold were significant (P < 0.05). Note that this scale colors only findings with changes greater than twofold. Detailed information on these changes can be found in Dataset S1 and S3.

Fig 4.

Alterations in metabolic pathways observed in cells expressing IDH1-R132H, expressing IDH2-R172K, or treated with 2HG. The fold difference in metabolite in each experiment relative to vector is indicated by the color of each box. (A) Amino acids and N-acetylated amino acids (IDH1-R132H, left boxes; IDH2-R172K, center boxes; 30 mM 2HG, right boxes). (B) Glutathione and metabolites involved in its regeneration. (C) BCAAs and catabolites. (D) Choline, GPC, and intermediates. (E) TCA and shuttling of citrate, isocitrate, and α-ketoglutarate to the cytosol. Dashed lines indicate exchange of a metabolite between the mitochondria and cytosol. cysH-gly, cysteinylglycine; γ-glu-aa, γ-glutamyl amino acids; γ-glu-cys, γ-glutamyl-cysteine.

Fig 5.

NAA and NAAG in cell lines containing IDH1-R132H determined by targeted LC-MS/MS. (A) NAA and NAAG levels in HOG cells expressing vector, IDH1-WT, or IDH1-R132H mock-treated or incubated in 100 μM NAA or 10 μM NAAG medium for 48 h. (B) NAA and NAAG in media incubated for 48 h with HOG cells expressing IDH1-R132H, IDH1-WT, or a vector control (v).