2HG Levels Research Articles

Roles of the oncometabolite enantiomers of 2-hydroxyglutarate and their metabolism by diverse dehydrogenases.

2-Hydroxyglutarate (2HG) is an oncometabolite that can contribute to tumor progression. Two enantiomer forms, L-2HG and D-2HG, arise from independent pathways starting from the precursor α-ketoglutarate (αKG). L-2HG production occurs through the promiscuous activities of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) under acidic and/or hypoxic conditions. D-2HG frequently accumulates by gain-of-function mutations in the genes encoding two isoforms of isocitrate dehydrogenase (IDH1 and IDH2). Cognate metabolite repair enzymes, L- and D-2-hydroxyglutarate dehydrogenases, oxidize the enantiomers and cause abnormally high 2HG accumulation and disease when mutated. Elevated levels of either oncometabolite affect redox homeostasis, metabolism, and immune system functioning. Moreover, the oncometabolites inhibit several α-ketoglutarate-dependent dioxygenases resulting in epigenetic changes such as DNA and histone hypermethylation as well as deficiencies in DNA repair. L-2HG, and D-2HG in some cases, inhibit degradation of hypoxia-inducible factor (HIF1α), a transcription factor that alters gene expression to adapt to hypoxic conditions, favoring tumorigenesis. Patients with the rare disease 2-hydroxyglutaric aciduria (2HGA) have exceedingly high levels of 2HG, which is neurotoxic, causing developmental delays and brain abnormalities. D-2HG also has specific effects on collagen production and NADPH pools. Recently, D-2HG has been targeted in new chemotherapies aimed at disrupting the gain-of-function IDH1 and IDH2 mutants, resulting in successful clinical trials for several cancers.

Abstract B040: NCI 10129: A Phase 2 Study of the PARP inhibitor Olaparib (AZD2281) in IDH1 and IDH2 mutant advanced solid tumors

Abstract Introduction: Cholangiocarcinoma (CCA) is a highly aggressive malignancy with mutations in Isocitrate dehydrogenase (IDH) identified in approximately 15% of patients. Neomorphic mutations in IDH 1/2 result in accumulation of the oncometabolite 2-hydroxyglutarate (2G), which has been implicated in tumor progression via inhibitory effects on alpha ketoglutarate. Moreover, the mutant IDH dependent accumulation of 2HG results in homologous recombination deficiency (HRD) and renders these cells exquisitely sensitive to Poly (ADP-Ribose) polymerase (PARP) inhibitors. NCI 10129 evaluated the efficacy of olaparib for IDH mutated solid tumors, and here we present the CCA cohort. Methods: NCI 10129 is a 3 arm, open label phase 2 clinical trial performed in the NCI National Clinical Trials Network evaluating olaparib 300 mg twice daily for IDH mutated solid tumors refractory to standard treatment. Patients with CCA were enrolled in 2 cohorts 1) IDHi pre-treated CCA and 2) IDHi untreated CCA. The primary endpoint was overall response rate (ORR) and secondary endpoints includes progression-free survival (PFS), and overall survival (OS). An exploratory analysis measured the impact of circulating 2HG levels, circulating tumor DNA, and tumor genomics on patient outcomes. Results: Thirty patients with CCA enrolled on NCI10129 (15 each IDHi pre-treated and IDHi treatment naïve), 18 patients received ≥ 2 prior systemic treatments, and 23 patients had IDH1 mutations and 7 patients IDH2 mutations. There were no objective responses seen in the first 15 CCA patients in either cohort and enrollment was closed. The median PFS was 2.4 months 95% CI (1.9 - 6.5) and median OS 12.9 months 95% CI (6.3 – not reached). The IDHi pre-treated cohort had improved median OS compared to IDHi treatment naïve (15.3 vs 7.3, P = 0.04). There were 8/30 (27%) patients with clinical benefit (CB) as defined by PFS > 6 months. Patients with CB had lower baseline mean D-2HG levels compared to those without CB (1.4 umol/L vs 5.9 umol/L, p = 0.02). Whole exome sequencing revealed the study population to be enriched for HRD by sum scores and HRD mutational signatures without alternative HRD mutations other than IDH1/2. No improvement in survival was noted based on HRD status. RNA sequencing Gene Set Enrichment Analysis revealed that cell cycle and interferon alpha pathways were activated in patients with CB (p < 0.01). Olaparib was tolerable with the majority of adverse events grade 1-2. Conclusion: Olaparib did not result in objective responses in IDH mutated CCA. However, a subgroup of patients did demonstrate prolonged PFS comparable to historical controls for refractory CCA as well that seen with targeted agents such as IDHi. Exploratory analysis revealed this subgroup to be enriched for lower baseline 2HG levels. Serial ctDNA analysis is ongoing and will be presented. Given the CB subgroup and OS data, additional clinical trials leveraging the HRD properties of IDH mutations is warranted. Future studies may benefit from DNA damage combination therapies thereby enhancing potency. Citation Format: Michael Cecchini, Mary Jo Pilat, Nataliya Uboha, Nilofer S Azad, May Cho, Elizabeth J Davis, Jordi Rodon Ahnert, Gabriel Tinoco, Geoff I Shapiro, Simon Khagi, Benjamin Powers, Roman Groisberg, Jan Drappatz, Li Chen, Biswajit Das, Xun Bao, Jing Li, Abhijit Patel, Monica Niger, Deborah Doroshow, Diane Durecki, Scott Boerner, Ranjit Bindra, Percy Ivy, Yu Shyr, Patricia LoRusso. NCI 10129: A Phase 2 Study of the PARP inhibitor Olaparib (AZD2281) in IDH1 and IDH2 mutant advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B040.

In Vivo 2-Hydroxyglutarate Monitoring With Edited MR Spectroscopy for the Follow-up of IDH-Mutant Diffuse Gliomas: The IDASPE Prospective Study.

