Mutations at IDH1 R100 and G97 result in 2HG production
A metabolite screening test of AML samples established that IDH2 mutations at R140 produce 2HG (Ward et al., 2010
). Since mutations at two other IDH residues which are analogous to each other, mitochondrial IDH2 R172 and cytosolic IDH1 R132, both lead to 2HG production, we asked whether the same relationship held between known neomorphic mutation s at IDH2 R140 and mutations at the uncharacterized analogous position in IDH1, R100. Like IDH2 R140, IDH1 R100 normally stabilizes isocitrate’s β-carboxyl group through a charge interaction from its guanidinium moiety (). We predicted that removing this stabilization of the β-carboxyl through mutation of R100 would hinder the enzyme’s ability to use isocitrate as a substrate and, like mutation of IDH2 R140, facilitate the non-carboxylating reduction of α-ketoglutarate to 2HG. The effect of IDH1 G97D mutation was also examined, as this alteration has been identified in colon cancer cell lines and pediatric glioblastoma ( Bleeker et al., 2009
;Paugh et al., 2010
), and we predicted that this mutation to a negatively charged aspartate would disfavor coordination of isocitrate’s negatively charged β-carboxyl.
Mutation of IDH1 R100 or G97 leads to 2HG production
To test our predictions, we expressed IDH1 R100A and IDH1 G97D mutants in cells, along with IDH1 WT and the established 2HG-producing IDH1 R132Hmutant (). We first found that, unlike overexpression of IDH1 WT, expression of either IDH1 R100A or G97D failed to increase isocitrate-dependent NADPH production in cell lysates above the levels invector-transfected cells. Thus, like the IDH1 R132H mutation as shown previously (Dang et al., 2009
;Yan et al., 2009
), IDH1 R100Aand G97D mutations are loss-of-function for generating NADPH in the presence of isocitrate. To determine if theR100A and G97D mutants also share with the R132H mutant the gain-of-function to produce 2HG, we extracted metabolites from parallel-transfected cells and measured 2HG elevation by GC-MS. 2HG levels were observed to be elevated in cells expressing either IDH1 R100A or G97D (). As confirmation we found that 2HG was also elevated in the HCT15 colon cancer line containing an endogenous G97D mutation (data not shown). Of note, following the initiation of this study, IDH1mutations at R100 were described in adult glioma( Pusch et al., 2011
), further supporting the importance of predicting and then characterizing additional 2HG-producing alleles.
Electrostatics analysis predicts an other 2HG-producing mutation:IDH1 Y139D
To better understand the loss of isocitrate utilization and gain of 2HG production from IDH1 R132H, R100A, and G97D mutations, we performed an electrostatics analysis to calculate the destabilizing effect of each mutation on isocitrate’s β-carboxyl (). Consistent with IDH1 R132H, R100A, and G97D mutations all favoring conversion of α-ketoglutarate to 2HG rather than inter conversion of α-ketoglutarate and isocitrate, all three mutations were calculated to be unfavorable for isocitrate β-carboxyl stabilization. While performing this analysis, we also examined if mutations at other IDH1 active site residues could be predicted to disfavor coordination of the isocitrate β-carboxyl. Surprisingly, we found that an additional mutation, Y139D, could be modeled to have an electrostatic effect comparable to that for the 2HG-producing mutant G97D. As Y139 islocated near the substrate in t he IDH1 active site (), w e investigated whether IDH1 Y139D could be yet another 2HG-producing neomorph. We first found thatY139D mutant overexpression failed to increase isocitrate-dependent NADPH production from cell lysates, similar to R132H mutant overexpression and in contrast to cells overexpressing IDH1 WT(). From parallel-transfected cells, we then assessed whether IDH1 Y139D could produce 2HG. Unlike cells expressing IDH1WT, cells expressing the predicted Y139D mutantdisplayed 2HG elevation().
