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Abstract 4325: 5-hydroxymethylation captures the heterogeneity in keratinization and cytoskeletal rearrangement processes in head & neck cancers

Infection with high-risk HPV, or human papillomavirus, has been shown to be a risk factor for head and neck squamous cell carcinoma (HNSCC) development in addition to the classical risk factors of tobacco and alcohol usage. Previous studies have identified epigenetic differences between these two classes of HNSCC, including DNA methylation, histone modifications and non-coding RNAs. 5-hydroxymethylation (5hmC) is an oxidative derivative of 5-methylcytosine (5mC) and is depleted in human cancers of many different origins. However, the roles of 5hmC in HNSCC is still unknown. Our study is the first to characterize 5hmC in HNSCC, which we profiled in 18 HPV(+) and 18 HPV(-) HNSCC tumors. Since previous studies found higher levels of 5hmC in more differentiated cells and lower levels in stem-like cells, we hypothesized an overall higher 5hmC level in HPV(-) tumors, which tend to be more differentiated in nature. Consistent with our hypothesis, results showed significant hyper-hydroxymethylation in HPV(-) tumors, and detected multiple cancer-related genes with hyper-hydroxymethylation in either HPV(+) and HPV(-) tumors. For example, CDKN2A (p16) and MTAP were the most hyper-methylated genes in HPV(+) tumors, while 5hmC levels of HPV(-) tumors were particularly high in CDH13, TIMP2, and BCAR1. We found that the two HPV(+) subtypes defined by Zhang et al., IMU (heightened immune response, more mesenchymal differentiation) and KRT (more keratinization, more likely viral integration), explained a high level of heterogeneity in 5hmC levels at promoter and enhancer regions. By testing for genes and pathways that demonstrate concordant changes in gene expression and 5hmC levels, we found that keratinocyte differentiation, cell-cell adhesion and adherens junction pathways are both up-regulated and have hyper-hydroxymethylation in HPV(-) tumors. Using predicted enhancers of epidermal keratinocytes, we found a much higher portion of hydroxymethylated regions fall in enhancer regions for HPV(-) samples compared with HPV(+) samples. The higher differential 5hmC in enhancer regions was further validated by stronger histone marks of H3K4me1 and H3K27ac in HPV(-) tumors. Since some of these enhancers can be linked to differentially expressed target genes, this result indicates that the enhancer hydryoxymethylation in HNSCC could also play an important role in downstream gene regulation. We hope that this study could partially explain the different mechanisms and cast light on potential targeted therapies in HNSCC both based on HPV status and different HPV(+) subtypes. Citation Format: Siyu Liu, Katie Zarins, Evan Fernandez, Laura Rozek, Maureen Sartor. 5-hydroxymethylation captures the heterogeneity in keratinization and cytoskeletal rearrangement processes in head & neck cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4325.