2HG Levels Research Articles

140 - Mitochondrial Deacetylase SIRT3 Regulates IDH2 Function in Breast Cancer Cells

Cancer metabolic alterations result from complex genetic and epigenetic adjustments which include mitochondrial pathways glutaminolysis, reductive carboxylation (RC), and 2-hydroxy-glutarate (2HG) production. We studied regulatory mechanisms that promote anabolic function of mitochondrial isocitrate dehydrogenase 2 (IDH2) towards RC and/or 2HG production in breast cancer cell lines, especially the activity of mitochondrial deacetylase sirtuin SIRT3, which was previously implicated in the regulation of IDH2 enzymatic activity. We demonstrated that IDH2 is a direct substrate of SIRT3 and that distinct regulation by SIRT3 of oxidative vs. reductive IDH2 activity exists. The analysis of metabolite labeling with stable isotope shows that mitochondrial production of 2HG by wild-type IDH2 is largely dependent on SIRT3 presence and activity, as assumed by overexpression of wild-type SIRT3 and SIRT3-inactive mutant in breast cancer cells. Moreover, we demonstrate that IDH2 activity produces 2HG in vitro, as assayed by the product analysis of the enzyme reaction of the isolated recombinant wild-type IDH2. The observed 2HG-producing activity is regulated by SIRT3, as an acetylation surrogate IDH2 mutant K413Q tend to decrease levels of 2HG in vitro. Our findings impact understanding of breast cancer etiology, since breast cancer cell lines exhibit a broad range of IDH2 expression and metabolic phenotypes, including 2HG levels.

Abstract 4647: Mass spectrometric analysis of oncometabolite 2-hydroxyglutarate in clinical gliomas with the isocitrate dehydrogenase-1 mutation

Abstract Introduction: Mutations of the isocitrate dehydrogenase (IDH)-1/2 genes frequently occur in certain malignancies, including gliomas. Mutant IDH-1/2 proteins gain a new ability to produce oncometabolite 2-hydroxiglutarate (2HG), which results in a different clinical impact on gliomas in comparison to those with a wild-type IDH. The status of IDH mutations has been determined by immunohistochemistry (IHC) using the IDH1-R132H antibody or through the DNA sequencing of IDH1-R132C/R132S; however, its practical applications are still under discussion, and the significance of the oncometabolite 2HG evaluation is not clear. In this study, we analyzed the serum and tissue levels of 2HG in clinical gliomas using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the matrix assisted laser desorption and ionization mass spectrometry imaging (MALDI-MSI) system. Method: Glioma samples (n = 78, matched pairs of serum and flash frozen tissue) were obtained between October 2007 to January 2015 at the National Cancer Center Hospital, Japan. High performance liquid chromatography (Nexera X2, Shimadzu) and triple quad mass spectrometry (QTRAP4500, AB SCIEX) were used for LC-MS/MS analysis. The two MALDI-MSI systems, quadrupole ion trap time-to-flight mass spectrometry imaging (iMScope, Shimadzu), and high-resolution atmospheric pressure mass spectrometry imaging (AP-SMALDI, TransMIT; Q-Exactive, Thermo Fisher Scientific) were used for analyzing the 2HG tissue distribution after coating with 9-aminoacridine or 2,5-dihydroxybenzoic acid. The IDH mutation was determined by direct sequencing and IHC. Results: The median 2HG concentration in serum was 21.43 ng/mL (range: 9.58 to 62.51) in this study, and no significant correlation was observed between the IDH mutation and the wild type status (mutation, n = 28, median 20.2 ng/mL, range 9.58 to 62.51 vs. wild type, n = 50, median 22.88 ng/mL, range 11.51 to 40.32; Wilcoxon test p = 0.1597). The median of the 2HG tissue concentration in the entire group was 0.16 ng/μg, and a significant difference was seen between the IDH mutant and wild type groups (mutation, median 4.86 ng/μg, range 0.51 to 49.86 vs. wild type, median 0.09 ng/μg, range 0.02 to 0.45; Wilcoxon test p = <0.0001). In the MALDI-MSI analysis, the average 2HG ion intensities were 381.62/mm2 in the IDH mutation and 8.25/mm2 in the wild-type IDH tissues (Wilcoxon test p = 0.016), which showed a positive correlation with the 2HG tissue concentration by LC-MSMS analyses (Spearman ρ = 0.87, p = 0.0003). The MSI results indicate a possible link between the 2HG tissue distributions and the histology of IDH mutant sections. Conclusion: Tissue 2HG measurement using mass spectrometric analyses may be a useful alternative in determining IDH mutations. Further research may provide a biological perspective and help gain some insights on the clinical significance of 2HG. Citation Format: Mitsuhiro Hayashi, Hiroaki Aikawa, Makoto Ohno, Koichi Ichimura, Yuko Matsushita, Mayu Ohuchi, Mariko Mizui, Akihiko Yoshida, Yoshitaka Narita, Akinobu Hamada. Mass spectrometric analysis of oncometabolite 2-hydroxyglutarate in clinical gliomas with the isocitrate dehydrogenase-1 mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4647. doi:10.1158/1538-7445.AM2017-4647

Replication Study: The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate.

