Rapid detection of IDH mutations in gliomas by intraoperative mass spectrometry.

The development and performance of two mass spectrometry (MS) workflows for the intraoperative diagnosis of isocitrate dehydrogenase (IDH) mutations in glioma is implemented by independent teams at Mayo Clinic, Jacksonville, and Huashan Hospital, Shanghai. The infiltrative nature of gliomas makes rapid diagnosis necessary to guide the extent of surgical resection of central nervous system (CNS) tumors. The combination of tissue biopsy and MS analysis used here satisfies this requirement. The key feature of both described methods is the use of tandem MS to measure the oncometabolite 2-hydroxyglutarate (2HG) relative to endogenous glutamate (Glu) to characterize the presence of mutant tumor. The experiments i) provide IDH mutation status for individual patients and ii) demonstrate a strong correlation of 2HG signals with tumor infiltration. The measured ratio of 2HG to Glu correlates with IDH-mutant (IDH-mut) glioma (P < 0.0001) in the tumor core data of both teams. Despite using different ionization methods and different mass spectrometers, comparable performance in determining IDH mutations from core tumor biopsies was achieved with sensitivities, specificities, and accuracies all at 100%. None of the 31 patients at Mayo Clinic or the 74 patients at Huashan Hospital were misclassified when analyzing tumor core biopsies. Robustness of the methodology was evaluated by postoperative re-examination of samples. Both teams noted the presence of high concentrations of 2HG at surgical margins, supporting future use of intraoperative MS to monitor for clean surgical margins. The power of MS diagnostics is shown in resolving contradictory clinical features, e.g., in distinguishing gliosis from IDH-mut glioma.

IDH1 mutation produces R-2-hydroxyglutarate (R-2HG) and induces mir-182-5p expression to regulate cell cycle and tumor formation in glioma.

BACKGROUND: Mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2), are present in most gliomas. IDH1 mutation is an important prognostic marker in glioma. However, its regulatory mechanism in glioma remains incompletely understood. RESULTS: miR-182-5p expression was increased within IDH1-mutant glioma specimens according to TCGA, CGGA, and online dataset GSE119740, as well as collected clinical samples. (R)-2-hydroxyglutarate ((R)-2HG) treatment up-regulated the expression of miR-182-5p, enhanced glioma cell proliferation, and suppressed apoptosis; miR-182-5p inhibition partially eliminated the oncogenic effects of R-2HG upon glioma cells. By direct binding to Cyclin Dependent Kinase Inhibitor 2 C (CDKN2C) 3'UTR, miR-182-5p inhibited CDKN2C expression. Regarding cellular functions, CDKN2C knockdown promoted R-2HG-treated glioma cell viability, suppressed apoptosis, and relieved cell cycle arrest. Furthermore, CDKN2C knockdown partially attenuated the effects of miR-182-5p inhibition on cell phenotypes. Moreover, CDKN2C knockdown exerted opposite effects on cell cycle check point and apoptosis markers to those of miR-182-5p inhibition; also, CDKN2C knockdown partially attenuated the functions of miR-182-5p inhibition in cell cycle check point and apoptosis markers. The engineered CS-NPs (antagomir-182-5p) effectively encapsulated and delivered antagomir-182-5p, enhancing anti-tumor efficacy in vivo, indicating the therapeutic potential of CS-NPs(antagomir-182-5p) in targeting the miR-182-5p/CDKN2C axis against R-2HG-driven oncogenesis in mice models. CONCLUSIONS: These insights highlight the potential of CS-NPs(antagomir-182-5p) to target the miR-182-5p/CDKN2C axis, offering a promising therapeutic avenue against R-2HG's oncogenic influence to glioma.

[Research Progress of Isocitrate Dehydrogenase Mutation-Positive Acute Myeloid Leukemia --Review].

Isocitrate dehydrogenase (IDH) is an enzymes involved in a variety of metabolic and epigenetic processes. IDH can be detected in approximately 20% of patients with acute myeloid leukemia (AML), the mutated IDH enzyme acquires new oncogenic enzyme activity and converts α-ketoglutaric acid (α-KG) to the tumor metabolite 2-hydroxyglutaric acid (2-HG), which accumulates at high levels in cells and hinders the function of αKG-dependent enzymes, including epigenetic regulators, resulting in DNA hypermethylation, abnormal gene expression, cell proliferation, and abnormal differentiation, and contributes to leukemia disease progression. IDH mutations have different effects on the prognosis of patients with AML depending on the location of the mutation and other co-occurring genomic abnormalities. This paper will review the latest research progress on the IDH positive AML gene changes, prognosis, and inhibitors.

