D-2-hydroxyglutarate Levels Research Articles

Catalytically distinct IDH1 mutants tune phenotype severity in tumor models.

Mutations in isocitrate dehydrogenase 1 (IDH1) impart a neomorphic reaction that produces D-2-hydroxyglutarate (D2HG), which can inhibit DNA demethylases to drive tumorigenesis. Mutations affect residue R132 and display distinct catalytic profiles for D2HG production. We show that catalytic efficiency of D2HG production is greater in IDH1 R132Q than R132H mutants, and expression of R132Q in cellular and xenograft models leads to higher D2HG concentrations in cells, tumors, and sera compared to R132H. Though expression of IDH1 R132Q leads to hypermethylation in DNA damage pathways, DNA hypomethylation is more notable when compared to R132H expression. Transcriptome analysis shows increased expression of many pro-tumor pathways upon expression of IDH1 R132Q versus R132H, including transcripts of EGFR and PI3K signaling pathways. Thus, IDH1 mutants appear to modulate D2HG levels via altered catalysis, resulting in distinct epigenetic and transcriptomic consequences where higher D2HG levels appear to be associated with more aggressive tumors.

Abstract C036: Investigating the phenotypic changes in in vitro and in vivo cancer models expressing isocitrate dehydrogenase 1 (IDH1) mutations R132H and R132Q

Abstract According to the National Cancer Institute, there were 25,050 new brain cancer cases in 2022, representing 1.3% of all cancer incidents in the United States. In the same year, the estimated death was 18,280 cases, representing 3.0% of all cancer deaths. Relative survival statistics between 2017 and 2018 showed that only 32.5% of patients would survive five years or more. Disparities in cancer incidence and outcomes, including brain cancers, can be due to biological, socioeconomic, and care access factors that often have roots in structural racism. Research shows lower three years survival rates of glioblastoma in the African American population by 28.8% compared to the Asian Pacific Islanders at 36.6% and Hispanic at 32.3%. Mutant isocitrate dehydrogenase 1 (IDH1) can lead to brain cancer, including > 80% of lower-grade gliomas and secondary glioblastomas. Mutant IDH1 catalyzes the NADPH-dependent conversion of α-ketoglutarate to D2-hydroxyglutarate (D2HG), an oncometabolite linked to many cancer types. IDH1 mutation occurs primarily at residue 132, and our lab has shown that the catalytic activity differs based on the amino acid substituted at that location. There is a lack of information describing the relation of D2HG produced to cancer cells’ phenotype severity. Understanding IDH1 mutation will clarify cancer progression, patients’ prognosis, and treatment responses. Here, we hypothesize that the toxic effect of D2HG will impair the viability of cells expressing mutant IDH1 and will cause a reduction in ROS and NADPH cellular levels. This work examines D2HG production efficiency in vivo and in vitro and the metabolic consequences of this oncometabolite. We created cell lines stably overexpressing IDH1-R132Q and IDH1-R132H to conduct this work. Our results showed higher levels of D2HG in cells expressing IDH1-R132Q compared to DH1-wild type and IDH1-R132H, supportive of our previous finding that IDH1-R132Q mutation has higher catalytic activities (higher efficiency in producing mutant IDH1). These higher D2HG levels caused cells to proliferate and migrate at lower rates. D2HG interferes with DNA demethylation by inhibiting the TET enzyme, and we observed higher levels of histone protein expression in IDH1-R132Q compared to the other modified cells. In in vivo studies, we identified elevated D2HG levels in mouse xenografts expressing IDH1-R132Q that affected tumor growth. Citation Format: Mowaffaq A.A. Adam, Mikella Robinson, An Hoang, Grace A. Wells, David A. Scott, Olga Zagnitko, Carrie House, Christal D. Sohl. Investigating the phenotypic changes in in vitro and in vivo cancer models expressing isocitrate dehydrogenase 1 (IDH1) mutations R132H and R132Q [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C036.

Inhibition of D-2HG leads to upregulation of a proinflammatory gene signature in a novel HLA-A2/HLA-DR1 transgenic mouse model of IDH1R132H-expressing glioma

BackgroundLong-term prognosis of WHO grade II, isocitrate dehydrogenase (IDH)-mutated low-grade glioma (LGG) is poor due to high risks of recurrence and malignant transformation into high-grade glioma. Immunotherapy strategies are attractive...

