Abstract TP63 is a member of the p53 family and is a master regulator of epithelial development and differentiation. Overexpression and/or genomic amplification of TP63 is commonly found in a large number of epithelial tumors, including the majority of head and neck squamous cell carcinomas (HNSCC). In many tumors, p63 appears to function as an oncogene, contributing to both increased tumor cell proliferation and survival. To address the mechanisms of p63 function, we studied the interplay of p63 with the chromatin microenvironment at its genomic targets, under distinct physiological and pathological states. We generated transcriptomic (RNA-Seq) and epigenomic (ChIP-Seq, ATAC-Seq, and DNase-Seq) datasets in representative normal oral and epidermal keratinocytes (HGEP, NHEK) and HNSCC (SCC25, SCC13) cell-lines, to examine whether there exists a HNSCC-specific molecular pattern of p63 targeting. We utilized machine learning methods including Random Forests for classification and regression, to determine the contribution of epigenetic states, transcriptional co-factors and the p63 DNA binding motif in differential p63 binding between normal and cancer states. We found that a core epigenomic signature (H3K4me1 and H3K27me3 binding status and chromatin accessibility) is highly predictive of p63 binding. Furthermore, our analysis revealed that SCC25-specific p63 targets (corresponding to 1239 genomic regions) are inaccessible in normal cells, but in SCC25 they are marked by H3K4me1 and H3K27ac marks, indicative of active regulatory regions. Our data suggests that the complex interplay between local chromatin microenvironment, presence and/or absence of distinct co-factors and potential cell-type specific p63 activity might be key drivers of the differential binding events between normal and cancer epithelial cells. Our investigation of SCC-specific p63 binding events also led to the identification of several oncogenic genes (for e.g. SOX2, TRIM32, HAS2, PRKCE) that are direct targets of p63. By performing Gene Set Enrichment Analysis we found that these p63 misregulated genes encompass both known (G-protein coupled receptor signaling pathway) and novel cancer pathways thus providing a framework for the oncogenic function of p63. To further evaluate the importance of these findings in the tumor context, we derived a p63 target genes signature and used it to cluster RNA-Seq data of HNSCC patients (obtained from The Cancer Genome Atlas). The gene signature could both classify normal samples away from tumors and importantly distinguish between different molecular subtypes of HNSCCs. Collectively our studies suggest that aberrant targeting of p63 leads to dynamic remodeling of the chromatin at its target sites, trigger the expression of a cascade of pro-tumorigenic genes, ultimately condemning the cell to an oncogenic fate. These novel insights potentially provide new avenues to be harnessed for tumor subtype classification, development of biomarkers and for targeted therapies. Citation Format: Isha Sethi, Maria Tsompana, Christian Gluck, Michael J. Buck, Satrajit Sinha. Genomic mistargeting of p63 drives the cancer phenotype in head and neck squamous cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B13.