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.
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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)