PROJECT SUMMARY Late-stage, HPV-negative (HPV-) head and neck squamous cell carcinoma (HNSCC) (or HNC-) is the most lethal form of HNSCC. HNC- occurs at the highest incidence in the Appalachian region, where a large segment of this population is rural, economically disadvantaged and medically underserved. The Appalachian population is the highest user of tobacco in the nation, a behavior directly linked to disparities in HNC- incidence and mortality. As such, there is a pressing need to identify the underlying genomic mechanisms responsible for the disparate survival in tobacco-positive Appalachian HNC- patients as an essential step towards improving medical outcomes for these patients. Single gene informatic analysis of national cohorts has identified elevated copies of multiple genes associated with smoking. The majority of these genes map to chromosome 11q13 cytobands, the most commonly amplified region in HNC- and long known to be associated with reduced survival. 13 smoking-correlated genes overexpressed from the 11q13 amplicon correspond with decreased survival, increased risk of death and elevated risk of lymph node metastasis. While some genes in this region have been well studied as drivers of HNC- progression, other genes identified by this analysis have unknown roles in cancer and may present new targets for therapeutic intervention. The overall goal of this proposal is to identify the roles of uncharacterized genes from the smoking-associated expression signature (SAES) in the 11q13 amplicon that contribute to neoplastic progression of Appalachian tobacco-positive HNC-. Our central hypothesis is that overexpression of novel 11q13 genes in the SAES contribute in driving reduced Appalachian HPV-survival. Oncogenic screening of uncharacterized SAES genes will be conducted to evaluate the individual contributions of each gene in promoting cancer hallmarks. SAES gene function will be assessed using cultured cells from Appalachian tobacco-positive patients and mouse orthotopic HNSCC models. Aim 1 will test the role of SAES genes in driving tumor cell growth, proliferation, invasion, and metabolic reprogramming in tobacco-induced 11q13 amplified HNC-. Aim 2 will utilize experimental and clinically-parallel imaging modalities to individually evaluate novel predicted drivers of lymph node metastasis. These genes will be tested for promoting tumor cell growth, nodal spread, altered metabolism and proliferation in 11q13 amplified Appalachian HNC- tumors. A comprehensive understanding of how each novel SAES gene contributes to enhancing this aggressive HPV- subtype will fill a key gap in our knowledge regarding how the 11q13 amplicon contributes to the overall poor outcomes seen in tobacco-associated HNC-. Results from this proposal will provide a foundation for future studies that will mechanistically address novel tumor-promoting SAES genes identified from this work as drivers of disease aggressiveness, potentially serving as new targets for th...