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R -2-hydroxyglutarate accumulates to millimolar levels in cancer cells with gain-of-function isocitrate dehydrogenase 1/2 mutations. These levels of R -2-hydroxyglutarate affect 2-oxoglutarate-dependent dioxygenases. Both metabolite enantiomers, R - and S -2-hydroxyglutarate, are detectible in healthy individuals, yet their physiological function remains elusive. Here we show that 2-hydroxyglutarate accumulates in mouse CD8 + T cells in response to T-cell receptor triggering, and accumulates to millimolar levels in physiological oxygen conditions through a hypoxia-inducible factor 1-alpha (HIF-1α)-dependent mechanism. S -2-hydroxyglutarate predominates over R -2-hydroxyglutarate in activated T cells, and we demonstrate alterations in markers of CD8 + T-cell differentiation in response to this metabolite. Modulation of histone and DNA demethylation, as well as HIF-1α stability, mediate these effects. S -2-hydroxyglutarate treatment greatly enhances the in vivo proliferation, persistence and anti-tumour capacity of adoptively transferred CD8 + T cells. Thus, S -2-hydroxyglutarate acts as an immunometabolite that links environmental context, through a metabolic–epigenetic axis, to immune fate and function. S -2-hydroxyglutarate produced by CD8+ T cells under hypoxic conditions affects locus-specific histone and DNA methylation patterns, which enhances T-cell proliferation, survival and recall responses. Broad immune stimulation by S -2-hydroxyglutarate Randall Johnson and colleagues demonstrate that CD8 + T cells produce the immunometabolite S -2-hydroxyglutarate ( S -2HG) in response to activation triggered by T-cell receptors under hypoxic conditions. The resulting S -2HG affects locus-specific histone and DNA methylation patterns, enhancing T-cell proliferation, survival and recall responses.

Key Points Cancer cells show increased consumption of and dependence on glutamine. Glutamine metabolism fuels the tricarboxylic acid (TCA) cycle, nucleotide and fatty acid biosynthesis, and redox balance in cancer cells. Glutamine activates mTOR signalling, suppresses endoplasmic reticulum stress and promotes protein synthesis. Cancer cells may metabolize glutamate to α-ketoglutarate through one of two different pathways (glutamate dehydrogenase or aminotransferases), with aminotransferases potentially supporting a more biosynthetic and pro-growth phenotype. Activation of oncogenic pathways and loss of tumour suppressors reprogramme glutamine metabolism in a tissue-dependent manner. Targeting glutamine metabolism shows promise as an anticancer therapy. Compensatory glutamine metabolism induced by cancer therapies suggests that targeting glutamine metabolism may be used in combination therapy. This Review provides an overview of glutamine metabolism and its involvement in tumorigenesis in vitro and in vivo , exploring the recent potential applications of basic science discoveries in the clinical setting. The resurgence of research into cancer metabolism has recently broadened interests beyond glucose and the Warburg effect to other nutrients, including glutamine. Because oncogenic alterations of metabolism render cancer cells addicted to nutrients, pathways involved in glycolysis or glutaminolysis could be exploited for therapeutic purposes. In this Review, we provide an updated overview of glutamine metabolism and its involvement in tumorigenesis in vitro and in vivo , and explore the recent potential applications of basic science discoveries in the clinical setting.

