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`Cancer-associated metabolite 2-
`hydroxyglutarate accumulates in acute
`myelogenous leukemia with isocitrate
`dehydrogenase 1 and 2 mutations
`
`Stefan Gross,1 Rob A. Cairns,2 Mark D. Minden,2 Edward M. Driggers,1
`Mark A. Bittinger,1 Hyun Gyung Jang,1 Masato Sasaki,2 Shengfang Jin,1
`David P. Schenkein,1 Shinsan M. Su,1 Lenny Dang,1 Valeria R. Fantin,1
`and Tak W. Mak2
`
`1Agios Pharmaceuticals Incorporated, Cambridge, MA 02139
`2The Campbell Family Institute for Breast Cancer Research at Princess Margaret Hospital, University Health Network, Toronto,
`Ontario M5G 2M9, Canada
`
`Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2), are present in most gliomas and
`secondary glioblastomas, but are rare in other neoplasms. IDH1/2 mutations are heterozy-
`gous, and affect a single arginine residue. Recently, IDH1 mutations were identified in 8%
`of acute myelogenous leukemia (AML) patients. A glioma study revealed that IDH1 muta-
`tions cause a gain-of-function, resulting in the production and accumulation of 2-hydroxy-
`glutarate (2-HG). Genotyping of 145 AML biopsies identified 11 IDH1 R132 mutant
`samples. Liquid chromatography-mass spectrometry metabolite screening revealed in-
`creased 2-HG levels in IDH1 R132 mutant cells and sera, and uncovered two IDH2 R172K
`mutations. IDH1/2 mutations were associated with normal karyotypes. Recombinant IDH1
`R132C and IDH2 R172K proteins catalyze the novel nicotinamide adenine dinucleotide
`phosphate (NADPH)–dependent reduction of -ketoglutarate (-KG) to 2-HG. The IDH1
`R132C mutation commonly found in AML reduces the affinity for isocitrate, and increases
`the affinity for NADPH and -KG. This prevents the oxidative decarboxylation of isocitrate
`to -KG, and facilitates the conversion of -KG to 2-HG. IDH1/2 mutations confer an
`enzymatic gain of function that dramatically increases 2-HG in AML. This provides an
`explanation for the heterozygous acquisition of these mutations during tumorigenesis.
`2-HG is a tractable metabolic biomarker of mutant IDH1/2 enzyme activity.
`
`CORRESPONDENCE
`Tak W. Mak:
`tmak@uhnres.utoronto.ca
`OR
`Valeria R. Fantin:
`Valeria.fantin@agios.com
`
`Abbreviations used: 2-HG, 2-
`hydroxyglutarate; -KG, -
`ketoglutarate; AML, acute
`myelogenous leukemia; CNS,
`central nervous system;
`IDH1/2, isocitrate dehydroge-
`nase 1 and 2; LC-MS, liquid
`chromatography mass spectrom-
`etry; MDS, myelodysplastic
`syndrome; NADPH, nicotin-
`amide adenine dinucleotide
`phosphate; NPM1,
`nucleophosmin 1.
`
`The Journal of Experimental Medicine
`
`Isocitrate dehydrogenase 1 and 2 (IDH1 and
`IDH2) are NADP-dependent enzymes that
`catalyze the oxidative decarboxylation of isoci-
`trate to -ketoglutarate (-KG) in the cyto-
`plasm/peroxisome and mitochondrial matrix,
`respectively, with the concomitant production
`of nicotinamide adenine dinucleotide phos-
`phate (NADPH). Their activities are distinct from
`the NAD-dependent enzyme IDH3, which func-
`tions in the tricarboxylic acid cycle to produce
`the NADH required to supply the electron
`transport chain.
