Vol 462 | 10 December 2009 | doi:10.1038/nature08617
`
`ARTICLES
`
`Cancer-associated IDH1 mutations
`produce 2-hydroxyglutarate
`
`Lenny Dang1, David W. White1, Stefan Gross1, Bryson D. Bennett2, Mark A. Bittinger1, Edward M. Driggers1,
`Valeria R. Fantin1, Hyun Gyung Jang1, Shengfang Jin1, Marie C. Keenan1, Kevin M. Marks1, Robert M. Prins3,
`Patrick S. Ward4, Katharine E. Yen1, Linda M. Liau3, Joshua D. Rabinowitz2, Lewis C. Cantley5, Craig B. Thompson4,
`Matthew G. Vander Heiden1{ & Shinsan M. Su1
`
`Mutations in the enzyme cytosolic isocitrate dehydrogenase 1 (IDH1) are a common feature of a major subset of primary
`human brain cancers. These mutations occur at a single amino acid residue of the IDH1 active site, resulting in loss of the
`enzyme’s ability to catalyse conversion of isocitrate to a-ketoglutarate. However, only a single copy of the gene is mutated in
`tumours, raising the possibility that the mutations do not result in a simple loss of function. Here we show that
`cancer-associated IDH1 mutations result in a new ability of the enzyme to catalyse the NADPH-dependent reduction of
`a-ketoglutarate to R(2)-2-hydroxyglutarate (2HG). Structural studies demonstrate that when arginine 132 is mutated to
`histidine, residues in the active site are shifted to produce structural changes consistent with reduced oxidative
`decarboxylation of isocitrate and acquisition of the ability to convert a-ketoglutarate to 2HG. Excess accumulation of 2HG
`has been shown to lead to an elevated risk of malignant brain tumours in patients with inborn errors of 2HG metabolism.
`Similarly, in human malignant gliomas harbouring IDH1 mutations, we find markedly elevated levels of 2HG. These data
`demonstrate that the IDH1 mutations result in production of the onco-metabolite 2HG, and indicate that the excess 2HG
`which accumulates in vivo contributes to the formation and malignant progression of gliomas.
`
`Mutations in the enzyme cytosolic isocitrate dehydrogenase 1 (IDH1)
`are found in approximately 80% of grade II–III gliomas and secondary
`glioblastomas in humans1–3. These mutations occur at a single amino
`acid residue of IDH1, arginine 132, which is most commonly mutated
`to histidine (R132H)1,3,4. Only a single copy of the gene has been found
`to be mutated in tumours1–6. Many of the high-grade gliomas with
`IDH1 mutations are secondary glioblastomas that have progressed
`from lower grade lesions1–3,5. When analysed in relation to other genes
`implicated in brain tumours, the compiled evidence suggests that
`IDH1 is often the first mutation that occurs2. Although these findings
`suggest that IDH1 mutations are selected for early during tumorigen-
`esis, why mutations in a single allele of IDH1 result in predilection for
`malignant progression is uncertain. It has been reported that the
`R132H mutation disrupts the ability of IDH1 to convert isocitrate
`to a-ketoglutarate3,7, but the consequences of this impaired enzymatic
`activity on cellular metabolism have not been systematically analysed.
`For example, although R132 IDH1 mutations might reduce the rate of
`cytosolic a-ketoglutarate production as suggested by others7, whether
`IDH1 mutations can influence the enzyme’s ability to act on a-
`ketoglutarate as a substrate has not been explored. This latter activity
`may be particularly important for the tumorigenic role of IDH1 muta-
`tions because cytosolic a-ketoglutarate is in equilibrium via transami-
`nation with glutamate that has a unique role in glia cell physiology,
`and IDH1 mutations are especially prevalent in malignant gliomas.
