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Cited by 1,035Cancer, Hypoxia, and MetabolismEpigenetics and DNA MethylationGlioma Diagnosis and Treatment

The oncometabolite 2-hydroxyglutarate inhibits histone lysine demethylases

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Chowdhury, Rasheduzzaman, Yeoh, Kar Kheng, Tian, Ya‐Min, Hillringhaus, Lars, Bagg, Eleanor A, Rose, Nathan R, Leung, Ivanhoe K H, Li, Xuan S +10 more·2011·The EMBO Reports
DOI 10.1038/embor.2011.43PMC PMC3090014
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Abstract

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(ɛ)-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.

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Crossref · 10.1038/embor.2011.43

Cites

31 of 31 references verified0 citation errors · 0 not checkable

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Nothing to recompute.

Claims the paper makes

0 of 22 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Crystal structures of FIH and JMJD2A with R-2HG or S-2HG show that both enantiomers can bind the active-site iron, or surrogate nickel, in a bidentate manner.

    “Structures of FIH and JMJD2A with R-or S-2HG show that both can bind to the active site iron, or surrogate nickel, in a bidentate manner (Fig 2).”
    Quote in the paperSubstantial claim
  • Findingown data

    For most 2OG oxygenases tested in vitro, S-2HG was a more potent inhibitor than R-2HG.

    “Of the two 2HG enantiomers, the S-form was the more-potent inhibitor.”
    • Also stated in: § DISCUSSION · plain
      “For most of the 2OG oxygenases tested in vitro, S-2HG was more potent than R-2HG (Table 1).”
    Quote in the paperSubstantial claim
  • Findingown data

    For R-2HG, IC50 values ranged from about 25 μM for JMJD2A to more than 5 mM for the HIF prolyl hydroxylase.

    “Half-maximal inhibitory concentration (IC 50 ) values for the R-form of 2HG varied from approximately 25 lM for the histone N e -lysine demethylase JMJD2A to more than 5 mM for the hypoxia-inducible factor (HIF) prolyl hydroxylase.”
    Quote in the paperSubstantial claim
  • Findingown data

    In RCC4 cells, S-2HG caused a greater reduction in HIF1α Asn-hydroxylation than R-2HG.

    “Consistent with in vitro data, we observed a clear reduction in HIF1a Asn-hydroxylation, that was greater with S-than R-2HG.”
    Quote in the paperSubstantial claim
  • Findingreasoning

    HIF1α induced by 2HG is not hydroxylated at the Asn site in the C-terminal activation domain.

    “We found this is the case (Fig 3F).”
    Quote in the paperSubstantial claim
  • May combine two claims. 19
  • Classified as probably not a substantial claim. 7

  • May combine two claims.

    Findingown data

    R-2HG weakly inhibited PHD2 and BBOX1, with IC50 values of approximately 7 mM and 13 mM, respectively.

    “By contrast, inhibition of PHD2 and BBOX1 by R-2HG was weak, with IC 50 values of approximately 7 and 13 mM, respectively, consistent with previous work with the PHDs (Mole et al, 2003).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Mole et al, 2003 source

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    The other C1-carboxylate oxygen of 2HG is positioned to hydrogen bond to N205 in FIH or S288 in JMJD2A, and the C5-carboxylate is positioned to hydrogen bond to K214, Y145 and T196 in FIH and to K206 and Y132 in JMJD2A.

    “The other oxygen of the 2HG C1-carboxylate is positioned to hydrogen bond to Nd2 of N205 FIH (Fig 2A,B) or to Og of S288 JMJD2A (Fig 2D,E); the 2HG C5-carboxylate is positioned to hydrogen bond to K214 FIH Nz, Y145 FIH OH and T196 FIH Og1 (Fig 2A,B) and to K206 JMJD2A Nz and Y132 JMJD2A OH (Fig 2D,E), as observed for the 2HG C5-carboxylate.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In VHL-competent cell lines, neither multiple 10 mM additions nor a single 20 mM addition of R-2HG methyl ester induced HIF1α, whereas S-2HG caused modest induction.