`The high-throughput sequencing of glio-
`blastoma multiforme tumors identified a novel
`mutation in IDH1 that was present in 12% of
`
`tumors from glioblastoma multiforme patients
`(Parsons et al., 2008). Further investigation has
`revealed that this mutation is present in a high
`proportion of gliomas and secondary glioblasto-
`mas, but not in other human malignancies (Balss
`et al., 2008; Bleeker et al., 2009; Hartmann et al.,
`2009; Kang et al., 2009; Sanson et al., 2009;
`Watanabe et al., 2009; Yan et al., 2009). Less
`common mutations in IDH2 have also been
`identified in gliomas, and are mutually exclusive
`with mutations in IDH1 (Hartmann et al.,
`2009; Sonoda et al., 2009; Yan et al., 2009). All
`
`© 2010 Gross et al. This article is distributed under the terms of an Attribu-
`tion–Noncommercial–Share Alike–No Mirror Sites license for the first six months
`after the publication date (see http://www.jem.org/misc/terms.shtml). After six
`months it is available under a Creative Commons License (Attribution–Noncom-
`mercial–Share Alike 3.0 Unported license, as described at http://creativecommons
`S. Gross and R.A. Cairns contributed equally to the paper.
`.org/licenses/by-nc-sa/3.0/).
`Supplemental material can be found at:
`http://doi.org/10.1084/jem.20092506
`
`The Rockefeller University Press $30.00
`J. Exp. Med. Vol. 207 No. 2 339-344
`www.jem.org/cgi/doi/10.1084/jem.20092506
`
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`mutations identified to date involve a single amino acid change
`at arginine 132 (R132) of IDH1, or the analogous residue in
`IDH2 (R172). This residue is located in the active site of the
`enzyme and participates in isocitrate binding (Xu et al., 2004).
`Interestingly, all mutations are heterozygous, suggesting that
`alteration of R132 of IDH1 or R172 of IDH2 causes an enzy-
`matic gain of function. Furthermore, these studies have pro-
`vided evidence that the IDH1 mutation is an early event in the
`pathogenesis of the disease (Watanabe et al., 2009).
`Recently, whole genome sequencing of a patient with
`acute myelogenous leukemia (AML) identified an R132 mu-
`tation in IDH1 (Mardis et al., 2009). Sequencing of addi-
`tional patients revealed that IDH1 is mutated at R132, mainly
`to histidine and cysteine, in 8% of AML patients, demon-
`strating that this mutation is not restricted to gliomas, as pre-
`viously thought (Mardis et al., 2009). More importantly, we
`recently reported that IDH1 R132 mutations confer a novel
`enzymatic activity. Surprisingly, the IDH1 R132H mutant
`protein was found to catalyze the NADPH-dependent re-
`duction of -KG to 2-hydroxyglutarate (2-HG), a rare me-
`tabolite normally present at very low levels in healthy cells
`and tissues (Dang et al., 2009). We set out to further investi-
`gate the role of IDH mutation in AML. Our work establishes
`that IDH1 R132 mutations cause production and accumula-
`tion of 2-HG in AML cells. Additionally, 2-HG screening
`uncovered previously unrecognized IDH2 mutations in AML.
`Overall, our data indicate that mutations at R132 and R172
`in the active sites of IDH1 and IDH2, respectively, lead to a
`change in the molecular mechanism of enzyme catalysis, re-
`sulting in elevated levels of 2-HG in AML.
`
`RESULTS AND DISCUSSION
`As IDH1 is a critical enzyme in cellular metabolism, the recent
`report of IDH1 mutations in AML is intriguing. To investigate
`the role of IDH1 R132 mutations in AML, we genotyped
`leukemic cells obtained at initial presentation, from a series of
`145 AML patients treated at the Princess Margaret Hospital
`with the aim of identifying mutant samples in our viable cell
`tissue bank. Heterozygous IDH1 R132 mutations were found
`in 11 (8%) of these patients (Table I). The spectrum of IDH1
`mutations in AML appears to differ from that seen in central
`nervous system (CNS) tumors. In the CNS, the majority of
`mutations (80–90%) are IDH1 R132H substitutions, whereas
`we observe 5, 4, and 2 patients with IDH1 R132H, R132C,
`and R132G mutations, respectively (Table I). This is consis-
`tent with the previous report in AML (Mardis et al., 2009) and
`suggests that there may be functional differences among these
`IDH1 mutants. In four cases, leukemic cells were also available
`from samples taken at the time of relapse. The IDH1 mutation
`was retained in 4/4 of these samples (Table I). One of the
`AML patients harboring an IDH1 mutation had progressed
`from an earlier myelodysplastic syndrome (MDS). When cells
`from the prior MDS biopsy were genotyped, IDH1 was found
`to be WT. Further sequencing of a large group of well-charac-
`terized MDS samples will be necessary to determine whether
`IDH1 mutations are a feature of this heterogeneous disease,
`and whether they contribute to progression to AML. In a sub-
`set of IDH1 mutant patients (n = 8), reverse transcribed RNA
`was used for genotyping to assess the relative expression of
`mutant and WT alleles. Sequenom genotyping showed bal-
`anced allele peaks for these samples, indicating that both the
`
`Table I.
