Abstract The number of known mutations and specific mutational hotspots in Head and Neck Squamous Cell Carcinomas (HNSCCs) only partially explain their biological complexity and limit the development of novel diagnostic markers and therapeutic agents. Tumor suppressor genes (TSGs) are commonly inactivated by somatic mutation and/or promoter hypermethylation yet recent high throughput genomic studies have not identified key TSGs inactivated by both mechanisms. Our aim was to perform a detailed genome wide analysis of the HNSCC methylome to study expression alterations associated with differential methylation patterns in the greater promoter region, focusing on key TSGs also inactivated by somatic mutations. Methylation Binding Domain Sequencing (MBD-seq), 450K Methylation arrays, whole exome sequencing and whole genome gene expression arrays in 41 primary HNSCC tumors and 16 matched uvulopalatopharyngoplasty tissue samples (UPPPs) were assessed. Results were validated in an independent cohort of 76 tumors and 19 UPPPs using quantitative methylation specific PCR (qMSP) and Sanger sequencing. A second external validation using 279 samples from the TCGA project was also performed. The integrated molecular analysis identified 186 downregulated genes harboring cancer specific promoter methylation including PAX1 and PAX5 and 10 key tumor suppressor genes inactivated by both promoter methylation and/or somatic mutation. PAX1, ZIC4, PLCB1 and PAX5 were selected for validation. Methylation frequencies in all of them could distinguish tumor from UPPP samples (PAX1, p <0.0001; ZIC4, p<0.0001; PLCB1, p<0.001; PAX5, p <0.0001). A gene panel combining promoter methylation results for these four genes had 96% sensitivity, 94% specificity, a 0.97 AUC and a PPV of 98.5%. Among the novel TSGs discovered with dual mechanisms of inactivation, a high frequency of genomic and epigenomic alterations was observed in the PAX gene family of transcription factors, which selectively impact canonical NOTCH and TP53 pathways to determine cell fate, cell survival, and genome maintenance. To our knowledge, our study consist the first detailed genome wide analysis of the HNSCC methylome covering expression alterations associated with differential methylation patterns in the greater promoter region, on key TSGs also inactivated by somatic mutations. Characterization of the complete HNSCC methylome has contributed insights into the clustering of specific genetic and epigenetic events, and highlights the importance of understanding the relative contribution of each to the overall frequency of TSG inactivation. Understanding the complete contribution of genomic and epigenomic alterations to specific genes and pathways in cancer will reveal novel high frequency specific markers for better risk assessment and can highlight the true frequency of therapeutic pathways to better target the disease at the molecular level. Citation Format: Rafael Guerrero-Preston, Christina Michailidi, Luigi Marchionni, Curtis Pickering, Mitchell Frederick, Jeffrey Myers, Srinivasan Yegnasubramanian, Tal Hadar, Maartje Noordhuis, Elana Fertig, Nishant Agrawal, William Westra, Wayne Koch, Joseph Califano, Victor Velculescu, David Sidransky. Key tumor suppressor genes inactivated by promoter methylation and somatic mutations in head and neck cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2482. doi:10.1158/1538-7445.AM2014-2482