The ( R )-enantiomer of 2-hydroxyglutarate, which is produced when IDH is mutated in human tumours, is shown to stimulate the activity of the EGLN prolyl 4-hydroxylases, leading to diminished levels of HIF and enhanced human astrocyte proliferation. Cancer induction by isocitrate dehydrogenase mutation Mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 have been identified in gliomas, the most common form of brain tumour, and in other cancers including leukaemias. The mutated enzymes produce 2-hydroxyglutarate (2HG), which is a potential oncometabolite. Three papers in this issue of Nature examine the mechanisms through which IDH mutations promote cancers. Lu et al . show that 2HG-producing IDH mutants can prevent the histone demethylation that is required for progenitor cells to differentiate, potentially contributing to tumour-cell accumulation. Turcan et al . show that IDH1 mutation in primary human astrocytes induces DNA hypermethylation and reshapes the methylome to resemble that of the CIMP phenotype, a common feature of gliomas and other solid tumours. Koivunen et al . show that the ( R )-enantiomer of 2HG (but not the ( S )-enantiomer) can stimulate the activity of the EGLN prolyl 4-hydroxylases, leading to diminished levels of hypoxia-inducible factor (HIF), which in turn can enhance cell proliferation. These papers establish a framework for understanding gliomagenesis and highlight the interplay between genomic and epigenomic changes in human cancers. The identification of succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) mutations in human cancers has rekindled the idea that altered cellular metabolism can transform cells. Inactivating SDH and FH mutations cause the accumulation of succinate and fumarate, respectively, which can inhibit 2-oxoglutarate (2-OG)-dependent enzymes, including the EGLN prolyl 4-hydroxylases that mark the hypoxia inducible factor (HIF) transcription factor for polyubiquitylation and proteasomal degradation 1 . Inappropriate HIF activation is suspected of contributing to the pathogenesis of SDH-defective and FH-defective tumours but can suppress tumour growth in some other contexts. IDH1 and IDH2, which catalyse the interconversion of isocitrate and 2-OG, are frequently mutated in human brain tumours and leukaemias. The resulting mutants have the neomorphic ability to convert 2-OG to the ( R )-enantiomer of 2-hydroxyglutarate (( R )-2HG) 2 , 3 . Here we show that ( R )-2HG, but not ( S )-2HG, stimulates EGLN activity, leading to diminished HIF levels, which enhances the proliferation and soft agar growth of human astrocytes. These findings define an enantiomer-specific mechanism by which the ( R )-2HG that accumulates in IDH mutant brain tumours promotes transformation and provide a justification for exploring EGLN inhibition as a potential treatment strategy.

The oncometabolite ( R )-2-hydroxyglutarate (R-2-HG) produced by isocitrate dehydrogenase ( IDH ) mutations promotes gliomagenesis via DNA and histone methylation. Here, we identify an additional activity of R-2-HG: tumor cell–derived R-2-HG is taken up by T cells where it induces a perturbation of nuclear factor of activated T cells transcriptional activity and polyamine biosynthesis, resulting in suppression of T cell activity. IDH1 -mutant gliomas display reduced T cell abundance and altered calcium signaling. Antitumor immunity to experimental syngeneic IDH1 -mutant tumors induced by IDH1-specific vaccine or checkpoint inhibition is improved by inhibition of the neomorphic enzymatic function of mutant IDH1 . These data attribute a novel, non-tumor cell-autonomous role to an oncometabolite in shaping the tumor immune microenvironment. An oncometabolite produced by tumor cells acts as a paracrine immunosuppressant dampening antitumor T cell responses in glioma.

d -2-Hydroxyglutarate ( d -2-HG) is a functional endogenous metabolite in various domains of life. Its abnormal accumulation promotes human tumorigenesis. Convenient d -2-HG testing for diagnosis and prognosis of d -2-HG-related diseases remains technically challenging, and there is no analytical method to directly detect d -2-HG in living cells. Here, we identify a d -2-HG-specific transcriptional activator, HgcR, and develop a d -2- H G sens or (DHOR) using HgcR as a sensing moiety. Then, we build a portable device adaptive with DHOR for rapid and low-cost point-of-care d -2-HG testing in serum, urine, and glioma tissue samples. DHOR also allow spatiotemporal resolution of d -2-HG in living bacteria and human cells. We use DHOR to identify d -2-HG transporters from Escherichia coli and human solute carrier 22 family. Overall, DHOR provides a powerful and versatile tool for in vitro and live-cell detection of d -2-HG, offering the opportunity to deepen our understanding about physiological and pathogenetic roles of d -2-HG. D-2-hydroxyglutarate (D-2-HG) is an endogenous metabolite that plays both physiological and pathological roles. Here, the authors report a genetically encoded D-2-HG biosensor based on the transcriptional activator HgcR, which enables rapid detection of D-2-HG in vitro and in live cells.