`Patient ID
`
`Identification of 13 AML patients bearing an IDH1 R132 or IDH2 R172 mutation
`Mutation
`Amino acid
`FAB subtype
`NPM1 and FLT3
`Cytogenetic profile
`change
`status
`
`Genotype at
`relapse
`
`2-HG level (ng/2
`× 106 cells)
`
`IDH1 mutations
`090108
`090356
`0034
`0086
`0488
`8587
`8665
`8741
`9544
`747762
`090148
`IDH2 mutations
`9382
`0831
`IDH1/2 WT
`090239
`090158
`090313
`
`G/A
`G/A
`C/T
`C/G
`C/T
`G/A
`C/T
`G/A
`C/G
`G/A
`C/T
`
`G/A
`G/A
`
`WT
`WT
`WT
`
`R132H
`R132H
`R132C
`R132G
`R132C
`R132H
`R132C
`R132H
`R132G
`R132H
`R132C
`
`R172K
`R172K
`
`WT
`WT
`WT
`
`M4
`na
`M5a
`M2
`M0
`na
`M1
`M4
`na
`M1
`M1
`
`M0
`M1
`
`na
`M2
`na
`
`na
`na
`Normal
`Normal
`Normal
`Normal
`na
`NPM1
`na
`NPM1
`na
`
`Normal
`Normal
`
`FLT3
`na
`na
`
`Normal
`na
`Normal
`Normal
`Normal
`Normal
`Normal
`Normal
`Normal
`Normal
`46, xx, i(7) (p10) [20]
`
`Normal
`Normal
`
`Normal
`46, XX [15]
`Normal
`
`na
`na
`na
`na
`R132C
`na
`na
`R132H
`R132G
`R132H
`na
`
`na
`na
`
`na
`na
`na
`
`2,090
`1,529
`10,285
`10,470
`13,822
`5,742
`7,217
`6,419
`4,962
`8,464
`na
`
`19,247
`15,877
`
`112
`411
`116
`
`FLT3, FMS-related tyrosine kinase 3 internal tandem duplications. Na indicates that some data were not available for some patients.
`
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`WT and mutant genes are expressed. 10 established AML cell
`lines were also genotyped (OCI/AML-1, OCI/AML-2, OCI/
`AML-3, OCI/AML-4, OCI/AML-5, HL-60, MV-4-11,
`THP-1, K562, and KG1A) and all were IDH1 WT. This find-
`ing is consistent with the failure of other investigators to iden-
`tify glioma or glioblastoma cell lines retaining the IDH1 R132
`mutation after long-term culture (Bleeker et al., 2009).
`Because gliomas carrying IDH1 mutations accumulate
`high levels of 2-HG (Dang et al., 2009), we measured 2-HG
`in this set of AML samples. Levels of 2-HG were 50-fold
`higher in samples harboring an IDH1 R132 mutation (Table I,
`Fig. 1 A, and Table S2). 2-HG was also elevated in the sera
`of patients with IDH1 R132 mutant AML (Fig. 1 B). There
`was no relationship between the specific amino acid substitu-
`tion at residue 132 and the level of 2-HG.
`Interestingly, two IDH1 WT samples also showed high
`levels of 2-HG (Table I). Mutation of arginine 172 in IDH2
`has been previously reported in gliomas (Ducray et al., 2009;
`Hartmann et al., 2009; Sonoda et al., 2009; Yan et al., 2009),
`but not in AML. The high 2-HG concentration prompted
`sequencing of the IDH2 gene in these two AML samples,
`and IDH2 R172K mutations were identified in both samples
`(Table I). This is the first report of IDH2 R172K mutations
`in AML. Subsequently, all samples were screened for IDH2
`mutations, but no further mutations were identified.
