RIPK3 deficiency blocks R-2-hydroxyglutarate-induced necroptosis in IDH-mutated AML cells.

Mutant isocitrate dehydrogenases (IDHs) produce R-2-hydroxyglutarate (R-2HG), which inhibits the growth of most acute myeloid leukemia (AML) cells. Here, we showed that necroptosis, a form of programmed cell death, contributed to the antileukemia activity of R-2HG. Mechanistically, R-2HG competitively inhibited the activity of lysine demethylase 2B (KDM2B), an α-ketoglutarate-dependent dioxygenase. KDM2B inhibition increased histone 3 lysine 4 trimethylation levels and promoted the expression of receptor-interacting protein kinase 1 (RIPK1), which consequently caused necroptosis in AML cells. The expression of RIPK3 was silenced because of DNA methylation in IDH-mutant (mIDH) AML cells, resulting in R-2HG resistance. Decitabine up-regulated RIPK3 expression and repaired endogenous R-2HG-induced necroptosis pathway in mIDH AML cells. Together, R-2HG induced RIPK1-dependent necroptosis via KDM2B inhibition in AML cells. The loss of RIPK3 protected mIDH AML cells from necroptosis. Restoring RIPK3 expression to exert R-2HG's intrinsic antileukemia effect will be a potential therapeutic strategy in patients with AML.

D-2-HG Inhibits IDH1mut Glioma Growth via FTO Inhibition and Resultant m6A Hypermethylation.

IDH1mut gliomas produce high levels of D-2-hydroxyglutarate (D-2-HG), an oncometabolite capable of inhibiting α-ketoglutarate-dependent dioxygenases critical to a range of cellular functions involved in gliomagenesis. IDH1mut gliomas also exhibit slower growth rates and improved treatment sensitivity compared with their IDH1wt counterparts. This study explores the mechanism driving apparent reduced growth in IDH1mut gliomas. Specifically, we investigated the relationship between IDH1mut and the RNA N6-methyladenosine (m6A) demethylases FTO and ALKBH5, and their potential for therapeutic targeting. We investigated the role of D-2-HG and m6A in tumor proliferation/viability using glioma patient tumor samples, patient-derived gliomaspheres, and U87 cells, as well as with mouse intracranial IDH1wt gliomasphere xenografts. Methylation RNA immunoprecipitation sequencing (MeRIP-seq) RNA sequencing was used to identify m6A-enriched transcripts in IDH1mut glioma. We show that IDH1mut production of D-2-HG is capable of reducing glioma cell growth via inhibition of the m6A epitranscriptomic regulator, FTO, with resultant m6A hypermethylation of a set of mRNA transcripts. On the basis of unbiased MeRIP-seq epitranscriptomic profiling, we identify ATF5 as a hypermethylated, downregulated transcript that potentially contributes to increased apoptosis. We further demonstrate how targeting this pathway genetically and pharmacologically reduces the proliferative potential of malignant IDH1wt gliomas, both in vitro and in vivo. Our work provides evidence that selective inhibition of the m6A epitranscriptomic regulator FTO attenuates growth in IDH1wt glioma, recapitulating the clinically favorable growth phenotype seen in the IDH1mut subtype. SIGNIFICANCE: We show that IDH1mut-generated D-2-HG can reduce glioma growth via inhibition of the m6A demethylase, FTO. FTO inhibition represents a potential therapeutic target for IDH1wt gliomas and possibly in conjunction with IDH1mut inhibitors for the treatment of IDH1mut glioma. Future studies are necessary to demonstrate the role of ATF5 downregulation in the indolent phenotype of IDH1mut gliomas, as well as to identify other involved gene transcripts deregulated by m6A hypermethylation.

The diagnostic efficiency of integration of 2HG MRS and IVIM versus individual parameters for predicting IDH mutation status in gliomas in clinical scenarios: A retrospective study.

