Neuro-Oncology Program, Department of Neurology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California.
Department of Pediatrics, Psychiatry and Biobehavioral Sciences, Pediatric Neurology, Semel Institute for Neuroscience and Human Behavior, Molecular & Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, California.
Mol Cancer Res. 2018 Jun;16(6):947-960. doi: 10.1158/1541-7786.MCR-17-0367. Epub 2018 Mar 15.
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. .
突变体异柠檬酸脱氢酶 (IDH) 1/2 将α-酮戊二酸 (α-KG) 转化为 D-2 羟戊二酸 (D-2-HG)，一种潜在的致癌代谢物，可抑制α-KG 依赖性酶，包括 ten-eleven 易位甲基胞嘧啶双加氧酶 (TET) DNA 去甲基酶。我们最近证实，miRNA 是 IDH1 突变相关神经胶质瘤 CpG 岛甲基化表型 (G-CIMP) 的组成部分，并特别确定 MIR148A 是 G-CIMP 内的肿瘤抑制性 miRNA。然而，突变 IDH 诱导 和其他 G-CIMP 启动子过度甲基化的确切机制仍有待阐明。在这项研究中，我们证明外源性 D-2-HG 处理可诱导人胚肾 293T（293T）细胞和原代正常人星形胶质细胞中的 启动子甲基化和转录沉默。相反，我们表明，通过突变 IDH1 生成 D-2-HG 的抑制剂 C227 处理，可消除突变 IDH1 过表达的 293T 细胞中 启动子甲基化的发展。通过点印迹分析进行 5-羟甲基胞嘧啶 (5-hmC) 的全局评估，我们表明 D-2-HG 处理降低 5-hmC 水平，而 C227 处理增加 5-hmC 水平，强烈表明 D-2-HG 抑制 TET。此外，我们表明，D-2-HG 处理的撤回会导致甲基化逆转，并伴有 MIR148A 转录本水平的升高和 5-hmC 的短暂产生。我们还证明 RNA 聚合酶 II 内源性结合到 的预测启动子区域，验证了其转录由独立启动子驱动的假设。将 D-2-HG 确立为突变体 IDH1 诱导 中 CpG 岛甲基化的必要和充分中间产物，将有助于理解选择性突变体 IDH1 抑制剂在临床上的疗效。