`Evaluation of the clinical characteristics of patients with
`or without IDH1/2 mutations revealed a significant associa-
`tion between IDH1/2 mutations and normal karyotype (P =
`0.05), but no other differences (Table S1). Notably, there
`was no difference in treatment response for a subgroup of pa-
`tients who received consistent treatment (n = 136). These
`findings are consistent with the initial study identifying IDH1
`mutations in AML (Mardis et al., 2009).
`
`Unlike in glioma, where IDH1 mutant patients have
`improved outcome, there does not seem to be an effect of
`IDH1/2 mutation status on most clinical characteristics or
`treatment response in AML. However, the studies reported to
`date are likely not sufficiently powered to adequately address
`these questions. Further studies using larger numbers of pa-
`tients will be required to determine whether IDH1 mutation
`status will be a clinically useful marker. Regardless of its effect
`on outcome, the finding that 4/4 patients retained the muta-
`tion upon relapse suggests that it is stable through the course of
`disease progression, and may be clinically useful as a marker of
`minimal residual disease, as has been shown for nucleophosmin
`1 (NPM1) mutations (Schnittger et al., 2009).
`To extend these findings, panels of AML cells from WT
`and IDH1 mutant patients were cultured in vitro. There was
`no difference in the growth rates or viability of the IDH1
`R132 mutant and WT cells, with both groups showing high
`variability in their ability to proliferate in culture, as is charac-
`teristic of primary AML cells (Fig. S1). There was no relation-
`ship between 2-HG levels in the IDH1 R132 mutant cells and
`their growth rate or viability in culture. After 14 d in culture,
`the mutant AML cells retained their IDH1 R132 mutations
`(11/11), and continued to accumulate high levels of 2-HG
`(Fig. 1 A), further confirming that IDH1 R132 mutations lead
`to the production and accumulation of 2-HG in AML cells.
`To investigate the effect of IDH1/2 mutations on the
`concentration of cellular metabolites proximal to the IDH
`reaction, -KG, succinate, malate, and fumarate levels were
`measured in AML cells with IDH1/2 mutations and in a set
`of WT AML cells matched for AML subtype and cytogenetic
`profile. None of the metabolites were found to be greatly al-
`tered in the IDH1 mutants compared with the IDH1 WT
`cells (Fig. 2 and Table S2). Of note, the mean level of -KG
`was not altered in the IDH1/2 mutant AML cells, suggesting
`
`IDH1/2 mutant AML cells and sera have increased levels
`Figure 1.
`of 2-HG. (A) Extracts from IDH1/2 WT (n = 10) and IDH1/2 mutant (n =
`16) patient leukemia cells obtained at presentation and relapse, and IDH1
`R132 mutant leukemia cells grown in culture for 14 d (n = 14) were ana-
`lyzed by LC-MS to measure levels of 2-HG. (B) 2-HG was measured in sera
`of patients with IDH1 WT or IDH1 R132 mutant leukemia. In A and B,
`each point represents an individual patient sample. Diamonds represent
`WT, circles represent IDH1 mutants, and triangles represent IDH2 mutants.
`Horizontal bars indicate the mean. * indicates a statistically significant
`difference relative to WT patient cells (P < 0.05).
`
`IDH1/2 mutant AML cells do not display altered levels
`Figure 2.
`of central carbon metabolites. Extracts from leukemia cells of AML
`patients carrying an IDH1/2 mutant allele (mutant; n = 16) or WT (n = 10)
`obtained at initial presentation and relapse were assayed by LC-MS for
`levels of -KG, succinate, malate, and fumarate. Each point represents an
`individual patient sample. Open circles represent WT, closed circles repre-
`sent IDH1 mutants, and triangles represent IDH2 mutants. Horizontal bars
`represent the mean. There were no statistically significant differences
`between the WT and IDH1/2 mutant AML samples.
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`that the mutation does not decrease the concentration of this
`metabolite as has been previously hypothesized (Zhao et al.,
`2009). These findings are consistent with measurements made
`in glioma tissue (Dang et al., 2009), and indicate that IDH1
`mutations do not cause large changes in the cellular concen-
`trations of these metabolites.