Molecular biology and novel therapeutics for IDH mutant gliomas: The new era of IDH inhibitors.

Gliomas with Isocitrate dehydrogenase (IDH) mutation represent a discrete category of primary brain tumors with distinct and unique characteristics, behaviors, and clinical disease outcomes. IDH mutations lead to aberrant high-level production of the oncometabolite D-2-hydroxyglutarate (D-2HG), which act as a competitive inhibitor of enzymes regulating epigenetics, signaling pathways, metabolism, and various other processes. This review summarizes the significance of IDH mutations, resulting upregulation of D-2HG and the associated molecular pathways in gliomagenesis. With the recent finding of clinically effective IDH inhibitors in these gliomas, this article offers a comprehensive overview of the new era of innovative therapeutic approaches based on mechanistic rationales, encompassing both completed and ongoing clinical trials targeting gliomas with IDH mutations.

Isocitrate Dehydrogenase Mutations in Cancer: Mechanisms of Transformation and Metabolic Liability.

Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are metabolic enzymes that interconvert isocitrate and 2-oxoglutarate (2OG). Gain-of-function mutations in IDH1 and IDH2 occur in a number of cancers, including acute myeloid leukemia, glioma, cholangiocarcinoma, and chondrosarcoma. These mutations cripple the wild-type activity of IDH and cause the enzymes to catalyze a partial reverse reaction in which 2OG is reduced but not carboxylated, resulting in production of the (R)-enantiomer of 2-hydroxyglutarate ((R)-2HG). (R)-2HG accumulation in IDH-mutant tumors results in profound dysregulation of cellular metabolism. The most well-characterized oncogenic effects of (R)-2HG involve the dysregulation of 2OG-dependent epigenetic tumor-suppressor enzymes. However, (R)-2HG has many other effects in IDH-mutant cells, some that promote transformation and others that induce metabolic dependencies. Herein, we review how cancer-associated IDH mutations impact epigenetic regulation and cellular metabolism and discuss how these effects can potentially be leveraged to therapeutically target IDH-mutant tumors.

The two enantiomers of 2-hydroxyglutarate differentially regulate cytotoxic T cell function.

2-Hydroxyglutarate (2HG) is a byproduct of the tricarboxylic acid (TCA) cycle and is readily detected in the tissues of healthy individuals. 2HG is found in two enantiomeric forms: S-2HG and R-2HG. Here, we investigate the differential roles of these two enantiomers in cluster of differentiation (CD)8+ T cell biology, where we find they have highly divergent effects on proliferation, differentiation, and T cell function. We show here an analysis of structural determinants that likely underlie these differential effects on specific α-ketoglutarate (αKG)-dependent enzymes. Treatment of CD8+ T cells with exogenous S-2HG, but not R-2HG, increased CD8+ T cell fitness in vivo and enhanced anti-tumor activity. These data show that S-2HG and R-2HG should be considered as two distinct and important actors in the regulation of T cell function.

Glutarate regulates T cell metabolism and anti-tumour immunity.

T cell function and fate can be influenced by several metabolites: in some cases, acting through enzymatic inhibition of α-ketoglutarate-dependent dioxygenases, in others, through post-translational modification of lysines in important targets. We show here that glutarate, a product of amino acid catabolism, has the capacity to do both, and has potent effects on T cell function and differentiation. We found that glutarate exerts those effects both through α-ketoglutarate-dependent dioxygenase inhibition, and through direct regulation of T cell metabolism via glutarylation of the pyruvate dehydrogenase E2 subunit. Administration of diethyl glutarate, a cell-permeable form of glutarate, alters CD8+ T cell differentiation and increases cytotoxicity against target cells. In vivo administration of the compound is correlated with increased levels of both peripheral and intratumoural cytotoxic CD8+ T cells. These results demonstrate that glutarate is an important regulator of T cell metabolism and differentiation with a potential role in the improvement of T cell immunotherapy.

