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Quantitative metabolome analysis profiles activation of glutaminolysis in glioma with IDH1 mutation

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Tumor Biology

Abstract

Isocitrate dehydrogenase 1 (IDH1), which localizes to the cytosol and peroxisomes, catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate (α-KG) and in parallel converts NADP+ to NADPH. IDH1 mutations are frequently detected in grades 2–4 gliomas and in acute myeloid leukemias (AML). Mutations of IDH1 have been identified at codon 132, with arginine being replaced with histidine in most cases. Mutant IDH1 gains novel enzyme activity converting α-KG to d-2-hydroxyglutarate (2-HG) which acts as a competitive inhibitor of α-KG. As a result, the activity of α-KG-dependent enzyme is reduced. Based on these findings, 2-HG has been proposed to be an oncometabolite. In this study, we established HEK293 and U87 cells that stably expressed IDH1-WT and IDH1-R132H and investigated the effect of glutaminase inhibition on cell proliferation with 6-diazo-5-oxo-l-norleucine (DON). We found that cell proliferation was suppressed in IDH1-R132H cells. The addition of α-KG restored cell proliferation. The metabolic features of 33 gliomas with wild type IDH1 (IDH1-WT) and with IDH1-R132H mutation were examined by global metabolome analysis using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). We showed that the 2-HG levels were highly elevated in gliomas with IDH1-R132H mutation. Intriguingly, in gliomas with IDH1-R132H, glutamine and glutamate levels were significantly reduced which implies replenishment of α-KG by glutaminolysis. Based on these results, we concluded that glutaminolysis is activated in gliomas with IDH1-R132H mutation and that development of novel therapeutic approaches targeting activated glutaminolysis is warranted.

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Acknowledgements

This work was supported in part by the Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Authors and Affiliations

  1. Department of Neurosurgery, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, 466-8550, Japan

    Fumiharu Ohka, Maki Ito, Melissa Ranjit, Ayako Motomura, Kazuya Motomura, Toshihiko Wakabayashi & Atsushi Natsume

  2. Division of Cancer Biology, Nagoya University School of Medicine, Nagoya, Japan

    Takeshi Senga

  3. Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan

    Yukinari Kato

  4. Institute for Advanced Bioscience, Keio University, Yamagata, Japan

    Kaori Saito, Keiko Kato & Tomoyoshi Soga

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  1. Fumiharu Ohka
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Correspondence to Fumiharu Ohka.

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Fumiharu Ohka and Maki Ito equally contributed to this work

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Supplemental Figure 1

Microscopic imaging of cells expressing IDH1-WT or IDH1-R132H We generate HEK293 and U87 cells, expressing IDH1-WT or IDH1-R132H fused with GFP protein. In both cell lines, exogenous IDH1-WT and IDH1-R132H with GFP are located in the cytoplasm. (PPTX 2432 kb)

Supplemental Figure 2

Expression of endogenous IDH2 in cells expressing IDH1-WT or IDH1-R132H We detected expression of endogenous IDH2 expressing IDH1-WT or IDH1-R132H cells, compared with those expressions of β-actin. (PPTX 42260 kb)

Supplemental Figure 3

Cell growth rate of cells expressing IDH1-WT or IDH1-R132H in normal condition MTT assay revealed that cell growth rate of control cells, IDH1-WT cells and IDH1-R132H cells are almost equal in HEK293 and U87. (PPTX 80 kb)

Supplemental Figure 4

Restoration of cell viability by adding α-KG in glutamine deprivation condition In glutamine free medium, we cultured cells for 72 h with or without addition of 1 mM α-KG. Addition of α-KG, in glutamine deprivation condition, significantly helped to recover the cell viability of IDH1-R132H cells compared to that of IDH1-WT cells in U87 cell lines. (*: p < 0.05) (PPTX 119 kb)

Supplemental Figure 5

Analysis of α-KG level in U87 cells expressing IDH1-WT or IDH1-R132H Using CE-TOFMS, we analyzed α-KG level in U87 cells expressing IDH1-WT or IDH1-R132H. α-KG level in IDH1-R132H cells are significantly decreased, compared with those of IDH1-WT cells. (*: p < 0.05) (PPTX 55 kb)

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Ohka, F., Ito, M., Ranjit, M. et al. Quantitative metabolome analysis profiles activation of glutaminolysis in glioma with IDH1 mutation. Tumor Biol. 35, 5911–5920 (2014). https://doi.org/10.1007/s13277-014-1784-5

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