`
`IDH1 mutant expressing cells have elevated 2HG levels
`To understand the impact of IDH1 mutation on cellular metabolism,
`we profiled metabolites to identify changes in metabolite levels in
`
`cells expressing R132 mutant IDH1 compared with cells expressing
`wild-type IDH1. To initiate these studies, we stably transfected
`U87MG glioblastoma cells, which are wild-type for IDH1, with
`Myc-tagged wild-type or R132H mutant IDH1. Cells expressing either
`Myc-tagged wild-type or mutant IDH1 were used for metabolite pro-
`filing experiments (Fig. 1a). Metabolites extracted from exponentially
`growing cells were profiled by liquid chromatography–electrospray
`ionization–mass spectrometry (LC–MS). In initial survey analyses,
`full-scan LC–MS in negative ion mode (exact mass) was used to
`examine differences in metabolite species with an m/z between 110
`and 1,000. Relative quantitative data were collected for approximately
`850 ions and identities were proposed by comparison with known
`human metabolites. Identities of .100 species identified by a com-
`bination of exact mass and retention time match to purified standards
`were assigned8. There were no significant differences between cells
`expressing wild-type IDH1 when compared with parental cells. The
`levels of most observed ions were also similar between wild-type and
`R132H mutant IDH1 expressing cells (Fig. 1b), with no significant
`changes found in canonical tricarboxylic acid (TCA) cycle species
`(P . 0.05 for citrate, isocitrate, a-ketoglutarate, succinate, fumarate
`and malate). However, three species were significantly more abundant
`in R132H mutant IDH1 expressing cells (P , 0.001 for each). The
`mass of one of these ions matched precisely to 2HG (expected m/z
`147.0299; measured 147.0299). The other ions co-eluted with 2HG,
`and had masses consistent with the sodium adduct and a dehydrated
`form of 2HG. Subsequent injection of 2HG standard confirmed a
`retention time match to the biological peak, and that it forms all
`three of the observed ions during LC–MS ionization (data not shown).
`The cellular accumulation of 2HG was quantified by targeted
`
`1Agios Pharmaceuticals, Cambridge, Massachusetts 02139, USA. 2Department of Chemistry and Integrative Genomics, Princeton University, Princeton, New Jersey 08544, USA.
`3Department of Neurosurgery, UCLA Medical School, Los Angeles, California 90095, USA. 4Abramson Cancer Center, University of Pennsylvania, Philadelphia, Pennsylvania 19104,
`USA. 5Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA. {Present address: Koch Institute for Integrative Cancer Research,
`Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
`
`
`
` ©2009
`
`Macmillan Publishers Limited. All rights reserved
`
`739
`
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`

`ARTICLES
`
`NATURE | Vol 462 | 10 December 2009
`
`B
`
`A
`
`C
`
`107
`
`106
`
`105
`
`R132H signal intensity
`
`b
`
`a
`
`Blot:
`anti-IDH1
`Blot:
`anti-Myc
`
`ID H1(R132H)– M ycID H1– M ycParental
`
`
`
`
`
`IDH1–Myc
`
`IDH1
`endogenous
`
`1.0 × 106
`7.5 × 105
`5.0 × 105
`2.5 × 105
`0
`
`(a.u.)
`
`Peak intensity
`
`A
`
`149.50
`
`m/z
`
`146.50
`
`11.5
`
`OH
`
`HO
`
`Time (min)
`
`14.0
`
`2HG
`
`OH
`
`O
`O
`m/z 147.0299 (expected 147.0299, C5H7O5)
`
`104
`
`104
`
`107
`106
`105
`Wild-type signal intensity
`
`Parental
`
`IDH1–Myc
`
`IDH1(R132H)–Myc
`
`4,000
`
`3,200
`
`2,400
`
`1,600
`
`800
`
`d
`
`2HG (ng ml–1)
`
`120
`100
`80
`60
`40
`20
`0
`
`2HG (fold change)
`
`c
`
`ID H1– M ycParental
`
`ID H1(R132H)– M yc
`
`0
`
`0.5 h
`Figure 1 | Cells expressing human R132H IDH1 contain markedly elevated
`levels of 2HG. a, Western blots for Myc-tagged human isocitrate
`dehydrogenase 1 (IDH1–Myc) or R132H mutant (IDH1(R132H)–Myc) in
`stably transfected U87MG human glioblastoma cells. b, Metabolite profiles
`from cells expressing R132H IDH1 or wild-type IDH1 detected by LC–MS
`scanning for species between 110–1,000 m/z (M-H1). Red spots labelled A, B
`and C represent species assigned to 2HG, dehydro-2HG and 2HG-sodium
`adduct, respectively. Spectrometric details supporting the identification of
`
`triple-quadrupole LC–MS–MS analysis of cell extracts (Fig. 1c). The
`structure of 2HG is close to a-ketoglutarate, the product of the IDH1
`enzyme. Thus, the sole metabolite identified by untargeted metabolite
`profiling to be markedly altered by R132H mutant IDH1 expression
`was also implicated by its structure to be IDH1-related.