PURPOSE: Currently, there remains a scarcity of established preoperative tests to accurately predict the isocitrate dehydrogenase (IDH) mutation status in clinical scenarios, with limited research has explored the potential synergistic diagnostic performance among metabolite, perfusion, and diffusion parameters. To address this issue, we aimed to develop an imaging protocol that integrated 2-hydroxyglutarate (2HG) magnetic resonance spectroscopy (MRS) and intravoxel incoherent motion (IVIM) by comprehensively assessing metabolic, cellular, and angiogenic changes caused by IDH mutations, and explored the diagnostic efficiency of this imaging protocol for predicting IDH mutation status in clinical scenarios. METHODS: Patients who met the inclusion criteria were categorized into two groups: IDH-wild type (IDH-WT) group and IDH-mutant (IDH-MT) group. Subsequently, we quantified the 2HG concentration, the relative apparent diffusion coefficient (rADC), the relative true diffusion coefficient value (rD), the relative pseudo-diffusion coefficient (rD*) and the relative perfusion fraction value (rf). Intergroup differences were estimated using t-test and Mann-Whitney U test. Finally, we performed receiver operating characteristic (ROC) curve and DeLong's test to evaluate and compare the diagnostic performance of individual parameters and their combinations. RESULTS: 64 patients (female, 21; male, 43; age, 47.0 ± 13.7 years) were enrolled. Compared with IDH-WT gliomas, IDH-MT gliomas had higher 2HG concentration, rADC and rD (P < 0.001), and lower rD* (P = 0.013). The ROC curve demonstrated that 2HG + rD + rD* exhibited the highest areas under curve (AUC) value (0.967, 95%CI 0.889-0.996) for discriminating IDH mutation status. Compared with each individual parameter, the predictive efficiency of 2HG + rADC + rD* and 2HG + rD + rD* shows a statistically significant enhancement (DeLong's test: P < 0.05). CONCLUSIONS: The integration of 2HG MRS and IVIM significantly improves the diagnostic efficiency for predicting IDH mutation status in clinical scenarios.

Systems engineering of Escherichia coli for high-level glutarate production from glucose.

Glutarate is a key monomer in polyester and polyamide production. The low efficiency of the current biosynthetic pathways hampers its production by microbial cell factories. Herein, through metabolic simulation, a lysine-overproducing E. coli strain Lys5 is engineered, achieving titer, yield, and productivity of 195.9 g/L, 0.67 g/g glucose, and 5.4 g/L·h, respectively. Subsequently, the pathway involving aromatic aldehyde synthase, monoamine oxidase, and aldehyde dehydrogenase (AMA pathway) is introduced into E. coli Lys5 to produce glutarate from glucose. To enhance the pathway's efficiency, rational mutagenesis on the aldehyde dehydrogenase is performed, resulting in the development of variant Mu5 with a 50-fold increase in catalytic efficiency. Finally, a glutarate tolerance gene cbpA is identified and genomically overexpressed to enhance glutarate productivity. With enzyme expression optimization, the glutarate titer, yield, and productivity of E. coli AMA06 reach 88.4 g/L, 0.42 g/g glucose, and 1.8 g/L·h, respectively. These findings hold implications for improving glutarate biosynthesis efficiency in microbial cell factories.

[Clinical and genetic analysis of two children with 3-hydroxy-3-methylglutaryl-CoA lyase deficiency].

OBJECTIVE: To explore the clinical characteristics and genetic variants of two children with 3-hydroxy-3-methylglutaryl-coenzyme A lyase deficiency (HMGCLD). METHODS: Two children with HMGCLD diagnosed at Henan Provincial Children's Hospital respectively in December 2019 and June 2022 were selected as the study subjects. Clinical data and results of laboratory testing were analyzed retrospectively. RESULTS: Both children had manifested with repeated convulsions, severe hypoglycemia, metabolic acidosis and liver dysfunction. Blood amino acids and acylcarnitine analysis showed increased 3-hydroxy-isovalyl carnitine (C5OH) and 3-hydroxy-isovalyl carnitine/capryloyl carnitine ratio (C5OH/C8), and urinary organic acid analysis showed increased 3-hydroxyl-3-methyl glutaric acid, 3-methyl glutaric acid, 3-methyl glutaconic acid, 3-hydroxyisoglycine and 3-methylprotarylglycine. Child 1 was found to harbor homozygous c.722C>T variants of the HMGCL gene, which was rated as uncertain significance (PM2_Supporting+PP3). Child 2 was found to harbor homozygous c.121C>T variants of the HMGCL gene, which was rated as pathogenic variant (PVS1+PM2_Supporting+PP4). CONCLUSION: Acute episode of HMGCLD is usually characterized by metabolic disorders such as hypoglycemia and metabolic acidosis, and elevated organic acids in urine may facilitate the differential diagnosis, though definite diagnosis will rely on genetic testing.

In Situ Monitoring of Competitive Coformer Exchange Reaction by 1H MAS Solid-State NMR.

