The potential for isocitrate dehydrogenase mutations to produce 2-hydroxyglutarate depends on allele specificity and subcellular compartmentalization

Abstract

Monoallelic point mutations in cytosolic isocitrate dehydrogenase 1 (IDH1) and its mitochondrial homolog IDH2 can lead to elevated levels of 2-hydroxyglutarate (2HG) in multiple cancers. Here we report that cellular 2HG production from cytosolic IDH1 mutation is dependent on the activity of a retained wild-type IDH1 allele. In contrast, expression of mitochondrial IDH2 mutations led to robust 2HG production in a manner independent of wild-type mitochondrial IDH function. Among the recurrent IDH2 mutations at Arg-172 and Arg-140, IDH2 Arg-172 mutations consistently led to greater 2HG accumulation than IDH2 Arg-140 mutations, and the degree of 2HG accumulation correlated with the ability of these mutations to block cellular differentiation. Cytosolic IDH1 Arg-132 mutations, although structurally analogous to mutations at mitochondrial IDH2 Arg-172, were only able to elevate intracellular 2HG to comparable levels when an equivalent level of wild-type IDH1 was co-expressed. Consistent with 2HG production from cytosolic IDH1 being limited by substrate production from wild-type IDH1, we observed 2HG levels to increase in cancer cells harboring an endogenous monoallelic IDH1 mutation when mitochondrial IDH flux was diverted to the cytosol. Finally, expression of an IDH1 construct engineered to localize to the mitochondria rather than the cytosol resulted in greater 2HG accumulation. These data demonstrate that allelic and subcellular compartment differences can regulate the potential for IDH mutations to produce 2HG in cells. The consequences of 2HG elevation are dose-dependent, and the non-equivalent 2HG accumulation resulting from IDH1 and IDH2 mutations may underlie their differential prognosis and prevalence in various cancers.

FIGURE 1.

Mitochondrial IDH2 Arg-140 mutations result in less cellular 2HG accumulation than IDH2 Arg-172 mutations, and 2HG accumulation from both mutations is insensitive to depletion of wild-type IDH. A, FLAG-tagged IDH2 R140Q and R172K cDNA constructs, or empty vector, were transfected into 293T cells at various doses. Cells were harvested 48 h post-transfection and assessed for 2HG accumulation by GC-MS (top) or protein expression by Western blot (bottom). Data are from a representative of 3 independent experiments. B, FLAG-tagged IDH2 R140Q and R172K constructs engineered to be resistant to siRNA knockdown, or empty vector, were transfected into cells along with 50 pmol of non-targeting siRNA (siCTRL) or 50 pmol of siRNA targeting only the endogenous wild-type IDH2 (siIDH2). C, siRNA-resistant IDH2 constructs were transfected into cells along with 60 pmol of siCTRL (2x siCTRL), 30 pmol of siRNA targeting the α subunit of IDH3 (siIDH3A) plus 30 pmol of siCTRL, or 30 pmol of siIDH3A plus 30 pmol of siIDH2. D, CS-1 chondrosarcoma cells with a naturally occurring, endogenous, monoallelic IDH2 R172S mutation were transfected with 30 pmol of siCTRL, siIDH2 (targeting both wild-type and mutant IDH2), one of two independent siRNAs targeting the mitochondrial aconitase ACO2 (siACO2#1 and #2), siIDH3A, or siIDH1. For B-D, data are representative of mean ± S.D. of 3 biological replicates from 2 independent experiments. *, p < 0.005; **, p < 0.001.

FIGURE 2.

Extent of 2HG accumulation correlates with the degree of differentiation blockade in non-transformed cells expressing IDH2 Arg-140 or Arg-172 mutations. A, Western blot of 3T3-L1 pre-adipocytes stably expressing additional IDH2 WT, IDH2 R172K, IDH2 R140Q, or empty vector. IDH1 protein levels were used as a loading control. B, cells were treated with a mixture to induce differentiation into mature adipocytes for 7 days. Oil Red O staining was used to assess the accumulation of lipid droplets. C, at day 4 following differentiation induction, RNA was extracted and the expression of adipocyte-specific gene and transcription factors was measured by quantitative PCR with reverse transcription (RT-qPCR). B and C, data are representative of 3 independent experiments. D, cellular 2HG accumulation was measured by GC-MS. Data are representative of mean ± S.D. of 3 biological replicates from 2 independent experiments. *, p < 0.001.