`The accumulation of 2HG was not restricted to cell extracts, as
`2HG was found to accumulate rapidly in the medium of cells expres-
`sing R132H mutant IDH1 (Fig. 1d). No appreciable 2HG could be
`found in the medium of wild-type cells or cells transfected with wild-
`type IDH1 (Fig. 1d). Isotope-labelling experiments on whole cells
`using uniformly labelled 13C-glutamine as a culture media nutrient
`demonstrated that the carbons in 2HG are derived from glutamine,
`with reasonably high overall pathway flux from glutamine through
`glutamate and a-ketoglutarate to 2HG. Moreover, labelling experi-
`ments did not demonstrate any other major alterations in central
`carbon metabolic flux in cells expressing R132H mutant 2HG (data
`not shown). The presence of a Myc epitope tag did not alter activity of
`R132H mutant IDH1. Despite being expressed at lower levels than
`the Myc-tagged R132H IDH1, cells transfected with untagged R132H
`IDH1 demonstrated a comparable increase in 2HG production
`(Supplementary Fig. 1). To determine whether 2HG production in
`cells expressing R132H mutant IDH1 is unique to U87MG cells, we
`stably expressed wild-type and R132H mutant IDH1 in wild-type
`IDH1-expressing LN-18 glioblastoma cells (Supplementary Fig.
`2a). Similar to results obtained with U87MG cells, the major differ-
`ence in metabolite levels observed in LN-18 cells expressing R132H
`mutant IDH1 was an increased level of 2HG (Supplementary Fig. 2b).
`
`Mutant IDH1 directly converts a-ketoglutarate to 2HG
`The R132H mutation has been reported to result in loss of function
`for enzyme activity3,7. However, in these studies only the NADP1-
`dependent oxidative decarboxylation of isocitrate to a-ketoglutarate
`was assessed. To understand how IDH1 activity is altered in cells by
`the presence of R132H mutant IDH1, we expressed increasing
`
`740
`
`4 h
`
`24 h
`
`species ‘A’ as 2HG are shown in the right panel. c, Cells expressing R132H
`IDH1 contain elevated levels of 2HG. Data were normalized by cell number
`and expressed as fold difference relative to parental values. Error bars depict
`one standard deviation (s.d.) from the mean of three independent
`experiments. d, Cells expressing R132H IDH1 display time-dependent
`accumulation of 2HG in cell culture media, normalization was as described
`in c. Errors bars depict one s.d. from the mean of four independent
`experiments.
`
`amounts of wild-type and R132H mutant IDH1 separately or in
`combination and assessed isocitrate-dependent NADPH production
`and a-ketoglutarate-dependent NADPH consumption in cell lysates.
`Consistent with published results, expression of R132H mutant
`IDH1 resulted in no measurable production of NADPH from isoci-
`trate, and isocitrate-dependent NADPH production increased with
`increasing amounts of wild-type enzyme (Supplementary Fig. 3a, b).
`The ability of the wild-type enzyme to generate NADPH was
`decreased slightly by co-expression of the R132H mutant IDH1.
`Opposite results were obtained, however, when NADPH consump-
`tion was measured in the presence of a-ketoglutarate. NADPH con-
`sumption by wild-type enzyme was not observed, whereas R132H
`mutant IDH1 expression resulted in a-ketoglutarate-dependent
`NADPH consumption (Supplementary Fig. 3c). Although the overall
`consumption of NADPH was slow, if anything co-expression of wild-
`type IDH1 with R132H mutant IDH1 facilitated the a-ketoglutarate-
`dependent consumption of NADPH. These findings demonstrate
`that in contrast to wild-type IDH1, R132H mutant IDH1 promotes
`an NADPH-dependent reduction of a-ketoglutarate. Furthermore,
`as this reduction was not inhibited by co-expression of wild-type
`IDH1, these data indicate that the novel activity of mutant IDH1
`can persist even in the presence of a wild-type IDH1 allele. In fact,
`it is possible that in the case of a heterodimer of wild-type and mutant
`IDH1, the a-ketoglutarate and NADPH produced locally by the wild-
`type subunit could be used as substrates for the mutant subunit,
`explaining the decrease in NADPH production observed in lysates
`when wild-type and mutant IDH1 are co-expressed.