D-2-hydroxyglutarate (2HG) characterizes IDH-mutant gliomas and can be detected and quantified with edited MRS (MEGA-PRESS). In this study, we investigated the clinical, radiologic, and molecular parameters affecting 2HG levels. MEGA-PRESS data were acquired in 71 patients with glioma (24 untreated, 47 treated) on a 3 T system. Eighteen patients were followed during cytotoxic (n = 12) or targeted (n = 6) therapy. 2HG was measured in tumor samples using gas chromatography coupled to mass spectrometry (GCMS). MEGA-PRESS detected 2HG with a sensitivity of 95% in untreated patients and 62% in treated patients. Sensitivity depended on tumor volume (>27 cm3; p = 0.02), voxel coverage (>75%; p = 0.002), and expansive presentation (defined by equal size of T1 and FLAIR abnormalities, p = 0.04). 2HG levels were positively correlated with IDH-mutant allelic fraction (p = 0.03) and total choline levels (p < 0.001) and were higher in IDH2-mutant compared with IDH1 R132H-mutant and non-R132H IDH1-mutant patients (p = 0.002). In patients receiving IDH inhibitors, 2HG levels decreased within a few days, demonstrating the on-target effect of the drug, but 2HG level decrease did not predict tumor response. Patients receiving cytotoxic treatments showed a slower decrease in 2HG levels, consistent with tumor response and occurring before any tumor volume change on conventional MRI. At progression, 1p/19q codeleted gliomas, but not the non-codeleted, showed detectable in vivo 2HG levels, pointing out to different modes of progression characterizing these 2 entities. MEGA-PRESS edited MRS allows in vivo monitoring of 2-hydroxyglutarate, confirming efficacy of IDH inhibition and suggests different patterns of tumor progression in astrocytomas compared with oligodendrogliomas.

IDH1 p.R132H ctDNA and D-2-hydroxyglutarate as CSF biomarkers in patients with IDH-mutant gliomas.

We aimed to evaluate IDH1 p.R132H mutation and 2-hydroxyglutarate (2HG) in cerebrospinal fluid (CSF) as biomarkers for patients with IDH-mutant gliomas. CSF was collected from patients with infiltrating glioma, and 2HG levels were measured by liquid chromatography-mass spectrometry. IDH1 p.R132H mutant allele frequency (MAF) in CSF-ctDNA was measured by digital droplet PCR (ddPCR). Tumor volume was measured from standard-of-care magnetic resonance images. The study included 48 patients, 6 with IDH-mutant and 42 with IDH-wildtype gliomas, and 57 samples, 9 from the patients with IDH-mutant and 48 from the patients with IDH-wildtype gliomas. ctDNA was detected in 7 of the 9 samples from patients with IDH-mutant glioma, and IDH1 p.R132H mutation was detected in 5 of the 7 samples. The MAF ranged from 0.3 to 39.95%. Total 2HG level, D-2HG level, and D/L-2HG ratio in CSF were significantly higher in patients with IDH-mutant gliomas than in patients with IDH-wildtype gliomas. D-2HG level and D/L-2HG ratio correlated with total tumor volume in patients with IDH-mutant gliomas but not in patients with IDH-wildtype gliomas. Our results suggest that detection of IDH1 p.R132H mutation by ddPCR and increased D-2HG level in CSF may help identify IDH-mutant gliomas. Our results also suggest that D-2HG level and D/L-2HG ratio correlate with tumor volume in patients with IDH-mutant gliomas. Further prospective studies with larger cohorts are needed to validate these findings.

Abstract 1812: Therapeutic targeting and development of IDH inhibitors for the treatment of chondrosarcoma

Abstract Chondrosarcomas are the second most frequently occurring type of bone malignancy, and account for approximately 25% of all bone sarcomas. They are often highly aggressive neoplasms that rapidly progress and eventually recur and give distant metastases. They are largely considered to be resistant to conventional chemotherapy and radiotherapy. Recurrent somatic mutations in the isocitrate dehydrogenase I (IDH1) genes have been identified in a spectrum of human malignancies, including chondrosarcoma. Mutations in these genes lead to impaired ability of IDH1 to catalyze the conversion of isocitrate to alpha ketoglutarate and to gain of a neomorphic enzymatic activity which results in production of oncometabolite 2-hydroxyglutarate (2HG). Our preliminary data indicated that compared to samples that were wild type, IDH1 mutant samples showed a significant increase in intra-tumoral 2HG levels. In the present study, we hypothesized that 2-HG accumulation induces DNA and histone hypermethylation and altered gene expression, ultimately resulting in a block in cellular differentiation by competitively inhibiting α-ketoglutarate-dependent dioxygenases involved in histone and DNA demethylation. Therefore, we proposed that inhibition of IDH should mediate its antitumor effects through the induction of differentiation. To test this hypothesis, we utilized human chondrosarcoma cell lines including IDH wild type (CH2879) and IDH1 mutant (JJ012) and the IDH1 inhibitor FT6535. Results from our in vitro cell viability assay and histone methylation showed that selective inhibition of IDH1 resulted in modest inhibition of cell proliferation and suppression of histone methylation in IDH1 mutant chondrosarcoma cells. Next we elected to test the IDH inhibitor in combination with differentiating agents including retinoic acid. Cell viability assays showed that when compared to either agent alone, the combination of retinoic acid and FT6535 resulted in a significant decrease in cellular proliferation. Western blot analysis showed induction of COL1A2 and SOX9 (differentiation markers) with suppression of histone methylation H3K27me3 or H3K9me3, Taken together, our data strongly suggests that combination treatment with the IDH1 inhibitor FT6535 and retinoic acid represents a novel approach to suppress cell growth by epigenetic modulation to induce differentiation and merits further evaluation as a potential treatment modality in chondrosarcoma. Citation Format: Tahir N. Sheikh, Chao Lu, Gary K. Schwartz. Therapeutic targeting and development of IDH inhibitors for the treatment of chondrosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1812.