CLRM-13. INTRAOPERATIVE MICRODIALYSIS: GLIOMA INTELLIGENCE FROM BEHIND ENEMY LINES

INTRODUCTIONGliomas present a formidable challenge for translational progress. Heterogeneity within and between tumors may demand empirically individualized and adaptive paradigms requiring rapid mechanistic feedback. We asked if tumor-associated metabolic biomarkers from glioma extracellular fluid could impart mechanistic “intelligence” reflecting intra- and inter-tumoral heterogeneity.METHODSFive live human gliomas (2 oligos; 2 IDH-WT GBMs; 1 IDH-mutant GBM), were evaluated in situ with high molecular weight (100kDA) intraoperative microdialysis using 3 disparately placed catheters. Isotonic 3% dextran perfusate was collected in 20 min (40mL) aliquots. CSF samples (n=21) were additionally evaluated from these and other patients with diverse brain tumors. The IDH-mutant glioma-associated oncometabolite D2-hydroxyglutarate (D2-HG) was quantified with targeted Liquid Chromotography-Mass Spectrometry (LC-MS). Over 200 metabolites were further evaluated via untargeted LC-MS using the Metabolon platform. Correlation, clustering, ROC and enrichment analyses were employed to identify correlations within and between patient samples.RESULTSCSF samples from patients with IDH-mutant gliomas contained over twenty-fold higher levels of D2-HG (median 4.1 mM, range 1.6-13.2, n=7) compared to those from IDH-wild type tumors (median 0.19 mM; range 0.89-0.35, n=14). Microdialysate from IDH-mutant gliomas contained 10-953mM D2-HG, 9-63x higher than paired CSF samples. Interestingly, IDH status failed to predict the global metabolic signature of microdialysate. Microdialysate samples clustered into 2 major metabolic phenotype clusters with IDH-WT and IDH-mutant gliomas in each cluster. A superimposed metabolic signature distinguishing enhancing from non-enhancing tumor, was conserved in both patient clusters. Amino acid and carnitine metabolism predominated in microdialysate signatures. TCA cycle and Warburg-associated metabolites were differentially enriched in CSF samples after prior therapy independent of tumor burden.CONCLUSIONSIntra-operative micro-dialysis may complement currently available “intelligence” regarding the phenotype, burden, and metabolism of live human gliomas and is feasible within standard-of-care surgical procedures. Future work will evaluate utility for pharmacodynamic feedback following novel early phase candidate therapies.

Single-cell profiling of D-2-hydroxyglutarate using surface-immobilized resazurin analogs

D-2-hydroxyglutarate (D2HG) is over-produced as an oncometabolite due to mutations in isocitrate dehydrogenases (IDHs). Accumulation of D2HG can cause the dysfunction of many enzymes and genome-wide epigenetic alterations, which can promote oncogenesis. Quantification of D2HG at single-cell resolution can help understand the phenotypic signatures of IDH-mutant cancers and identify effective therapeutics. In this study, we developed an analytical method to detect D2HG levels in single cancer cells by adapting cascade enzymatic reactions on a resazurin-based fluorescence reporter. The resazurin probe was immobilized to the sensing surface via biotin-streptavidin interaction. This surface chemistry was rationally optimized to translate the D2HG levels to sensitive fluorescence readouts efficiently. This D2HG assay demonstrated good selectivity and high sensitivity toward D2HG, and it was compatible with the previously developed single-cell barcode chip (SCBC) technology. Using the SCBC platform, we performed simultaneous single-cell profiling of D2HG, glucose uptake, and critical oncogenic signaling proteins in single IDH-mutant glioma cells. The results unveiled the complex interplays between metabolic and oncogenic signaling and led to the identification of effective combination targeted therapy against these IDH-mutant glioma cells.

A Phase Ib Clinical Trial of Metformin and Chloroquine in Patients with IDH1-Mutated Solid Tumors.