`To confirm that the R132C mutation of IDH1, and the
`R172K mutation of IDH2 confer a novel enzymatic activity
`that produces 2-HG, recombinant mutant enzymes were as-
`sayed for the NADPH-dependent reduction of -KG. When
`samples were analyzed by liquid chromatography mass spec-
`trometry (LC-MS) upon completion of the enzyme assay,
`2-HG was identified as the end product for both the IDH1
`R132C and IDH2 R172K mutant enzymes (Fig. 3 A). No
`isocitrate was detectable by LC-MS, indicating that 2-HG is
`the sole product of this reaction (Fig. 3 A). This observation
`held true even when the reductive reaction was performed in
`buffer containing NaHCO3 saturated with CO2 (unpublished
`data), which would be expected to favor the formation of
`isocitrate via the canonical reverse reaction.
`As discussed above, a large proportion of IDH1 mutant
`patients in AML have an IDH1 R132C mutation (Table I;
`Mardis et al., 2009). To biochemically characterize mutant
`IDH1 R132C, the enzymatic properties of recombinant
`
`R132C protein were assessed in vitro. Kinetic analyses
`showed that the R132C substitution severely impairs the ox-
`idative decarboxylation of isocitrate to -KG, with a signifi-
`cant decrease in kcat, even though the affinity for the cofactor
`NADP+ remains essentially unchanged (Table II). However,
`unlike the R132H mutant enzyme described previously
`(Dang et al., 2009), the R132C mutation leads to a dramatic
`loss of affinity for isocitrate (KM), and a drop in net isocitrate
`metabolism efficiency (kcat/KM) of more than six orders of
`magnitude (Table II). This suggests a potential difference in
`the substrate-level regulation of enzyme activity in the con-
`text of AML. Although substitution of cysteine at R132 in-
`activates the canonical conversion of isocitrate to -KG, the
`IDH1 R132C mutant enzyme acquires the ability to catalyze
`the reduction of -KG to 2-HG in an NADPH dependent
`manner (Fig. 3 B). This reductive reaction of mutant IDH1
`R132C is highly efficient (kcat/KM) compared with the WT
`enzyme because of the considerable increase in binding affin-
`ity of both the NADPH and -KG substrates (KM; Table II).
`There is a group of rare, inherited, neurometabolic disor-
`ders called 2-hydroxyglutaric acidurias, in which 2-HG levels
`are dramatically increased in the CNS and sera of patients
`(Rzem et al., 2007; Struys, 2006). These diseases are caused
`by homozygous loss of function mutations in the 2-HG
`
`Figure 3. Recombinant IDH1 R132C and IDH2 R172K produce 2-HG. (A) LC-MS analysis of in vitro reactions using recombinant IDH1 R132C and
`IDH2 R172K confirms that 2-HG and not isocitrate is the end product of the mutant enzyme reactions. Reactions were performed in triplicate in each
`of two independent experiments; typical chromatograms are presented. (B) The WT IDH1 enzyme catalyzes the oxidative decarboxylation of isocitrate to
`-KG, with the concomitant reduction of NADP to NADPH. The IDH1 R132C and IDH2 R172K mutants reduce -KG to 2-HG while oxidizing NADPH to
`NADP. These are referred to in the text as the “forward” and “partial reverse” reactions, respectively.
`
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`Mutations in the analogous arginine residue in the active sites
`of the two enzymes leads to the same biochemical gain of
`function. Further functional and mechanistic work will be
`required to understand the underlying biology driving the
`acquisition of these mutations, and to determine whether
`mutants of IDH1 R132 and IDH2 R172 may be useful ther-
`apeutic targets.
`
`MATERIALS AND METHODS
`Patients and clinical data. Peripheral blood and bone marrow were col-
`lected from AML patients at the time of diagnosis and at relapse, after in-
`formed consent and approval from the Research Ethics Board of the
`University Health Network. Sample identification numbers are unique,
`anonymous identifiers generated by the leukemia bank at the Princess Mar-
`garet Hospital, and are independent of true patient ID numbers. The cells
`were separated by ficoll hypaque centrifugation, and stored at 150°C as
`described previously (Miyauchi et al., 1987). Patient sera were stored at
`80°C. Cytogenetics and molecular testing were performed in the diagnos-
`tic laboratory of the University Health Network (Toronto, Canada). A sub-
`group of patients (n = 132) was given consistent treatment using a standard
`induction and consolidation chemotherapy regimen consisting of daunoru-
`bicin and cytarabine.