In a competitive coformer exchange reaction, a recent topic of interest in pharmaceutical research, the coformer in a pharmaceutical cocrystal is exchanged with another coformer that is expected to form a cocrystal that is more stable. There will be a competition between coformers to form the most stable product through the formation of hydrogen bonds. This will cause destabilization of the pharmaceutical products during processing or storage. Therefore, it is important to develop a mechanistic understanding of this transformation by monitoring each and every step of the reaction, employing a technique such as 1H nuclear magnetic resonance (NMR). In this study, an in situ monitoring of a coformer exchange reaction is carried out by 1H magic angle spinning (MAS) solid-state NMR (SSNMR) at a spinning frequency of 60 kHz. The changes in caffeine maleic acid cocrystals on addition of glutaric acid and caffeine glutaric cocrystals on addition of maleic acid were monitored. In all of the reactions, it has been observed that caffeine glutaric acid Form I is formed. When glutaric acid was added to 2:1 caffeine maleic acid, the formation of metastable 1:1 caffeine glutaric acid Form I was observed at the start of the experiment, indicating that the centrifugal pressure is enough for the formation. The difference in the end product of the reactions with a similar reaction pathway of 1:1 and 2:1 reactant stoichiometry indicates that a complete replacement of maleic acid has occurred only in the 1:1 stoichiometry of the reactants. The polymorphic transition of caffeine glutaric acid Form II to Form I at higher temperatures was a crucial reason that triggered the exchange of glutaric acid with maleic acid in the reaction of caffeine glutaric acid and maleic acid. Our results are novel since the new reaction pathways in competitive coformer exchange reactions enabled understanding the remarkable role of stoichiometry, polymorphism, temperature, and centrifugal pressure.

Focused Screening Identifies Different Sensitivities of Human TET Oxygenases to the Oncometabolite 2-Hydroxyglutarate.

Ten-eleven translocation enzymes (TETs) are Fe(II)/2-oxoglutarate (2OG) oxygenases that catalyze the sequential oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine in eukaryotic DNA. Despite their roles in epigenetic regulation, there is a lack of reported TET inhibitors. The extent to which 2OG oxygenase inhibitors, including clinically used inhibitors and oncometabolites, modulate DNA modifications via TETs has been unclear. Here, we report studies on human TET1-3 inhibition by a set of 2OG oxygenase-focused inhibitors, employing both enzyme-based and cellular assays. Most inhibitors manifested similar potencies for TET1-3 and caused increases in cellular 5hmC levels. (R)-2-Hydroxyglutarate, an oncometabolite elevated in isocitrate dehydrogenase mutant cancer cells, showed different degrees of inhibition, with TET1 being less potently inhibited than TET3 and TET2, potentially reflecting the proposed role of TET2 mutations in tumorigenesis. The results highlight the tractability of TETs as drug targets and provide starting points for selective inhibitor design.

Robust aortic media adhesion using hydrophobically modified Alaska pollock gelatin-based adhesive for aortic dissections.

Type-A aortic dissection is an acute injury involving the delamination of the aorta at the parts of the aortic media. Aldehyde crosslinker-containing glues have been used to adhere to the media of the dissected aorta before joining an artificial graft. These glues effectively adhere to the aortic media; however, they show low biocompatibility due to the release of aldehyde compounds. In this study, we report innovative adhesives based on hydrophobically modified Alaska pollock gelatin (hm-ApGltn) with different alkyl or cholesteryl (Chol) groups that adhere to the media of the dissected aorta by combining hm-ApGltns with a biocompatible crosslinker, pentaerythritol poly(ethylene glycol) ether tetrasuccinimidyl glutarate. The modification of alkyl or Chol groups contributed to enhanced adhesion strength between porcine aortic media. The adhesion strength increased with increasing modification ratios of alkyl groups from propanoyl to dodecanoyl groups and then decreased at a modification ratio of ~20 mol %. Porcine aortic media adhered using 7.5Chol-ApGltn adhesive showed stretchability even when expanded and shrunk vertically by 25% at least five times. Hm-ApGltn adhesives subcutaneously injected into the backs of mice showed no severe inflammation and were degraded during the implantation period. These results indicated that hm-ApGltn adhesives have potential applications in type-A aortic dissection.