`To understand how R132 mutations alter the enzymatic properties
`of IDH1, wild-type and R132H mutant IDH1 proteins were pro-
`duced and purified from Escherichia coli. When NADP1-dependent
`oxidative decarboxylation of isocitrate was measured using purified
`wild-type or R132H mutant IDH1 protein, it was confirmed that
`R132H mutation impairs the ability of IDH1 to catalyse this reac-
`tion3,7, as evident by the loss in binding affinity for both isocitrate and
`
`
`
` ©2009
`
`Macmillan Publishers Limited. All rights reserved
`
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`

`NATURE | Vol 462 | 10 December 2009
`
`ARTICLES
`
`Table 1 | R132H mutation alters the enzymatic properties of IDH1
`Wild-type IDH1
`R132H IDH1
`Kinetic parameter of reaction
`
`O
`
`O
`
`HO
`
`OH
`
`OH
`
`2HG control
`
`Reductive rxn
`product
`
`0
`
`5
`HPLC elution time (min)
`
`10
`
`S(+)-2HG
`
`rxn + racemate
`
`2HG racemate
`
`R(–)-2HG
`
`O
`
`O
`
`HO
`
`OH
`
`OH
`
`rxn + R(–)-2HG
`R(–)-2HG std
`
`Reductive rxn
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`150
`
`100
`
`50
`
`0
`
`a
`
`LC–MS–MS signal (normalized)
`
`b
`
`LC–MS–MS signal (normalized)
`
`10
`0
`HPLC elution time (min)
`
`20
`
`Figure 2 | R132H mutation in IDH1 results in production of R(2)-2HG.
`a, 2HG was identified as the reductive reaction product of recombinant
`human R132H mutant IDH1 using LC–MS as shown. b, The chirality of
`2HG produced by R132H mutant IDH1 was assessed as by diacetyl-L-tartaric
`anhydride derivatization and LC–MS analysis. Normalized LC–MS signal
`for the reductive reaction (rxn) product alone, an R(2)-2HG standard
`alone, and the two together (rxn 1 R(2)-2HG) are shown, as is the signal for
`a racemic mixture of R(2) and S(1) forms (2HG racemate) alone or with the
`reaction products (rxn 1 racemate).
`
`Two important features were noted by the change of R132 to
`histidine: the effect on conformation equilibrium and the reorganization
`of the active site. Located atop a b-sheet in the relatively rigid small
`domain, R132 acts as a gate-keeper residue and seems to orchestrate
`the hinge movement between the open and closed conformations. The
`guanidinium moiety of R132 swings from the open to the closed con-
`formation with a distance of nearly 8 A˚ . Substitution of histidine for
`arginine is likely to change the equilibrium in favour of the closed
`conformation that forms the catalytic cleft for cofactor and substrate
`to bind efficiently, which partly explains the high affinity for NADPH
`shown by the R132H mutant enzyme. This feature may be advantageous
`for the NADPH-dependent reduction of a-ketoglutarate to R(2)-2HG
`in an environment where NADPH concentrations are low. Second,
`closer examination of the catalytic pocket of the mutant IDH1 structure
`in comparison to the wild-type enzyme showed not only the expected
`loss of key salt-bridge interactions between the guanidinium of R132 and
`the a/b carboxylates of isocitrate, as well as changes in the network that
`coordinates the metal ion, but also an unexpected reorganization of the
`active site. Mutation to histidine resulted in a significant shift in position
`of the highly conserved residues Y139 from the A subunit and K2129
`from the B subunit (Fig. 3b, c), both of which are thought to be critical
`for catalysis by this enzyme family11. In particular, the hydroxyl moiety of
`Y139 now occupies the space of the b-carboxylate of isocitrate.
