(A-D) MIRA profiles of strong and weak enhancers in the NHEK cell line (A), JunD binding sites in the K562 cell line (B), NFKB binding sites in the GM10847 cell line, and STAT3 binding sites in the HeLaS3 cell line (D) Samples are colored by HPV tumor subtype. (E) Correlations between CTCF MIRA scores and Strong Enhancer MIRA scores. (F) Box plot of MIRA scores separated by subtype for EZH2 binding sites and JunB binding sites from NHEK and K562 cell lines, respectively. (G) Correlations between JunD MIRA scores and gene expression-based keratinization scores. All scatter plots separate subtype by color and display the Pearson correlation coefficient (R) along with the corresponding p-value.
ARTICLE ABSTRACTDNA methylation is a vital early step in carcinogenesis. Most findings of aberrant DNA methylation in head and neck squamous cell carcinomas (HNSCC) are array-based with limited coverage and resolution, and mainly explored by human papillomavirus (HPV) status, ignoring the high heterogeneity of this disease. In this study, we performed whole-genome bisulfite sequencing (WGBS) on a well-studied HNSCC cohort (n=36) and investigated the methylation changes between fine-scaled HNSCC subtypes in relation to genomic instability, repetitive elements, gene expression, and key carcinogenic pathways. The previously observed hypermethylation phenotype in HPV-positive (HPV+) tumors compared to HPV-negative tumors was robustly present in the immune-strong (IMU) HPV+ subtype but absent in the highly keratinized (KRT) HPV+ subtype. Methylation levels of IMU tumors were significantly higher in repetitive elements, and methylation showed a significant correlation with genomic stability, consistent with the IMU subtype having more genomic stability and better prognosis. Expression quantitative trait methylation (cis-eQTM) analysis revealed extensive functionally-relevant differences, and differential methylation pathway analysis recapitulated gene expression pathway differences between subtypes. Consistent with their characteristics, KRT and HPV-negative tumors had high regulatory potential for multiple regulators of keratinocyte differentiation, which positively correlated with an expression-based keratinization score. Together, our findings revealed distinct mechanisms of carcinogenesis between subtypes in HPV-positive HNSCC and uncovered previously ignored epigenomic differences and clinical implications, illustrating the importance of fine-scale subtype analysis in cancer.