Spectroscopic imaging of D-2-hydroxyglutarate and other metabolites in pre-surgical patients with IDH-mutant lower-grade gliomas

PurposePrognostically favorable IDH-mutant gliomas are known to produce oncometabolite D-2-hydroxyglutarate (2HG). In this study, we investigated metabolite-based features of patients with grade 2 and 3 glioma using 2HG-specific in vivo MR spectroscopy, to determine their relationship with image-guided tissue pathology and predictive role in progression-free survival (PFS).MethodsForty-five patients received pre-operative MRIs that included 3-D spectroscopy optimized for 2HG detection. Spectral data were reconstructed and quantified to compare metabolite levels according to molecular pathology (IDH1R132H, 1p/19q, and p53); glioma grade; histological subtype; and T2 lesion versus normal-appearing white matter (NAWM) ROIs. Levels of 2HG were correlated with other metabolites and pathological parameters (cellularity, MIB-1) from image-guided tissue samples using Pearson’s correlation test. Metabolites predictive of PFS were evaluated with Cox proportional hazards models.ResultsQuantifiable levels of 2HG in 39/42 (93%) IDH+ and 1/3 (33%) IDH– patients indicated a 91.1% apparent detection accuracy. Myo-inositol/total choline (tCho) showed reduced values in astrocytic (1p/19q-wildtype), p53-mutant, and grade 3 (vs. 2) IDH-mutant gliomas (p < 0.05), all of which exhibited higher proportions of astrocytomas. Compared to NAWM, T2 lesions displayed elevated 2HG+ γ-aminobutyric acid (GABA)/total creatine (tCr) (p < 0.001); reduced glutamate/tCr (p < 0.001); increased myo-inositol/tCr (p < 0.001); and higher tCho/tCr (p < 0.001). Levels of 2HG at sampled tissue locations were significantly associated with tCho (R = 0.62; p = 0.002), total NAA (R = − 0.61; p = 0.002) and cellularity (R = 0.37; p = 0.04) but not MIB-1. Increasing levels of 2HG/tCr (p = 0.0007, HR 5.594) and thresholding (≥ 0.905, median value; p = 0.02) predicted adverse PFS.ConclusionIn vivo 2HG detection can reasonably be achieved on clinical scanners and increased levels may signal adverse PFS.

NIMG-41. PH-WEIGHTED MOLECULAR MRI AS AN EARLY BIOMARKER OF METABOLIC RESPONSE TO IDH INHIBITION IN IDH MUTANT GLIOMAS

Abstract The current study tested the hypotheses that (1) pH-weighted MRI measurements of tumor acidity reflect glycolytic activity in human gliomas, (2) that tumor acidity, and thus glycolytic activity, differs between human IDH mutant (mIDH) and wild type gliomas, and (3) that an increase in tumor acidity, suggestive of increased glycolytic activity, occurs after inhibition of mutant IDH enzyme activity and reduction in 2HG, an oncometabolite. To test these hypotheses, we employed a custom pH-weighted amine chemical exchange saturation transfer echoplanar (CEST-EPI) technique in 152 patients with IDH mutant or wildtype glioma prior to surgery and 11 patients before and after treatment with AG120 or AG881 enrolled at our institution in a phase 1 perioperative study in patients with recurrent, non-enhancing, IDH mutant low-grade gliomas (NCT03343197). Results from image-guided biopsies in more than 100 patients demonstrated a significant correlation between MTRasym at 3ppm, a measure of tumor acidity from pH-weighted amine CEST-EPI, and expression of key glycolytic proteins including GLUT3 (R2=0.2188,P=0.0105), HK2 (R2=0.1788,P=0.0314), LDHA (R2=0.1111,P=0.0071), and MCT1 (R2=0.1228,P=0.0039) as well as ex vivo extracellular flux analysis estimates of ATP consumption from glycolysis (R2=0.6684,P=0.0021). Data suggests a significantly lower acidity (MTRasym@3ppm) within non-enhancing tumor in IDH mutant gliomas when compared to IDH wild type gliomas (P&amp;lt; 0.0001). Patients in a phase 1 perioperative study showed a shift toward higher tumor acidity (i.e. higher glycolytic activity) following inhibition of IDH based on 2HG suppression in resected tumors, but at levels below that of IDH wild type gliomas. Levels of 2HG within the tumor after IDH inhibition were inversely correlated with post-treatment tumor acidity (R2=0.6342, P=0.0180). Overall, results suggest mIDH gliomas have low levels of glycolytic activity, and successful inhibition of the mutant IDH enzyme results in reduction in 2HG and a measurable metabolic shift toward elevated glycolysis as evidenced using pH-weighted molecular MRI.

Efficacy of 5-Azacitidine on IDH1/2 Acute Myeloid Leukemia/Myelodysplastic Syndromes and Correlation with 2-Hydroxyglutarate Production