Simple SummaryMutations in the isocitrate dehydrogenase 1 (IDH1) gene occur in high-grade chondrosarcoma, high-grade glioma and intrahepatic cholangiocarcinoma. Due to the lack of effective treatment options, these aggressive types of cancer have a dismal outcome. The metabolism of IDH1-mutated cancer cells is reprogrammed in order to produce the oncometabolite D-2-hydroxyglutarate (D-2HG). In this clinical trial, we used the oral antidiabetic drug metformin and the oral antimalarial drug chloroquine to disrupt the vulnerable metabolism of IDH1-mutated solid tumors. We found that the combination regimen of metformin and chloroquine is well tolerated, but the combination did not induce a clinical response in this patient population. Secondly, we confirmed the clinical usefulness of D/L-2HG ratios in serum as a biomarker and the ddPCR-facilitated detection of an IDH1 mutation in circulating DNA from peripheral blood.Background: Mutations in isocitrate dehydrogenase 1 (IDH1) occur in 60% of chondrosarcoma, 80% of WHO grade II-IV glioma and 20% of intrahepatic cholangiocarcinoma. These solid IDH1-mutated tumors produce the oncometabolite D-2-hydroxyglutarate (D-2HG) and are more vulnerable to disruption of their metabolism. Methods: Patients with IDH1-mutated chondrosarcoma, glioma and intrahepatic cholangiocarcinoma received oral combinational treatment with the antidiabetic drug metformin and the antimalarial drug chloroquine. The primary objective was to determine the occurrence of dose-limiting toxicities (DLTs) and the maximum tolerated dose (MTD). Radiological and biochemical tumor responses to metformin and chloroquine were investigated using CT/MRI scans and magnetic resonance spectroscopy (MRS) measurements of D-2HG levels in serum. Results: Seventeen patients received study treatment for a median duration of 43 days (range: 7–74 days). Of twelve evaluable patients, 10 patients discontinued study medication because of progressive disease and two patients due to toxicity. None of the patients experienced a DLT. The MTD was determined to be 1500 mg of metformin two times a day and 200 mg of chloroquine once a day. A serum D/L-2HG ratio of ≥4.5 predicted the presence of an IDH1 mutation with a sensitivity of 90% and a specificity of 100%. By utilization of digital droplet PCR on plasma samples, we were able to detect tumor-specific IDH1 hotspot mutations in circulating tumor DNA (ctDNA) in investigated patients. Conclusion: Treatment of advanced IDH1-mutated solid tumors with metformin and chloroquine was well tolerated but did not induce a clinical response in this phase Ib clinical trial.

Enasidenib for the treatment of relapsed or refractory acute myeloid leukemia with an isocitrate dehydrogenase 2 mutation

ABSTRACT Introduction Isocitrate dehydrogenase 2 (IDH2) is a key metabolic enzyme that converts isocitrate to α-ketoglutarate (αKG). Somatic point mutations in IDH2 confer a gain-of-function in blast cells resulting in overproduction of D-2-hydroxyglutarate (D-2HG). High intracellular concentrations of D-2HG inhibit α-ketoglutarate-dependent dioxygenases including histone, DNA and RNA demethylases, leading to histone, DNA and RNA hypermethylation, and cell differentiation blockade. In vitro and in vivo preclinical studies have demonstrated that inhibition of IDH2-mutant enzymes with enasidenib decrease intracellular D-2HG levels, reverse epigenetic dysregulation, and release the differentiation block. The US Food and Drug Administration (FDA) approved enasidenib, a mutant-IDH2 enzyme inhibitor for patients with relapsed or refractory (R/R) IDH2-mutated AML. Areas covered We review the biology and prognostic significance of IDH2 mutations in AML and discuss the pharmacology, clinical efficacy, and toxicity profile of enasidenib. We highlight areas of ongoing preclinical and clinical research. Expert opinion Enasidenib was FDA approved due to high response rates, durability of the responses that translated into an impressive OS in that heavily pretreated population. Promising ongoing clinical trials are evaluating combination therapies with enasidenib frontline.

Ivosidenib to treat adult patients with relapsed or refractory acute myeloid leukemia.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that convert isocitrate to alpha-ketoglutarate (alphaKG). Somatic point mutations in IDH1/2 that are found in rare distinct subsets of cancers confer a gain of function in cancer cells which results in the accumulation and secretion in vast excess of the oncometabolite D-2-hydroxyglutarate (D-2HG). Overproduction of D-2HG interferes with cellular metabolism and epigenetic regulation, contributing to oncogenesis. High levels of D-2HG inhibit alphaKG-dependent dioxygenases including histone, DNA and RNA demethylases, resulting in histone, DNA and RNA hypermethylation and cell differentiation blockade. In addition, D-2HG is a biomarker suitable for the detection of IDH1/2 mutations at diagnosis, and is also predictive of clinical response. The U.S. Food and Drug Administration (FDA) approved ivosidenib, a mutant-IDH1 enzyme inhibitor, for patients with relapsed or refractory IDH1-mutated acute myeloid leukemia (AML) in 2018, and also as front-line therapy for newly diagnosed elderly patients 75 years or older or who are ineligible to receive intensive chemotherapy in 2019. Ivosidenib represents a novel drug class for targeted therapy in AML.