`
`IDH1 and IDH2 genotyping. DNA was extracted from leukemic cells
`and cell lines using the Puregene kit (QIAGEN). For a subset of samples
`(n = 96), RNA was extracted using a Qiagen RNeasy kit, and reverse tran-
`scribed into cDNA for genotyping. IDH1 and IDH2 genotype was deter-
`mined at the Analytical Genetics Technology Centre at the University
`Health Network using a Sequenom MassARRAY platform (Sequenom).
`Positive results were confirmed by direct sequencing.
`
`Cell lines. AML cell lines and 5637 cells were obtained from the labora-
`tory of M. Minden (Ontario Cancer Institute, Toronto, Canada). Primary
`AML cells were cultured in -MEM media with 20% fetal bovine serum
`and 10% 5637 cell-conditioned media as previously described (Miyauchi et
`al., 1987). Growth curves were generated by counting viable cells as as-
`sessed by trypan blue exclusion on a Vi-CELL automated cell counter
`(Beckman Coulter).
`
`Expression/purification of IDH1 and IDH2 proteins. The human
`IDH1 cDNA (GenBank/EMBL/DDBJ accession no. NM_005896) and
`IDH2 cDNA (GenBank/EMBL/DDBJ accession no. NM_002168) were
`purchased from OriGene Technologies. IDH1 (full length) and IDH2 (resi-
`dues 40–452) were subcloned into vector pET41a (EMD Biosciences) to
`enable the E. coli expression of C-terminal His8-tagged proteins. Site-
`directed mutagenesis was performed using the QuikChange Lightning kit
`(Stratagene) to change C394 to T in IDH1 and G515 to A in IDH2, result-
`ing in the R132C and R172K mutations, respectively. WT and mutant
`IDH1 proteins were expressed in and purified from the E. coli Rosetta (DE3)
`strain (Invitrogen). Overexpression of IDH2 protein was accomplished by
`co-transfection of plasmids encoding respective IDH2 clones and pG-KJE8–
`expressing chaperone proteins (Nishihara et al., 1998, 2000).
`
`IDH1/2 activity assays. Enzymatic activity was assessed by following the
`change in NADPH absorbance at 340 nm in an SFM-400 stopped-flow
`spectrophotometer (Biological) in the presence of isocitrate and NADP (for-
`ward reaction) or -KG and NADPH (reverse reaction) in standard reaction
`buffer (150 mM NaCl, 20 mM Tris-Cl, pH 7.5, 10 mM MgCl2, and 0.03%
`[wt/vol] bovine serum albumin). To measure kinetic parameters, sufficient
`enzyme was added to give a linear reaction for 1–5 s. Enzymatic binding
`constants were determined using curve-fitting algorithms to standard kinetic
`models with SigmaPlot software (Systat Software). For determination of kcat,
`enzyme was incubated with 5X Km of substrate and cofactor. An extinction
`coefficient of 6,200 M1 cm1 was used for NADPH.
`
`dehydrogenase enzymes responsible for converting 2-HG back
`into -KG. Although the phenotypes are diverse, some of
`these patients are at higher risk of developing brain tumors.
`Whether 2-HG contributes directly to tumor development is
`unknown. The existence of these diseases raises the possibility
`that mutations in genes other than IDH1 could result in an
`increase in 2-HG in tumors. 2-HG–based metabolite screening
`as performed in this study might facilitate rapid and sensitive
`identification of these mutations.
`The physiological effects of elevated 2-HG are poorly
`understood. It has been reported that 2-HG can increase lev-
`els of reactive oxygen species, which may drive tumor pro-
`gression (Latini et al., 2003a,b). Alternatively, because of its
`high degree of structural similarity to -KG, 2-HG may bind
`and inhibit specific -KG–dependent enzymes such as the
`prolyl hydroxylases that control the stability of the hypoxia-
`inducible factor transcription factors. This could explain the
`finding that hypoxia-inducible factor target genes have been
`reported to be elevated in IDH1 mutant glioma tissue (Zhao
`et al., 2009), whereas levels of -KG are not reduced in AML
`or glioma tumor cells harboring IDH1 mutations.