Background. Isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) mutations are found in 10 and 15% of acute myeloid leukemia (AML) patients, respectively. IDH1/2 mutants' proteins produce high 2-hydroxyglutarate (2HG) levels that inhibit α-ketoglutarate-dependent dioxygenases including histone and DNA demethylases, as TET2 proteins. Furthermore, serum 2HG can act as a biomarker predictive of the presence of IDH1 and IDH2 mutations at diagnosis (cut-off value >2µM) but also correlate with response after intensive chemotherapy. 5-azacitidine (AZA) is a DNA methyltransferase inhibitor that demonstrated significant clinical benefit not only in high-risk MDS but also in AML. Hypomethylating agents yield higher response rate in the context of CpG island hypermethylation phenotype induced by TET2 mutations in myelodysplastic syndrome (MDS). Thus, we evaluate AZA response in the context of IDH1/2 mutations and the effect of AZA on serum 2HG production.Patients and methods. The present study retrospectively focused on IDH1/2 mutant AML/MDS patients treated in our center with at least 1 cycle of AZA. IDH1/2 mutational status was evaluated in all patients with Sanger or Next-Generation sequencing before therapy. All patients received subcutaneous AZA 75mg/m2/d for 7 days every 28 days. Response to therapy was evaluated according to modified 2003 IWG criteria's for AML. Hematological improvement (HI) was evaluated according to IWG 2006 criteria's for MDS. Overall survival (OS) was censored in case of IDH1/2 inhibitor therapy after AZA. Serum samples were analyzed for total 2HG by reverse-phase liquid chromatography coupled to mass spectrometry.Results. Thirty-six patients (34 AML, 2 MDS) were retrospectively analyzed. Initial characteristics of the patients are shown on Table 1. Median age was 74.3 years (range, 48.4-86). Thirteen patients (36%) had IDH1 mutations (R132C: 10, R132H: 2, R132G: 1) and 22 patients (61%) had IDH2 mutations (R140Q: 19, R172K: 3). One patient had both IDH1 R132H and IDH2 R140Q mutations. Twenty-nine patients had intermediate cytogenetic (including normal karyotype in 20 patients) and 6/36 unfavorable. IDH1/2 mutations were associated with SRSF2 mutations in 13 patients, DNMT3A in 11 patients, NPM1 mutation in 9 patients, ASXL1 in 5 patients and FLT3 -ITD in 5 patients. Ten patients (28%) were treated frontline with AZA while the others received AZA as second line (50%, including 6 patients with primary refractory AML to intensive chemotherapy) or subsequent lines (22%). Five patients received IDH1/2 inhibitors (AG221: 3, AG120: 2) prior to AZA treatment. Patients received a median of 4.5 cycles of AZA (range, 1-23). Four patients (11%) achieved complete remission (CR) and 7 (19%) obtained HI with persistent excess of blasts (overall response rate: 30%). Median OS was 9.5 months (FU range, 0.2-41.6) with a trend toward shorter OS in IDH1 mutant AML patient (6.4 vs 10.1; p≤0.0764) (Figure 1). No difference was observed between patients in 1st or subsequent treatment lines (p≤0.7265).Median 2HG concentration (available in 25/36 patients) before the onset of AZA treatment was 9.7µM. Seventeen patients had available serum during follow-up (median: 2 measures/patient). When 2HG values in responding and non-responding patients were compared at different time-points, 2HG values remained elevated in responders (median 2HG: 6.8µM; range 0.99-13.5) without significant difference compared to baseline or relapse values (Figure 2). All responders to AZA ultimately relapsed or progressed during follow-up. IDH1 or IDH2 mutations persisted at relapse along with elevated 2HG levels (median 2HG: 4.5µM).Discussion. In the present series, IDH1/2 mutations were not associated with an increased sensitivity to AZA, as opposed to TET2 mutations. AZA may decrease production of 2HG in responders compared to diagnosis values. However, unlike intensive chemotherapy, AZA is unable to normalize 2HG values in responding patients. This suggests that 2HG cannot be used as a surrogate marker of AZA response and AZA efficacy might not be related to inhibition of 2HG production in IDH1/2 AML/MDS patients. Thus, combinations of AZA with IDH1/2 mutant proteins inhibitors might lead to an additive or synergistic antitumor effect. [Display omitted] DisclosuresRenneville:CELGENE: Research Funding. Ribrag:Roche: Honoraria, Other: travel, accommodation, expenses; BMS: Consultancy, Honoraria; MSD: Consultancy, Honoraria; Infinity: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Nanostring: Consultancy, Honoraria; Epizyme: Consultancy, Honoraria; Pharmamar: Consultancy; Esai: Honoraria, Research Funding; ArgenX: Research Funding; Servier: Consultancy, Honoraria. de Botton:Celgene Corporatation: Honoraria; Novartis: Honoraria; Pfizer: Honoraria; Servier: Honoraria; Agios Pharmaceuticals, Inc.: Honoraria, Research Funding.

Characterization of Acute Myeloid Leukaemia (AML) Patients with Elevated Peripheral Blood Plasma D-2-Hydroxyglutarate (D-2HG) and/or Isocitrate Dehydrogenase (IDH) Mutational Status

Isocitrate dehydrogenase (IDH) gene mutations are identifiable in AML preleukemic stems cells that persist during post-induction clinical remission, justifying their targeted therapy. Recent monotherapy trials with mutant IDH1 and IDH2 oral inhibitors have shown a manageable safety profile, durable responses of greater than 6 months, and evidence of induced differentiation of malignant cells. IDH mutations commonly lead to an increased oncogenic metabolite, D-hydroxyglutarate (D-2HG). This is a single institution, multi-site study to examine pre-treatment plasma 2HG levels and corresponding IDH mutational status in patients diagnosed with de novo or recurrent AML (n=50) or high grade myelodysplastic syndromes (n=1). D- and L-2HG were quantified by Chiral chromatography coupled mass-spectrometry (2D GCxGC-TOP-MS). Elevated D-2HG in the peripheral blood with increased D/L 2HG ratio was detected in 17% (9 of 51) of patients having 25% to <1% peripheral blood blasts. DNA sequence analyses were performed by Sanger chain-terminating dideoxynucleotide labelling and the droplet digital PCR approach. SNP-specific digital PCR was markedly more sensitive, effectively identifying 10 IDH mutations in the peripheral blood, including one AML patient with <1 % blast load. These include 9 patients with IDH1R132 point mutations and one localized at IDH2R140. Overall prevalence of detectable IDH mutations (19.6%) was consistent with previously described findings, although detectable IDH2 mutation was under-represented in this limited study. D-2HG positivity correlated with IDH mutational status (p=0.0005, Fisher exact test) particularly among older patients (p=0.048, linear regression). The mean clinical follow-up for all patients was 292.6 ± 292.2 days (median 189 days, range 4 - 1201 days). Median time to progression was 248 days and median survival was 461 days. No significant association was observed between overall survival and plasma D-2HG level (p=0.7896) nor IDH mutational status (p=0.7964, log rank test). Progression-free survival also did not differ significantly among patients with or without an elevated plasma D-2 HG level (p=0.9227) nor IDH mutational status (p=0.5522, log rank test). For patients without the benefit of bone marrow archiving, plasma 2HG quantification represents a rapid and cost-effective surrogate indicator of IDH mutational status (sensitivity 0.8, specificity 0.93). Similar to reports by others, we found that not all patients with IDH1/2 mutations displayed elevated D-2HG. Nonetheless, for patients with baseline elevation of this oncometabolite, longitudinal, plasma-based determination of its trajectory may be effective for post-induction clinical prognostication, and/or as a trigger for second line IDH-targeted intervention following mutational status confirmation. DisclosuresNo relevant conflicts of interest to declare.