`The electron density in the active site was not sufficient to assign
`a-ketoglutarate and its orientation unambiguously. We have mod-
`elled the substrate based on the available electron density, taking into
`consideration the coordination between the carbonyl oxygen of
`a-ketoglutarate and the calcium ion as well as an orientation of
`a-ketoglutarate that would produce R(2)-2HG, the experimental
`product. The model required a significant repositioning of a-keto-
`glutarate compared to isocitrate, such that the distal carboxylate of
`
`741
`
`Oxidative (RNADPH)
`1 (mM)
`Km,NADP
`Km,isocitrate (mM)
`Km,MgCl2 (mM)
`Ki,a-ketoglutarate (mM)
`kcat (s21)
`Reductive (RNADP1)
`Km,NADPH (mM)
`Km,a-ketoglutarate (mM)
`kcat (s21)
`Kinetic parameters of oxidative and reductive reactions as measured for wild-type and R132H
`IDH1 enzymes are shown. Km (Michaelis constant) and kcat values for the reductive activity of
`the wild-type enzyme were unable to be determined as no measurable enzyme activity was
`detectable at any substrate concentration. Ki, inhibition constant.
`* No measurable enzymatic activity.
`
`49
`65
`29
`1.9 3 103
`4.4 3 104
`
`n/a*
`n/a
`n/a
`
`84
`370
`1.0 3 104
`24
`37.5
`
`0.44
`965
`1.0 3 103
`
`MgCl2 along with a 1,000-fold decrease in catalytic turnover (Table 1
`and Supplementary Fig. 4a). In contrast, when NADPH-dependent
`reduction of a-ketoglutarate was assessed using either wild-type or
`R132H mutant IDH1 protein, only R132H mutant could catalyse this
`reaction (Table 1 and Supplementary Fig. 4b). Part of this increased
`rate of a-ketoglutarate reduction results from an apparent increase in
`affinity for both the cofactor NADPH and substrate a-ketoglutarate
`in the R132H mutant IDH1 (Table 1). Taken together, these data
`demonstrate that whereas the R132H mutation leads to a loss of
`enzymatic function for oxidative decarboxylation of isocitrate, this
`mutation also results in a gain of enzyme function for the NADPH-
`dependent reduction of a-ketoglutarate.
`Reduction of the a-ketone in a-ketoglutarate can result in 2HG.
`To determine whether R132H mutant protein directly produced
`2HG from a-ketoglutarate we examined the product of the mutant
`IDH1 reaction using negative ion mode triple-quadrupole electro-
`spray LC–MS. These experiments confirmed that 2HG was the direct
`product of NADPH-dependent a-ketoglutarate reduction by the
`purified R132H mutant protein through comparison with known
`metabolite standards (Fig. 2a). Conversion of a-ketoglutarate to iso-
`citrate was not observed. To determine the chirality of the 2HG
`produced, we derivatized the products of the R132H reaction with
`diacetyl-L-tartaric anhydride, which allowed us to separate the (S)
`and (R) enantiomers of 2HG by simple reverse-phase LC and detect
`the products by tandem mass spectrometry9 (Fig. 2b). The peaks
`corresponding to the (S) and (R) isomers of 2HG were confirmed
`using racemic and R(2)-2HG standards. The reaction product from
`R132H co-eluted with the R(2)-2HG peak, demonstrating that the
`R(2) stereoisomer is the product produced from a-ketoglutarate by
`R132H mutant IDH1.
`To determine whether the altered enzyme properties resulting
`from the R132H mutation were shared by other R132 mutations
`found in human gliomas, recombinant R132C, R132L and R132S
`mutant IDH1 proteins were generated and the enzymatic properties
`assessed. Similar to R132H mutant protein, R132C, R132L and
`R132S mutations all result in a gain-of-function for NADPH-
`dependent reduction of a-ketoglutarate (Supplementary Fig. 4).
`Thus, in addition to impaired oxidative decarboxylation of isocitrate,
`one common feature shared among the IDH1 mutations found in
`human gliomas is the ability to catalyse direct NADPH-dependent
`reduction of a-ketoglutarate.
`
`X-ray structure reveals a distinct active site of mutant IDH1
`To define how R132 mutations alter the enzymatic properties of
`IDH1, the crystal structure of R132H mutant IDH1 bound to a-
`ketoglutarate, NADPH and Ca21 was solved at 2.1 A˚ resolution (see
`Supplementary Table 1 for crystallographic data and refinement sta-
`tistics). The overall quaternary structure of the homodimeric R132H
`mutant enzyme adopts the same closed catalytically competent
`conformation (shown as a monomer in Fig. 3a) that has been prev-
`iously described for the wild-type enzyme10.