FIGURE 3.

Cellular 2HG accumulation from cytosolic IDH1 mutation depends on maintained expression of wild-type IDH1. A, depiction of the open reading frames of the homologous proteins cytosolic IDH1 and mitochondrial IDH2. Recurrently mutated amino acid residues are marked with an asterisk (*). Arg-132 of cytosolic IDH1 is structurally analogous to Arg-172 of mitochondrial IDH2. B, FLAG-tagged IDH2 R140Q, IDH2 R172K, and IDH1 R132H (at various doses), or empty vector were transfected into 293T cells. Forty-eight hours post-transfection, cells were harvested and assessed for 2HG accumulation by GC-MS (top) or protein expression by Western blot (bottom). Data are representative of mean ± S.D. of 3 biological replicates from 2 independent experiments. C, a similar transfection as in B was performed with or without the co-transfection of Myc-tagged wild-type IDH1. D, Myc-tagged IDH1 R132H engineered to be siRNA-resistant, with or without FLAG-tagged IDH1 WT also engineered to be siRNA resistant, or empty vector, were transfected into 293T cells along with siCTRL or siRNA targeting only endogenous wild-type IDH1 (siIDH1). siIDH1 transfection to knockdown endogenous IDH1 levels was reproducibly associated with greater expression levels of Myc-tagged mutant IDH1, but this was still accompanied by a significant decrease in 2HG accumulation unless FLAG-tagged wild-type IDH1 was co-transfected. Data are representative of mean ± S.D. of 3 biological replicates from 4 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 4.

Impairing mitochondrial IDH flux results in increased 2HG accumulation in cells with endogenous cytosolic IDH1 mutation. A, model for metabolism in a cell harboring a monoallelic IDH1 mutation. B, JJ012 chondrosarcoma cells with a naturally occurring, endogenous, monoallelic IDH1 R132G mutation were transfected with 30 pmol of siCTRL, siIDH1 (targeting both wild-type and mutant IDH1), siIDH2, siIDH3A, or one of two independent siRNAs against ACO2. Forty-eight hours post-transfection cells were harvested and assessed for cellular 2HG accumulation by GC-MS (top) or protein expression by Western blot (bottom, images presented are panels from different areas of the same gel). Data are representative of mean ± S.D. of 3 biological replicates from 3 independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with siCTRL.

FIGURE 5.

Mitochondrial localization of cytosolic IDH1 mutant results in greater accumulation of 2HG in cells. A, An IDH1 construct designed to mislocalize to the mitochondria was engineered by placing the mitochondrial targeting sequence (MTS) of IDH2 at the N terminus of IDH1. Ten additional amino acids immediately following the MTS were also included to ensure proper processing, similar to that performed for previous IDH domain-swapping experiments in yeast (29). B, the desired localization of the mito-IDH1 construct was confirmed by Western blot of non-heavy and heavy membrane fractions of 293T cells 48 h following transfection of the indicated tagged constructs. C, Myc-tagged versions of the appropriately localized cytosolic IDH1 R132H or the mislocalized mito-IDH1 R132H were transfected into 293T cells at the doses indicated. Forty-eight hours post-transfection, cells were harvested and assessed for cellular 2HG accumulation by GC-MS (top) or IDH1 expression by Western blot (bottom). Data are representative of 3 independent experiments. D, FLAG-tagged versions of mitochondrial IDH2 R172K, cytosolic IDH1 R132H, and mito-IDH1 R132H were transfected into 293T cells. Forty-eight hours post-transfection, cells were harvested and assessed for 2HG accumulation by GC-MS (top) or protein expression by Western blot (bottom, images presented are panels from different areas of the same gel). Data are mean ± S.D. of 3 biological replicates from a representative experiment. *, p < 0.01.