Biochemical Background in Mitochondria Affects 2HG Production by IDH2 and ADHFE1 in Breast Carcinoma.

Mitochondrial production of 2-hydroxyglutarate (2HG) can be catalyzed by wild-type isocitrate dehydrogenase 2 (IDH2) and alcohol dehydrogenase, iron-containing 1 (ADHFE1). We investigated whether biochemical background and substrate concentration in breast cancer cells promote 2HG production. To estimate its role in 2HG production, we quantified 2HG levels and its enantiomers in breast cancer cells using analytical approaches for metabolomics. By manipulation of mitochondrial substrate fluxes using genetic and pharmacological approaches, we demonstrated the existence of active competition between 2HG producing enzymes, i.e., IDH2 and ADHFE1. Moreover, we showed that distinct fractions of IDH2 enzyme molecules operate in distinct oxido-reductive modes, providing NADPH and producing 2HG simultaneously. We have also detected 2HG release in the urine of breast cancer patients undergoing adjuvant therapy and detected a correlation with stages of breast carcinoma development. In summary, we provide a background for vital mitochondrial production of 2HG in breast cancer cells with outcomes towards cancer biology and possible future diagnosis of breast carcinoma.

BIMG-09. GLUTAMINE AND GLYCINE BY MR SPECTROSCOPY IDENTIFY AGGRESSIVE GLIOMAS

Cancers reprogram their metabolism and the resulting alterations in metabolite abundance can be monitored in patients noninvasively using proton magnetic resonance spectroscopy (MRS). We evaluated glutamine, glycine and 2-hydroxyglutarate (2HG) in 27 adult subjects with gliomas (17 male and 10 female; age 22 - 69, median 39 years) using optimized MRS at 3T (PRESS TE 97ms) and examined their association with post-gadolinium enhancement, cell proliferation rate (MIB-1 labeling index), and overall survival of patients. The tumors included 9 glioblastomas (3 IDH mutated and 6 IDH wildtype), 10 astrocytomas (7 IDH mutated and 3 IDH wildtype), and 8 oligodendrogliomas (IDH mutated). The concentrations of glutamine and glycine were both significantly higher in enhancing tumors than in non-enhancing tumors (p=0.001 and 0.0001, respectively). The concentrations of glutamine and glycine were both positively correlated with MIB-1 (p=4E-5 and 1E-7, respectively). The sum of glutamine and glycine levels showed stronger association with MIB-1 (p=5E-10, r=0.89). In the Kaplan-Meier overall survival analysis, the survival was significantly shorter in patients with glutamine levels higher than 4.1 mM than those with concentrations less than 4.1 mM (p=0.02). For glycine, the patients with higher than 2.4 mM showed association with poor survival (p=0.03). The sum of glutamine and glycine levels showed stronger association with overall survival (p=0.008, cutoff 8.5mM). 2HG level greater than 0.5 mM was associated with long survival (p=0.01). We tested metabolic ratios to 2HG, in which 2HG estimates less than 1 mM were put as 1 mM (avoiding infinite ratios arising from null 2HG cases). The glutamine/2HG, glycine/2HG, and (glutamine+glycine)/2HG showed strong association with overall survival (p=2E-4, 2E-5 and 4.5E-7, respectively). Our data suggest that increased metabolism of glutamine and glycine is closely associated with rapid cell proliferation and poor survival, suggesting the metabolites are imaging biomarkers of glioma aggressiveness.

NIMG-24. GLYCINE AND GLUTAMINE BY MR SPECTROSCOPY ARE IMAGING BIOMARKERS OF GLIOMA AGGRESSIVENESS

Abstract Cancers reprogram their metabolism and the resulting alterations in metabolite concentrations may be closely related to the clinical behavior of the tumors. We evaluated glycine, glutamine and 2-hydroxyglutarate (2HG) in 16 adult subjects with glioblastomas (9 male and 7 female; age 43-64 years, median 58) noninvasively using proton magnetic resonance spectroscopy and examined their association with the cell proliferation rate (MIB-1 labeling index) and overall survival. MRS was acquired using point-resolved spectroscopy (PRESS TE 97ms) at 3T. Metabolite levels were quantified with reference to water. The concentrations of glycine and glutamine were both positively correlated with MIB-1 (p=.002 and .0008 respectively). The sum of glycine and glutamine levels showed stronger association with MIB-1 (p&amp;lt; .0001). In the Kaplan-Meier overall survival analysis, the median survival was significantly shorter in patients with glycine levels higher than 2.3 mM than those with concentrations less than 2.3 mM. For glutamine, the patients with higher than 5.7 mM showed association with poor survival. The log-rank p value was substantially smaller in glutamine compared to glycine (p=.008 vs .04). The sum of glycine and glutamine levels showed stronger association with overall survival. 2HG level greater than 1 mM was associated with long survival, which was as expected since elevation of 2HG represents IDH mutant tumors and IDH mutation carries favorable prognosis. Given the association of low 2HG with poor survival, we tested metabolic ratios to 2HG, in which 2HG estimates &amp;lt; 1 mM were put as 1 mM. The glycine/2HG, glutamine/2HG, and (glycine+glutamine)/2HG showed stronger association with overall survival, compared to glycine, glutamine, and glycine+glutamine. Our data suggested that increased metabolism of glycine and glutamine is closely associated with rapid cell proliferation and poor clinical outcome, suggesting the metabolites as an MRS imaging biomarker of glioma aggressiveness.

