Introduction: Gliomas demonstrate epigenetic dysregulation highlighted by the Glioma CpG-Island Methylator Phenotype (G-CIMP) seen in IDH1 mutant tumors. IDH1 mutation perturbs the balance between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) by inhibiting TET-mediated active demethylation. 5mC has been firmly implicated in oncogenesis. More recently, 5hmC has been identified as dysregulated and contributing to the malignant phenotype in human cancers. Despite this, the role 5hmC plays in IDH1 mutant gliomas remains poorly understood.
Methods: We examined for 5hmC profiles in high grade (WHO III/IV) IDH1 mutant (n = 12) and IDH1 wild-type (n = 9) tumors through parallel processing of samples using bisulfite (BS) and oxidative bisulfite (OxBS) conversion, with subsequent analysis on the Illumina MethylationEPIC Beadchip platform. Probes within the highest top 1% beta-value as well as differentially hydroxymethylated regions (DHMR) between IDH1 cohorts were identified. Hydroxymethylation profiles were correlated with gene expression measured using the Affymetrix Human Gene 2.0 ST array platform.
Results: Mean 5hmC beta-values were 4.6%% and 3.8% for IDH1 mutant and wild-type tumors, respectively. Top 1% and DHMR probes demonstrated increased 5hmC among IDH1 mutants. 5hmC enriched for enhancer and super-enhancers. Among G-CIMP target genes, 22/50 were hydroxymethylated in our IDH1 mutant cohort, suggesting that 5hmC contributes to their overall methylation. Gene expression was strongly associated with gene body 5hmC. 48 genes differentially expressed between IDH1 cohorts showed a positive Spearman correlation between 5hmC and gene expression, in particular for genes upregulated in IDH1 mutant tumors.
Conclusions: Our study represents the first quantitative genome-wide locus-specific study examining 5hmC comparing IDH1 mutant versus wild-type gliomas. Focal gains of 5hmC targeting gene bodies and regulatory regions, and associated with over-expressed genes, implicates 5hmC as an epigenetic mark significant in IDH1 mutant HGG pathogenesis.
Patient Care: Hydroxymethylation has been identified as a novel epigenetic mechanism that is disrupted in many human cancers, contributing to their malignant phenotype. Its role in gliomas is only beginning to be understood. Epigenetic changes are potentially reversible, and thus further understanding of their contribution to gliomagenesis may identify novel therapeutic targets.
Learning Objectives: By the conclusion of this session, participants should be able to: 1) distinguish between DNA cytosine methylation and its variant hydroxymethylation as unique epigenetic marks implicated in tumorigenesis, 2) understand the unique role hydroxymethylation plays in regulating gene expression, 3) appreciate the differential targeting of hydroxymethylation in IDH1 mutant versus wild-type high-grade gliomas, including its contribution to the G-CIMP hypermethylator phenotype.