Structure and biochemical characterization of l-2-hydroxyglutarate dehydrogenase and its role in the pathogenesis of l-2-hydroxyglutaric aciduria.

l-2-hydroxyglutarate dehydrogenase (L2HGDH) is a mitochondrial membrane-associated metabolic enzyme, which catalyzes the oxidation of l-2-hydroxyglutarate (l-2-HG) to 2-oxoglutarate (2-OG). Mutations in human L2HGDH lead to abnormal accumulation of l-2-HG, which causes a neurometabolic disorder named l-2-hydroxyglutaric aciduria (l-2-HGA). Here, we report the crystal structures of Drosophila melanogaster L2HGDH (dmL2HGDH) in FAD-bound form and in complex with FAD and 2-OG and show that dmL2HGDH exhibits high activity and substrate specificity for l-2-HG. dmL2HGDH consists of an FAD-binding domain and a substrate-binding domain, and the active site is located at the interface of the two domains with 2-OG binding to the re-face of the isoalloxazine moiety of FAD. Mutagenesis and activity assay confirmed the functional roles of key residues involved in the substrate binding and catalytic reaction and showed that most of the mutations of dmL2HGDH equivalent to l-2-HGA-associated mutations of human L2HGDH led to complete loss of the activity. The structural and biochemical data together reveal the molecular basis for the substrate specificity and catalytic mechanism of L2HGDH and provide insights into the functional roles of human L2HGDH mutations in the pathogeneses of l-2-HGA.

Circulating Oncometabolite 2-hydroxyglutarate as a Potential Biomarker for Isocitrate Dehydrogenase (IDH1/2) Mutant Cholangiocarcinoma.

Isocitrate dehydrogenase (IDH) enzymes catalyze the decarboxylation of isocitrate to alpha-ketoglutarate (αKG). IDH1/2 mutations preferentially convert αKG to R-2-hydroxyglutarate (R2HG), resulting in R2HG accumulation in tumor tissues. We investigated circulating 2-hydroxyglutate (2HG) as potential biomarkers for patients with IDH-mutant (IDHmt) cholangiocarcinoma (CCA). R2HG and S-2-hydroxyglutarate (S2HG) levels in blood and tumor tissues were analyzed in a discovery cohort of patients with IDHmt glioma and CCA. Results were validated in cohorts of patients with CCA and clear-cell renal cell carcinoma. The R2HG/S2HG ratio (rRS) was significantly elevated in tumor tissues, but not in blood for patients with IDHmt glioma, while circulating rRS was elevated in patients with IDHmt CCA. There were overlap distributions of circulating R2HG and total 2HG in patients with both IDHmt and wild-type (IDHwt) CCA, while there was minimal overlap in rRS values between patients with IDHmt and IDHwt CCA. Using the rRS cut-off value of 1.5, the sensitivity of rRS was 90% and specificity was 96.8%. Circulating rRS is significantly increased in patients with IDHmt CCA compare with patients with IDHwt CCA. Circulating rRS is a sensitive and specific surrogate biomarker for IDH1/2 mutations in CCA. It can potentially be used as a tool for monitoring IDH-targeted therapy.

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.

2-hydroxyglutarate rides the cancer-immunity cycle.

2-hydroxyglutarate (2HG) is a biproduct of the Krebs cycle, which exists in a D- and L- enantiomer and is structurally similar to α-ketoglutarate. Both 2HG enantiomers have been described to accumulate in diverse cancer and immune cells and can influence cell fate and function. While D-2HG was originally considered as an 'oncometabolite' that aberrantly builds up in certain cancers, it is becoming clear that it also physiologically accumulates in immune cells and regulates immune function. Conversely, L-2HG is considered as an 'immunometabolite' due to its induction and regulatory function in T cells, but it can also be induced in certain cancers. Here, the authors review the effects of both 2HG enantiomers on immune cells within the tumor microenvironment.

Nrf2 sensitizes ferroptosis through l-2-hydroxyglutarate-mediated chromatin modifications in sickle cell disease.

Sickle cell disease (SCD) is a chronic hemolytic and systemic hypoxia condition with constant oxidative stress and significant metabolic alterations. However, little is known about the correlation between metabolic alterations and the pathophysiological symptoms. Here, we report that Nrf2, a master regulator of cellular antioxidant responses, regulates the production of the metabolite l-2-hydroxyglutarate (L2HG) to mediate epigenetic histone hypermethylation for gene expression involved in metabolic, oxidative, and ferroptotic stress responses in SCD. Mechanistically, Nrf2 was found to regulate the expression of L2HG dehydrogenase (L2hgdh) to mediate L2HG production under hypoxia. Gene expression profile analysis indicated that reactive oxygen species (ROS) and ferroptosis responses were the most significantly affected signaling pathways after Nrf2 ablation in SCD. Nrf2 silencing and L2HG supplementation sensitize human sickle erythroid cells to ROS and ferroptosis stress. The absence of Nrf2 and accumulation of L2HG significantly affect histone methylation for chromatin structure modification and reduce the assembly of transcription complexes on downstream target genes to regulate ROS and ferroptosis responses. Furthermore, pharmacological activation of Nrf2 was found to have protective effects against ROS and ferroptosis stress in SCD mice. Our data suggest a novel mechanism by which Nrf2 regulates L2HG levels to mediate SCD severity through ROS and ferroptosis stress responses, suggesting that targeting Nrf2 is a viable therapeutic strategy for ameliorating SCD symptoms.