    “Neither dose of R-2HG induced HIF1a.Modest induction was observed with S-2HG (Fig 3E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    PHD2 is poorly inhibited in vitro by R-2HG, and the authors could not demonstrate PHD inhibition sufficient to substantially upregulate HIF in cells.

    “Nevertheless, PHD2 is poorly inhibited in vitro by R-2HG (Fig 1; Table 1), and we were unable to demonstrate inhibition of the PHDs sufficient to substantially upregulate HIF in cells (Fig 3C-E).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Under standard conditions, neither R-2HG nor S-2HG was converted to 2OG by PHD2 or FIH, and neither was a cosubstrate for PHD2.

    “We investigated whether R-or S-2HG is oxidized to 2OG by PHD2 and FIH; in our standard conditions, we did not find conversion of 2HG to 2OG by NMR analysis (data not shown).Neither R-nor S-2HG was a cosubstrate for PHD2 (supplementary Fig S1 online).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    At high concentrations, both R-2HG and S-2HG inhibited JMJD2A in HeLa cells in a dose-dependent manner, with similar extent of inhibition for the two enantiomers.

    “At high concentrations (8 Â 5 mM or 10-mM additions at 30-min intervals), both enantiomers inhibited JMJD2A in a dose-dependent manner (Fig 3B); consistent with the in vitro data, the extent of inhibition was similar for S”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The cell-penetrating ester forms of R-2HG and S-2HG are hydrolysed inside cells to deliver sufficient 2HG to inhibit JMJD2A.

  • May combine two claims.

    Findingown data

    Both R-2HG and S-2HG coordinate the metal with the 2-hydroxyl group trans to D201 in FIH or E190 in JMJD2A, and one oxygen of the C1-carboxylate trans to H199 in FIH or H188 in JMJD2A.

    “Similarly to 2OG and NOG (Elkins et al, 2003;Ng et al, 2007), both R-and S-2HG coordinate the metal with the 2-hydroxyl group trans to D201 FIH /E190 JMJD2A and one oxygen of the the C1-carboxylate trans to H199 FIH /H188 JMJD2A (Fig 2A,B,D,E).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites Elkins et al, 2003 source

      Not checked in the free run.

    • cites Ng et al, 2007

      Not checked in the free run.

  • May combine two claims.

    Findingown data

    Only JMJD2A and JMJD2C showed similar potency for both enantiomers, and JMJD2A was the only enzyme tested for which R-2HG had potency similar to NOG.

    “Only with the histone N e -methyl lysine demethylases JMJD2A/C were both enantiomers of similar potency, and of the enzymes tested, JMJD2A was the only one for which R-2HG showed similar potency to NOG, a 'generic' 2OG oxygenase inhibitor.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    At the same doses that inhibited JMJD2A, R-2HG caused no inhibition and S-2HG caused only minimal inhibition of HIF Pro-hydroxylation.

    “In contrast to these results and consistent with the in vitro data, we observed no R-2HG and only minimal S-2HG inhibition of Pro-hydroxylation, when using the same doses at which JMJD2A was inhibited (exposure to higher 2HG doses was toxic).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both R-2HG and S-2HG inhibit 2OG-dependent oxygenases, but with different potencies.

    “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.”
    • Also stated in: § RESULTS · plain
      “these results show that both R-and S-2HG inhibit the 2OG oxygenases, but with different potencies.”
    • Also stated in: § DISCUSSION · hedged
      “Overall, our results demonstrate that R-and S-2HG have the potential to inhibit a range of human 2OG oxygenases”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    R-2HG most significantly inhibited JMJD2A, with an IC50 of 24 μM, similar to the 17 μM observed for NOG.

    “For R-2HG, the most significant inhibition was of JMJD2A, for which the IC 50 (24 mM) was similar to that observed for NOG (17 mM).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    The lack of a substantial IC50 increase at higher iron concentrations for JMJD2A and BBOX1, together with different values for the two enantiomers, implies that inhibition is not predominantly due to iron sequestration.