Exceptionally rare IDH1-mutant adult medulloblastoma with concurrent GNAS mutation revealed by in vivo magnetic resonance spectroscopy and deep sequencing.

Medulloblastoma (MB) is the most common malignant brain tumor occurring in childhood and rarely found in adults. Based on transcriptome profile, MB are currently classified into four major molecular groups reflecting a considerable biological heterogeneity: WNT-activated, SHH-activated, group 3 and group 4. Recently, DNA methylation profiling allowed the identification of additional subgroups within the four major molecular groups associated with different clinic-pathological and molecular features. Isocitrate dehydrogenase-1 and 2 (IDH1 and IDH2) mutations have been described in several tumors, including gliomas, while in MB are rarely reported and not routinely investigated. By means of magnetic resonance spectroscopy (MRS), we unequivocally assessed the presence the oncometabolite D-2-hydroxyglutarate (2HG), a marker of IDH1 and IDH2 mutations, in a case of adult MB. Immunophenotypical work-up and methylation profiling assigned the diagnosis of MB, subclass SHH-A, and molecular testing revealed the presence of the non-canonical somatic IDH1(p.R132C) mutation and an additional GNAS mutation, also rarely described in MB. To the best of our knowledge, this is the first reported case of MB simultaneously harboring both mutations. Of note, tumor exhibited a heterogeneous phenotype with a tumor component displaying glial differentiation, with robust GFAP expression, and a component with conventional MB features and selective presence of GNAS mutation, suggesting co-existence of two different major tumor subclones. These findings drew attention to the need for a deeper genetic characterization of MB, in order to get insights into their biology and improve stratification and clinical management of the patients. Moreover, our results underlined the importance of performing MRS for the identification of IDH mutations in non-glial tumors. The use of throughput molecular profiling analysis and advanced medical imaging will certainly increase the frequency with which tumor entities with rare molecular alterations will be identified. Whether these findings have any specific therapeutic implications or prognostic relevance requires further investigations.

Recommendations for diagnosing and managing individuals with glutaric aciduria type 1: Third revision.

Glutaric aciduria type 1 is a rare inherited neurometabolic disorder of lysine metabolism caused by pathogenic gene variations in GCDH (cytogenic location: 19p13.13), resulting in deficiency of mitochondrial glutaryl-CoA dehydrogenase (GCDH) and, consequently, accumulation of glutaric acid, 3-hydroxyglutaric acid, glutaconic acid and glutarylcarnitine detectable by gas chromatography/mass spectrometry (organic acids) and tandem mass spectrometry (acylcarnitines). Depending on residual GCDH activity, biochemical high and low excreting phenotypes have been defined. Most untreated individuals present with acute onset of striatal damage before age 3 (to 6) years, precipitated by infectious diseases, fever or surgery, resulting in irreversible, mostly dystonic movement disorder with limited life expectancy. In some patients, striatal damage develops insidiously. In recent years, the clinical phenotype has been extended by the finding of extrastriatal abnormalities and cognitive dysfunction, preferably in the high excreter group, as well as chronic kidney failure. Newborn screening is the prerequisite for pre-symptomatic start of metabolic treatment with low lysine diet, carnitine supplementation and intensified emergency treatment during catabolic episodes, which, in combination, have substantially improved neurologic outcome. In contrast, start of treatment after onset of symptoms cannot reverse existing motor dysfunction caused by striatal damage. Dietary treatment can be relaxed after the vulnerable period for striatal damage, that is, age 6 years. However, impact of dietary relaxation on long-term outcomes is still unclear. This third revision of evidence-based recommendations aims to re-evaluate previous recommendations (Boy et al., J Inherit Metab Dis, 2017;40(1):75-101; Kolker et al., J Inherit Metab Dis 2011;34(3):677-694; Kolker et al., J Inherit Metab Dis, 2007;30(1):5-22) and to implement new research findings on the evolving phenotypic diversity as well as the impact of non-interventional variables and treatment quality on clinical outcomes.

Dysregulated Lipid Synthesis by Oncogenic IDH1 Mutation Is a Targetable Synthetic Lethal Vulnerability.

