PROJECT SUMMARY Globally, more than 900,000 people are diagnosed with head and neck squamous cell carcinoma (HNSCC) each year, with more that 250,000 dying annually from this cancer. Infection with human papilloma virus (HPV), a known risk factor for developing HNSCC, significantly impacts clinical prognostication. Specifically, for HPV- negative HNSCC, the most lethal subtype of head and neck cancer, less than 30% of those diagnosed survive for more than five years. The most common and lethal HPV-negative HNSCC subtype is oral cavity squamous cell carcinoma (OSCC). Importantly, most OSCCs are preceded by morphologically distinguishable pre- cancerous lesions which are readily accessible for histological and molecular evaluation. This provides a unique opportunity for intercepting this deadly cancer in the earliest stages of its development by halting the conversion of oral precancer into invasive OSCC. Oral leukoplakia, the focus of this application, represents the most frequent type of oral premalignancy, with one in every fifty people is expected to develop OL in their lifetime. OL’s low malignant transformation rate of ~3.3% progressing to oral cancer and its highly variable natural history poses a major challenge for surveying OLs and for intercepting their malignant conversion into invasive oral cancers. To address this challenge, we hypothesize that the evolutionary transition from an OL into an OSCC is due to the immuno-genomic interactions encompassing the acquisition of somatic driver events, the gain of chromosomal instability, and the loss of effective immunosurveillance. We further hypothesize that the genomic and immune landscapes of OL in patients who subsequently develop oral cancer (progressors) will differ from those that do not develop oral cancer (non-progressors). The overall objective of this project is to elucidate the molecular and immune mechanisms by which OLs progress to OSCCs, and to develop actionable and predictive biomarkers. To achieve this objective, we will leverage well-annotated OL cohorts to generate the largest whole- exome and whole-transcriptome atlas encompassing 300 OLs, including at least 100 cancer progressors and 100 non-progressors. Further, by utilizing a spatial multiplex immuno-fluorescence platform and an unbiased RNA-sequencing approach for immuno-profiling, we will comprehensively map the immune landscapes of these 300 OLs and associate distinct immuno-genetic features with likely progression to OSCC. Lastly, our state-of- the-art oral carcinogenesis mouse model will be used to model the transition of OL to OSCC at the single cell resolution in order to understand the role of common genomic alterations and immune surveillance in this process. Overall, this project will reveal the compendium of immuno-genetic changes that drive the evolutionary transition from an OL to an OSCC and elucidate a set of targetable immune cell population(s) and novel immune surveillance mechanisms, which can likely halt this malignant transformation.