Effects of azacitidine in 93 patients with IDH1/2 mutated acute myeloid leukemia/myelodysplastic syndromes: a French retrospective multicenter study

Isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) mutations in Myeloid Neoplams (MNs) exhibit DNA hypermethylation via 2-hydroxyglutarate (2HG) over-production. Clinical impact of azacitidine (AZA) remains inconsistent in IDH1/2-mutated MNs and the potential of serum 2HG as a suitable marker of response to AZA is unknown. To address these questions, we retrospectively analyzed 93 MNs patients (78 AML, 11 MDS, 4 CMML) with IDH1/2 mutations treated with AZA. After a median of 5 cycles of AZA, overall response rate was 28% (including 15% complete remission) and median OS was 12.3 months (significantly shorter in AML compared to MDS/CMML patients). In multivariate analysis of AML patients, DNMT3A mutation was associated with shorter OS while IDH1/2 mutation subtypes had no independent impact. No difference was observed in serum 2HG levels upon AZA treatment between responding and refractory patients suggesting that serum 2HG cannot be used as a surrogate marker of AZA response.

2-Hydroxyglutarate in Cancer Cells.

Significance: Cancer cells are stabilized in an undifferentiated state similar to stem cells. This leads to profound modifications of their metabolism, which further modifies their genetics and epigenetics as malignancy progresses. Specific metabolites and enzymes may serve as clinical markers of cancer progression.Recent Advances: Both 2-hydroxyglutarate (2HG) enantiomers are associated with reprogrammed metabolism, in grade III/IV glioma, glioblastoma, and acute myeloid leukemia cells, and numerous other cancer types, while acting also in the cross talk of tumors with immune cells. 2HG contributes to specific alternations in cancer metabolism and developed oxidative stress, while also inducing decisions on the differentiation of naive T lymphocytes, and serves as a signal messenger in immune cells. Moreover, 2HG inhibits chromatin-modifying enzymes, namely 2-oxoglutarate-dependent dioxygenases, and interferes with hypoxia-inducible factor (HIF) transcriptome reprogramming and mammalian target of rapamycin (mTOR) pathway, thus dysregulating gene expression and further promoting cancerogenesis.Critical Issues: Typically, heterozygous mutations within the active sites of isocitrate dehydrogenase isoform 1 (IDH1)R132H and mitochondrial isocitrate dehydrogenase isoform 2 (IDH2)R140Q provide cells with millimolar r-2-hydroxyglutarate (r-2HG) concentrations, whereas side activities of lactate and malate dehydrogenase form submillimolar s-2-hydroxyglutarate (s-2HG). However, even wild-type IDH1 and IDH2, notably under shifts toward reductive carboxylation glutaminolysis or changes in other enzymes, lead to “intermediate” 0.01–0.1 mM 2HG levels, for example, in breast carcinoma compared with 10−8 M in noncancer cells.Future Directions: Uncovering further molecular metabolism details specific for given cancer cell types and sequence-specific epigenetic alternations will lead to the design of diagnostic approaches, not only for predicting patients' prognosis or uncovering metastases and tumor remissions but also for early diagnostics.

Abstract 1266: Mutant IDH1 alters epigenetic regulation to promote stemness in low grade glioma: Deciphering signaling pathways altered by mIDH1 inhibition

Abstract Greater than 75% of Low Grade Gliomas (LGG) harbor a mutation in isocitrate dehydrogenase 1 (IDH1) and this molecular subgroup also exhibits mutations in TP53 and ATRX. Mutant IDH1 often recurs and presents with a more aggressive phenotype that is refractory to existing therapies. The production of an oncometabolite, 2-hydroxyglutarate by mutant IDH1 (mIDH1) has been shown to induce histone and DNA hypermethylation. Pharmacological inhibition of mIDH1 (AGI-5198) can restore expression of genes involved in neural differentiation. We hypothesize that mIDH1 remodels chromatin to promote a stem cell-like transcriptional state. Recent single cell RNA-seq data from primary mIDH1 astrocytoma and oligodendroglioma has identified an undifferentiated pool of tumor cells with a neural stem/progenitor transcriptional profile. We aim to identify unique regulatory networks in SF10602, a human primary glioma culture derived from a surgical biopsy, which endogenously expresses mIDH1.We treated SF10602 with AGI-5198 every 2 days for 1 week. We observed a global reduction in histone marks: H3K4me3, H3K36me3, and H3K27me3. This reversibility of histone hypermethylation has previously been described in mIDH1 tumor lines treated with mIDH1 inhibitors that block 2HG production. We confirmed that mIDH1 inhibition reduced 2HG levels by collecting condition media and performing liquid chromatography mass spectrometry. We performed a temporal micrococcal nuclease (Mnase) digestion to assess chromatin accessibility and we observed enhanced chromatin condensation following mIDH1 inhibition. To evaluate if the reversion of chromatin organization resulting from mIDH1 inhibition impacts neural differentiation pathways we performed RNA-seq. We identified 496 differentially up-regulated and 893 down-regulated genes (log2&amp;gt;+/-0.6) comparing SF10602 with/out AGI-5198. Following gene set enrichment analysis, we observed upregulation of pathways involved in epigenetic regulation, DNA conformation and neuronal development. Additionally, we observed downregulation of pathways involved in cell polarity and transcription factor activity. In future work, we will perform ChIP-seq to define enhancers and promoters that regulate stem cell-like characteristics. These findings will reveal novel genomic loci unique to mIDH1 glioma. SC is supported by the T32 Cancer Biology Training Grant, the work is supported by grants from NIH/NINDS to MGC and PRL. Citation Format: Stephen Carney, Brandon Zhu, Padma Kadiyala, Maria Garcia-Fabiani, Preethi Gorla, Felipe Nunez, Pedro R. Lowenstein, Maria G. Castro. Mutant IDH1 alters epigenetic regulation to promote stemness in low grade glioma: Deciphering signaling pathways altered by mIDH1 inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1266.

