Advancing Education, Research, and Quality of Care for the Head and Neck oncology patient.
BACKGROUND: DOT1L is the only known histone methyltransferase for the histone H3 lysine 79 (H3K79) residue. DOT1L and H3K79 methylation have diverse functions in cells, including maintenance of telomeres, transcriptional activation, DNA damage response, and regulation of pluripotency in stem cells. While DOT1L is an oncogene in leukemia, its role in solid tumors is less defined. We identified significant up-regulation of DOT1L in HPV+ oropharyngeal squamous cell carcinoma (OPSCC) and studied the effects of knockout.
METHODS: DOT1L knockout (KO) was performed in the HPV+ cell lines SCC-2, SCC-47, and SCC-90 via Crispr/Cas9 tranduction and cells were assessed for proliferation, transwell migration, colony formation, and cisplatin resistance. The Cancer Genome Atlas (TCGA) was mined for clinical associations with RNA-seq data and DOT1L expression.
RESULTS: In TCGA, DOT1L is significantly up-regulated in HPV+ OPSCC relative to normal tissue (p < 0.001) and HPV- SCC (p < 0.001). DOT1L over-expression is associated with significantly improved overall survival for HPV+ OPSCC (n = 98, HR = 0.548, p = 0.001, stratification at mean) but there is no difference is survival in HPV- SCC. Given these findings, we hypothesized that DOT1L functions as a tumor suppressor in HPV+ OPSCC. DOT1L KO led to complete absence of H3K79me2 and H3K79me3 in all cell lines on Western blot. DOT1L KO in SCC90 and SCC2 cells led to significant increase in proliferation in cell culture as well as a significant increase in resistance to cisplatin as assessed by IC50 curves. In SCC47, DOT1L KO led to a significant increase in migration on the transwell migration assay and colony formation on clonogenic assay. We used CIBERSORT deconvolution of TCGA RNA-seq data to identify immune cell subpopulations based on DOT1L expression. In HPV+ OPSCC, there was a significant positive correlation between DOT1L and CD8+ T-cells, CD4+ T-cells, and NK cells, suggesting increasing immune infiltration of the tumor microenvironment with higher DOT1L expression.
CONCLUSIONS: DOT1L likely functions as a major tumor suppressor in HPV+ OPSCC and its expression is associated with a substantial difference in overall survival in patients. Exclusion of DOT1L from the nucleus is necessary to maintain pluripotency of stem cells and it appears that DOT1L KO leads to de-differentiation of OPSCC cells to increase proliferation, motility, and chemoresistance. We are undertaking chromatin and gene sequencing studies to identify the epigenetic mediators of this effect.