P11.47 Generation, characterisation and drug screening of patient-derivedIDH1-mutated glioma cell lines

Abstract BACKGROUND Despite considerable scientific efforts, endogenous in vitro isocitrate dehydrogenase (IDH)-mutated glioma models remain scarce. Availability of these models is key to understanding underlying molecular mechanisms and vital for the development of new therapeutic interventions. We established and characterized a set of seven cell lines derived from IDH1-mutated gliomas and utilized them to investigate IDH-mutant glioma drug-response in vitro. MATERIAL AND METHODS Fresh tumor material was collected directly from the operating room, mechanically and enzymatically dissociated and cultured under serum-free culture conditions. IDH mutation status was verified at several passages with Sanger sequencing, as well as with whole exome sequencing. D-2-hydroxyglutarate (D2-HG) levels were determined with mass-spectrometry. Genome-wide methylation profiling was performed using the Infinium MethylationEpic BeadChip array. Cell viability was measured using the ATP-based CellTiter-Glo assay. Cell proliferation was determined through cell counting. Drug screens were performed with an FDA-approved anti-cancer drug set from the NIH as well as two IDH-mutant specific inhibitors. RESULTS Over the last decade our lab processed over 800 glioma samples from all WHO grades and IDH mutational status. Optimisation of culture conditions led to the establishment of seven IDH1-mutant glioma cell lines. The IDH1-mutation was present during all tested passages in these cell lines. Whole exome sequencing showed a high concordance between tumor and cell lines with regard to driver gene mutations. Similarly, copy-number changes based on genome-wide methylation data also show a close resemblance between the parental tumor and resulting cell lines. The Heidelberg methylation profiler classified each cell line and its parental tumor as IDH mutant glioma. When exposed to an IDH1-mutant specific inhibitor, all cell lines showed a concentration-dependent decrease of D2-HG levels. The inhibitors showed little to no effect on viability up to 10uM during 8 days, however, long term treatment up to 4 weeks revealed a decrease in cell doubling time in 2 of 6 cultures. Drug screen results either alone or in combination with an IDH1-mutant specific inhibitor identified several interesting candidates that are currently followed up in additional experiments. CONCLUSION We established a unique set of patient-derived IDH1-mutant glioma cell lines that closely resemble their respective tumors and can be used to identify new therapeutic strategies.

Biochemical characterization of human D-2-hydroxyglutarate dehydrogenase and two disease related variants reveals the molecular cause of D-2-hydroxyglutaric aciduria

D-2-hydroxyglutaric aciduria is a neurometabolic disorder, characterized by the accumulation of D-2-hydroxyglutarate (D-2HG) in human mitochondria. Increased levels of D-2HG are detected in humans exhibiting point mutations in the genes encoding isocitrate dehydrogenase, citrate carrier, the electron transferring flavoprotein (ETF) and its downstream electron acceptor ETF-ubiquinone oxidoreductase or D-2-hydroxyglutarate dehydrogenase (hD2HGDH). However, while the pathogenicity of several amino acid replacements in the former four proteins has been studied extensively, not much is known about the effect of certain point mutations on the biochemical properties of hD2HGDH.Therefore, we recombinantly produced wild type hD2HGDH as well as two recently identified disease-related variants (hD2HGDH-I147S and -V444A) and performed their detailed biochemical characterization. We could show that hD2HGDH is a FAD dependent protein, which is able to catalyze the oxidation of D-2HG and D-lactate to α-ketoglutarate and pyruvate, respectively. The two variants were obtained as apo-proteins and were thus catalytically inactive. The addition of FAD failed to restore enzymatic activity of the variants, indicating that the cofactor binding site is compromised by the single amino acid replacements. Further analyses revealed that both variants form aggregates that are apparently unable to bind the FAD cofactor.Since, D-2-hydroxyglutaric aciduria may also result from a loss of function of either the ETF or its downstream electron acceptor ETF-ubiquinone oxidoreductase, ETF may serve as the cognate electron acceptor of reduced hD2HGDH. Here, we show that hD2HGDH directly reduces recombinant human ETF, thus establishing a metabolic link between the oxidation of D-2-hydroxyglutarate and the mitochondrial electron transport chain.