`
`
`
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`
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`NATURE | Vol 462 | 10 December 2009
`
`a
`
`b
`
`c
`
`Closed
`(R132H)
`
`vs
`
`Closed
`(WT)
`
`Closed
`(R132H)
`
`vs
`
`Open
`(WT)
`
`WT
`
`Arg 132
`
`AArg 100
`
`Tyr 139
`
`AAAAsn 96
`
`Ser 94
`
`
`
`Arg AA 109Arg 109
`
`Asp 275
`
`Ca2+
`
`Thr 77
`
`Thr 214′
`
`Lys 212′
`Asp 252′
`
`NADP+
`
`Ser 94
`
`Thr 77
`Thr 214′
`
`NADPH
`
`Tyr 139
`
`
`
`96AAAsnAAsn 9
`
`
`
`R132H
`
`His 132
`
`Arg rg 100
`
`
`
`Arg gArg 10
`
`9
`
`Asp 275
`
`Ca2+
`
`Lys 212′
`Asp 252′
`
`′
`
`′
`
`Figure 3 | Structural analysis of R132H mutant IDH1. a, On the left is shown
`an overlay of R132H mutant IDH1 (green) and wild-type IDH1 (grey)
`structures in the ‘closed’ conformation. On the right is shown an overlay of
`wild-type IDH1 (blue) structure in the ‘open’ conformation with mutant
`IDH1 (green) for comparison. b, Close-up comparison of the R132H IDH1
`active site (left) with a-ketoglutarate (yellow) and NADPH (grey) and the
`wild-type IDH1 active site (right) with isocitrate (yellow) and NADP (grey).
`
`Residues coming from the other monomer are denoted with a prime (9)
`symbol. In addition to the mutation at residue 132, the major changes are the
`positions of the catalytic residues Tyr 139 and Lys 2129. c, Wall-eyed stereo
`image showing the composite omit map for a-ketoglutarate, NADPH,
`calcium ion, His 132 and other key catalytic residues in the R132H mutant
`active site contoured at the 1s level.
`
`a-ketoglutarate now points upward to make new contacts with N96
`and S94. Overall, this single R132H mutation results in formation of
`a distinct active site compared to wild-type IDH1.
`
`2HG levels are elevated in human glioma samples
`Our data demonstrate that mutation of R132 can result in the ability
`of IDH1 to generate R(2)-2HG from a-ketoglutarate. To determine
`if 2HG production is characteristic of tumours harbouring muta-
`tions in IDH1, metabolites were extracted from human malignant
`gliomas that were either wild-type or mutant
`for IDH1 (see
`Supplementary Table 2 for summary of tumour characteristics). It
`has been suggested that a-ketoglutarate levels are decreased in cells
`transfected with mutant IDH17. We observe that the average a-keto-
`glutarate level from 12 tumour samples harbouring various R132
`mutations was slightly less than the average a-ketoglutarate level
`observed in 10 tumours which are wild type for IDH1. This difference
`
`in a-ketoglutarate was not statistically significant, and a range of
`a-ketoglutarate levels was observed in both wild-type and mutant
`tumours (Fig. 4). Similarly, a range of levels was observed for other
`proximal TCA cycle metabolites with no significant differences
`observed between wild-type IDH1 tumours and tumours with
`R132 IDH1 mutations. In contrast, increased 2HG levels were found
`in all tumours that contained an R132 IDH1 mutation (Fig. 4 and
`Supplementary Table 2). All R132 mutant IDH1 tumours examined
`had between 5 and 35 mmol of 2HG per gram of tumour, whereas
`tumours with wild-type IDH1 had over 100-fold less 2HG. This
`increase in 2HG in R132 mutant tumours was statistically significant
`(P , 0.0001). We confirmed that (R)-2HG was the isomer present in
`tumour samples (data not shown). Together these data establish that
`the novel enzymatic activity associated with R132 mutations in IDH1
`results in the production of 2HG in human brain tumours that har-
`bour these mutations.