Predicted Y139D mutation of IDH1 is another 2HG-producing allele
While mutation of IDH1 Y139 has yet to be described in any cancer, the residue does lie outside IDH1’s fourth exon, which has been the region of exclusive focus in many sequencing studies. The lack of samples to date with reported mutation of IDH1 Y139 (or the analogous IDH2 Y179)m ay also be structurally explained by the strict requirement for this position to incorporate a negatively charged residue in order to facilitate neomorphic enzyme activity (Supplementary Table 1
). In contrast, for the commonly affected residues IDH1 R132, IDH2 R172, and IDH2 R140, the diversity of substitutions observed suggests a less specific structural requirement. Still, specific substitutions of IDH1 R132H/C, IDH2 R172K, and IDH2 R140Q are observed with higher frequency. These can all arise through C→T or G→Atransitions in CpG dinucelotides at these codons, potentially from the frequent methylation-induced deamination of 5-methylcytosine (Cooper and Youssoufian 1988
), in contrast to IDH1 Y139D where a T→G transversion must occur at a non-CpG site.
Rare recurring mutations in lymphoma and thyroid cancer do not produce 2HG, but can result in loss of function
At the time of this article’s submission, over 100 papers had been published that reported patient samples containing tumor-specific IDH mutations. Greater than 99% of these patient samples have an IDH1/2 mutation that has now been confirmed to be R
(-)-2HG-producing, either from this or prior studies(Supplementary Table 2
). However, several reports have recently identified IDH1/2 variants which have yet to be enzymatically characterized(). We first examined the effects of reported IDH1 SNPs V71I and V178I, as well as non-arginine variants only reported in a single sample to date( Hemerly et al
.,2010 ; Marcucci et al., 2010
; Ho et al., 2010
; Murugan et al., 2010
; Zou et al., 2010
; Forbes et al., 2011
; Rakheja et al., 2011
). All were expressed at comparable levels to IDH1/2 WT in cells (Supplementary Figure 1
). None were observed to increase intracellular 2HG, while all retained the ability to increase isocitrate-dependent NADPH production from cell lysates.
Summary of the effect of IDH mutations on enzyme expression and activity
In addition to these variants which we found to behave like wild-type IDH enzymes, we also investigated several rare yet recurring somatic mutations. Although previously undescribed in cancer, we have found somatic mutations at IDH2 F394 in two T-cell angioimmunoblastic lymphoma (AILT) samples(one sample with IDH2 F394I, and one with F394V). By transfection we were unable to express these mutants, despite achieving comparable levels of IDH2 mRNA in the transfected cells (Supplementary Figure 2
). Transfection of these mutants did not elevate intracellular 2HG, and we confirmed that their transfection did not increase isocitrate-dependent NADPH production.
We then investigated IDH1 G70D and A134D mutations described in six and two thyroid cancer samples, respectively, as well as an IDH1 R49C mutation described in one pediatric glioblastoma (Hemerly et al., 2010
, Paugh et al., 2010
). All of these mutations have been demonstrated to occur at a single allele in a somatic manner. Only the IDH1 A134D mutant was able to be effectively overexpressed to levels comparable to IDH1 WT or R132H, despite a clear increase in IDH1 mRNA levels for all mutant-transfected cells (Supplementary Figure 3
). While not producing 2HG, the expressed A134D mutant also failed to increase isocitrate-dependent NADPH production. Together these data suggest that there is a subset of rare IDH somatic mutations that result in decreased wild-type activity without a concomitant increase in 2HG production.
Since retention of at least one wild-type IDH allele is important for proliferation in MYC-driven cancer cells and for IDH1/2-mediated shuttling of NADPH from the mitochondria to cytoplasm (Ward et al., 2010
; Lemons et al., 2010
), we examined if the expressed but loss-of-function A134D mutant IDH1 could dominantly inhibit IDH1 WT activity in cells. For the 2HG-producing mutant IDH1 R132H, it was shown first in Dang et al. (2009)
, testing a range of physiological isocitrate concentrations up to 100 μM, that R132H mutant expression does not substantially inhibit the isocitrate-dependent NADPH production of IDH1WT, a finding recently confirmed extensively by an independent group (Jin et al., 2011
). We repeated this analysis for IDH1 A134D. Co-transfecting IDH1 A134D with IDH1WT at either a 1:1 or 3:1 ratio, we did not observe a decrease in isocitrate-dependent NADPH production from transfection of the A134D mutant (Supplementary Figure 4
For the IDH1 G70D, IDH1 R49C, and IDH2 F394I/V mutants that were unable to be effectively overexpressed in cells, we performed in vitro
transcription coupled translation with [35
S] methionine and rabbit reticulocyte lysates (Supplementary Figure 5
). We found that in vitro
transcription/translation of IDH1 G70D or IDH1 R49Cgenerated a major band corresponding to the predicted IDH1 molecular weight of 47 kD, matching the product observed from IDH1 WT. Similarly, we found that in vitro
transcription/translation of IDH2 F394I or F394V generated a major band matching that produced from IDH2 WT. These results further indicate that the inability of these mutants to be effectively overexpressed following transfection reflects an impairment in cellular accumulation.