Abstract 16708: Inhibition of Alpha-Ketoglutarate Dehydrogenase Activates Autophagy

Introduction: Metabolic reprogramming is a hallmark of cancer and heart failure. In isocitrate dehydrogenase 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) inhibit α-ketoglutarate dehydrogenase (α-KGDH) in the heart. This inhibition is associated with muscle atrophy and reduced ATP and NADH provision. The connection between metabolic changes and proteome remodeling are still poorly understood. Hypothesis: Inhibition of α-KGDH promotes autophagy activation through the sirtuin family of NAD + -dependent deacetylases. Methods: We treated both male and female wild-type C57BL/J6 mice (n=12) with D2-HG in vivo, and cultured L6 myocytes (L6Ms) and adult mouse ventricular cardiomyocytes (AMVMs) with D2-HG. We constructed LC3 mutants with a lysine-to-arginine (GFP-LC3K49R-K51R) and lysine-to-glutamine (GFP-LC3K49Q-K51Q) replacement at Lys49 and Lys51 mimicking deacetylated and acetylated LC3, respectively. Results: Inhibition of α-KGDH by D2-HG is accompanied by heart and skeletal muscle atrophy, as well as increased LC3 and p62 expression in mice treated over 30-days. In vivo and in vitro treatment with D2-HG for 24h, increases phosphorylation and activation of AMPK, while phosphorylation of mTOR and p70S6K decreases. Thus, acute α-KGDH inhibition impacts protein turnover. Autophagy activation is highly dependent on D2-HG concentration and duration. ATP levels significantly decrease in differentiated L6Ms and AMVMs when treated with D2-HG levels above 0.13 mM over 24 to 96 h. GFP-tagged LC3 co-localizes with lysosomes labeled with LysoTracker Blue dye and mitochondria labeled with MitoTracker Red CMXRos dye upon α-KGDH inhibition, which indicates the presence of autophagosomes and mitophagosomes. We show that upon α-KGDH inhibition and subsequent NAD + increase, Sirt1 forms a molecular complex with LC3 and activates autophagy through deacetylation of LC3. In these conditions, LC3K49R-K51R mutants show puncta formation in the cytoplasm indicating that in addition to NAD + increase other factors are driving autophagy activation. Mass spectrometry-based proteomics and metabolomics revealed that α-KGDH inhibition induces upregulation of tropomyosin, NADH dehydrogenase and proteins involved in glycolysis and pentose phosphate pathway. Conclusions: Inhibition of α-KGDH promotes autophagy activation through deacetylation of LC3 by Sirt1 and culminates in proteome remodeling.

IDH1-mutated transgenic zebrafish lines: An in-vivo model for drug screening and functional analysis.

IntroductionThe gene encoding isocitrate dehydrogenase 1 (IDH1) is frequently mutated in several tumor types including gliomas. The most prevalent mutation in gliomas is a missense mutation leading to a substitution of arginine with histidine at the residue 132 (R132H). Wild type IDH1 catalyzes oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) whereas mutant IDH1 converts α-KG into D2-hydroxyglutarate (D2HG). Unfortunately, there are few in vivo model systems for IDH-mutated tumors to study the effects of IDH1 mutations in tumor development. We have therefore created transgenic zebrafish lines that express various IDH1 mutants.Materials and methodsIDH1 mutations (IDH1R132H, IDH1R132C and loss-of-function mutation IDH1G70D), IDH1wildtype or eGFP were cloned into constructs with several brain-specific promoters (Nestin, Gfap or Gata2). These constructs were injected into fertilized zebrafish eggs at the one-cell stage.ResultsIn total more than ten transgenic zebrafish lines expressing various brain-specific IDH1 mutations were created. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing IDH1wildtype, IDH1G70D or eGFP. No differences in 5-hydroxymethyl cytosine and mature collagen IV levels were observed between wildtype and mutant IDH1 transgenic fish. To our surprise, we failed to identify any strong phenotype, despite increased levels of the oncometabolite D2HG. No tumors were observed, even when backcrossing with tp53-mutant fish which suggests that additional transforming events are required for tumor formation. Elevated D2HG levels could be lowered by treatment of the transgenic zebrafish with an inhibitor of mutant IDH1 activity.ConclusionsWe have generated a transgenic zebrafish model system for mutations in IDH1 that can be used for functional analysis and drug screening. Our model systems help understand the biology of IDH1 mutations and its role in tumor formation.