Regulatory T cells (T reg cells), a distinct subset of CD4 + T cells, are necessary for the maintenance of immune self-tolerance and homeostasis 1 , 2 . Recent studies have demonstrated that T reg cells exhibit a unique metabolic profile, characterized by an increase in mitochondrial metabolism relative to other CD4 + effector subsets 3 , 4 . Furthermore, the T reg cell lineage-defining transcription factor, Foxp3, has been shown to promote respiration 5 , 6 ; however, it remains unknown whether the mitochondrial respiratory chain is required for the T cell-suppression capacity, stability and survival of T reg cells. Here we report that T reg cell-specific ablation of mitochondrial respiratory chain complex III in mice results in the development of fatal inflammatory disease early in life, without affecting T reg cell number. Mice that lack mitochondrial complex III specifically in T reg cells displayed a loss of T cell-suppression capacity without altering T reg cell proliferation and survival. T reg cells deficient in complex III showed decreased expression of genes associated with T reg function, whereas Foxp3 expression remained stable. Loss of complex III in T reg cells increased DNA methylation as well as the metabolites 2-hydroxyglutarate (2-HG) and succinate that inhibit the ten-eleven translocation (TET) family of DNA demethylases 7 . Thus, T reg cells require mitochondrial complex III to maintain immune regulatory gene expression and suppressive function. Specific ablation of mitochondrial complex III subunits in T reg cells in mice results in inflammatory disease, altered T reg gene expression and defective T reg function, indicating a key functional role for mitochondrial complex III in T reg cells.

Cancer-associated IDH mutants that produce 2-hydroxyglutarate are shown to prevent the histone demethylation that is required for lineage-specific progenitor cells to differentiate into terminally differentiated cells. Cancer induction by isocitrate dehydrogenase mutation Mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 have been identified in gliomas, the most common form of brain tumour, and in other cancers including leukaemias. The mutated enzymes produce 2-hydroxyglutarate (2HG), which is a potential oncometabolite. Three papers in this issue of Nature examine the mechanisms through which IDH mutations promote cancers. Lu et al . show that 2HG-producing IDH mutants can prevent the histone demethylation that is required for progenitor cells to differentiate, potentially contributing to tumour-cell accumulation. Turcan et al . show that IDH1 mutation in primary human astrocytes induces DNA hypermethylation and reshapes the methylome to resemble that of the CIMP phenotype, a common feature of gliomas and other solid tumours. Koivunen et al . show that the ( R )-enantiomer of 2HG (but not the ( S )-enantiomer) can stimulate the activity of the EGLN prolyl 4-hydroxylases, leading to diminished levels of hypoxia-inducible factor (HIF), which in turn can enhance cell proliferation. These papers establish a framework for understanding gliomagenesis and highlight the interplay between genomic and epigenomic changes in human cancers. Recurrent mutations in isocitrate dehydrogenase 1 (IDH1) and IDH2 have been identified in gliomas, acute myeloid leukaemias (AML) and chondrosarcomas, and share a novel enzymatic property of producing 2-hydroxyglutarate (2HG) from α-ketoglutarate 1 , 2 , 3 , 4 , 5 , 6 . Here we report that 2HG-producing IDH mutants can prevent the histone demethylation that is required for lineage-specific progenitor cells to differentiate into terminally differentiated cells. In tumour samples from glioma patients, IDH mutations were associated with a distinct gene expression profile enriched for genes expressed in neural progenitor cells, and this was associated with increased histone methylation. To test whether the ability of IDH mutants to promote histone methylation contributes to a block in cell differentiation in non-transformed cells, we tested the effect of neomorphic IDH mutants on adipocyte differentiation in vitro . Introduction of either mutant IDH or cell-permeable 2HG was associated with repression of the inducible expression of lineage-specific differentiation genes and a block to differentiation. This correlated with a significant increase in repressive histone methylation marks without observable changes in promoter DNA methylation. Gliomas were found to have elevated levels of similar histone repressive marks. Stable transfection of a 2HG-producing mutant IDH into immortalized astrocytes resulted in progressive accumulation of histone methylation. Of the marks examined, increased H3K9 methylation reproducibly preceded a rise in DNA methylation as cells were passaged in culture. Furthermore, we found that the 2HG-inhibitable H3K9 demethylase KDM4C was induced during adipocyte differentiation, and that RNA-interference suppression of KDM4C was sufficient to block differentiation. Together these data demonstrate that 2HG can inhibit histone demethylation and that inhibition of histone demethylation can be sufficient to block the differentiation of non-transformed cells.