`The surprising finding that the IDH1 R132 mutant pro-
`tein acquires a novel enzymatic function provides a new ex-
`planation for the heterozygous acquisition of this mutation
`during tumorigenesis (Dang et al., 2009). We have shown
`that the IDH1 R132C mutant commonly found in AML has
`the ability to reduce -KG to 2-HG in an NADPH-depen-
`dent manner, and that the usually scarce -KG derivative 2-
`HG is dramatically elevated in leukemia cells and sera of
`patients carrying IDH1 mutations. Our previous work has
`also shown high levels of 2-HG in glioma tissues harboring
`IDH1 R132 mutations, suggesting that this is a consistent
`feature of IDH1 R132 mutant cells and tissues (Dang et al.,
`2009). In addition, we identified two IDH2 R172K muta-
`tions and corresponding elevated 2-HG levels for the first
`time in AML, and showed directly that IDH2 R132K mu-
`tant protein produces 2-HG in vitro. The observation that the
`mutation of IDH2 arginine 172 leads to production of 2-HG
`in AML represents an example of metabolic convergence.
`
`Table II. Kinetic parameters of the IDH1 R132C mutant
`enzyme
`Oxidative (→NADPH)
`KM,NADP+ (µM)
`KM,isocitrate (µM)
`KM,MgCl2 (µM)
`Ki,KG (µM)
`kcat (s1)
`kcat /KM,isoc(M1.s1)
`Reductive (→ NADP+)
`KM,NADPH (µM)
`KM,KG (µM)
`kcat (s1)
`Na indicates no measureable activity.
`
`R132C
`
`21
`8.7 × 104
`4.5 × 102
`61
`7.1 × 102
`8.2 × 103
`R132C
`0.3
`295
`5.5 × 102
`
`WT
`
`49
`57
`29
`6.1 × 102
`1.3 × 105
`2.3 × 109
`WT
`na
`na
`7
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`2-HG and metabolite analysis. Metabolites were extracted from cells and
`sera using 80% aqueous methanol, as previously described (Lu et al., 2006).
`For cell extraction, 2 × 106 cells were suspended in 80°C 80% methanol.
`For serum extraction, 1 ml of serum was mixed with 4 ml 80°C methanol.
`All extracts were spun at 13,000 rpm at 4°C to remove precipitate, dried at
`room temperature, and stored at 80°C. Metabolite levels were determined
`by ion-paired reverse-phase LC coupled to negative mode electrospray
`triple-quadrupole mass spectrometry using multiple reaction monitoring,
`and integrated elution peaks were compared with metabolite standard curves
`for absolute quantification (Dang et al., 2009).
`
`Statistical analysis. Fisher’s exact test was used to test for differences in
`categorical variables between IDH1/2 WT and IDH1/2 mutant patients.
`One-way analysis of variance followed by a Student’s t test with correction
`for multiple comparisons was used to test for differences in IDH1 activity
`and metabolite concentrations. Differences with P < 0.05 were considered
`significant.
`
`Online supplemental material. Fig. S1 shows growth of IDH1 R132
`mutant cells in vitro. Table S1 shows characteristics of IDH1/2 mutant and
`WT patients. Table S2 shows metabolite concentrations in individual
`IDH1/2 mutant and WT AML cells. Online supplemental material is avail-
`able at http://www.jem.org/cgi/content/full/jem.20092506/DC1.
`
`We thank Shaohui Wang at ChemPartner for assistance with biochemical
`experiments. We also thank Katherine Yen, Kevin Marks, Francisco Salituro, Jeffrey
`Sauders, Craig Thompson, and Lewis Cantley for discussion and comments on the
`manuscript.
`Tak Mak is supported by grants from the Canadian Institutes of health
`Research, the Canadian Cancer Society, the Terry Fox Foundation, and the Leukemia
`and Lymphoma Society.
`Stefan Gross, Edward M. Driggers, Mark A. Bittinger, Hyun Gyung Jang, Shenfang
`Jin, David P. Shenkein, Shinsan M. Su, Lenny Dang, and Valeria Fantin disclose financial
`interests, as employees of Agios Pharmaceuticals. Tak Mak owns stock options in
`Agios Pharmaceuticals. The authors have no further conflicts of interest.
`
`Submitted: 23 November 2009
`Accepted: 8 January 2010
`
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