Isocitrate dehydrogenase 1 and 2 (IDH) are mutated in multiple cancers and drive production of (R)-2-hydroxyglutarate (2HG). We identified a lipid synthesis enzyme [acetyl CoA carboxylase 1 (ACC1)] as a synthetic lethal target in mutant IDH1 (mIDH1), but not mIDH2, cancers. Here, we analyzed the metabolome of primary acute myeloid leukemia (AML) blasts and identified an mIDH1-specific reduction in fatty acids. mIDH1 also induced a switch to b-oxidation indicating reprogramming of metabolism toward a reliance on fatty acids. Compared with mIDH2, mIDH1 AML displayed depletion of NADPH with defective reductive carboxylation that was not rescued by the mIDH1-specific inhibitor ivosidenib. In xenograft models, a lipid-free diet markedly slowed the growth of mIDH1 AML, but not healthy CD34+ hematopoietic stem/progenitor cells or mIDH2 AML. Genetic and pharmacologic targeting of ACC1 resulted in the growth inhibition of mIDH1 cancers not reversible by ivosidenib. Critically, the pharmacologic targeting of ACC1 improved the sensitivity of mIDH1 AML to venetoclax. SIGNIFICANCE: Oncogenic mutations in both IDH1 and IDH2 produce 2-hydroxyglutarate and are generally considered equivalent in terms of pathogenesis and targeting. Using comprehensive metabolomic analysis, we demonstrate unexpected metabolic differences in fatty acid metabolism between mutant IDH1 and IDH2 in patient samples with targetable metabolic interventions. See related commentary by Robinson and Levine, p. 266. This article is highlighted in the In This Issue feature, p. 247.

Metabolite-driven antitumor immunity.

An oncometabolite blocks T cell killing by inhibiting glycolysis.

Oncometabolite d-2HG alters T cell metabolism to impair CD8+ T cell function.

Gain-of-function mutations in isocitrate dehydrogenase (IDH) in human cancers result in the production of d-2-hydroxyglutarate (d-2HG), an oncometabolite that promotes tumorigenesis through epigenetic alterations. The cancer cell-intrinsic effects of d-2HG are well understood, but its tumor cell-nonautonomous roles remain poorly explored. We compared the oncometabolite d-2HG with its enantiomer, l-2HG, and found that tumor-derived d-2HG was taken up by CD8+ T cells and altered their metabolism and antitumor functions in an acute and reversible fashion. We identified the glycolytic enzyme lactate dehydrogenase (LDH) as a molecular target of d-2HG. d-2HG and inhibition of LDH drive a metabolic program and immune CD8+ T cell signature marked by decreased cytotoxicity and impaired interferon-γ signaling that was recapitulated in clinical samples from human patients with IDH1 mutant gliomas.

D2HGDH-mediated D2HG catabolism enhances the anti-tumor activities of CAR-T cells in an immunosuppressive microenvironment.

The effect of immunotherapy is limited by oncometabolite D-2-hydroxyglutarate (D2HG). D2HGDH is an inducible enzyme that converts D2HG into the endogenous metabolite 2-oxoglutarate. We aimed to evaluate the impairment of CD8 T lymphocyte function in the high-D2HG environment and to explore the phenotypic features and anti-tumor effect of D2HGDH-modified CAR-T cells. D2HG treatment inhibited the expansion of human CD8 T lymphocytes and CAR-T cells, increased their glucose uptake, suppressed effector cytokine production, and decreased the central memory cell proportion. D2HGDH-modified CAR-T cells displayed distinct phenotypes, as D2HGDH knock-out (KO) CAR-T cells exhibited a significant decrease in central memory cell differentiation and intracellular cytokine production, while D2HGDH over-expression (OE) CAR-T cells showed predominant killing efficacy against NALM6 cancer cells in high-D2HG medium. In vivo xenograft experiments confirmed that D2HGDH-OE CAR-T cells decreased serum D2HG and improved the overall survival of mice bearing NALM6 cancer cells with mutation IDH1. Our findings demonstrated that the immunosuppressive effect of D2HG and distinct phenotype of D2HGDH modified CAR-T cells. D2HGDH-OE CAR-T cells can take advantage of the catabolism of D2HG to foster T cell expansion, function, and anti-tumor effectiveness.

Diagnostic performance of edited 2HG MR spectroscopy of central glioma in the clinical environment.