Abstract 2508: Profiling the metabolic effects of AGI-5198 treatment on IDH1-mutated gliomas

Abstract Background: AGI-5198 (a specific IDH1mut inhibitor) inhibits the formation of D-2-hydroxyglutarate (D-2HG) typically produced in millimolar quantities by the R132H mutation of isocitrate dehydrogenase I (IDH1). AGI-5198 was found to delay growth, to promote differentiation in cells and to decrease tumor volume in IDH1-mutant (IDH1mut) mice xenographs, and as a result, has been advanced to clinical trials in humans with IDH1mut glioblastomas. However, no metabolic study of this drug has been reported to date. Here, we investigated the metabolic changes induced by AGI-5198 treatment in order to determine if the drug restores the normal metabolic status once D-2HG concentration is decreased. Methods: We treated glioma cells carrying an IDH1 mutation with 10 μM AGI-5198 for 72 h. Metabolic changes were explored using an untargeted metabolomics approach. Cell viability analysis verified that the concentration of drug does not lead to significant cell death. NMR and MS-linked metabolomics together with in vivo cell imaging using RAMAN were used. Principal component analysis was employed to explore the differences in the metabolic profiles comparing treated and untreated glioma cells. Results and Conclusions: We confirmed that AGI5198 treatment reduced D-2HG levels in the cell lines investigated, validating previous reports. Moreover, we observed a decrease in phospholipid levels upon AGI-5198 treatment, which suggests a decreased proliferative potential for these cells; of particular importance for clinical application. Our preliminary analysis also suggests that metabolites involved in one carbon metabolism are altered upon AGI-5198 treatment. This pathway provides the methyl group for the DNA methylation by DNA methylases producing a hypermethylated phenotype. Our findings suggest that combining the AGI-5198 treatment with a DNA methylase inhibitor may improve outcomes in IDH1-mutated gliomas. Citation Format: Victor Ruiz Rodado, Adrian Lita, Tyrone Dowdy, Mark R. Gilbert, Mioara Larion. Profiling the metabolic effects of AGI-5198 treatment on IDH1-mutated gliomas [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 2508. doi:10.1158/1538-7445.AM2017-2508

Colonic inflammation and cancer are driven by elevated D‐2‐hydroxyglutarate levels in an EglN1‐dependent mechanism

Emerging evidence suggests a crucial role for altered expression of enzymes with functions in cellular metabolism in the pathogenesis of inflammation and cancer. But the detailed molecular mechanisms that link these alterations to carcinogenesis remain elusive. Here, we identify a novel role for altered metabolism of D‐2‐hydroxyglutarate (D2HG) in colitis and colitis‐associated colon cancer. We found that the levels of D2HG were elevated in the urine of mice during dextran sodium sulfate (DSS)‐induced colitis. In a mouse model of colitis‐associated cancer, intraperitoneal injection of D2HG resulted in delayed recovery from colitis and severe tumorigenesis. The colonic expression of D‐2‐hydroxyglutarate dehydrogenase (D2HGDH), an enzyme that eliminates D2HG, was decreased during DSS colitis and human ulcerative colitis (UC) but unaltered in mouse or UC tumors. Mechanistically, we demonstrate that elevated expression of Egg‐laying defective Nine 1 (EglN1) inhibited hypoxia inducible factor (Hif)‐1a, which in turn attenuated D2HGDH transcription, thereby resulting in elevated D2HG. Furthermore, pharmacological inhibition of EglN1 ameliorated colitis‐associated cancer in mice. Thus, we have identified a novel regulatory mechanism where D2HG promotes colonic inflammation and carcinogenesis, which may provide functional therapeutic targets to inhibit the progression from colitis to cancer.Support or Funding InformationCancer Prevention & Research Institute of Texas grant RP150638 (A.L.T) and Charles A. Sammons Cancer Center, Baylor University Medical Center grant (A.L.T).

Selective Detection of the D-enantiomer of 2-Hydroxyglutarate in the CSF of Glioma Patients with Mutated Isocitrate Dehydrogenase.

Elevation in D-2-Hydroxyglutarate (D-2HG) has recently emerged as a mandatory byproduct of mutated Isocitrate Dehydrogenase (IDH) genes 1 and 2 in glioma patients. The goal of the present study was to demonstrate the feasibility of detection of elevated levels of D-2HG in the cerebrospinal fluid (CSF) of glioma patients that carry point substitutions in the IDH gene. We developed a mass spectrometry (MS)-based platform to detect and quantify the D- and L-forms of 2HG in the CSF of glioma patients. Three independent cohorts of patients were analyzed, comprising a total of 176 samples derived from 84 patients. The levels of D- and L-2HG were used to stratify patients into IDH wild-type or IDH-mutated groups using an empirically obtained threshold of 0.69 μmol/L. Using this platform, a greater than 17-fold mean increase in D-2HG was observed in the CSF of patients with IDH mutant versus wild-type gliomas. The means for the D-2HG levels in CSF were 0.427 μmol/L in wild-type and 7.439 μmol/L in mutant groups. The C statistic for the receiver operator curve was 0.938, with 84% sensitivity, 90% specificity, and 89% accuracy to detect D-2HG. The levels of D- and L-2HG in CSF from wild-type patients varied by location of CSF draw (cisternal > ventricular > lumbar). Our findings demonstrate that the CSF of patients harboring IDH mutant gliomas contain increased levels of D-2HG, which can be reliably detected with a MS-based platform. Clin Cancer Res; 22(24); 6256-65. ©2016 AACR.

