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. 2011 Feb 4;6(2):e16812.
doi: 10.1371/journal.pone.0016812.

2-hydroxyglutarate production, but not dominant negative function, is conferred by glioma-derived NADP-dependent isocitrate dehydrogenase mutations

Genglin Jin  1 , Zachary J Reitman, Ivan Spasojevic, Ines Batinic-Haberle, Jian Yang, Oleg Schmidt-Kittler, Darell D Bigner, Hai Yan
Affiliations

2-hydroxyglutarate production, but not dominant negative function, is conferred by glioma-derived NADP-dependent isocitrate dehydrogenase mutations

Genglin Jin et al. PLoS One. .

Abstract

Background: Gliomas frequently contain mutations in the cytoplasmic NADP(+)-dependent isocitrate dehydrogenase (IDH1) or the mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDH2). Several different amino acid substitutions recur at either IDH1 R132 or IDH2 R172 in glioma patients. Genetic evidence indicates that these mutations share a common gain of function, but it is unclear whether the shared function is dominant negative activity, neomorphic production of (R)-2-hydroxyglutarate (2HG), or both.

Methodology/principal findings: We show by coprecipitation that five cancer-derived IDH1 R132 mutants bind IDH1-WT but that three cancer-derived IDH2 R172 mutants exert minimal binding to IDH2-WT. None of the mutants dominant-negatively lower isocitrate dehydrogenase activity at physiological (40 µM) isocitrate concentrations in mammalian cell lysates. In contrast to this, all of these mutants confer 10- to 100-fold higher 2HG production to cells, and glioma tissues containing IDH1 R132 or IDH2 R172 mutations contain high levels of 2HG compared to glioma tissues without IDH mutations (54.4 vs. 0.1 mg 2HG/g protein).

Conclusions: Binding to, or dominant inhibition of, WT IDH1 or IDH2 is not a shared feature of the IDH1 and IDH2 mutations, and thus is not likely to be important in cancer. The fact that the gain of the enzymatic activity to produce 2HG is a shared feature of the IDH1 and IDH2 mutations suggests that this is an important function for these mutants in driving cancer pathogenesis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. IDH1 R132 mutants bind IDH1-WT, but IDH2 R172 mutants poorly bind IDH2-WT.
(a) Whole cell lysates of HOG cells overexpressing FLAG-MYC-tagged IDH1 proteins contain comparable amounts of endogenous IDH1. FLAG-IP of these lysates coprecipitates endogenous IDH1-WT (lower anti-IDH1 band) along with the FLAG-MYC-tagged IDH1 proteins (anti-MYC band and upper anti-IDH1 band). (b) HOG cells were transfected with FLAG-MYC-tagged IDH1-WT, IDH1-R132H, or a vector control (V) in combination with EGFP-tagged IDH1-WT or IDH1-R132H. As expected, FLAG-IPs of these cells pull down FLAG-MYC-tagged IDH1 (middle band). Endogenous IDH1 (lower band) coprecipitates with FLAG-MYC-tagged IDH1. EGFP-tagged IDH1 (upper band) also coprecipitates with FLAG-MYC-tagged IDH1. (c) Whole cell lysates of HOG cells overexpressing FLAG-MYC-tagged IDH2 contain comparable amounts of endogenous IDH2. FLAG-IP of these lysates coprecipitates endogenous IDH2 (anti-IDH2, lower band) along with the FLAG-MYC-tagged IDH2 (anti-IDH2, upper band and anti-MYC band). However, IDH2 R172 mutants pull down endogenous IDH2-WT poorly compared to IDH2-WT. (d) FLAG-MYC-tagged IDH1-WT, IDH1-R132H, IDH2-WT, and IDH2-R172K were transfected into HOG cells. FLAG-IP of lysates pulled down FLAG-MYC-tagged IDH1-WT, IDH1-R132H, IDH2-WT, and IDH2-R172K (anti-MYC bands). FLAG-MYC-tagged IDH1-WT and IDH1-R132H also stained with anti-IDH1 (upper anti-IDH1 band). Endogenous IDH1-WT coprecipitated with FLAG-MYC-tagged IDH1-WT and IDH1-R132H, but not with FLAG-MYC-tagged IDH2-WT and IDH2-R172K (lower anti-IDH1 band).
Figure 2
Figure 2. Overexpression of IDH1 R132 and IDH2 R172 mutants does not reduce cellular NADP+-IDH activity.
Total NADP+-IDH reaction rate, which consists of the combined rates of IDH1 and IDH2, was measured in lysates by measuring the rate of conversion of NADP+ to NADPH in the presence of isocitrate. (a) Lysates of stable, clonal HOG cell lines containing an shRNA sequence targeted against IDH1 have lowered NADP+-IDH activity compared to a sister cell line expressing scrambled RNA from the same vector (V). (b) HOG cells were transfected with a vector control, IDH1-WT, an IDH1 R132 mutant, IDH2-WT, or an IDH2 R172 mutant, lysates were collected after 48 h, and total cellular NADP+-IDH reaction rate at 40 µM isocitrate was assayed. (c) The experiment in B was repeated with 293T cells. (d) 293T lysates transfected with vector control, IDH1-R132H, or IDH2-R172K from C were assayed for NADP+-IDH reaction rate at 0 to 80 µM isocitrate.
Figure 3
Figure 3. IDH1 R132 and IDH2 R172 mutants produce 2HG.
(a) HOG cells were transfected with an empty vector (V), IDH1-WT, the indicated IDH1 R132 mutant, IDH2-WT, or the indicated IDH2 R172 mutant. The media was changed after 24 h. 2HG was analyzed in samples of media taken at 0, 24, and 48 h after the media was changed. (b) 2HG was measured in lysates of HOG cells expressing the indicated IDH variant 48 hours after transfection. (c) 2HG levels in human gliomas with either no IDH mutation (WT) or the indicated mutation.
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