SIRT3 and GCN5L regulation of NADP+- and NADPH-driven reactions of mitochondrial isocitrate dehydrogenase IDH2

Wild type mitochondrial isocitrate dehydrogenase (IDH2) was previously reported to produce oncometabolite 2-hydroxyglutarate (2HG). Besides, mitochondrial deacetylase SIRT3 has been shown to regulate the oxidative function of IDH2. However, regulation of 2HG formation by SIRT3-mediated deacetylation was not investigated yet. We aimed to study mitochondrial IDH2 function in response to acetylation and deacetylation, and focus specifically on 2HG production by IDH2. We used acetylation surrogate mutant of IDH2 K413Q and assayed enzyme kinetics of oxidative decarboxylation of isocitrate, 2HG production by the enzyme, and 2HG production in cells. The purified IDH2 K413Q exhibited lower oxidative reaction rates than IDH2 WT. 2HG production by IDH2 K413Q was largely diminished at the enzymatic and cellular level, and knockdown of SIRT3 also inhibited 2HG production by IDH2. Contrary, the expression of putative mitochondrial acetylase GCN5L likely does not target IDH2. Using mass spectroscopy, we further identified lysine residues within IDH2, which are the substrates of SIRT3. In summary, we demonstrate that 2HG levels arise from non-mutant IDH2 reductive function and decrease with increasing acetylation level. The newly identified lysine residues might apply in regulation of IDH2 function in response to metabolic perturbations occurring in cancer cells, such as glucose-free conditions.

Phase II basket trial of olaparib and durvalumab in patients (pts) with isocitrate dehydrogenase (IDH) mutated solid tumors.

TPS3167 Background: Somatic IDH mutations are common in low grade gliomas, and rare in other solid tumors with the exception of intrahepatic cholangiocarcinoma (ICA) and certain subtypes of chondrosarcoma. IDH mutations confer a gain-of-function neomorphic activity, such that mutant IDH enzymes preferentially convert αKG to 2-hydroxyglutarate (2HG), resulting in abnormal accumulation of 2HG. 2HG competitively inhibits αKG-dependent dioxygenases, many of which are involved in DNA repair. Preclinical studies show that IDH mutated cancer cells have defective homologous recombination repair and are exquisitely sensitive to poly (adenosine 5’-diphophate-ribose) polymerase (PARP) inhibition. Methods: This is a single arm phase II basket study (NCT03991832). Pts with IDH mutated solid tumors are divided into three cohorts; A: low-grade glioma; B: cholangiocarinoma; C: all other solid tumors. Pts are treated with olaparib 300 mg twice daily continuously and durvalumab 1500 mg every 4 weeks until progression, intolerable toxicity or consent withdrawal. Radiological assessment is performed after every 2 cycles of study treatments. Major eligibility criteria include IDH mutation by immunohistochemistry or sequencing, up to 2 lines of systemic therapy for advanced disease, performance status 0 – 2 and adequate organ function. Pts are excluded if they received prior PARP inhibitors and anti-PD-1/PD-L1 antibody. The Simon’s optimal 2-stage design is applied for Cohorts A and B. 10 pts will be enrolled in each of Cohort A and B initially. If 2 or more partial responses (PR) are seen in these 10 pts, additional 19 pts will be enrolled for a total of 29 pts in that cohort. The combination is considered to be of clinical interest for further development if ≥ 6 PRs are seen in each cohort. Cohort C will enroll 20 pts. Archival tumor tissues and serial blood samples will be collected on study. 2HG levels will be measured and correlated with responses. The study was activated in December 2019. 3 pts have been enrolled into Cohort B. Clinical trial information: NCT03991832 .

Characterisation of isocitrate dehydrogenase 1/isocitrate dehydrogenase 2 gene mutation and the d-2-hydroxyglutarate oncometabolite level in dedifferentiated chondrosarcoma.

Dedifferentiated chondrosarcoma (DDCHS) is an aggressive type of chondrosarcoma that results from high-grade transformation of a low-grade chondrosarcoma. Mutations in the isocitrate dehydrogenase (IDH) 1 gene and the IDH2 gene that lead to increased d-2-hydroxyglutarate (2HG) oncometabolite production, promoting tumorigenesis, have been recently described in low-grade cartilaginous neoplasms. The aims of this study were to examine the prevalence of IDH mutations in a single-institution cohort of DDCHS cases and correlate 2HG levels with mutation status. We examined a series of 21 primary DDCHS cases by using Sanger sequencing and quantitative polymerase chain reaction genotyping to look for IDH1/IDH2 mutations, and evaluated the 2HG levels in formalin-fixed paraffin-embedded tumour and matched normal tissue samples by using a fluorometric assay. Seventy-six per cent of DDCHS cases (16/21) harboured a heterozygous IDH1 or IDH2 mutation. Six of 14 IDH-mutated DDCHS cases showed elevated 2HG levels in tumour tissue relative to matched normal tissue. There were no consistent histological or disease-specific survival differences between IDH-mutated tumours and wild-type tumours. Our study confirms the frequent presence of a variety of IDH1 and IDH2 mutation variants, indicating that a sequencing-based approach is required for DDCHS if IDH is to be used as a diagnostic marker. Similarly to other IDH-mutated tumour types, IDH-mutated DDCHS cases show elevated 2HG levels, indicating that the oncometabolite activity of 2HG may contribute to DDCHS oncogenesis and progression.

Intraoperative assessment of isocitrate dehydrogenase mutation status in human gliomas using desorption electrospray ionization-mass spectrometry.

