Supplementary Figure S1: Treatment with octyl-D-2-HG-induces MIR148A methylation; Supplementary Figure S2: Octyl-D-2-HG-induced MIR148A methylation status remains constant at late passages; Supplementary Figure S3: Octyl-D-2-HG induces CpG island methylation of the RBP1 promoter in 293T cells; Supplementary Figure S4: Octyl-D-2-HG induces CpG island methylation of the MIR148A promoter in normal human astrocytes (NHA); Supplementary Figure S5: MIR148A promoter CpG methylation in IDH1MUT-transfected 293T cells; Supplementary Figure S6: C227 prevents IDH1 mutant-induced MIR148A promoter methylation; Supplementary Figure S7: C227 prevents IDH1 mutant-induced RBP1 promoter methylation; Supplementary Figure S8: Schematic graph of MIR148A genomic location, CpG island, and promoter-reporter construct; Supplementary Figure S9: MIR148a expression is unchanged by octyl-D-2-HG treatment in early passages in 293T and NHA cells; Supplementary Figure S10: Octyl-D-2-HG withdrawal restores MIR148A promoter to an unmethylated state
ARTICLE ABSTRACT
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 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. ©2018 AACR.