OBJECTIVE: Oncometabolite D-2-hydroxyglutarate (2HG) is pooled in isocitrate dehydrogenase (IDH)-mutant glioma cells. Detecting 2HG by MR spectroscopy (MRS) has been proven viable in the last decade but has not entirely found its way into the clinical routine. This study aimed to explore the adoption of 2HG MRS while acknowledging factors that influence its performance in the clinical environment. METHODS: Thirty-nine MR spectra were acquired and reported prospectively in patients with suspected glioma using a 3 T system with Mescher-Garwood point-resolved spectroscopy (MEGA-PRESS) sequence utilizing averaged free induction decay (FID) signals. Postprocessing and evaluation of spectra were performed with jMRUI and LCModel. 2HG concentration estimates, 2HG/Cr ratio, together with quality measures, including Cramér-Rao lower bounds (CRLBs), full-width at half-maximum (FWHM) values, and signal-to-noise ratio (SNR) were calculated using LCModel. Immunohistochemistry and genomic analysis results used as a ground truth were available for 15 patients. RESULTS: The threshold for test positivity was set according to the ROC curve at 1 mM. Calculated sensitivity was 57.14% (95% CI 0.20-0.88), specificity 87.5% (95% CI 0.46-0.99), positive predictive value 80%, and negative predictive value 70%. Overall diagnostic accuracy was 73.33% (95% CI 0.45-0.92). The 2HG/Cr ratio with the cutoff value 0.085 significantly improved sensitivity and overall diagnostic accuracy [85.71%, 95% CI 0.42-1.00 and 86.67%, (95% CI 0.60-0.98), respectively]. CONCLUSION: Multiple factors compromising spectral quality in the clinical adoption of edited 2HG MRS resulted in diminished sensitivity but clinically acceptable specificity. Furthermore, the 2HG/Cr ratio performs better than the sole 2HG concentration estimate in the pre-operative setting.

2-Hydroxyglutarate destabilizes chromatin regulatory landscape and lineage fidelity to promote cellular heterogeneity.

The epigenome delineates lineage-specific transcriptional programs and restricts cell plasticity to prevent non-physiological cell fate transitions. Although cell diversification fosters tumor evolution and therapy resistance, upstream mechanisms that regulate the stability and plasticity of the cancer epigenome remain elusive. Here we show that 2-hydroxyglutarate (2HG) not only suppresses DNA repair but also mediates the high-plasticity chromatin landscape. A combination of single-cell epigenomics and multi-omics approaches demonstrates that 2HG disarranges otherwise well-preserved stable nucleosome positioning and promotes cell-to-cell variability. 2HG induces loss of motif accessibility to the luminal-defining transcriptional factors FOXA1, FOXP1, and GATA3 and a shift from luminal to basal-like gene expression. Breast tumors with high 2HG exhibit enhanced heterogeneity with undifferentiated epigenomic signatures linked to adverse prognosis. Further, ascorbate-2-phosphate (A2P) eradicates heterogeneity and impairs growth of high 2HG-producing breast cancer cells. These findings suggest 2HG as a key determinant of cancer plasticity and provide a rational strategy to counteract tumor cell evolution.

Beyond Isocitrate Dehydrogenase Mutations: Emerging Mechanisms for the Accumulation of the Oncometabolite 2-Hydroxyglutarate.

2-Hydroxyglutarate (2-HG) is an unconventional oncometabolite of α-ketoglutarate. Isocitrate dehydrogenase mutation is generally acknowledged to be the main cause of 2-HG accumulation. In isocitrate dehydrogenase mutant tumors, 2-HG accumulation inhibits α-ketoglutarate/Fe(II)-dependent dioxygenases, resulting in epigenetic alterations. Recently, the increase of 2-HG has also been observed in the cases of mitochondrial dysfunction and hypoxia. In these cases, 2-HG not only inhibits α-ketoglutarate/Fe(II)-dependent dioxygenases to regulate epigenetics but also affects other cellular pathways, such as regulating hypoxia-inducible transcription factors and glycolysis. These provide a new perspective for the study of 2-HG.

Signaling metabolite L-2-hydroxyglutarate activates the transcription factor HIF-1α in lipopolysaccharide-activated macrophages.

