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

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.