`
`742
`
`
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`
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`NATURE | Vol 462 | 10 December 2009
`
`ARTICLES
`
`catalytic activity, are not found. Finally, these findings have clinical
`implications in that they suggest that 2HG production will identify
`patients with IDH1 mutant brain tumours. This will be important for
`prognosis as patients with IDH1 mutations live longer than patients
`with gliomas characterized by other mutations5. In addition, patients
`with lower-grade gliomas may benefit by the therapeutic inhibition
`of 2HG production. Inhibition of 2HG production by mutant IDH1
`might slow or halt conversion of lower-grade glioma into lethal sec-
`ondary glioblastoma, changing the course of the disease.
`
`METHODS SUMMARY
`R132H, R132C, R132L and R132S mutations were introduced into human IDH1
`by standard molecular biology techniques. 293T and human glioma U87MG and
`LN-18 cell lines were transfected using standard techniques. Protein expression
`levels were determined by western blot. Metabolites were extracted from cul-
`tured cells and from tissue samples using 80% aqueous methanol (280 uC) as
`previously reported8. Metabolite levels in samples were determined by negative
`mode electrospray LC–MS. For untargeted profiling, extract components were
`resolved using reverse-phase high-performance liquid chromatography (HPLC)
`and metabolites were detected in ultra-high resolution mode (resolution
`,100,000) by stand alone orbitrap MS, collecting at one scan per second over
`an m/z range of 110–1,100. For targeted evaluation of 2HG, a-ketoglutarate and
`other TCA intermediates, extracts were resolved by reverse-phase HPLC system
`and metabolites detected by triple-quadrupole mass spectrometry, using
`multiple-reaction monitoring. Enzymatic activity in cell lysates was assessed
`by following a change in NADPH fluorescence over time in the presence of
`isocitrate and NADP, or a-ketoglutarate and NADPH. For enzyme assays using
`recombinant IDH1 enzyme, proteins were purified from E. coli using Ni affinity
`and size-exclusion chromatography. Enzymatic activity for recombinant IDH1
`protein was assessed by following a change in NADPH absorbance at 340 nm
`using a stop-flow spectrophotometer. Chirality of 2HG was determined as
`described previously9. For crystallography studies, purified R132H IDH1 was
`pre-incubated with NADPH, calcium chloride and a-ketoglutarate. Crystals
`were obtained at 20 uC by vapour diffusion equilibration using 3 ml drops mixed
`2:1 (protein:precipitant) against a well-solution of MES pH 6.5 and PEG 6000.
`Patient tumour samples were obtained after informed consent as part of a UCLA
`IRB-approved research protocol, collected by surgical resection, snap frozen in
`isopentane cooled by liquid nitrogen and stored at 280 uC. The IDH1 mutation
`status of each sample was determined as described previously3.
`
`Full Methods and any associated references are available in the online version of
`the paper at www.nature.com/nature.
`
`Received 15 July; accepted 29 October 2009.
`Published online 22 November 2009.
`
`α-
`Ketoglutarate Malate
`
`2HG
`
`Fumarate
`
`Succinate
`
`Isocitrate
`
`Mutant
`Wild type
`
`Mutant
`Wild type
`
`Mutant
`Wild type
`
`Mutant
`Wild type
`
`Mutant
`Wild type
`
`Mutant
`Wild type
`
`100.00
`
`10.00
`
`1.00
`
`0.10
`
`0.01
`
`Metabolite concentration (μmol g–1)
`
`Figure 4 | Human malignant gliomas containing R132 mutations in IDH1
`contain increased concentrations of 2HG. Human glioma samples obtained by
`surgical resection were snap frozen, genotyped to stratify as wild type (n 5 10)
`or carrying an R132 mutant allele (mutant) (n 5 12) and metabolites extracted
`for LC–MS analysis. Among the 12 mutant tumours, 10 carried a R132H
`mutation, one an R132S mutation, and one an R132G mutation. Each symbol
`represents the amount of the listed metabolite found in each tumour sample.
`Horizontal lines indicate the group sample means. The difference in 2HG
`observed between wild-type and R132 mutant IDH1 tumours was statistically
`significant by Student’s t-test (P , 0.0001). There were no statistically
`significant differences in a-ketoglutarate, malate, fumarate, succinate, or
`isocitrate levels between the wild-type and R132 mutant IDH1 tumours.
