An IDH-independent mechanism of DNA hypermethylation upon VHL inactivation in cancer

Abstract

Hypermethylation of tumour suppressors and other aberrations of DNA methylation in tumours 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 transcriptional program associated with HIF1A transcription factor. Inactivation of Von Hippel-Lindau Tumour Suppressor gene (VHL) by genetic or epigenetic events, which also induces aberrant activation of HIF1A, is the most common driver event for renal cancer. With whole-genome bisulphite 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 showed that global genome hypermethylation in VHL mutants can be explained by transcriptional changes in MDH and L2HGDH genes that cause the accumulation of 2-hydroxyglutarate - a metabolite that inhibits DNA demethylation by TET enzymes. Unlike the known cases of DNA hypermethylation in cancer, 2-hydroxyglutarate was accumulated in the cells with the wild-type isocitrate dehydrogenases.

Keywords: DNA methylation; HIF1A; VHL; hypoxia; kidney cancer.

Figure 1.

Whole-genome methylation in Caki-1 cells, three clones with inactivated VHL and the same clones with VHL expression rescued by exogenous construct. (a). Design of the experiment. VHL was inactivated independently in three clones of Caki-1 human kidney cancer cell line. In each clone, the mutation was rescued by exogenous expression of VHL. DNA methylation and metabolites were profiled in these cells. (b). Global DNA methylation levels estimated from bisulphite sequencing data. (c). Global DNA methylation levels measured by mass-spectrometry. (d). Global DNA hydroxymethylation (hmC) measured with mass-spectrometry. (e). For each transcription factor, we estimated the fraction of hypermethylated sites among all CpG sites with significantly altered methylation. (f). DNA methylation changes in CpGs with significant difference between wild-type Caki-1 cells and the clones with inactivated VHL. G. While all transcription factors tend to be hypermethylated in VHL mutants, HIF1A sites to a great extent avoid hypermethylation.

Figure 2.

(a). Changes of gene expressions of MDH and L2HGDH enzymes responsible for 2-hydroxyglutarate synthesis and degradation. Y-axis indicates log fold-change of gene expression in VHL mutants, rescued VHL mutants with exogenic expression of VHL and Caki-1 cells in hypoxic conditions, all compared to Caki-1 cells in normoxic conditions. DESeq2 p-values corrected for multiple testing (FDR) are indicated as follows: **** P < 0.0001; *** P < 0.001; ** P < 0.01; * P < 0.05. (b). Concentrations of L and D isomers of 2-hydroxyglutarate measured by mass-spectrometry. (c). Proposed mechanism of DNA hypermethylation in VHL mutants. Green arrows indicate the increase of gene expression of all enzymes involved in glycolysis and MDH after VHL inactivation. Red arrow indicates decreased expression of L2HGDH. Increased concentrations of L- and D- hydroxyglutarate in VHL mutants lead to the inhibition of TET enzymes and DNA hypermethylation.

Figure 3.

(a). Mass of the tumours formed by cells injected into immunodeficient mice. VHL mutant Caki-1 cells formed significantly bigger tumours. (b, c). DNA methylation in CpG islands increases in VHL mutants compared to VHL-wild-type Caki-1 cells. Each promoter CpG island was assigned to a gene. CpG islands were significantly hypermethylated in VHL mutants. (d). Genes with significantly hypermethylated CpG islands in their promoters were enriched among negative regulators of cell proliferation.