In 2016, as part of the Reproducibility Project: Cancer Biology, we published a Registered Report (Fiehn et al., 2016), that described how we intended to replicate selected experiments from the paper "The common feature of leukemia-associated IDH1 and IDH2 mutations is a neomorphic enzyme activity converting alpha-ketoglutarate to 2-hydroxyglutarate" (Ward et al., 2010). Here, we report the results of those experiments. We found that cells expressing R172K mutant IDH2 did not display isocitrate-dependent NADPH production above vector control levels, in contrast to the increased production observed with wild-type IDH2. Conversely, expression of R172K mutant IDH2 resulted in increased alpha-ketoglutarate-dependent consumption of NADPH compared to wild-type IDH2 or vector control. These results are similar to those reported in the original study (Figure 2; Ward et al., 2010). Further, expression of R172K mutant IDH2 resulted in increased 2HG levels within cells compared to the background levels observed in wild-type IDH2 and vector control, similar to the original study (Figure 3D; Ward et al., 2010). In primary human AML samples, the 2HG levels observed in samples with mutant IDH1 or IDH2 status were higher than those observed in samples without an IDH mutation, similar to what was observed in the original study (Figure 5C; Ward et al., 2010). Finally, we report meta-analyses for each result.

Comparison of 2-hydroxyglutarate (2HG) levels in tissue and serum of isocitrate dehydrogenase (IDH)-mutated (MUT) versus wild-type (WT) gliomas.

2037 Background: IDH mutations are common in low-grade gliomas and confer significantly improved prognosis. IDH catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate, and subsequently to the oncometabolite 2HG. Mutant IDH leads to preferential accumulation of the R relative to the S enantiomer of 2HG. We analyzed the ratio of R to S enantiomers (rRS) in glioma tissues and matched serum samples, and correlated findings with IDH status, 1p19q codeletion status and survival. Methods: Fresh frozen glioma tissues and matched serum samples were obtained from the University of Toronto Brain Tumor Bank. IDH status was determined by immunohistochemistry and confirmed by 450K methylation profile or direct sequencing. 1p19q codeletion status was determined by loss-of-heterozygosity PCR analysis. R-2HG and S-2HG levels were quantified using HPLC tandem mass spectrometry coupled with a CHIROBIOTIC column. Results: Glioma tissues from 70 patients were analyzed – 52 with IDH MUT and 18 WT. 30 had matched serum samples. Using glioma tissues, rRS clearly distinguished MUT vs WT (median 574 vs 1.3, p < 1x10-9) with only three outliers. In contrast, rRS was not elevated in serum samples (median 1.5 vs 1.2, p = 0.13). Overall survival (OS) was significantly longer for MUT vs WT (median 178 vs 33 months, p < 1×10-7). Stratifying MUT by tissue rRS, median OS was 197, 178, 178 and 122 months for lowest to highest quartiles of rRS respectively. 1p19q codeletion status and tumor latency did not explain this trend, given rRS was similar in codeleted vs non-codeleted MUT, and similar in MUT operated < 3 vs ≥3 months from diagnosis. Progression-free survival results corresponded to OS. Conclusions: rRS from glioma tissues effectively differentiated MUT vs WT, whereas serum samples were unreliable. Unlike current methods, tissue rRS enables real-time determination of IDH status, and thus may guide clinical practice such as extent of surgical resection intraoperatively and upfront selection of adjuvant therapy. rRS potentially stratifies survival within MUT patients, providing detailed correlative information.

Noninvasive assessment of isocitrate dehydrogenase mutation status in cerebral gliomas by magnetic resonance spectroscopy in a clinical setting.

OBJECTIVE Mutations in the isocitrate dehydrogenase (IDH) genes are of proven diagnostic and prognostic significance for cerebral gliomas. The objective of this study was to evaluate the clinical feasibility of using a recently described method for determining IDH mutation status by using magnetic resonance spectroscopy (MRS) to detect the presence of 2-hydroxyglutarate (2HG), the metabolic product of the mutant IDH enzyme. METHODS By extending imaging time by 6 minutes, the authors were able to include a point-resolved spectroscopy (PRESS) MRS sequence in their routine glioma imaging protocol. In 30 of 35 patients for whom this revised protocol was used the lesions were subsequently diagnosed histologically as gliomas. Of the remaining 5 patients, 1 had a gangliocytoma, 1 had a primary CNS lymphoma, and 3 had nonneoplastic lesions. Immunohistochemistry and/or polymerase chain reaction were used to detect the presence of IDH mutations in the glioma tissue resected. RESULTS In vivo MRS for 2HG correctly identified the IDH mutational status in 88.6% of patients. The sensitivity and specificity was 89.5% and 81.3%, respectively, when using 2 mM 2HG as threshold to discriminate IDH-mutated from wildtype tumors. Two glioblastomas that had elevated 2HG levels did not have detectable IDH mutations, and in 2 IDH-mutated gliomas 2HG was not reliably detectable. CONCLUSIONS The noninvasive determination of the IDH mutation status of a presumed glioma by means of MRS may be incorporated into a routine diagnostic imaging protocol and can be used to obtain additional information for patient care.

