MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG

Abstract

α-Ketoglutarate (αKG) is a key node in many important metabolic pathways. The αKG analog N-oxalylglycine (NOG) and its cell-permeable prodrug dimethyloxalylglycine (DMOG) are extensively used to inhibit αKG-dependent dioxygenases. However, whether NOG interference with other αKG-dependent processes contributes to its mode of action remains poorly understood. Here we show that, in aqueous solutions, DMOG is rapidly hydrolyzed, yielding methyloxalylglycine (MOG). MOG elicits cytotoxicity in a manner that depends on its transport by monocarboxylate transporter 2 (MCT2) and is associated with decreased glutamine-derived tricarboxylic acid–cycle flux, suppressed mitochondrial respiration and decreased ATP production. MCT2-facilitated entry of MOG into cells leads to sufficiently high concentrations of NOG to inhibit multiple enzymes in glutamine metabolism, including glutamate dehydrogenase. These findings reveal that MCT2 dictates the mode of action of NOG by determining its intracellular concentration and have important implications for the use of (D)MOG in studying αKG-dependent signaling and metabolism.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: DMOG induces cytotoxicity that correlates with MCT2 expression and is not explained by differential inhibition of oxygen-sensitive dioxygenases.
Fig. 2: The methyl oxoacetate ester of DMOG is rapidly hydrolyzed to MOG in cell culture medium.
Fig. 3: MOG is sufficient to cause cytotoxicity in an MCT2-dependent manner.
Fig. 4: MOG inhibits glutamine catabolism in an MCT2-dependent manner.
Fig. 5: Evidence that inhibition of GDH-mediated glutamine carbon flux accounts for MOG-induced metabolic changes associated with cytotoxicity.
Fig. 6: NOG binds GDH and inhibits its enzymatic activity.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request. Accession codes and relevant web links can be found in the respective legends and methods sections.

Change history

  • 23 October 2019

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

References

  1. 1.

    Zhang, J., Pavlova, N. N. & Thompson, C. B. Cancer cell metabolism: the essential role of the nonessential amino acid, glutamine. EMBO J. 36, 1302–1315 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  2. 2.

    Altman, B. J., Stine, Z. E. & Dang, C. V. From Krebs to clinic: glutamine metabolism to cancer therapy. Nat. Rev. Cancer 16, 619–634 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. 3.

    Still, E. R. & Yuneva, M. O. Hopefully devoted to Q: targeting glutamine addiction in cancer. Br. J. Cancer 116, 1375–1381 (2017).

    PubMed  PubMed Central  Article  Google Scholar 

  4. 4.

    Zdzisińska, B., Żurek, A. & Kandefer-Szerszeń, M. Alpha-ketoglutarate as a molecule with pleiotropic activity: well-known and novel possibilities of therapeutic use. Arch. Immunol. Ther. Exp. (Warsz.) 65, 21–36 (2017).

    Article  CAS  Google Scholar 

  5. 5.

    Anastasiou, D. & Cantley, L. C. Breathless cancer cells get fat on glutamine. Cell Res. 22, 443–446 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  6. 6.

    Jiang, L. et al. Reductive carboxylation supports redox homeostasis during anchorage-independent growth. Nature 532, 255–258 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7.

    Loenarz, C. & Schofield, C. J. Physiological and biochemical aspects of hydroxylations and demethylations catalyzed by human 2-oxoglutarate oxygenases. Trends Biochem. Sci. 36, 7–18 (2011).

    CAS  PubMed  Article  Google Scholar 

  8. 8.

    Ivan, M. & Kaelin, W. G. Jr. The EGLN-HIF O2-sensing system: multiple inputs and feedbacks. Mol. Cell 66, 772–779 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  9. 9.

    Chan, M. C., Holt-Martyn, J. P., Schofield, C. J. & Ratcliffe, P. J. Pharmacological targeting of the HIF hydroxylases: a new field in medicine development. Mol. Aspects Med. 47-48, 54–75 (2016).

    CAS  PubMed  Article  Google Scholar 

  10. 10.

    Son, J. et al. Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway. Nature 496, 101–105 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  11. 11.

    Jin, L. et al. Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth. Cancer Cell 27, 257–270 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  12. 12.

    Mariño, G. et al. Regulation of autophagy by cytosolic acetyl-coenzyme A. Mol. Cell 53, 710–725 (2014).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  13. 13.