Separation and quantification of the urinary enantiomers of 2-hydroxyglutaric acid by capillary electrophoresis with capacitively coupled contactless conductivity detection: Application to the diagnosis of D- and L-2-hydroxyglutaric aciduria.

2-hydroxyglutaric aciduria is an inherited neurometabolic disorder with two major types: D-2-hydroxyglutaric aciduria and L-2-hydroxyglutaric aciduria. An easy and fast capillary electrophoresis system combined with a capacitively coupled contactless conductivity detection method was developed for the enantioseparation and determination of D- and L-2-hydroxyglutaric acid in urine. D- and L-2-hydroxyglutaric acids were separated using vancomycin as the chiral selector. The optimal separation conditions for enantiomers were achieved by the use of a buffer containing 50 mM 4-(N-morpholino) butane sulfonic acid solution (pH 6.5), an electroosmotic flow modifier (0.001% [w/v] polybrene), and 30 mM vancomycin as chiral selector. The analysis time was 6 min under optimal conditions. The optimized and validated method was successfully implemented for quantifying D- and L-2-hydroxyglutaric aciduria in patients' urine, without any pretreatment step. The linearity of the method was determined to be in the range of 2-100 mg/L for D- and L-2-hydroxyglutaric acid in urine. The precision (relative standard deviation%) was obtained at about 7%. For D- and L-2-hydroxyglutaric acids, the limits of detection were 0.567 and 0.497 mg/L, respectively.

An on-line heart-cutting two-dimensional liquid chromatography method for intracellular 2-hydroxyglutarate enantiomers.

D-2-Hydroxyglutarate (D-2-HG) is an oncometabolite that induces cancer cell survival and growth. D-2-HG is produced by mutations in isocitrate dehydrogenases 1 and 2. L-2-HG has different roles than the D-form, and chiral discrimination is important for elucidating the exact roles of the 2-HG enantiomers. In this study, an analytical method for 2-HG enantiomers was developed using on-line heart-cutting two-dimensional liquid chromatography (2D-LC) with fluorescence detection. Fluorescence derivatization of 2-HG with 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was performed using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, a hydrophilic condensing reagent, at 70 °C for 30 min. The first dimension on the octadecylsilyl column was aimed at separating NBD-PZ-2-HG from other compounds obtained via derivatization or from biological fluids. The NBD-PZ-2-HG peak was fractionated into a sample loop and automatically injected into the second dimension. In the second dimension, a CHIRALPAK IC column separated NBD-PZ-D- and L-2-HG with a resolution of 2.14. The limits of quantification were 0.25 pmol per injection for NBD-PZ-D-2-HG and-L-2-HG. The precision values were below 6.58%, and the accuracies were 88.2-92.8%. The intracellular concentrations of D-2-HG and L-2-HG in the cancer cells were 13.5 ± 0.4 and 9.9 ± 0.3 pmol per 1.0 × 106 cells, respectively. The developed method will be useful for elucidating the role of 2-HG enantiomers in cancer cells.

Phenotypic prediction in glutaric aciduria type 1 combining in silico and in vitro modeling with real-world data.

Glutaric aciduria type 1 (GA1) is caused by inherited deficiency of glutaryl-CoA dehydrogenase (GCDH). To further understand the unclear genotype-phenotype correlation, we transfected mutated GCDH into COS-7 cells resembling known biallelic GCDH variants of 47 individuals with GA1. In total, we modeled 36 genotypes with 32 missense variants. Spectrophotometry demonstrated an inverse correlation between residual enzyme activity and the urinary concentration of glutaric acid and 3-hydroxyglutaric acid, confirming previous studies (Pearson correlation, r = -0.34 and r = -0.49, p = 0.045 and p = 0.002, respectively). In silico modeling predicted high pathogenicity for all genotypes, which caused a low enzyme activity. Western blotting revealed a 2.6-times higher GCDH protein amount in patients with an acute encephalopathic crisis (t-test, p = 0.015), and high protein expression correlated with high in silico protein stability (Pearson correlation, r = -0.42, p = 0.011). The protein amount was not correlated with the enzyme activity (Pearson correlation, r = 0.09, p = 0.59). To further assess protein stability, proteolysis was performed, showing that the p.Arg88Cys variant stabilized a heterozygous less stable variant. We conclude that an integration of different data sources helps to predict the complex clinical phenotype in individuals with GA1.