The IDH2 R172K mutation associated with angioimmunoblastic T-cell lymphoma produces 2HG in T cells and impacts lymphoid development

Oncogenic isocitrate dehydrogenase (IDH)1 and IDH2 mutations at three hotspot arginine residues cause an enzymatic gain of function that leads to the production and accumulation of the metabolite 2-hydroxyglutarate (2HG), which contributes to the development of a number of malignancies. In the hematopoietic system, mutations in IDH1 at arginine (R) 132 and in IDH2 at R140 and R172 are commonly observed in acute myeloid leukemia, and elevated 2HG is observed in cells and serum. However, in angioimmunoblastic T-cell lymphoma (AITL), mutations are almost exclusively restricted to IDH2 R172, and levels of 2HG have not been comprehensively measured. In this study, we investigate the expression pattern of mutant IDH2 in the AITL tumor microenvironment and measure levels of 2HG in tissue and serum of AITL patients. We find that mutant IDH2 expression is restricted to the malignant T-cell component of AITL, and that 2HG is elevated in tumor tissue and serum of patients. We also investigate the differences between the three hotspot mutation sites in IDH1 and IDH2 using conditional knock-in mouse models. These studies show that in the lymphoid system, mutations in IDH2 at R172 produce high levels of 2HG compared with mutations at the other two sites and that lymphoid development is impaired in these animals. These data provide evidence that IDH2 R172 mutations may be the only variants present in AITL because of their capacity to produce significant amounts of the oncometabolite 2HG in the cell of origin of this disease.

MPTH-22. SERUM 2-HYDROXYGLUTARATE (2HG) LEVELS ARE CORRELATED WITH TISSUE 2HG LEVELS IN ISOCITRATE DEHYDROGENASE 1 AND 2 GENES (IDH1/2)-MUTATED GLIOMAS BUT NOT IN IDH1/2-WILDTYPE GLIOMAS

Mutations in isocitrate dehydrogenase 1 and 2 genes (IDH1/2) frequently occur in lower-grade gliomas and secondary glioblastomas. Mutant IDH1/2 proteins gain a new ability to produce the oncometabolite 2-hydroxyglutarate (2HG), however, the clinical impact of 2HG is not clear. In this study, we analyzed 2HG levels in both serum and tumor tissue and evaluated the role of 2HG in diffuse gliomas. We measured 2HG concentrations in 123 serums and in 78 tumor tissues by using liquid chromatography-tandem mass spectrometry method. IDH1/2 mutations status were determined by direct sequencing and/or immunohistochemistry. O-6-methylguanine DNA methyltransferase (MGMT) promoter methylations were analyzed using pyrosequencing and defined the mean level of methylation at the 16 CpG sites.Serum levels of 2HG in 42 patients with IDH-Mut tumors did not differ from those in 81 patients with IDH-WT tumors (median: 19.9 ng/mL for IDH-Mut vs 22.8 ng/mL for IDH-WT, p=0.86), however, tissue levels of 2HG in 28 patients with IDH-Mut tumors were significantly higher than those in 50 patients with IDH-WT tumors (median: 4.9 ug/mg for IDH-Mut vs 0.09 ug/mg for IDH-WT, p<0.0001). In 28 patients with IDH-Mut tumors, serum levels of 2HG were significantly correlated with tissue levels of 2HG (R2 = 0.39, p=0.0004), but in 50 patients with IDH-WT tumors, serum and tissue levels of 2HG did not show the relationship (R2 = 0.067, p=0.070). Tissue levels of 2HG showed a significant correlation with MGMT promoter methylations (R2= 0.15, p=0.0024).Our results suggest that tumor-derived 2HG is released in serum in the patients with IDH1/2 Mut tumors, however, their amounts are too small to detect the difference of serum 2HG levels in between patients with IDH-Mut and IDH-WT tumors. Accumulation of 2HG in tumor tissue may have an impact on MGMT promoter methylations.

In-vivo brain H1-MR-Spectroscopy identification and quantification of 2-hydroxyglutarate in L-2-Hydroxyglutaric aciduria

L-2-Hydroxyglutaric aciduria (L2HGA) is an extremely rare hereditary neurometabolic disease, characterized by increased L-2-hydroxyglutarate (L2HG) levels in the brain and biological fluids. 24-h urine 2HG level remains the biochemical hallmark for the diagnosis of L2HGA, whereas it is unknown the feasibility to measure in vivo the intracerebral levels of 2HG by using magnetic resonance spectroscopy (MRS). Patients and methodsWe used at 3T H1-MRS Single-Voxel (SV) PRESS sequences tailored to detect 2HG, in three adult patients with the diagnosis of L2HGA and in healthy controls. We also used mass spectrometric methods to measure the levels of 2HG in plasma and serum. Results2HG peak was detected and quantified in the white matter (WM) of the three L2HGA patients, while it was absent in controls. All patients showed also high levels of 2HG in plasma and serum. ConclusionsBrain 2HG detected by MRS may play a role in the diagnosis and follow-up of L2HGA, besides circulating plasma/serum 2HG levels by mass spectrometric assays, although studies on a large cohort of patients are required to confirm these observations.