    “The lack of a substantial increase in IC 50 values at higher (10-fold) iron concentrations (for JMJD2A and BBOX-1), and different values for the two enantiomers imply that the mode of inhibition is not predominantly iron sequestration (Table 1).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    2HG, and in particular the R-enantiomer produced by IDH1 mutants, is probably an ineffective inhibitor of HIF Pro-hydroxylation and would be relatively ineffective at inducing HIF1α.

    “These results suggest that 2HG-and in particular the R-enantiomer produced by IDH1 mutants (Dang et al, 2009)-is probably an ineffective inhibitor of HIF Pro-hydroxylation, and predict that it would be relatively ineffective in inducing HIF1a.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Dang et al, 2009 source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The crystallographic analyses indicate that both R-2HG and S-2HG have the potential to bind active-site Fe(II) and compete with 2OG for binding.

    “Overall, the crystallographic analyses indicte that, similarly to succinate and fumarate (Fig 2C,F; Hewitson et al, 2007), both R-and S-2HG have the potential to bind to the active site Fe(II) and compete with 2OG for binding.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Hewitson et al, 2007 source

      Not checked in the free run.

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    HIF hydroxylases are Fe(II)- and 2OG-dependent oxygenases that produce succinate and CO2; PHD/EGLN enzymes signal HIF degradation, while FIH is an asparaginyl hydroxylase that reduces HIF activity.

    “The HIF hydroxylases are Fe(II)-and 2OG-dependent oxygenases that produce succinate and CO 2 as coproducts; the HIF prolyl hydroxylases (PHD/EGLN enzymes) signal for HIF degradation, whereas factor inhibiting-HIF (FIH) is an asparaginyl hydroxylase that reduces HIF activity (Kaelin & Ratcliffe, 2008).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Kaelin & Ratcliffe, 2008 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationcitation

    Inhibition of Asn-hydroxylation in the C-terminal transactivation domain of HIFα might enhance transcriptional activity through recruitment of p300/CBP coactivators.

    “Inhibition of Asn-hydroxylation in the C-terminal transactivation domain of HIFa polypeptides might be predicted to enhance transcriptional activity through the recruitment of p300/ CBP coactivators to the C-terminal activation domain (Kaelin & Ratcliffe, 2008).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Kaelin & Ratcliffe, 2008 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All assays were performed in triplicate and are shown as averages with standard error of the mean.

    “All assays were performed in triplicate and are shown as averages with s.e.m.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    HIF is stabilized in IDH-associated gliomas, and this has been proposed to result from intracellular 2OG depletion following IDH loss of function.

    “HIF is stabilized in IDH-associated gliomas; this has been proposed to be a consequence of intracellular depletion of 2OG following IDH loss of function (Zhao et al, 2009).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Zhao et al, 2009 source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Interpretationreasoning

    Because HIF transcriptional target genes differ in their dependence on this domain, such an action might affect hypoxia pathways.

    “As HIF transcriptional target genes are differently dependent on the activity of this domain, such an action might affect hypoxia pathways.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Data were normalized by setting DMSO-treated JMJD2A-transfected cells to 100% activity and a catalytically inactive JMJD2A H188A mutant to 0% activity.

    “Data were normalized by setting DMSO-treated JMJD2A-transfected cells to 100% activity and a catalytically inactive JMJD2A H188A mutant to 0% activity.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The JMJD2A H188A mutant is catalytically inactive and therefore provides a valid 0% activity baseline.

  • Classified as probably not a substantial claim.

    Premisecitation

    Other 2OG oxygenases, including JmjC histone demethylases, are associated with cancer.

    “Other 2OG oxygenases are associated with cancer, including the JmjC histone demethylases (Varier & Timmers, 2010).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Varier & Timmers, 2010

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    The results indicate that 2HG is a relatively poor inhibitor of HIF hydroxylases compared with 2OG-dependent histone lysyl demethylases, suggesting that candidate oncogenic pathways involving 2HG elevation might involve chromatin modifications.

