Abstract Head and neck cancer squamous cell carcinomas (HNSCC) are the seventh most common cancers globally and are associated with poor survival rates. Pharmacological treatments for HNSCC remain largely ineffective and improving drug therapies requires the identification of novel factors that modulate not just the tumor but also the surrounding permissive oral microenvironment. One such novel factor may be the aging of the tissue. While older age is associated with worse five-year survival for HNSCC, almost all preclinical cancer model studies focus on outcomes in young animals. This discordance between preclinical adolescent in vivo models and largely elderly target patient populations may underlie the poor efficacy of treatment for HNSCC. The goal of this proposal is to define molecular events associated with HNSCC progression in aged tissues with the hope to uncover targetable mechanisms that may offer new therapeutic avenues. To test the influence of age on HNSCC, we optimized syngeneic orthotopic tongue xenograft models of HNSCC. We observed more rapid tumor growth in old animals, with tumors exhibiting an age-associated, immune evasive, transcriptionally-distinct tumor cell states as well as an age-associated collagen-secreting/modifying fibroblast population. A critical factor that may be involved in these age-associated alterations is the transcriptional regulator Yes-associated protein (YAP), a Hippo signaling effector that has a pro-tumorigenic role in HNSCC, including modulating the expression of genes that track with disease progression and treatment resistance. YAP mediates transcriptional changes that promote aggressive cell behavior and immune evasion, dynamics that may preferentially promote disease severity in older patients. Furthermore, the transcriptome of old animals revealed distinct elevation of the Runt- related transcription factor 1 (RUNX1) in aged oral tumor cells. RUNX1 is a YAP DNA-binding partner and has been implicated as a biomarker of worse HNSCC survival. We hypothesize that the aged oral tissue microenvironment is permissive to tumorigenesis in part due to extracellular matrix dynamics that drive elevated YAP and RUNX1 activity. In Aim 1, we will examine YAP-depleted and RUNX1-depleted tumor cell behavior in old and young mice using genomic, epigenomic and transcriptomic analyses in our syngeneic orthotopic xenograft models. Data collected from this aim will offer a comprehensive comparison of the role of YAP and RUNX1 on the mediating age-associated alterations observed in HNSCC. In Aim 2, we will define age-associated changes to lymphocytic and myeloid immune populations in the aged oral tumor niche and test the impact of matrix stiffness on conferring these age-associated alterations to immune and tumor cell populations in our syngeneic xenograft models. Data collected from this aim will provide a comprehensive assessment of age- associated immune and mechanical tissue targets for potential future therapeut...