A small molecule inhibitor of mutant IDH2 rescues cardiomyopathy in a D-2-hydroxyglutaric aciduria type II mouse model.

D-2-hydroxyglutaric aciduria (D2HGA) type II is a rare neurometabolic disorder caused by germline gain-of-function mutations in isocitrate dehydrogenase 2 (IDH2), resulting in accumulation of D-2-hydroxyglutarate (D2HG). Patients exhibit a wide spectrum of symptoms including cardiomyopathy, epilepsy, developmental delay and limited life span. Currently, there are no effective therapeutic interventions. We generated a D2HGA type II mouse model by introducing the Idh2R140Q mutation at the native chromosomal locus. Idh2R140Q mice displayed significantly elevated 2HG levels and recapitulated multiple defects seen in patients. AGI-026, a potent, selective inhibitor of the human IDH2R140Q-mutant enzyme, suppressed 2HG production, rescued cardiomyopathy, and provided a survival benefit in Idh2R140Q mice; treatment withdrawal resulted in deterioration of cardiac function. We observed differential expression of multiple genes and metabolites that are associated with cardiomyopathy, which were largely reversed by AGI-026. These findings demonstrate the potential therapeutic benefit of an IDH2R140Q inhibitor in patients with D2HGA type II.Electronic supplementary materialThe online version of this article (doi:10.1007/s10545-016-9960-y) contains supplementary material, which is available to authorized users.

Abstract 3101: The mutant IDH1 inhibitor prevents growth of glioblastoma with IDH1 mutation in patient-derived xenograft (PDX) model

Abstract Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in acute myeloid leukemia (AML), glioma, and many other cancers. While wild-type IDHs convert isocitrate to α-ketoglutarate (α-KG), mutant IDHs convert α-KG to oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases, such as TETs, histone demethylases, EGLNs, and other enzymes. Because the role of mutant IDH is not necessary for normal cells, inhibitors directed against mutant IDH are expected to have minimum side effects as those of anti-cancer agents. We established a mouse AML model harboring IDH mutations by co-transducing four mutant genes that frequently occur simultaneously in human AML patients. Conditional deletion of the IDH mutant blocked 2HG production and maintenance of leukemia stem cells, resulting in survival of the AML mice. These results indicate that the IDH mutations are critical for the development and maintenance of AML stem cells. Based on these findings, we developed potent and specific inhibitors of mutant IDH1 and tested their effects on the mutant IDH1-dependent AML mouse model, created by introducing four mutant genes including mutant IDH1. The 2HG level was promptly and dramatically decreased in AML cells soon after treatment with the mutant IDH1 inhibitors, and the number of leukemia cells was reduced after a 4-week treatment. To test the effect of the mutant IDH1 inhibitor on glioblastoma, we have established a patient-derived xenograft (PDX) model of glioblastoma carrying IDH1 mutation, and tested the effect of the mutant IDH1 inhibitor on this model. The inhibitor reduced 2HG levels in tumors as well as in plasma. The inhibitor prevented the growth of the tumors with IDH1 mutation. On the other hand, the inhibitor did not affect the growth of the glioblastoma with wild type IDH1. Gene expression analysis identified a set of genes, of which expressions is changed by the IDH1 inhibitor. The most strongly upregulated gene is Glial fibrillary acidic protein (GFAP), which is a differentiation marker for astrocytes. Up-regulation of GFAP is confirmed by immune-staining and quantitative RT-PCR analyses. These results suggest that IDH inhibitor induces differentiation of the glioblastoma with IDH1 mutation. We have found that the glioblastoma is transplantable in mouse brain. We are now looking at effects of the inhibitor on this model. Taken together, the results indicate that IDH1 mutant inhibitors are effective for the treatment for AML as well as glioblastoma with IDH1 mutations. Citation Format: Yukino Machida, Yoko Ogawara, Koichi Ichimura, Hironori Matsunaga, Seki Takahiko, Kazushi Araki, Issay Kitabayashi. The mutant IDH1 inhibitor prevents growth of glioblastoma with IDH1 mutation in patient-derived xenograft (PDX) model. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3101.

Comparison of oncometabolite 2-hydroxyglutarate (2HG) levels in mutant isocitrate dehydrogenase (IDH) versus wild-type (WT) glioma tissues.

2028Background: IDH mutations are common and prognostic in low-grade gliomas and secondary glioblastomas. IDH catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (αKG), and sub...

