2-hydroxyglutarate drives whole-genome hypermethylation in kidney cancer cells with inactivated VHL

Hypermethylation of tumor suppressors and other aberrations of DNA methylation in tumors play a significant role in cancer progression. DNA methylation can be affected by various environmental conditions including hypoxia. The response to hypoxia is mainly achieved through activation of the transcription program associated with HIF1a transcription factor. VHL inactivation by genetic or epigenetic events, which also induces aberrant activation of HIF1a, is the most common driver event for renal cancer. With whole-genome bisulfite sequencing and LC-MS, we demonstrated that VHL inactivation induced global genome hypermethylation in human kidney cancer cells under normoxic conditions. This effect was reverted by exogenous expression of wild-type VHL. We show that global genome hypermethylation in VHL mutants can be explained by the accumulation of 2-hydroxyglutarate -- a metabolite that inhibits DNA demethylation by Tet enzymes. We profiled genome-wide DNA methylation with whole-genome bisulfite sequencing (wgBS) in three clones with independent VHL inactivation and observed global genome hypermethylation. Reexpression of exogenous wild-type VHL has partially rescued DNA methylation phenotype to wild-type state.Hypermethylawe profiled genome-wide DNA methylation with bisulfite sequencing (wgBS) in three clones with independent VHL inactivation and observed global genome hypermethylation. Reexpression of exogenous wild-type VHL has partially rescued DNA methylation phenotype to wild-type state. tion of tumor suppressors and other aberrations of DNA methylation in tumors play a significant role in cancer progression. DNA methylation can be affected by various environmental conditions including hypoxia. The response to hypoxia is mainly achieved through activation of the transcription program associated with HIF1a transcription factor. VHL inactivation by genetic or epigenetic events, which also induces aberrant activation of HIF1a, is the most common driver event for renal cancer. With whole-genome bisulfite sequencing and LC-MS, we demonstrated that VHL inactivation induced global genome hypermethylation in human kidney cancer cells under normoxic conditions. This effect was reverted by exogenous expression of wild-type VHL. We show that global genome hypermethylation in VHL mutants can be explained by the accumulation of 2-hydroxyglutarate -- a metabolite that inhibits DNA demethylation by Tet enzymes.

肿瘤抑制基因的高甲基化以及肿瘤中其他DNA甲基化异常在癌症进展中发挥关键作用。DNA甲基化可受包括缺氧在内的多种环境因素影响。机体对缺氧的应答主要通过激活与缺氧诱导因子1α（hypoxia-inducible factor 1α，HIF1α）相关的转录程序实现。由遗传或表观遗传事件导致的VHL失活，同时会诱导HIF1α的异常激活，是肾癌最常见的驱动性事件。我们借助全基因组亚硫酸氢盐测序（whole-genome bisulfite sequencing）与液相色谱-质谱联用法（liquid chromatography-mass spectrometry，LC-MS）证实，常氧条件下VHL失活会诱导人类肾癌细胞产生全基因组高甲基化。外源性表达野生型VHL可逆转这一效应。我们发现，VHL突变体中的全基因组高甲基化可通过2-羟基戊二酸（2-hydroxyglutarate）的积累得到解释——该代谢物能够抑制Tet酶介导的DNA去甲基化。我们通过全基因组亚硫酸氢盐测序（wgBS）对3株独立发生VHL失活的克隆进行了全基因组DNA甲基化谱分析，观察到了全基因组高甲基化现象。外源性表达野生型VHL可部分将DNA甲基化表型恢复至野生型状态。我们对全基因组DNA甲基化进行了谱分析，采用亚硫酸氢盐测序（wgBS）对3株独立发生VHL失活的克隆进行检测，同样观察到全基因组高甲基化现象。外源性表达野生型VHL可部分将DNA甲基化表型恢复至野生型状态。肿瘤抑制基因的高甲基化以及肿瘤中其他DNA甲基化异常在癌症进展中发挥关键作用。DNA甲基化可受包括缺氧在内的多种环境因素影响。机体对缺氧的应答主要通过激活与缺氧诱导因子1α相关的转录程序实现。由遗传或表观遗传事件导致的VHL失活，同时会诱导HIF1α的异常激活，是肾癌最常见的驱动性事件。我们借助全基因组亚硫酸氢盐测序与LC-MS证实，常氧条件下VHL失活会诱导人类肾癌细胞产生全基因组高甲基化。外源性表达野生型VHL可逆转这一效应。我们发现，VHL突变体中的全基因组高甲基化可通过2-羟基戊二酸的积累得到解释——该代谢物能够抑制Tet酶介导的DNA去甲基化。