    “The results indicate that 2HG is a relatively poor inhibitor of the HIF hydroxylases in comparison to the 2OG-dependent histone lysyl demethylases, suggesting that candidate oncogenic pathways involving 2HG elevation might involve chromatin modifications.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    IDH mutations occur at substrate-binding arginine residues and inactivate IDH for conversion of isocitrate to 2OG.

    “The IDH mutations occur at substrate-binding arginine residues (Arg 132 in IDH1; Arg 140 and Arg 172 in IDH2) and inactivate IDH for conversion of isocitrate to 2-oxoglutarate (2OG; Zhao et al, 2009).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Zhao et al, 2009 source

      Not checked in the free run.

23 of 23

  • The paper relies on this without support.

    The cell-penetrating ester forms of R-2HG and S-2HG are hydrolysed inside cells to deliver sufficient 2HG to inhibit JMJD2A.

    “A cell-penetrating ester form of R-/S-2HG ABH2, AlkB homologue 2; BBOX1, g-butyrobetaine hydroxylase 1; FIH, factor inhibiting-hypoxia-inducible factor; HIF, hypoxia-inducible factor; NOG, N-oxalylglycine; 2OG, 2-oxoglutarate; PHD2, prolyl hydroxylase 2; R-2HG, R-enantiomer of 2-hydroxyglutarate; IC 50 , half-maximal inhibitory concentration.”
    “At high concentrations (8 Â 5 mM or 10-mM additions at 30-min intervals), both enantiomers inhibited JMJD2A in a dose-dependent manner (Fig 3B); consistent with the in vitro data, the extent of inhibition was similar for S”

  • The paper relies on this without support.

    IC50 values obtained under different assay conditions for different enzymes can be compared to rank their relative potency.

    “Although care must be taken in comparing IC 50 values determined with different assay conditions for the various oxygenases, these results show that both R-and S-2HG inhibit the 2OG oxygenases, but with different potencies.”

  • The paper relies on this without support.

    The JMJD2A H188A mutant is catalytically inactive and therefore provides a valid 0% activity baseline.

    “Data were normalized by setting DMSO-treated JMJD2A-transfected cells to 100% activity and a catalytically inactive JMJD2A H188A mutant to 0% activity.”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • D-2HG can accumulate to millimolar levels in cancer cells expressing mutant IDH and at these concentrations inhibits alpha-ketoglutarate-dependent dioxygenases, including enzymes involved in histone and DNA demethylation.

    “Though it is found at low levels in normal cells, D-2HG can accumulate to millimolar levels in cancer cells expressing mutant IDH. At these high concentrations, D-2HG can inhibit αKG-dependent dioxygenases, including enzymes involved in histone and DNA demethylation (Chowdhury et al., 2011; Janke et al., 2017; Koivunen et al., 2012; Xu et al., 2011).”

    From Targeting Metabolism for Cancer Therapy (2017) · cites this paper as Chowdhury et al., 2011

  • Intracellular 2-oxoglutarate levels are estimated to be in the low millimolar range, well above the Km values of JmjC histone demethylases and TET proteins determined to date.

    “Intracellular 2-oxoglutarate levels are estimated to be in the low millimolar range, well above the 2-oxoglutarate Km values of the JmjC histone demethylases and TET proteins determined to date (Chowdhury et al., 2011; Pritchard, 1995).”

    From Influence of Metabolism on Epigenetics and Disease (2013) · cites this paper as Chowdhury et al., 2011

  • R-2HG, succinate, and fumarate can inhibit multiple 2-oxoglutarate-dependent dioxygenases, including JmjC histone demethylases and TET proteins, at sufficiently high concentrations.

