D-2-hydroxyglutarate Is necessary and sufficient for isocitrate dehydrogenase 1 mutant-induced MIR148A promoter methylation

Tie Li, Christopher D. Cox, Byram Ozer, Nhung T. Nguyen, Huytram N. Nguyen, Thomas J. Lai, Sichen Li, Fei Liu, Harley I. Kornblum, Linda M. Liau, Phioanh L. Nghiemphu, Timothy F. Cloughesy, Albert Lai

Research output: Contribution to journalArticle

Abstract

Mutant isocitrate dehydrogenase (IDH) 1/2 converts a-ketoglutarate (a-KG) to D-2 hydroxyglutarate (D-2-HG), a putative oncometabolite that can inhibit a-KG-dependent enzymes, including ten-eleven translocation methylcytosine dioxygenase (TET) DNA demethylases. We recently established that miRNAs are components of the IDH1 mutant-associated glioma CpG island methylator phenotype (G-CIMP) and specifically identified MIR148A as a tumor-suppressive miRNA within G-CIMP. However, the precise mechanism by which mutant IDH induces hypermethylation of MIR148A and other G-CIMP promoters remains to be elucidated. In this study, we demonstrate that treatment with exogenous D-2-HG induces MIR148A promoter methylation and transcriptional silencing in human embryonic kidney 293T (293T) cells and primary normal human astrocytes. Conversely, we show that the development of MIR148A promoter methylation in mutant IDH1-overexpressing 293T cells is abrogated via treatment with C227, an inhibitor of mutant IDH1 generation of D-2-HG. Using dot blot assays for global assessment of 5-hydroxymethylcytosine (5-hmC), we show that D-2-HG treatment reduces 5-hmC levels, whereas C227 treatment increases 5-hmC levels, strongly suggesting TET inhibition by D-2-HG. Moreover, we show that withdrawal of D-2-HG treatment reverses methylation with an associated increase in MIR148A transcript levels and transient generation of 5-hmC. We also demonstrate that RNA polymerase II binds endogenously to the predicted promoter region of MIR148A, validating the hypothesis that its transcription is driven by an independent promoter. Implications: Establishment of D-2-HG as a necessary and sufficient intermediate by which mutant IDH1 induces CpG island methylation of MIR148A will help with understanding the efficacy of selective mutant IDH1 inhibitors in the clinic. Mol Cancer Res; 16(6); 947-60.

Original languageEnglish (US)
Pages (from-to)947-960
Number of pages14
JournalMolecular Cancer Research
Volume16
Issue number6
DOIs
StatePublished - Jun 1 2018
Externally publishedYes

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Isocitrate Dehydrogenase
Methylation
CpG Islands
Glioma
HEK293 Cells
MicroRNAs
Phenotype
Dioxygenases
RNA Polymerase II
alpha-hydroxyglutarate
Genetic Promoter Regions
Astrocytes
Neoplasms
Kidney
5-hydroxymethylcytosine
DNA
Enzymes

ASJC Scopus subject areas

  • Molecular Biology
  • Oncology
  • Cancer Research

Cite this

D-2-hydroxyglutarate Is necessary and sufficient for isocitrate dehydrogenase 1 mutant-induced MIR148A promoter methylation. / Li, Tie; Cox, Christopher D.; Ozer, Byram; Nguyen, Nhung T.; Nguyen, Huytram N.; Lai, Thomas J.; Li, Sichen; Liu, Fei; Kornblum, Harley I.; Liau, Linda M.; Nghiemphu, Phioanh L.; Cloughesy, Timothy F.; Lai, Albert.

In: Molecular Cancer Research, Vol. 16, No. 6, 01.06.2018, p. 947-960.

Research output: Contribution to journalArticle

Li, T, Cox, CD, Ozer, B, Nguyen, NT, Nguyen, HN, Lai, TJ, Li, S, Liu, F, Kornblum, HI, Liau, LM, Nghiemphu, PL, Cloughesy, TF & Lai, A 2018, 'D-2-hydroxyglutarate Is necessary and sufficient for isocitrate dehydrogenase 1 mutant-induced MIR148A promoter methylation', Molecular Cancer Research, vol. 16, no. 6, pp. 947-960. https://doi.org/10.1158/1541-7786.MCR-17-0367
Li, Tie ; Cox, Christopher D. ; Ozer, Byram ; Nguyen, Nhung T. ; Nguyen, Huytram N. ; Lai, Thomas J. ; Li, Sichen ; Liu, Fei ; Kornblum, Harley I. ; Liau, Linda M. ; Nghiemphu, Phioanh L. ; Cloughesy, Timothy F. ; Lai, Albert. / D-2-hydroxyglutarate Is necessary and sufficient for isocitrate dehydrogenase 1 mutant-induced MIR148A promoter methylation. In: Molecular Cancer Research. 2018 ; Vol. 16, No. 6. pp. 947-960.
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abstract = "Mutant isocitrate dehydrogenase (IDH) 1/2 converts a-ketoglutarate (a-KG) to D-2 hydroxyglutarate (D-2-HG), a putative oncometabolite that can inhibit a-KG-dependent enzymes, including ten-eleven translocation methylcytosine dioxygenase (TET) DNA demethylases. We recently established that miRNAs are components of the IDH1 mutant-associated glioma CpG island methylator phenotype (G-CIMP) and specifically identified MIR148A as a tumor-suppressive miRNA within G-CIMP. However, the precise mechanism by which mutant IDH induces hypermethylation of MIR148A and other G-CIMP promoters remains to be elucidated. In this study, we demonstrate that treatment with exogenous D-2-HG induces MIR148A promoter methylation and transcriptional silencing in human embryonic kidney 293T (293T) cells and primary normal human astrocytes. Conversely, we show that the development of MIR148A promoter methylation in mutant IDH1-overexpressing 293T cells is abrogated via treatment with C227, an inhibitor of mutant IDH1 generation of D-2-HG. Using dot blot assays for global assessment of 5-hydroxymethylcytosine (5-hmC), we show that D-2-HG treatment reduces 5-hmC levels, whereas C227 treatment increases 5-hmC levels, strongly suggesting TET inhibition by D-2-HG. Moreover, we show that withdrawal of D-2-HG treatment reverses methylation with an associated increase in MIR148A transcript levels and transient generation of 5-hmC. We also demonstrate that RNA polymerase II binds endogenously to the predicted promoter region of MIR148A, validating the hypothesis that its transcription is driven by an independent promoter. Implications: Establishment of D-2-HG as a necessary and sufficient intermediate by which mutant IDH1 induces CpG island methylation of MIR148A will help with understanding the efficacy of selective mutant IDH1 inhibitors in the clinic. Mol Cancer Res; 16(6); 947-60.",
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T1 - D-2-hydroxyglutarate Is necessary and sufficient for isocitrate dehydrogenase 1 mutant-induced MIR148A promoter methylation

AU - Li, Tie

AU - Cox, Christopher D.

AU - Ozer, Byram

AU - Nguyen, Nhung T.

AU - Nguyen, Huytram N.

AU - Lai, Thomas J.

AU - Li, Sichen

AU - Liu, Fei

AU - Kornblum, Harley I.

AU - Liau, Linda M.

AU - Nghiemphu, Phioanh L.

AU - Cloughesy, Timothy F.

AU - Lai, Albert

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