    Taniguchi, C. M. et al. Cross-talk between hypoxia and insulin signaling through Phd3 regulates hepatic glucose and lipid metabolism and ameliorates diabetes. Nat. Med. 19, 1325–1330 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  14. 14.

    Aragonés, J. et al. Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism. Nat. Genet. 40, 170–180 (2008).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  15. 15.

    Olenchock, B. A. et al. EGLN1 inhibition and rerouting of α-ketoglutarate suffice for remote ischemic protection. Cell 164, 884–895 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16.

    Eltzschig, H. K., Bratton, D. L. & Colgan, S. P. Targeting hypoxia signalling for the treatment of ischaemic and inflammatory diseases. Nat. Rev. Drug Discov. 13, 852–869 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  17. 17.

    Rose, N. R., McDonough, M. A., King, O. N., Kawamura, A. & Schofield, C. J. Inhibition of 2-oxoglutarate dependent oxygenases. Chem. Soc. Rev. 40, 4364–4397 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  18. 18.

    Cunliffe, C. J., Franklin, T. J., Hales, N. J. & Hill, G. B. Novel inhibitors of prolyl 4-hydroxylase. 3. Inhibition by the substrate analogue N-oxaloglycine and its derivatives. J. Med. Chem. 35, 2652–2658 (1992).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  19. 19.

    Jaakkola, P. et al. Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation. Science 292, 468–472 (2001).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  20. 20.

    Ivan, M. et al. HIFα targeted for VHL-mediated destruction by proline hydroxylation: implications for O2 sensing. Science 292, 464–468 (2001).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  21. 21.

    Hamada, S. et al. Synthesis and activity of N-oxalylglycine and its derivatives as Jumonji C-domain-containing histone lysine demethylase inhibitors. Bioorg. Med. Chem. Lett. 19, 2852–2855 (2009).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. 22.

    Baader, E., Tschank, G., Baringhaus, K. H., Burghard, H. & Günzler, V. Inhibition of prolyl 4-hydroxylase by oxalyl amino acid derivatives in vitro, in isolated microsomes and in embryonic chicken tissues. Biochem. J. 300, 525–530 (1994).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23.

    Fraisl, P., Aragonés, J. & Carmeliet, P. Inhibition of oxygen sensors as a therapeutic strategy for ischaemic and inflammatory disease. Nat. Rev. Drug Discov. 8, 139–152 (2009).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  24. 24.

    Leite de Oliveira, R. et al. Gene-targeting of Phd2 improves tumor response to chemotherapy and prevents side-toxicity. Cancer Cell 22, 263–277 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  25. 25.

    Fukuda, R. et al. HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells. Cell 129, 111–122 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  26. 26.

    Kim, J. W., Tchernyshyov, I., Semenza, G. L. & Dang, C. V. HIF-1-mediated expression of pyruvate dehydrogenase kinase: a metabolic switch required for cellular adaptation to hypoxia. Cell Metab. 3, 177–185 (2006).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  27. 27.

    Sun, R. C. & Denko, N. C. Hypoxic regulation of glutamine metabolism through HIF1 and SIAH2 supports lipid synthesis that is necessary for tumor growth. Cell Metab. 19, 285–292 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  28. 28.

    Garnett, M. J. et al. Systematic identification of genomic markers of drug sensitivity in cancer cells. Nature 483, 570–575 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  29. 29.

    Halestrap, A. P. The SLC16 gene family: structure, role and regulation in health and disease. Mol. Aspects Med. 34, 337–349 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. 30.

    Pérez-Escuredo, J. et al. Monocarboxylate transporters in the brain and in cancer. Biochim. Biophys. Acta 1863, 2481–2497 (2016).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  31. 31.

    Hudson, R. F. The perturbation treatment of chemical reactivity. Angew. Chem. Int. Edn Engl. 12, 36–56 (1973).

    Article  Google Scholar 

  32. 32.

    Sekine, N. et al. Low lactate dehydrogenase and high mitochondrial glycerol phosphate dehydrogenase in pancreatic beta-cells: potential role in nutrient sensing. J. Biol. Chem. 269, 4895–4902 (1994).

    CAS  PubMed  Google Scholar 

  33. 33.

    Bröer, S. et al. Characterization of the high-affinity monocarboxylate transporter MCT2 in Xenopus laevis oocytes. Biochem. J. 341, 529–535 (1999).

    PubMed  PubMed Central  Google Scholar 

  34. 34.