Highlights of This Issue

BRAF and MEK inhibitors have improved the clinical management of BRAF-mutant melanoma; however, patients often develop drug resistance. MITF is a transcription factor required for melanoma formation. Rose and colleagues demonstrate that clinically approved MAPK inhibitors cause the MITF-dependent upregulation of GPNMB. GPNMB is the target of an antibody-drug conjugate (CDX-011) currently in clinical trials. Combining BRAF and/or MEK inhibitors with CDX-011 enhanced melanoma tumor regression and delayed emergence of acquired resistance when compared with treatment with MAPK pathway inhibitors alone. These findings support the initiation of clinical trials to assess the efficacy of MAPK pathway inhibitors combined with CDX-011 for the treatment of melanoma.Osswald and colleagues develop an in vivo two-photon microscopy approach to study blood brain barrier (BBB) permeability properties, tumor growth kinetics, and response to PI3K/mTOR inhibitors in metastatic melanoma of the brain. A BBB permeable inhibitor, but not an impermeable inhibitor, reduced the growth rate of impermeable metastases. These results provide evidence that nonpermeable brain micro- and macrometastases can effectively be targeted by drugs designed to cross the BBB.Patient-derived xenograft (PDX) models have been proved to be effective in prioritizing therapeutic regimens in personalized cancer medicine; however, establishing and testing transplantable tumorgrafts are expensive and time-consuming. In this study, Roife and colleagues developed an ex vivo drug efficacy testing method, Live Tissue Sensitivity Assay (LTSA), for patients with pancreatic ductal adenocarcionma. The test enables efficient testing of PDX tumor in 96-well plates against standard of care chemotherapeutic agents in 2 to 3 days. This system is highly reflective of patients' responses to conventional therapeutic agents and has significant potential for guiding personalized cancer treatment design.There is a pressing need for biomarkers to assist with the clinical management of brain tumor patients. The majority of WHO grade II–III gliomas express mutant forms of Isocitrate Dehydrogenases (IDH) 1 and 2, resulting in excessive production of D-2-Hydroxyglutarate (D-2HG) in the tumor cells. Kalinina and colleagues examined whether this oncometabolite is shed into the cerebrospinal fluid (CSF) and if it can potentially serve as a biomarker. Using mass spectrometry the authors demonstrated high levels of D-2HG in the CSF of IDH1-mutant glioma patients, suggesting that this assay may be developed into a clinical test for diagnosis, prognosis, and follow-up of glioma patients.

Oncometabolite D-2-Hydroxyglurate Directly Induces Epithelial-Mesenchymal Transition and is Associated with Distant Metastasis in Colorectal Cancer.

Deranged metabolism is a hallmark of cancer, playing a significant role in driving the disease process. One such example is the induction of carcinogenesis by the oncometabolite D-2 hydroxyglutarate (D-2HG), which is produced by the mutated enzyme isocitrate dehydrogenase (IDH) occurring in subsets of leukaemias and brain tumours. The oncogenic property of D-2HG appears to stem from its ability to interfere with the activities of α-ketoglutarate-dependent dioxygenases, including the Jumonji family histone demethylases. Here, we find in colorectal cancer cells that even in the absence of IDH mutation, the levels of D-2HG and its enantiomer L-2HG were elevated through glutamine anaplerosis. D-2HG, but not L-2HG, increased the trimethylation of histone H3 lysine 4 of the promoter region of ZEB1, a master regulator of epithelial-mesenchymal transition (EMT), and increased the expression of the ZEB1 gene to directly induce EMT in colorectal cancer cells. EMT promotes the ability of cancer cells to invade the local tissue and enter into the bloodstream, leading to distant organ metastasis. D-2HG levels were elevated in colorectal cancer specimens, particularly in those associated with distant metastasis, supporting the observations in vitro and implicating the contribution of D-2HG in metastasis, the major cause of death in this disease.