No IDH mutations producing 2HG have been confirmed to date in thyroid cancer. Conversely, none of the recurring but non-2HG-producingsomatic mutations found in thyroid cancer or AILT have ever been described in leukemia or adult glioma. We further confirmed in this study, in 973 myeloid hematologic malignancy samples, that no recurring somatic mutations were found between IDH1 residues41–438 or IDH2 residues 125–226 except for the previously characterized 2HG-producing IDH1 R132, IDH2 R172K, and IDH2 R140Q alleles. Full-length sequencing of the entire IDH1/2coding regions in 20 samples also failed to detect any additional somatic alterations.
These data support the neomorphic activity converting α-ketoglutarate to R
(-)-2HG, rather than solely the loss of normal IDH function, being the common feature selected for in adult leukemia, glioma, and the vast majority of other IDH1/2 mutant cancers. This is further evidenced by recent work demonstrating the clustering of R
(-)-2HG-producing IDH mutant cases in a distinct DNA hypermethylation signature in tumor samples from both glioma and AML patients (Figueroa et al., 2010
, Noushmehr et al., 2010
). While those rare IDH mutations indicative of IDH haploinsufficiency in AILT and thyroid cancer require further investigation and may potentially result in impairment of cytosolic NADPH production and redox control, any of the residues in cytosolic IDH1 or mitochondrial IDH2 that can be altered to produce the R
(-)-2HG oncometabolite can be screened for their mutation by a GC-MS metabolite assay.
The data presented here suggest that a screening and diagnostic approach based on elevated oncometabolite levels may not be of the highest utility for thyroid cancer, as IDH mutations found to date in thyroid cancer do not produce 2HG and may function to promote tumorigenesis in an alternative manner. However, based on this study there are at least five reproducible cancer-associated mutations that can result in 2HG production, and these appear to be selected for in several well-characterized tumor types including AML, gliomas, chondrosarcomas, and gastrointestinal cancers. Thus, a screening and diagnostic approach for these malignancies based on elevated 2HG levels may be of substantial value. Firstly, given the rarity and marked developmental consequences of inborn errors of metabolism leading to elevated 2HG, tumors displaying increased levels of 2HG are unlikely to be false positives, in contrast to conventional gene sequencing in which numerous SNPs and uncharacterized sequencing artifacts and/or passenger alterations that have no effect on IDH enzyme activity are detected. Screening for elevated 2HG levels may also be a more sensitive test, as it can allow for the detection of neomorphic mutations at residues like IDH1 R100, G97, and Y139 that are not normally examined by sequencing or mutation-specific antibody approaches focused on the most common alleles. Finally, screening for 2HG can be non-invasive: patient sera/plasma can be assayed in the case of leukemia, while radiologic approaches for 2HG detection can be refined in the case of glioma and other solid tumors, and urinalysis may also be employed.
Overall, the data presented here demonstrate the complexity inherent in correlating the genetic alterations in the IDH1/2 enzymes found in sequencing studies with altered metabolic activity. Most IDH mutant tumor samples reported to date harbor a mutation which has now been shown to be R(-)-2HG producing, based on the work of this study and others. Yet we also report here the existence of rare subsets of IDH loss-of-function mutations which do not produce 2HG. Taken together, these data highlight the value of metabolite screening approaches to more specifically and sensitively identify those IDH mutant tumors which harbor elevations in the level of the R(-)-2HG oncometabolite.