Post-translational modifications (PTMs) dynamically regulate cellular processes. Lysine undergoes a range of acylations, including malonylation, succinylation (SucK) and glutarylation (GluK). These PTMs increase the size of the lysine side chain and reverse its charge from +1 to −1 under physiological conditions, probably impacting protein structure and function. To understand the functional roles of these PTMs, homogeneously modified proteins are required for biochemical studies. While the site-specific encoding of PTMs and their mimics via genetic code expansion has facilitated the characterization of the functional roles of many PTMs, negatively charged lysine acylations have defied this approach. Here we describe site-specific incorporation of SucK and GluK into proteins via temporarily masking their negative charge through thioester derivatives. We prepare succinylated and glutarylated bacterial and mammalian target proteins, including non-refoldable multidomain proteins. This allows us to study how succinylation and glutarylation impact enzymatic activity of metabolic enzymes and regulate protein–DNA and protein–protein interactions in biological processes from replication to ubiquitin signalling. Negatively charged lysine acylations—malonylation, succinylation and glutarylation—impact protein structure and function, which can affect cellular processes. Now temporarily masked thioester derivatives of succinylation and glutarylation can be used for site-specific modification of diverse bacterial and mammalian proteins, which can facilitate the study of how these lysine modifications impact enzymatic activity and control protein–protein and protein–DNA interactions.

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.

Cancer-associated mutations in IDH are associated with multiple types of human malignancies, which exhibit distinctive metabolic reprogramming, production of oncometabolite D-2-HG, and shifted epigenetic landscape. IDH mutated malignancies are signatured with “BRCAness”, highlighted with the sensitivity to DNA repair inhibitors and genotoxic agents, although the underlying molecular mechanism remains elusive. In the present study, we demonstrate that D-2-HG impacts the chromatin conformation adjustments, which are associated with DNA repair process. Mechanistically, D-2-HG diminishes the chromatin interactions in the DNA damage regions via revoking CTCF binding. The hypermethylation of cytosine, resulting from the suppression of TET1 and TET2 activities by D-2-HG, contributes to the dissociation of CTCF from DNA damage regions. CTCF depletion leads to the disruption of chromatin organization around the DNA damage sites, which abolishes the recruitment of essential DNA damage repair proteins BRCA2 and RAD51, as well as impairs homologous repair in the IDH mutant cancer cells. These findings provide evidence that CTCF-mediated chromatin interactions play a key role in DNA damage repair proceedings. Oncometabolites jeopardize genome stability and DNA repair by affecting high-order chromatin structure. The oncometabolite D-2-HG has been implicated in compromising DNA repair pathway. Here the authors show D-2-HG leads to genome-wide DNA hypermethylation and loss of CTCF coverage, which impairs the assembly of the homologous recombination DNA repair machinery and associated topology adjustment.