`
`Discussion
`2HG is known to accumulate in the inherited metabolic disorder
`2-hydroxyglutaric aciduria. This disease is caused by deficiency in
`the enzyme 2-hydroxyglutarate dehydrogenase, which converts 2HG
`to a-ketoglutarate12. Patients with 2-hydroxyglutarate dehydrogen-
`ase deficiencies accumulate 2HG in the brain as assessed by MRI and
`CSF analysis, develop leukoencephalopathy, and have an increased
`risk of developing brain tumours13–15. Furthermore, elevated brain
`levels of 2HG result in increased ROS levels16,17, potentially contrib-
`uting to an increased risk of cancer, and alterations in NADPH meta-
`bolism resulting from mutant IDH1 expression could further
`exacerbate this effect. The ability of 2HG to act as an NMDA (N-
`methyl-D-aspartate) receptor agonist may contribute to this effect16.
`2HG may also be toxic to cells by competitively inhibiting glutamate
`and/or a-ketoglutarate using enzymes. These include transaminases,
`which allow utilization of glutamate nitrogen for amino and nucleic
`acid biosynthesis, and a-ketoglutarate-dependent prolyl hydroxy-
`lases, such as those that regulate Hif1a levels. Alterations in Hif1a
`have been reported to result from mutant IDH1 protein expression7.
`Regardless of the mechanism, it seems likely that the gain-of-function
`ability of cells to produce 2HG as a result of R132 mutations in IDH1
`contributes to tumorigenesis. Patients with 2-hydroxyglutarate
`dehydrogenase deficiency have a high risk of central nervous system
`(CNS) malignancy15. The ability of mutant IDH1 to act directly on
`a-ketoglutarate may explain the prevalence of IDH1 mutations in
`tumours from CNS tissue, which are unique in their high level of
`glutamate uptake and its ready conversion to a-ketoglutarate in the
`cytosol18, thereby providing high levels of substrate for 2HG produc-
`tion. Myeloid cells also display a high ability to metabolize glutamine
`and recently R132 IDH1 mutations have also been described in a
`subset of acute myelogenous leukaemia (AML)19. The apparent co-
`dominance of the activity of mutant IDH1 with that of the wild-type
`enzyme is consistent with the genetics of the disease, in which only a
`single copy of the gene is mutated. As discussed above, the wild-type
`IDH1 could directly provide NADPH and a-ketoglutarate to the
`mutant enzyme. These data also demonstrate that mutation of
`R132 to histidine, serine, cysteine, glycine, or leucine shares a com-
`mon ability to catalyse the NADPH-dependent conversion of a-keto-
`glutarate to 2HG. These findings help clarify why mutations at other
`amino acid residues of IDH1, including other residues essential for
`
`1.
`
`5.
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`6.
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`Balss, J. et al. Analysis of the IDH1 codon 132 mutation in brain tumors. Acta
`Neuropathol. 116, 597–602 (2008).
`2. Watanabe, T., Nobusawa, S., Kleihues, P. & Ohgaki, H. IDH1 mutations are early
`events in the development of astrocytomas and oligodendrogliomas. Am. J.
`Pathol. 174, 1149–1153 (2009).
`3. Yan, H. et al. IDH1 and IDH2 mutations in gliomas. N. Engl. J. Med. 360, 765–773
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`4. Hartmann, C. et al. Type and frequency of IDH1 and IDH2 mutations are related to
`astrocytic and oligodendroglial differentiation and age: a study of 1,010 diffuse
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`7. Zhao, S. et al. Glioma-derived mutations in IDH1 dominantly inhibit IDH1 catalytic
`activity and induce HIF-1a. Science 324, 261–265 (2009).
`Lu, W., Kimball, E. & Rabinowitz, J. D. A high-performance liquid chromatography-
`tandem mass spectrometry method for quantitation of nitrogen-containing
`intracellular metabolites. J. Am. Soc. Mass Spectrom. 17, 37–50 (2006).
`Struys, E. A., Jansen, E. E., Verhoeven, N. M. & Jakobs, C. Measurement of urinary
`D- and L-2-hydroxyglutarate enantiomers by stable-isotope-dilution liquid
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`tartaric anhydride. Clin. Chem. 50, 1391–1395 (2004).
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`the metal ion-dependent pyridine dinucleotide-linked b-hydroxyacid oxidative
`decarboxylases. Biochemistry 48, 3565–3577 (2009).
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` ©2009
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`Macmillan Publishers Limited. All rights reserved
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`ARTICLES
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`NATURE | Vol 462 | 10 December 2009
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