    Fan, J. et al. Glutamine-driven oxidative phosphorylation is a major ATP source in transformed mammalian cells in both normoxia and hypoxia. Mol. Syst. Biol. 9, 712 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  35. 35.

    Zhdanov, A. V., Okkelman, I. A., Collins, F. W., Melgar, S. & Papkovsky, D. B. A novel effect of DMOG on cell metabolism: direct inhibition of mitochondrial function precedes HIF target gene expression. Biochim. Biophys. Acta 1847, 1254–1266 (2015).

    CAS  PubMed  Article  Google Scholar 

  36. 36.

    Rendina, A. R. et al. Mutant IDH1 enhances the production of 2-hydroxyglutarate due to its kinetic mechanism. Biochemistry 52, 4563–4577 (2013).

    CAS  PubMed  Article  Google Scholar 

  37. 37.

    Kell, D. B. Finding novel pharmaceuticals in the systems biology era using multiple effective drug targets, phenotypic screening and knowledge of transporters: where drug discovery went wrong and how to fix it. FEBS J. 280, 5957–5980 (2013).

    CAS  PubMed  Article  Google Scholar 

  38. 38.

    Gong, L., Goswami, S., Giacomini, K. M., Altman, R. B. & Klein, T. E. Metformin pathways: pharmacokinetics and pharmacodynamics. Pharmacogenet. Genomics 22, 820–827 (2012).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. 39.

    Gan, L. et al. Metabolic targeting of oncogene MYC by selective activation of the proton-coupled monocarboxylate family of transporters. Oncogene 35, 3037–3048 (2016).

    CAS  PubMed  Article  Google Scholar 

  40. 40.

    Pértega-Gomes, N. et al. Monocarboxylate transporter 2 (MCT2) as putative biomarker in prostate cancer. Prostate 73, 763–769 (2013).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  41. 41.

    Christen, S. et al. Breast cancer-derived lung metastases show increased pyruvate carboxylase-dependent anaplerosis. Cell Rep. 17, 837–848 (2016).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  42. 42.

    Avril, N. GLUT1 expression in tissue and (18)F-FDG uptake. J. Nucl. Med. 45, 930–932 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Kulkarni, A. et al. Glucose metabolism and oxygen availability govern reactivation of the latent human retrovirus HTLV-1. Cell Chem. Biol. 24, 1377–1387.e1373 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44.

    Daberkow, R. L., White, B. R., Cederberg, R. A., Griffin, J. B. & Zempleni, J. Monocarboxylate transporter 1 mediates biotin uptake in human peripheral blood mononuclear cells. J. Nutr. 133, 2703–2706 (2003).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  45. 45.

    Overington, J. P., Al-Lazikani, B. & Hopkins, A. L. How many drug targets are there? Nat. Rev. Drug Discov. 5, 993–996 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  46. 46.

    Hopkins, A. L., Mason, J. S. & Overington, J. P. Can we rationally design promiscuous drugs? Curr. Opin. Struct. Biol. 16, 127–136 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  47. 47.

    Knight, Z. A., Lin, H. & Shokat, K. M. Targeting the cancer kinome through polypharmacology. Nat. Rev. Cancer 10, 130–137 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  48. 48.

    Zhang, J. et al. EglN2 associates with the NRF1-PGC1α complex and controls mitochondrial function in breast cancer. EMBO J. 34, 2953–2970 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  49. 49.

    Papandreou, I., Cairns, R. A., Fontana, L., Lim, A. L. & Denko, N. C. HIF-1 mediates adaptation to hypoxia by actively downregulating mitochondrial oxygen consumption. Cell Metab. 3, 187–197 (2006).

    CAS  PubMed  Article  Google Scholar 

  50. 50.

    Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  51. 51.

    Starck, S. R., Green, H. M., Alberola-Ila, J. & Roberts, R. W. A general approach to detect protein expression in vivo using fluorescent puromycin conjugates. Chem. Biol. 11, 999–1008 (2004).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  52. 52.

    Allen, A., Kwagh, J., Fang, J., Stanley, C. A. & Smith, T. J. Evolution of glutamate dehydrogenase regulation of insulin homeostasis is an example of molecular exaptation. Biochemistry 43, 14431–14443 (2004).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  53. 53.

    Frezza, C., Cipolat, S. & Scorrano, L. Organelle isolation: functional mitochondria from mouse liver, muscle and cultured fibroblasts. Nat. Protoc. 2, 287–295 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  54. 54.