    “R-2HG, succinate, and fumarate are all capable of inhibiting multiple 2-oxoglutarate-dependent dioxygenases, including the JmjC histone demethylases and TET proteins, when present at sufficiently high concentrations (Figure 6B) (Cervera et al., 2009; Chowdhury et al., 2011; Figueroa et al., 2010; Koivunen et al., 2012; Lu et al., 2012; Smith et al., 2007; Turcan et al., 2012; Xiao et al., 2012; Xu”

    From Influence of Metabolism on Epigenetics and Disease (2013) · cites this paper as Chowdhury et al., 2011

  • The weight of evidence suggests that regulation of HIF-1 stability through decreased PHD2 activity is not the primary effect of IDH mutations.

    “The weight of the evidence now suggests that regulation of HIF-1 stability through decreased PHD2 activity is not the primary effect of IDH mutations (Chowdhury et al., 2011; Dang et al., 2009; Jin et al., 2011; Mardis et al., 2009; Metellus et al., 2011; Williams et al., 2011).”

    From Metabolic Reprogramming: A Cancer Hallmark Even Warburg Did Not Anticipate (2012) · cites this paper as Chowdhury et al., 2011

  • TH17 cell differentiation can be initiated by exogenous addition of D-2-HG to naïve CD4+ T cells or by genetic silencing of TET1 and TET2.

    “Furthermore, TH17 cell differentiation could be initiated by exogenous addition of D-2-HG to naïve CD4+ T cells or by genetic silencing of TET1 and TET2, established targets of 2-HG143-145.”

    From Coupling Krebs cycle metabolites to signalling in immunity and cancer (2018) · cites this paper as [144]

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Authors
Chowdhury, R., Yeoh, K. K., Tian, Y., Hillringhaus, L., Bagg, E. A., Rose, N. R., Leung, I. K. H., Li, X. S., Woon, E. C. Y., Yang, M., McDonough, M. A., King, O. N., Clifton, I. J., Klose, R. J., Claridge, T. D. W., Ratcliffe, P. J., Schofield, C. J., & Kawamura, A.
Affiliations
University of Oxford (GB)
Corresponding author
  • Schofield, Christopher J
  • Kawamura, Akane
ORCID
Venue
The EMBO Reports
Publisher
Springer Science and Business Media LLC
Published
2011-04-01
Volume
12
Issue
5
Pages
463-469
ISSN
  • 1469-221X (ISSN-L)
  • 1469-3178
DOI
10.1038/embor.2011.43
PMC
PMC3090014
OpenAlex
W2015755329
Semantic Scholar
e6bd34bdf5282202be386b2f353e072be7387c86
Cited by
1,035
References
31
Citations by year
2026: 38 · 2025: 55 · 2024: 63 · 2023: 64 · 2022: 56 · 2021: 98 · 2020: 79 · 2019: 78 · 2018: 76 · 2017: 69 · 2016: 74 · 2015: 73 · 2014: 70 · 2013: 62 · 2012: 64
FWCI
23.23
Type
journal article
Language
en
Open access
Licence
Locations
  • EMBO Reports (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Wellcome Trust
  • Deutscher Akademischer Austauschdienst
  • Directorate for Biological Sciences
  • Biotechnology and Biological Sciences Research Council
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Cancer, Hypoxia, and Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Health Sciences › Medicine › Genetics › Glioma Diagnosis and Treatment
Keywords
2-hydroxyglutarate, oncometabolites, IDH mutation, histone lysine demethylases, JMJD2A, 2-oxoglutarate-dependent dioxygenase, HIF prolyl hydroxylase, histone methylation, enzyme inhibition
MeSH terms
Crystallography, Glutarates, Humans, Mixed Function Oxygenases, Isocitrate Dehydrogenase, Models, Molecular, Mutation, Neoplasms, Magnetic Resonance Spectroscopy, Procollagen-Proline Dioxygenase, Repressor Proteins, Mass Spectrometry, Signal Transduction, Inhibitory Concentration 50, Cell Line, Tumor, Histone Demethylases, Jumonji Domain-Containing Histone Demethylases

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