Activated macrophages undergo metabolic reprogramming, which not only supports their energetic demands but also allows for the production of specific metabolites that function as signaling molecules. Several Krebs cycles, or Krebs-cycle-derived metabolites, including succinate, α-ketoglutarate, and itaconate, have recently been shown to modulate macrophage function. The accumulation of 2-hydroxyglutarate (2HG) has also been well documented in transformed cells and more recently shown to play a role in T cell and dendritic cell function. Here we have found that the abundance of both enantiomers of 2HG is increased in LPS-activated macrophages. We show that L-2HG, but not D-2HG, can promote the expression of the proinflammatory cytokine IL-1ß and the adoption of an inflammatory, highly glycolytic metabolic state. These changes are likely mediated through activation of the transcription factor hypoxia-inducible factor-1α (HIF-1α) by L-2HG, a known inhibitor of the HIF prolyl hydroxylases. Expression of the enzyme responsible for L-2HG degradation, L-2HG dehydrogenase (L-2HGDH), was also found to be decreased in LPS-stimulated macrophages and may therefore also contribute to L-2HG accumulation. Finally, overexpression of L-2HGDH in HEK293 TLR4/MD2/CD14 cells inhibited HIF-1α activation by LPS, while knockdown of L-2HGDH in macrophages boosted the induction of HIF-1α-dependent genes, as well as increasing LPS-induced HIF-1α activity. Taken together, this study therefore identifies L-2HG as a metabolite that can regulate HIF-1α in macrophages.

A D-2-hydroxyglutarate biosensor based on specific transcriptional regulator DhdR.

D-2-Hydroxyglutarate (D-2-HG) is a metabolite involved in many physiological metabolic processes. When D-2-HG is aberrantly accumulated due to mutations in isocitrate dehydrogenase or D-2-HG dehydrogenase, it functions in a pro-oncogenic manner and is thus considered a therapeutic target and biomarker in many cancers. In this study, DhdR from Achromobacter denitrificans NBRC 15125 is identified as an allosteric transcriptional factor that negatively regulates D-2-HG dehydrogenase expression and responds to the presence of D-2-HG. Based on the allosteric effect of DhdR, a D-2-HG biosensor is developed by combining DhdR with amplified luminescent proximity homogeneous assay (AlphaScreen) technology. The biosensor is able to detect D-2-HG in serum, urine, and cell culture medium with high specificity and sensitivity. Additionally, this biosensor is used to identify the role of D-2-HG metabolism in lipopolysaccharide biosynthesis of Pseudomonas aeruginosa, demonstrating its broad usages.

Identification of Isocitrate Dehydrogenase 2 (IDH2) Mutation in Carotid Body Paraganglioma.

Carotid body paragangliomas (PGLs) are rare neuroendocrine tumors that develop within the adventitia of the medial aspect of the carotid bifurcation. Carotid body PGLs comprise about 65% of head and neck paragangliomas, however, their genetic background remains elusive. In the present study, we report one case of carotid body PGL with a somatic mutation in the gene encoding isocitrate dehydrogenase 2 (IDH2). The missense mutation in IDH2 resulted in R172G amino acid substitution, which exhibits neomorphic activity and production of D-2-hydroxyglutarate.

Cancer-associated IDH mutations induce Glut1 expression and glucose metabolic disorders through a PI3K/Akt/mTORC1-Hif1α axis.

Isocitrate dehydrogenase 1 and 2 (IDH1/2) mutations and their key effector 2-hydroxyglutarate (2-HG) have been reported to promote oncogenesis in various human cancers. To elucidate molecular mechanism(s) associated with IDH1/2 mutations, we established mouse embryonic fibroblasts (MEF) cells and human colorectal cancer cells stably expressing cancer-associated IDH1R132C or IDH2R172S, and analyzed the change in metabolic characteristics of the these cells. We found that IDH1/2 mutants induced intracellular 2-HG accumulation and inhibited cell proliferation. Expression profile analysis by RNA-seq unveiled that glucose transporter 1 (Glut1) was induced by the IDH1/2 mutants or treatment with 2-HG in the MEF cells. Consistently, glucose uptake and lactate production were increased by the mutants, suggesting the deregulation of glucose metabolism. Furthermore, PI3K/Akt/mTOR pathway and Hif1α expression were involved in the up-regulation of Glut1. Together, these results suggest that Glut1 is a potential target regulated by cancer-associated IDH1/2 mutations.