2-Hydroxyglutarate Production, but Not Dominant Negative Function, Is Conferred by Glioma-Derived NADP+-Dependent Isocitrate Dehydrogenase Mutations

PLOS ONE, Feb 2011

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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2-Hydroxyglutarate Production, but Not Dominant Negative Function, Is Conferred by Glioma-Derived NADP+-Dependent Isocitrate Dehydrogenase Mutations

Is Conferred by Glioma-Derived NADP+-Dependent Isocitrate Dehydrogenase Mutations. PLoS ONE 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 Zachary J. Reitman Ivan Spasojevic Ines Batinic-Haberle Jian Yang Oleg Schmidt- Kittler Darell D. Bigner Hai Yan Maciej Lesniak, The University of Chicago, United States of America 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)-2hydroxyglutarate (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 dominantnegatively lower isocitrate dehydrogenase activity at physiological (40 mM) 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. - Funding: This project was supported by American Cancer Society Research Scholar Grant 10-126-01-CCE (www.cancer.org), The Pediatric Brain Tumor Foundation Institute Grant (http://www.pbtfus.org/), and 1R01CA140316 (www.nih.gov). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Heterozygous point mutations in IDH1 and IDH2 occur in a significant portion of human cancers. Affected cancer types include gliomas of intermediate malignant grade (7394%) [1,2] and acute myeloid leukemias (AMLs, 1622%) [3,4,5,6], and cases of IDH1 mutations have been reported in prostate cancer [7], acute lymphoblastic leukemia, B type [7], colorectal cancer [8], and melanoma [9]. The IDH1 mutations observed in cancer tissue are specific for R132, a residue in the enzyme active site. R132H is the most common IDH1 substitution in gliomas, followed by R132C, R132S, R132L, and R132G. IDH2 is homologous to IDH1, and IDH2 mutations in glioma are specific for R172, the residue that is analogous to IDH1 R132. R172K, R172M, and R172G are the IDH2 substitions observed in gliomas. The IDH2 R172 mutations are rarer than the IDH1 R132 mutations in gliomas, and mutations in either gene are mutually exclusive in cancer (for reviews, see [10,11]). The frequent observation of heterozygous hotspot mutations in IDH1 and IDH2 suggests that they are protooncogenes that are activated by these mutations in cancer. In line with this, two different molecular gains-of-function have been demonstrated for several of the mutated forms of IDH1 and IDH2. The first proposed function for IDH mutants is dominant negative inhibition of WT IDH enzymes. IDH1 R132 and IDH2 R172 mutations inactivate the normal NADP+-IDH activity of IDH1 and IDH2 to convert isocitrate to a-ketoglutarate [2,12]. Furthermore, IDH1-R132H can bind to IDH1-WT, and the resulting WT:mutant heterodimer has markedly lowered isocitrate dehydrogenase activity at physiological isocitrate concentrations (,80 mM) compared to WT:WT homodimers in vitro [13]. Since the IDH mutations observed in cancer are heterozygous, it has been speculated that IDH1 and IDH2 mutants bind the remaining IDH1-WT or IDH2-WT molecules in cells and exert a dominant negative function. However, it is unclear whether binding to IDH1-WT or IDH2-WT is shared by all of the gliomaderived IDH mutants, or whether IDH mutants actually bind a significant portion of IDH1-WT or IDH2-WT molecules to exert this dominant negative function in cells. In one study, tumor tissue from glioblastomas with IDH1 R132 mutations had 38% lower NADP+-IDH activity on average than tissue from glioblastomas without IDH mutations [14]. However, it is unclear whether this lowered activity reflects the loss of activity from the mutated IDH1 allele, or if it also reflects dominant neg (...truncated)


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Genglin Jin, Zachary J. Reitman, Ivan Spasojevic, Ines Batinic-Haberle, Jian Yang, Oleg Schmidt-Kittler, Darell D. Bigner, Hai Yan. 2-Hydroxyglutarate Production, but Not Dominant Negative Function, Is Conferred by Glioma-Derived NADP+-Dependent Isocitrate Dehydrogenase Mutations, PLOS ONE, 2011, Volume 6, Issue 2, DOI: 10.1371/journal.pone.0016812