IDH1 and IDH2 mutations as novel therapeutic targets: current perspectives.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are key metabolic enzymes that convert isocitrate to α-ketoglutarate. IDH1/2 mutations define distinct subsets of cancers, including low-grade gliomas and secondary glioblastomas, chondrosarcomas, intrahepatic cholangiocarcinomas, and hematologic malignancies. Somatic point mutations in IDH1/2 confer a gain-of-function in cancer cells, resulting in the accumulation and secretion in vast excess of an oncometabolite, the D-2-hydroxyglutarate (D-2HG). Overproduction of D-2HG interferes with cellular metabolism and epigenetic regulation, contributing to oncogenesis. Indeed, high levels of D-2HG inhibit α-ketoglutarate-dependent dioxygenases, including histone and DNA demethylases, leading to histone and DNA hypermethylation and finally a block in cell differentiation. Furthermore, D-2HG is a biomarker suitable for the detection of IDH1/2 mutations at diagnosis and predictive of the clinical response. Finally, mutant-IDH1/2 enzymes inhibitors have entered clinical trials for patients with IDH1/2 mutations and represent a novel drug class for targeted therapy.

Abstract 4170: A transgenic zebrafish model for gliomas with mutations in isocitrate dehydrogenase 1

Abstract The gene encoding Isocitrate dehydrogenase 1 (IDH1) is frequently mutated in gliomas, chondrosarcomas, acute myeloid leukemia and intrahepatic cholangiocarcinomas. As there are few in-vivo model systems for IDH-mutated tumors we have created a transgenic zebrafish (Danio rerio) model expressing mutant IDH1. We have chosen the zebrafish as a model because they are transparent (allowing monitoring of the transgene in-vivo) and drug screening assays are straightforward (they are simply added to the aquarium). IDH1R132H and IDH1R132C, mutations found in tumors that both produce D-2-hydroxyglutarate (D2HG) instead of alpha ketoglutarate, were cloned into an expression construct that is driven either by the Nestin or GFAP promoter. IDH1G70D (a loss of function mutation), IDH1wildtype and GFP were used as control. All IDH1 constructs were fused to GFP for visualization. These constructs were injected into fertilized zebrafish eggs at the one-cell stage. All of our transgenic zebrafish lines remain healthy and produce offspring. Transgene expression was detected in the mid/hindbrain of the central nervous system by immunohistochemistry, Western blot and RT-QPCR. A significant increase in the level of D2HG was observed in all transgenic lines expressing IDH1R132C or IDH1R132H, but not in any of the lines expressing control constructs (IDH1wildtype, IDH1G70D or GFP). In contrast to reported, we failed to detect any differences in hydroxymethyl cytosine (the first step in DNA-demethylation) and mature collagen IV levels between wildtype and mutant IDH1 transgenic fish. We also performed microinjections on fertilized eggs to screen for early developmental effects of IDH1R132H and IDH1R132C. Despite of the high expression of the transgene, no developmental effects were found. Our observations therefore suggest that elevated levels of D2HG are insufficient to initiate tumorigenesis or other phenotypic effects in our fish. Treatment of the transgenic zebrafish with an IDH1 mutant inhibitor, AGI-5198, resulted in a reduction in the D2HG level in the mutant zebrafish. The L2HG level was not affected by AGI-5198. As no tumors were formed in our transgenic zebrafish lines, we backcrossed them with Tp53 mutant fish. Analysis of these lines is currently being performed. In summary, we have generated a transgenic zebrafish model system that expresses mutated IDH1 that can be used to study effects of mutant IDH1 (or elevated levels of D2HG) in vivo and can be used for drug screening. Citation Format: Ya D. Gao, Maurice de Wit, Eduard A. Struys, Martine L.M. Lamfers, Gajja S. Salomons, Peter A.E. Sillevis Smitt, Pim J. French. A transgenic zebrafish model for gliomas with mutations in isocitrate dehydrogenase 1. [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 4170.

Saccharomyces cerevisiae Forms d-2-Hydroxyglutarate and Couples Its Degradation to d-Lactate Formation via a Cytosolic Transhydrogenase

The D or L form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders, and high D-2-hydroxyglutarate (D-2HG) levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here, we show that S. cerevisiae actively forms the D enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human D-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3Δ knock-out strain accumulates millimolar levels of D-2HG, whereas a dld2Δ knock-out strain displayed only very moderate increases in D-2HG. Recombinant Dld2 and Dld3, both currently annotated as D-lactate dehydrogenases, efficiently oxidized D-2HG to α-ketoglutarate. Depletion of D-lactate levels in the dld3Δ, but not in the dld2Δ mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert D-2HG to α-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce α-ketoglutarate to D-2HG using NADH and represent major intracellular sources of D-2HG in yeast. Based on our observations, we propose that D-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples D-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the D-lactate dehydrogenase Dld1.