D-2-Hydroxyglutarate Is Necessary and Sufficient for Isocitrate Dehydrogenase 1 Mutant-Induced Promoter Methylation.

Mutant isocitrate dehydrogenase (IDH) 1/2 converts α-ketoglutarate (α-KG) to D-2 hydroxyglutarate (D-2-HG), a putative oncometabolite that can inhibit α-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 and other G-CIMP promoters remains to be elucidated. In this study, we demonstrate that treatment with exogenous D-2-HG induces promoter methylation and transcriptional silencing in human embryonic kidney 293T (293T) cells and primary normal human astrocytes. Conversely, we show that the development of 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 , validating the hypothesis that its transcription is driven by an independent promoter. Establishment of D-2-HG as a necessary and sufficient intermediate by which mutant IDH1 induces CpG island methylation of will help with understanding the efficacy of selective mutant IDH1 inhibitors in the clinic. .