Diagnostic value of glutamate with 2-hydroxyglutarate in magnetic resonance spectroscopy forIDH1mutant glioma

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene that are frequently observed in low-grade glioma are strongly associated with the accumulation of 2-hydroxyglutarate (2HG), which is a valuable diagnostic and prognostic biomarker of IDH1 mutant glioma. However, conventional MR spectroscopy (MRS)-based noninvasive detection of 2HG is challenging. In this study, we aimed to determine the additional value of other metabolites in predicting IDH1 mutations with conventional MRS. Forty-seven patients with glioma underwent conventional single voxel short echo time MRS prior to surgery. A stereotactic navigation-guided operation was performed to resect tumor tissues in the center of the MRS voxel. MRS-based measurements of metabolites were validated with gas chromatography-mass spectrometry. We also conducted integrated analyses of glioma cell lines and clinical samples to examine the other metabolite levels and molecular findings in IDH1 mutant gliomas. A metabolomic analysis demonstrated higher levels of 2HG in IDH1 mutant glioma cells and surgical tissues. Interestingly, glutamate levels were significantly decreased in IDH1 mutant gliomas. Through an analysis of metabolic enzyme genes in glutamine pathways, it was shown that the expressions of branched-chain amino acid transaminase 1 were reduced and glutamate dehydrogenase levels were elevated in IDH1 mutant gliomas. Conventional MRS detection of glutamate and 2HG resulted in a high diagnostic accuracy (sensitivity 72%, specificity 96%) for IDH1 mutant glioma. IDH1 mutations alter glutamate metabolism. Combining glutamate levels optimizes the 2HG-based monitoring of IDH1 mutations via MRS and represents a reliable clinical application for diagnosing IDH1 mutant gliomas.

Treatment Response Assessment in IDH-Mutant Glioma Patients by Noninvasive 3D Functional Spectroscopic Mapping of 2-Hydroxyglutarate.

Measurements of objective response rates are critical to evaluate new glioma therapies. The hallmark metabolic alteration in gliomas with mutant isocitrate dehydrogenase (IDH) is the overproduction of oncometabolite 2-hydroxyglutarate (2HG), which plays a key role in malignant transformation. 2HG represents an ideal biomarker to probe treatment response in IDH-mutant glioma patients, and we hypothesized a decrease in 2HG levels would be measureable by in vivo magnetic resonance spectroscopy (MRS) as a result of antitumor therapy. We report a prospective longitudinal imaging study performed in 25 IDH-mutant glioma patients receiving adjuvant radiation and chemotherapy. A newly developed 3D MRS imaging was used to noninvasively image 2HG. Paired Student t test was used to compare pre- and posttreatment tumor 2HG values. Test-retest measurements were performed to determine the threshold for 2HG functional spectroscopic maps (fSM). Univariate and multivariate regression were performed to correlate 2HG changes with Karnofsky performance score (KPS). We found that mean 2HG (2HG/Cre) levels decreased significantly (median = 48.1%; 95% confidence interval = 27.3%-56.5%;P= 0.007) in the posttreatment scan. The volume of decreased 2HG correlates (R(2)= 0.88,P= 0.002) with clinical status evaluated by KPS. We demonstrate that dynamic measurements of 2HG are feasible by 3D fSM, and the decrease of 2HG levels can monitor treatment response in patients with IDH-mutant gliomas. Our results indicate that quantitative in vivo 2HG imaging may be used for precision medicine and early response assessment in clinical trials of therapies targeting IDH-mutant gliomas.

Association of isocitrate dehydorgenase-1 (IDH-1) mutations with elevated oncometabolite 2-hydroxyglutarate (2HG) in advanced colorectal cancer.

627 Background: IDH-1 mutations are common in AML, gliomas, and intrahepatic cholangiocarcinoma. The enzyme product of the IDH-1 R132 variant preferentially catalyzes the NADPH-dependent reduction of alpha-ketoglutarate to the oncometabolite R-2-Hydroxyglutarate (R-2HG), resulting in accumulation of R-2HG, relative to its enantiomer, S-2HG. Elevated plasma levels of 2HG and/or higher ratios of R/S have been observed in AML and intrahepatic cholangiocarinoma, but not in gliomas. Only a few cases of IDH-1/2 mutations have been reported in advanced colorectal cancers (CRC). We investigated plasma levels of 2HG and relative ratios of R/S in CRC patients harboring IDH-1/2mutations. Methods: Between 2012 and 2015, 428 patients with advanced CRC were molecularly profiled through the COMPACT and IMPACT programs at the Princess Margaret Cancer Centre. Tumor DNAs were isolated from FFPE archived samples and genotyped using a customized Sequenom panel or Illumina MiSeq TruSeq Amplicon Cancer Panel in a CLIA-certified laboratory. Plasma samples from patients harboring IDH-1/2mutations and 4 gender/age matched control patients were analyzed for 2HG, R-2HG and S-2HG using HPLC tandem mass spectrometry coupled with a CHIROBIOTIC R column. Results: Of 428 patients with advanced CRC, 4 (0.9%) patients were identified to harbor IDH-1 mutation. No IDH-2 mutations were detected. The variants identified were R132C (2 patients), R132S, and R132H. 3 of 4 patients also harbored KRAS mutations. Patients with IDH-1 mutations had higher levels of 2HG (319 ± 102 ng/ml vs 195 ± 43 ng/ml, p = 0.043). The ratio of R/S were 0.44, 0.75, 1.59, and 2.60 in 4 patients with IDH-1 mutations and 1.3, 0.90, 0.41 and 0.67 in 3 patients without IDH-1mutations. Conclusions: IDH-1 mutations are rare in advanced CRC. 3 of 4 patients in our study had concurrent KRAS mutations. Patients with IDH-1 mutations had higher levels of oncometabolite 2HG compared with controls with no difference in R/S ratio.

IDH1-Mutant Cancers Are Susceptible to NAD+ Depletion

Abstract IDH1-mutant cancers are addicted to NAD+ and grow independent of 2HG levels.

Photoactivation of Mutant Isocitrate Dehydrogenase 2 Reveals Rapid Cancer-Associated Metabolic and Epigenetic Changes.

Isocitrate dehydrogenase is mutated at a key active site arginine residue (Arg172 in IDH2) in many cancers, leading to the synthesis of the oncometabolite (R)-2-hydroxyglutarate (2HG). To investigate the early events following acquisition of this mutation in mammalian cells we created a photoactivatable version of IDH2(R172K), in which K172 is replaced with a photocaged lysine (PCK), via genetic code expansion. Illumination of cells expressing this mutant protein led to a rapid increase in the levels of 2HG, with 2HG levels reaching those measured in patient tumor samples, within 8 h. 2HG accumulation is closely followed by a global decrease in 5-hydroxymethylcytosine (5-hmC) in DNA, demonstrating that perturbations in epigenetic DNA base modifications are an early consequence of mutant IDH2 in cells. Our results provide a paradigm for rapidly and synchronously uncloaking diverse oncogenic mutations in live cells to reveal the sequence of events through which they may ultimately cause transformation.

Integration of 2-hydroxyglutarate-proton magnetic resonance spectroscopy into clinical practice for disease monitoring in isocitrate dehydrogenase-mutant glioma.

The majority of WHO grades II and III gliomas harbor a missense mutation in the metabolic gene isocitrate dehydrogenase (IDH) and accumulate the metabolite R-2-hydroxyglutarate (R-2HG). Prior studies showed that this metabolite can be detected in vivo using proton magnetic-resonance spectroscopy (MRS), but the sensitivity of this methodology and its clinical implications are unknown. We developed an MR imaging protocol to integrate 2HG-MRS into routine clinical glioma imaging and examined its performance in 89 consecutive glioma patients. Detection of 2-hydroxyglutarate (2HG) in IDH-mutant gliomas was closely linked to tumor volume, with sensitivity ranging from 8% for small tumors (<3.4 mL) to 91% for larger tumors (>8 mL). In patients undergoing 2HG-MRS prior to surgery, tumor levels of 2HG corresponded with tumor cellularity but not with tumor grade or mitotic index. Cytoreductive therapy resulted in a gradual decrease in 2HG levels with kinetics that closely mirrored changes in tumor volume. Our study demonstrates that 2HG-MRS can be linked with routine MR imaging to provide quantitative measurements of 2HG in glioma and may be useful as an imaging biomarker to monitor the abundance of IDH-mutant tumor cells noninvasively during glioma therapy and disease monitoring.

Use of 2HG Levels in the Serum, Urine, or Bone Marrow to Predict IDH Mutations in Adults with Acute Myeloid Leukemia

Mutations in genes encoding the isocitrate dehydrogenase 1/2 (IDH 1/2) enzymes increase production of the oncometabolite 2-hydroxyglutarate (2HG), resulting in elevated 2HG in patients with IDH-mutant AML. This may allow for non-invasive diagnostic and predictive markers of disease; however, the optimal threshold of 2HG levels to predict IDH mutation status, or whether 2-HG measurements in different compartments are equally predictive, is unknown. We measured 2-HG levels in serum, urine, bone marrow aspirate, and bone marrow cell pellet to determine the optimal predictive value of 2HG levels for IDH mutations at AML diagnosis.Patients with newly diagnosed AML had prospective measurements of 2HG levels at diagnosis by liquid chromatography-tandem mass spectrometry in serum, urine, marrow aspirate, and marrow pellet samples. Patients analyzed had IDH1 R132 and IDH2 R140 and R172 testing. Hotspot mutational profiling was performed for AKT1, APC, BRAF, CTNNB1, EGFR, ERBB2, KIT, KRAS, MAP2K1, NOTCH1, NRAS, PIK3CA, P53, and PTEN; patients were also tested for FLT3, NPM1, and CEBPA mutations. IDH1/2 mutant patients were compared to wildtype (WT) patients using a Wilcoxon rank sum, Fisher's exact, or Kruskal Wallis test, as appropriate. Performance characteristics of 2HG to predict the presence of IDH1/2 mutations were done using a recursive partitioning algorithm in R version 3.2.1, with the rpart package.228 patients with newly diagnosed AML had 2HG levels in serum, urine, marrow aspirate, and marrow pellet samples. All patients had testing for IDH1 R132 and IDH2 R140 and R172 mutations. The patients were 56% male; 13 patients had APL, none with IDH mutations. IDH mutations were identified in 23% (n=52) of the cohort: IDH1 R132C (n=12), IDH2 R172 (n=9), and IDH2 R140Q (n=29), and 2 additional patients had mutations in both IDH1 R132 and IDH2 R140 (Table 1).The optimal test cut-off of 2HG to predict IDH status was 534.5 ng/mL in the serum (n=221), 16650 ng/mL in the urine (n=213), 2210 ng/mL in the marrow aspirate (n=190), and 1146 ng/2*10^6 cells in the pellet (n=159). Serum and pellet values had the greatest specificity for the presence of an IDH1/2 mutation (0.9882 and 1.000, respectively; Table 2). The positive predictive value of an elevated serum or pellet 2HG level at these cut-offs was 95.4% and 100%, given a 23% IDH mutation prevalence in this study population. The marrow aspirate had the greatest sensitivity (0.8837) and negative predictive value (96.6%). Urine 2HG levels were less sensitive than serum 2HG levels, although above the urine cut-off the specificity for IDH mutations was similar (Figure 1).2HG levels in the serum, urine, and marrow can be used to identify IDH mutations in AML. Serum 2HG testing is an effective non-invasive mechanism to predict IDH1/2 mutation status. A serum cut-off of 534.5 ng/mL has a specificity of 0.9882 and, with an IDH mutation prevalence of 23%, was associated with a PPV of 0.9535. Table 1Patient CharacteristicsNo IDH mutationIDH mutationTotalp-valueMale sex101 (57%)26 (50%)127 (56%)0.43Cytogenetics0.0002Favorable26 (15%)026 (12%)Intermediate95 (54%)41 (82%)136 (61%)Adverse52 (30%)9 (18%)61 (27%)Age (median, range)66 (20, 87)66.5 (41, 86)66 (20, 87)0.27WBC (median, range)5.35 (0.60, 315.4)3.65 (0.20, 333.2)5.25 (0.20, 333.2)0.33No IDH mutationIDH mutationNumber assessedp-valueNRAS27 (17%)9 (19%)2100.83KRAS16 (10%)0 (0%)2100.03TP5315 (9%)1 (2%)2100.13KIT2 (1%)1 (2%)2100.54FLT3ITD34 (20%)4 (8%)2180.06FLT3TKD10 (6%)2(4%)2180.74NPM129 (17%)15 (30%)2190.07CEBPA13 (13%)3 (10%)1021.002HG MeasurementsNo IDH mutationIDH mutationTotalSerum (ng/mL)79.5 [52,123] n=1701420 [675,2735] n=51101 [58,101] n=221Urine (ng/mL)3590 [2230,6220] n=16318300 [7260,59500] n=504330 [2450,8580] n=213Marrow aspirate (ng/mL)BQL [BQL,BQL] n=14718400 [4270,43100] n=43BQL [BQL,1430] n=190Pellet (1000 ng/2*10^6 cells)64 [BQL,169] n=1271420 [675,2735] N=32107 [BQL,500] n=159Table 2Test characteristics based on optimal 2HG cut-off, by compartment.Compartment2HG cut-offSensitivitySpecificity23% prevalencePPVNPVSerum534.5 ng/mL0.80390.98820.95350.9438Urine16650 ng/mL0.56000.98770.93330.8798Marrow Aspirate2210 ng/mL0.88370.96600.88370.9660Marrow Pellet1146 ng/2*10^6 cells0.78131.0001.0000.9478 [Display omitted] [Display omitted] DisclosuresChen:Bayer: Consultancy, Research Funding. Stone:Agios: Consultancy; Celator: Consultancy; Pfizer: Consultancy; Merck: Consultancy; AROG: Consultancy; Abbvie: Consultancy; Celgene: Consultancy; Roche/Genetech: Consultancy; Karyopharm: Consultancy; Amgen: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Juno: Consultancy; Novartis: Research Funding. Fathi:Exelexis: Research Funding; Agios: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Membership on an entity's Board of Directors or advisory committees; Ariad: Consultancy; Takeda Pharmaceuticals International Co.: Research Funding.

The Role of IDH Mutants, Which Are Promising Therapeutic Targets for Acute Myeloid Leukemia

Mutations in isocitrate dehydrogenase (IDH) 1 and 2 are frequently observed in acute myeloid leukemia (AML), glioma, and many other cancers. While wild-type IDHs convert isocitrate to α-ketoglutarate (α-KG), mutant IDHs convert α-KG to oncometabolite 2-hydroxyglutarate (2-HG), which dysregulates a set of α-KG-dependent dioxygenases, such as TETs, histone demethylases, EGLNs, and other enzymes. Because the role of mutant IDH is not necessary for normal cells, inhibitors directed against mutant IDH are not expected to have the side effects as those of anti-cancer agents.To determine whether mutant IDH enzymes are valid targets for cancer therapy, we created a mouse model of mutant IDH-dependent AML. Previously, the IDH mutation alone was shown to be insufficient for the induction of AML, and IDH mutations occur simultaneously with mutations in other genes such as NPM, DNMT3A, and FLT3. In accordance with these observations, we found that NPM+/- hematopoietic progenitor cells transduced with IDH2/R140Q, NPMc, DNMT3A/R882H, and FLT3/ITD cooperatively induced AML in a mouse model. However, when only three of these mutant genes were transduced, myeloproliferative neoplasms (MPNs) rather than AML was more frequently induced and their onset was delayed in any combinations of the mutant genes. These results clearly indicate that all four mutations are necessary for the efficient induction of AML. By using a combination of AML model mice with cre-loxp, we conditionally deleted IDH2/R140Q from AML mice, which blocked 2-HG production and resulted in the loss of leukemia stem cells. Accordingly, the progression of AML was significantly delayed. These results indicate that the function of IDH2 mutation is critical for the development and maintenance of AML stem cells, and that mutant IDHs are promising targets for anticancer therapy. Based on these findings, we developed potent and specific inhibitors of mutant IDH1 and tested their effects in the mutant IDH1-dependent AML mouse model, created by introducing four mutant genes including mutant IDH1. The 2HG level was promptly and dramatically decreased in AML cells soon after treatment with the mutant IDH1 inhibitors, and the number of leukemia cells was reduced after a 4-week treatment. These results indicate that IDH1 mutant inhibitors are effective for the treatment for AML.Because IDH mutations and TET2 mutations are mutually exclusive in AML, the inhibition of TET-mediated conversion of 5mC to 5hmC is considered one of the main roles of mutant IDH. We found that levels of 5hmC on differentiation-inducing genes, such as Ebf1, Spib and Pax5 were decreased in AML cells with IDH2/R140Q and recovered by conditional deletion of IDH2/R140Q. In consistent with levels of 5hmC, expressions of these genes are downregulated in the AML cells and increased by deletion of IDH2/R140Q.Gene expression analysis revealed that IDH2/R140Q up-regulates a set of genes that is activated in response to hypoxia as well as Meis1. As 2HG inhibits EGLN that hydroxylates and marks HIF1α for ubiquitin-proteasomal degradation, it is probable that mutant IDH2-produced 2HG stabilizes HIF1α through inhibition of EGLN. Furthermore, it was reported that Meis1 activates the transcription of HIF1α. In consistent with these information, we showed that IDH2/R140Q increased the protein levels of HIF1α in cultured cells. DisclosuresMatsunaga:Daiichi Sankyo Co., Ltd.: Employment. Seki:Daiichi Sankyo Co., Ltd.: Employment. Araki:Daiichi Sankyo Co., Ltd.: Employment. Kitabayashi:Daiichi Sankyo Co., Ltd.: Research Funding.

CBIO-32MUTANT IDH EXPRESSION DRIVES hTERT REACTIVATION AS PART OF THE CELLULAR TRANSFORMATION PROCESS

IDH1 mutations in lower grade glioma result in the production of 2-hydroxyglutarate (2HG), disrupted patterns of histone methylation, altered gene expression, and cellular transformation. IDH1 mutations, however, are also linked to mutations in the ATRX gene and the hTERT promoter, suggesting that IDH1 mutation contributes to events that stabilize telomeres and help drive the cellular immortalization and transformation processes. To determine if and how IDH1 mutation influences telomeric stabilization and cellular immortalization, pRb/p53-deficient normal human astrocytes (E6/E7 NHA) were lentivirally infected with constructs encoding WT (IDH1 WT) or R132H mutant IDH 1 (IDH1mut). Following selection, the cells were then monitored for 2HG levels, histone modifications, hTERT promoter mutations, ATRX and telomerase expression, and telomere length as they progressed through their lifespan. Introduction of IDH1mut, but not IDH1 WT, resulted in 2HG production and alterations in histone methylation within 20 population doublings (PD). Neither IDH1mut nor IDH1 WT expression, however, had any direct effect on telomerase expression or telomere length, and cells expressing either IDH1 WT or IDH1mut entered a telomere-induced crisis at PD 70. While cells expressing IDH1 WT failed to reactivate telomerase and escape from crisis, cells expressing IDH1mut emerged from crisis after 51 days, grew indefinitely in culture, and formed colonies in soft agar and tumors in vivo. The transformed IDH1mut cells did not exhibit the ALT phenotype or hTERT promoter mutations, but rather exhibited telomerase expression and activity, and shortened but stabilized telomeres. These results show that while IDH1 mutations do not directly contribute to ATRX gene mutation, hTERT promoter mutation, or TERT reactivation, they are permissive for the events that indirectly drive the TERT reactivation that contributes to both the immortalization and transformation of astrocytic cells.