    Zhang, T., Creek, D. J., Barrett, M. P., Blackburn, G. & Watson, D. G. Evaluation of coupling reversed phase, aqueous normal phase, and hydrophilic interaction liquid chromatography with Orbitrap mass spectrometry for metabolomic studies of human urine. Anal. Chem. 84, 1994–2001 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  55. 55.

    Schleucher, J. et al. A general enhancement scheme in heteronuclear multidimensional NMR employing pulsed field gradients. J. Biomol. NMR 4, 301–306 (1994).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  56. 56.

    Bax, A. & Summers, M. F. H-1 and C-13 assignments from sensitivity-enhanced detection of heteronuclear multiple-bond connectivity by 2D multiple quantum NMR. J. Am. Chem. Soc. 108, 2093–2094 (1986).

    CAS  Article  Google Scholar 

  57. 57.

    Hwang, T. L. & Shaka, A. J. Water suppression that works: excitation sculpting using arbitrary wave-forms and pulsed-field gradients. J. Magn. Reson. 112, 275–279 (1995).

    CAS  Article  Google Scholar 

  58. 58.

    Dalvit, C., Fogliatto, G., Stewart, A., Veronesi, M. & Stockman, B. WaterLOGSY as a method for primary NMR screening: practical aspects and range of applicability. J. Biomol. NMR 21, 349–359 (2001).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  59. 59.

    London, R. E. & Gabel, S. A. Determination of membrane potential and cell volume by 19F NMR using trifluoroacetate and trifluoroacetamide probes. Biochemistry 28, 2378–2382 (1989).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank all members of the laboratory of D.A. for valuable discussions and input throughout this work, particularly J. Macpherson and N. Bevan for technical help. We are grateful to L. Cantley for advice during early stages of this work. We acknowledge S. O’Callaghan (Bio21 Institute, University of Melbourne) for the algorithm to correct for natural isotope abundance in metabolomics data. We thank J. Kleinjung for advice with statistical methods, M. Howell for advice and help with cell proliferation and viability measurements, J. Cerveira for help with flow cytometry measurements and A. Gould for comments on the manuscript. We are grateful to the staff at the Medical Research Council National Biomedical NMR Centre at the Francis Crick Institute, where NMR data were obtained. This work was funded by the MRC (MC_UP_1202/1) and by the Francis Crick Institute, which receives its core funding from Cancer Research UK (FC001033), the UK Medical Research Council (FC001033) and the Wellcome Trust (FC001033) to D.A.

Author information

Affiliations

Authors

Contributions

P.C.D. and T.J.R. performed NMR experiments; F.G. generated HIF1α-mutant cell lines and wrote scripts for metabolomics data analysis and visualization; A.J. and P.M.N. performed respiration experiments; M.G. assisted with the development of LC–MS analytical methods; G.D. generated and characterized cell lines; M.S.d.S. and J.I.M. assisted with and advised on metabolomics experiments; A.J.W. did flux modeling; G.P. and N.O’R. synthesized DM-[13C5]αKG; S.C. and D.H. synthesized MOG and advised on chemistry; C.H.B. contributed to the large-scale DMOG sensitivity screen; K.D.C. performed experiments and analyzed data; and L.F. and D.A. designed and performed experiments, analyzed data and wrote the manuscript. All authors reviewed and commented on the manuscript. Work by A.J. was supported by the MetaRNA Marie Skłodowska-Curie Innovative Training Nerwork (642738).

Corresponding author

Correspondence to Dimitrios Anastasiou.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1 and 2, and Supplementary Figures 1–13

Reporting Summary

Supplementary Note

Synthetic Procedures

Supplementary Dataset 1

Spearman’s rank correlation coefficients of gene transcripts with IC50DMOG, determined as described in Methods. Genes identified as positively or negatively correlating with sensitivity (as determined by a 5% FDR) are highlighted.

Supplementary Dataset 2

Spearman’s rank correlation coefficients of gene transcripts with IC50DMOG, using only the top quartile of SLC16A7-expressing cell lines (213 lines) used in Fig. 6e, f. Genes identified as positively or negatively correlating with sensitivity (as determined by a 5% FDR) are highlighted.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Fets, L., Driscoll, P.C., Grimm, F. et al. MCT2 mediates concentration-dependent inhibition of glutamine metabolism by MOG. Nat Chem Biol 14, 1032–1042 (2018). https://doi.org/10.1038/s41589-018-0136-y

Download citation

Further reading

Search

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing