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2-Oxoglutarate (2-OG) is a tricarboxylate cycle intermediate that can be biologically converted into several industrially important compounds. However, studies on the fermentative production of compounds synthesized from 2-OG, but not via glutamate (defined as 2-OG derivatives), have been limited. Herein, a system that can efficiently produce 2-hydroxyglutarate (2-HG), a 2-OG derivative biosynthesized by the hgdH-encoded NADH-dependent 2-HG dehydrogenase of Acidaminococcus fermentans, was developed as a model using Corynebacterium glutamicum. First, the D3 strain, which lacked the two NADH-consuming enzymes, lactate dehydrogenase and malate dehydrogenase, as well as isocitrate lyase, was constructed as a starting strain. Next, the growth conditions that induced the accumulation of 2-OG were investigated, and it was found that the biotin- and nitrogen-limited (B/N-limited) aerobic growth conditions were suitable for this purpose. Finally, the hgdH gene of A. fermentans became overexpressed in the D3 strain by inserting it into the intergenic regions with the strong constitutive promoter of the tuf gene of C. glutamicum; the engineered strain was cultured under the B/N-limited aerobic growth conditions. The engineered strain produced 80.1 mM 2-HG with a yield of 0.390 mol/mol glucose, which are the highest titer and yield reported thus far, to the best of our knowledge. Furthermore, reverse genetics showed that the produced 2-HG was partially exported via the YggB protein (NCgl1221 protein, a mechanosensitive channel) known as an exporter for glutamate under the conditions used herein.Key points• An efficient 2-HG production system was developed with Corynebacterium glutamicum.• Biotin- and nitrogen-limited aerobic growth conditions induced 2-OG production.• Produced 2-HG was partially excreted via the glutamate exporter, YggB.

Oncogenic isocitrate dehydrogenase (IDH)1 and IDH2 mutations at three hotspot arginine residues cause an enzymatic gain of function that leads to the production and accumulation of the metabolite 2-hydroxyglutarate (2HG), which contributes to the development of a number of malignancies. In the hematopoietic system, mutations in IDH1 at arginine (R) 132 and in IDH2 at R140 and R172 are commonly observed in acute myeloid leukemia, and elevated 2HG is observed in cells and serum. However, in angioimmunoblastic T-cell lymphoma (AITL), mutations are almost exclusively restricted to IDH2 R172, and levels of 2HG have not been comprehensively measured. In this study, we investigate the expression pattern of mutant IDH2 in the AITL tumor microenvironment and measure levels of 2HG in tissue and serum of AITL patients. We find that mutant IDH2 expression is restricted to the malignant T-cell component of AITL, and that 2HG is elevated in tumor tissue and serum of patients. We also investigate the differences between the three hotspot mutation sites in IDH1 and IDH2 using conditional knock-in mouse models. These studies show that in the lymphoid system, mutations in IDH2 at R172 produce high levels of 2HG compared with mutations at the other two sites and that lymphoid development is impaired in these animals. These data provide evidence that IDH2 R172 mutations may be the only variants present in AITL because of their capacity to produce significant amounts of the oncometabolite 2HG in the cell of origin of this disease.

L-2-hydroxyglutarate (L-2HG) has emerged as a putative oncometabolite that is capable of inhibiting enzymes involved in metabolism, chromatin modification, and cell differentiation. However, despite the ability of L-2HG to interfere with a broad range of cellular processes, this molecule is often characterized as a metabolic waste product. Here, we demonstrate that Drosophila larvae use the metabolic conditions established by aerobic glycolysis to both synthesize and accumulate high concentrations of L-2HG during normal developmental growth. A majority of the larval L-2HG pool is derived from glucose and dependent on the Drosophila estrogen-related receptor (dERR), which promotes L-2HG synthesis by up-regulating expression of the Drosophila homolog of lactate dehydrogenase (dLdh). We also show that dLDH is both necessary and sufficient for directly synthesizing L-2HG and the Drosophila homolog of L-2-hydroxyglutarate dehydrogenase (dL2HGDH), which encodes the enzyme that breaks down L-2HG, is required for stage-specific degradation of the L-2HG pool. In addition, dLDH also indirectly promotes L-2HG accumulation via synthesis of lactate, which activates a metabolic feed-forward mechanism that inhibits dL2HGDH activity and stabilizes L-2HG levels. Finally, we use a genetic approach to demonstrate that dLDH and L-2HG influence position effect variegation and DNA methylation, suggesting that this compound serves to coordinate glycolytic flux with epigenetic modifications. Overall, our studies demonstrate that growing animal tissues synthesize L-2HG in a controlled manner, reveal a mechanism that coordinates glucose catabolism with L-2HG synthesis, and establish the fly as a unique model system for studying the endogenous functions of L-2HG during cell growth and proliferation.

Mutations in isocitrate dehydrogenases (IDHs) have a gain‐of‐function effect leading to R (−)‐2‐hydroxyglutarate ( R‐ 2HG) accumulation. By using biochemical, structural and cellular assays, we show that either or both R ‐ and S ‐2HG inhibit 2‐oxoglutarate (2OG)‐dependent oxygenases with varying potencies. Half‐maximal inhibitory concentration (IC 50 ) values for the R ‐form of 2HG varied from approximately 25 μM for the histone N ε ‐lysine demethylase JMJD2A to more than 5 mM for the hypoxia‐inducible factor (HIF) prolyl hydroxylase. The results indicate that candidate oncogenic pathways in IDH‐associated malignancy should include those that are regulated by other 2OG oxygenases than HIF hydroxylases, in particular those involving the regulation of histone methylation. The oncometabolite 2‐hydroxyglutarate (2‐HG) inhibits chromatin‐modifying oxygenases (as histone lysine demethylases) with greater potency than HIF hydroxylases. This suggests that 2‐HG‐associated oncogenic pathways involve the regulation of histone methylation, rather than an elevated HIF response.

Mutations in isocitrate dehydrogenase (IDH) 1 and 2, originally discovered in 2008, occur in the vast majority of low-grade gliomas and secondary high-grade gliomas. These mutations, which occur early in gliomagenesis, change the function of the enzymes, causing them to produce 2-hydroxyglutarate, a possible oncometabolite, and to not produce NADPH. IDH mutations are oncogenic, although whether the mechanism is through alterations in hydroxylases, redox potential, cellular metabolism, or gene expression is not clear. The mutations also drive increased methylation in gliomas. Gliomas with mutated IDH1 and IDH2 have improved prognosis compared with gliomas with wild-type IDH. Mutated IDH can now be detected by immunohistochemistry and magnetic resonance spectroscopy. No drugs currently target mutated IDH, although this remains an area of active research.

Standard treatment for patients with IDH-mutant gliomas with radiation therapy and chemotherapy is non-curative and associated with long-term neurotoxicity. This has created intense interest in targeted therapeutic strategies that are specifically designed of IDH-mutant tumors. Much progress has been made in understanding the unique biology of IDH-mutant gliomas, and now various IDH-mutant-specific targeting strategies are in various phases of development. Here, we will review a range of IDH-mutant targeting treatments being explored, including direct IDH inhibitors, as well as strategies that take advantage of IDH-mutant-specific vulnerabilities.

2-Hydroxyglutarate (2-HG) is structurally similar to α-ketoglutarate (α-KG), which is an intermediate product of the tricarboxylic acid (TCA) cycle; it can be generated by reducing the ketone group of α-KG to a hydroxyl group. The significant role that 2-HG plays has been certified in the pathophysiology of 2-hydroxyglutaric aciduria (2HGA), tumors harboring mutant isocitrate dehydrogenase 1/2 (IDH1/2mt), and in clear cell renal cell carcinoma (ccRCC). It is taken as an oncometabolite, raising much attention on its oncogenic mechanism. In recent years, 2-HG has been verified to accumulate in the context of hypoxia or acidic pH, and there are also researches confirming the vital role that 2-HG plays in the fate decision of immune cells. Therefore, 2-HG not only participates in tumorigenesis. This text will also summarize 2-HG’s identities besides being an oncometabolite and will discuss their enlightenment for future research and clinical treatment.

Purpose Metabolic aberrations have been described in neoplasms with pathogenic variants (PV) in the Krebs cycle genes encoding succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH). In turn, accumulation of oncometabolites succinate, fumarate, and 2-hydroxyglutarate can be employed to identify tumors with those PV . Additionally, such metabolic readouts may aid in genetic variant interpretation and improve diagnostics. Methods Using liquid chromatography–mass spectrometry, 395 pheochromocytomas and paragangliomas (PPGLs) from 391 patients were screened for metabolites to indicate Krebs cycle aberrations. Multigene panel sequencing was applied to detect driver PV in cases with indicative metabolite profiles but undetermined genetic drivers. Results Aberrant Krebs cycle metabolomes identified rare cases of PPGLs with germline PV in FH and somatic PV in IDHx and SDHx , including the first case of a somatic IDH2 PV in PPGL. Metabolomics also reliably identified PPGLs with SDHx loss-of-function (LOF) PV. Therefore we utilized tumor metabolite profiles to further classify variants of unknown significance in SDHx , thereby enabling missense variants associated with SDHx LOF to be distinguished from benign variants. Conclusion We propose incorporation of metabolome data into the diagnostics algorithm in PPGLs to guide genetic testing and variant interpretation and to help identify rare cases with PV in FH and IDHx .

In solid tumours, cancer cells exist within hypoxic microenvironments, and their metabolic adaptation to this hypoxia is driven by HIF-1 transcription factor, which is overexpressed in a broad range of human cancers. HIF inhibitors are under pre-clinical investigation and clinical trials, but there is evidence that hypoxic cancer cells can adapt metabolically to HIF-1 inhibition, which would provide a potential route for drug resistance. Here, we review accumulating evidence of such adaptions in carbohydrate and creatine metabolism and other HIF-1-independent mechanisms that might allow cancers to survive hypoxia despite anti-HIF-1 therapy. These include pathways in glucose, glutamine, and lipid metabolism; epigenetic mechanisms; post-translational protein modifications; spatial reorganization of enzymes; signalling pathways such as Myc, PI3K-Akt, 2-hyxdroxyglutarate and AMP-activated protein kinase (AMPK); and activation of the HIF-2 pathway. All of these should be investigated in future work on hypoxia bypass mechanisms in anti-HIF-1 cancer therapy. In principle, agents targeted toward HIF-1β rather than HIF-1α might be advantageous, as both HIF-1 and HIF-2 require HIF-1β for activation. However, HIF-1β is also the aryl hydrocarbon nuclear transporter (ARNT), which has functions in many tissues, so off-target effects should be expected. In general, cancer therapy by HIF inhibition will need careful attention to potential resistance mechanisms.

SummaryBackground Mutant isocitrate dehydrogenase 1 and 2 (IDH1/IDH2) enzymes produce the oncometabolite D-2-hydroxyglutarate (2-HG). Ivosidenib (AG-120) is a targeted mutant IDH1 inhibitor under evaluation in a phase 1 dose escalation and expansion study of IDH1-mutant advanced solid tumors including cholangiocarcinoma, chondrosarcoma, and glioma. We explored the pharmacokinetic (PK) and pharmacodynamic (PD) profiles of ivosidenib in these populations. Methods Ivosidenib was administered orally once (QD) or twice (BID) daily in continuous 28-day cycles; 168 patients received ≥1 dose within the range 100 mg BID to 1200 mg QD. PK and PD were assessed using validated liquid chromatography-tandem mass spectrometry assays. Results Ivosidenib demonstrated good oral exposure after single and multiple doses, was rapidly absorbed, and had a long terminal half-life (mean 40–102 h after single dose). Exposure increased less than dose proportionally. Steady state was reached by day 15, with moderate accumulation across all tumors (1.5- to 1.7-fold for area-under-the-curve at 500 mg QD). None of the intrinsic and extrinsic factors assessed affected ivosidenib exposure, including patient/disease characteristics and concomitant administration of weak CYP3A4 inhibitors/inducers. After multiple doses in patients with cholangiocarcinoma or chondrosarcoma, plasma 2-HG was reduced by up to 98%, to levels seen in healthy subjects. Exposure-response relationships for safety and efficacy outcomes were flat across the doses tested. Conclusions Ivosidenib demonstrated good oral exposure and a long half-life. Robust, persistent plasma 2-HG inhibition was observed in IDH1-mutant cholangiocarcinoma and chondrosarcoma. Ivosidenib 500 mg QD is an appropriate dose irrespective of various intrinsic and extrinsic factors. Trial RegistrationClinicalTrials.gov (NCT02073994).