The authors describe a rapid intraoperative ambient ionization mass spectrometry (MS) method for determining isocitrate dehydrogenase (IDH) mutation status from glioma tissue biopsies. This method offers new glioma management options and may impact extent of resection goals. Assessment of the IDH mutation is key for accurate glioma diagnosis, particularly for differentiating diffuse glioma from other neoplastic and reactive inflammatory conditions, a challenge for the standard intraoperative diagnostic consultation that relies solely on morphology. Banked glioma specimens (n = 37) were analyzed by desorption electrospray ionization-MS (DESI-MS) to develop a diagnostic method to detect the known altered oncometabolite in IDH-mutant gliomas, 2-hydroxyglutarate (2HG). The method was used intraoperatively to analyze tissue smears obtained from glioma patients undergoing resection and to rapidly diagnose IDH mutation status (< 5 minutes). Fifty-one tumor core biopsies from 25 patients (14 wild type [WT] and 11 mutant) were examined and data were analyzed using analysis of variance and receiver operating characteristic curve analysis. The optimized DESI-MS method discriminated between IDH-WT and IDH-mutant gliomas, with an average sensitivity and specificity of 100%. The average normalized DESI-MS 2HG signal was an order of magnitude higher in IDH-mutant glioma than in IDH-WT glioma. The DESI 2HG signal intensities correlated with independently measured 2HG concentrations (R2 = 0.98). In 1 case, an IDH1 R132H-mutant glioma was misdiagnosed as a demyelinating condition by frozen section histology during the intraoperative consultation, and no resection was performed pending the final pathology report. A second craniotomy and tumor resection was performed after the final pathology provided a diagnosis most consistent with an IDH-mutant glioblastoma. During the second craniotomy, high levels of 2HG in the tumor core biopsies were detected. This study demonstrates the capability to differentiate rapidly between IDH-mutant gliomas and IDH-WT conditions by DESI-MS during tumor resection. DESI-MS analysis of tissue smears is simple and can be easily integrated into the standard intraoperative pathology consultation. This approach may aid in solving differential diagnosis problems associated with low-grade gliomas and could influence intraoperative decisions regarding extent of resection, ultimately improving patient outcome. Research is ongoing to expand the patient cohort, systematically validate the DESI-MS method, and investigate the relationships between 2HG and tumor heterogeneity.

Functional Interrogation of Variants of Undetermined Significance of the Isocitrate Dehydrogenase 1 and 2 Genes in Myeloid Neoplasms

Introduction: Isocitrate dehydrogenase 1 and 2 (IDH1/2) are metabolic enzymes in the citric acid cycle, producing alpha-ketoglutarate (αKG). Mutations in specific regions of these genes have been characterized in gliomas, AML and chronic myeloid neoplasms such as MDS and MPN. These mutations produce an oncometabolite 2-hydroxyglutarate (2HG) that disrupts the epigenetic phenotype of myeloid cells promoting oncogenesis. While this process has been characterized for arginine hot spot (R132/R140) mutations, there exist variants of unclear significance (IDHVUS), with potential pathogenic predictions using in silico assessment models. With the availability of mutant IDH inhibitors in practice, it has become important to characterize these VUS. We carried out this study to functionally interrogate potentially pathogenic IDHVUS in MDS and MPN. Methods: After institutional approval, bone marrow (BM) DNA of patients with MDS and MPN, including overlap syndromes were subjected to next generation sequencing (29-gene, including the entire coding region of IDH1/2) by previously described methods (Patnaik et al., BCJ 2016). A sub-set of patients with available plasma [IDH wild type (IDHWT), bona fide IDH mutations (IDHMT) and IDHVUS with predicted pathogenicity (≤0.01% minor allele frequency, and predicted damaging in silico scores)] were submitted for mass spectrometry (LC-MS) based detection of IDH-related metabolites; D and L isomers of 2HG, αKG, and total 2HG. The impact of variations on protein secondary structure was assessed by the GorIV prediction algorithm. Potential alterations within the tertiary structure of the proteins was evaluated by in silico mutagenesis. Results: One hundred and seventeen patients were included in the study; median age 68 years (range, 39-94), 68% male. Of these, 40 (34%) had MDS, 42 (36%) MPN, and 35 (30%) MDS/MPN overlap syndromes. Eighty six (74%) had IDHMT, including n=28 (24%) with IDH1MT (R132C/G/H/S) (11 MDS, 8 MPN, 9 MDS/MPN) and n=59 (50%) with IDH2MT (R140G/Q/W) (22 MDS, 20 MPN, 17 MDS/MPN). IDHVUS were detected in 32 (27%) patients, including 1 IDH1MT patient, and involved IDH1 in 14 (12%) cases and IDH2 in 18 (15%) cases. All IDHVUS were missense, except for a 2 amino acid insertion in IDH2, with the most frequent VUS being IDH1Y183C (7%) and IDH2T352P (7%). While there were no phenotypic differences between IDH1/2MUT and IDH1/2VUS, in comparison to patients with IDH1/2MT, those with IDH1VUS were more likely to have higher WBC counts (median 8.1 vs 3.6 x109/L, p=0.0322), higher neutrophil counts (median 3.3 vs 1.3 x109/L, p=0.0203), and lower BM blast % (median 1 vs 3%, p=0.02061). Compared to IDH1/2MT patients, those with sole IDH1/2VUS more frequently had mutations in SF3B1 (p=0.0167), while they were associated with a lower frequency of SRSF2 mutations (p=0.0002). Interestingly, TET2 mutations were detected in only 5% of patients with IDHMT and 16% of IDHVUS patients, although without reaching statistical significance. LC-MS analysis on 75 plasma samples (n=43 IDHMT, n=9 IDHVUS, n=20 IDHWT, 3 normal) revealed a significant increase in total 2HG levels in IDHMT vs IDHVUS (p=0.0089), and IDHWT (p=0.0020) cases (Figure 2A-D). While there were no significant differences in αKG levels between the groups, the D-2HG enantiomer levels were higher in IDHMT samples vs IDHVUS (p=0.0199) and IDHWT (p=0.0055). On examination of the crystal structures of the IDH genes using PDB, while we observed a direct interaction between R132/R140/R172 and the catalytic core, the IDHVUS were located away from this active center and were hence thought to be allosteric in nature (Figure 2F). These VUS however, did demonstrate predictive steric clashes in the tertiary structure context of IDH1 using the PyMol artificial mutagenesis tool. The absence of wild-type crystal structure for IDH2precluded a similar analysis for IDH2. Median overall survival (OS) of the entire cohort (Figure 1) was 36 (range, 0-190) months, with OS being inferior in IDH1/2MT patients (34 months) vs those with IDH1/2VUS (50 months, p=0.0255). Conclusion: Our study demonstrates that in spite of predictive pathogenicity of IDH1/2VUS using in silico predictive tools and crystal studies, the lack of involvement of the arginine hot spot precludes IDH neomorphic activity, as shown by normal 2HG levels. In addition, in comparison to IDHMT patients, those with IDHVUS had fewer BM blasts and a better survival. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.