PROJECT SUMMARY Oral squamous cell carcinoma (OSCC) is the most common type of head and neck cancer. Most patients with OSCC are diagnosed at advanced stages and have significant mortality and recurrence rates. In addition to PD- 1-based immunotherapy, cetuximab (CTX) is the only FDA-approved anti-epidermal growth factor receptor (EGFR) monoclonal antibody to treat this disease. However, only a small proportion of OSCC patients benefit from CTX monotherapy or CTX-based combination therapies, and most of them eventually develop resistance. These clinical challenges underscore the need to develop safer and more effective treatment strategies to improve the efficacy of CTX therapy in OSCC. Through an unbiased genome-wide CRISPR/Cas9 knockout screening, we have identified TNF-mediated pro-survival pathway as the dominant driver of reduced CTX efficacy in OSCC cells. Strikingly, IACS-010759 (IACS), an orally bioavailable mitochondrial complex I inhibitor, redirects TNF signaling from cell survival to gasdermin D (GSDMD)-mediated pyroptosis in CTX-treated OSCC cells via a reactive oxygen species (ROS)-dependent mechanism. Our pilot studies in a human EGFR (hEGFR)- expressing murine oral cancer model further demonstrated that CD8+ T cell-mediated anti-tumor immunity is essential for CTX/IACS-induced remission of GSDMD+ oral tumors. Based on these novel and significant findings, we formulated our central hypothesis that the therapeutic efficacy of CTX/IACS combination is not only due to its ability to suppress tumor growth, but also critically depends on the interplay between therapy-induced pyroptotic tumor cells and the host immune system, leading to a durable and more effective elimination of OSCC. This mechanism-driven project will integrate unbiased molecular and immunological technologies and multiple preclinical models to advance our understanding of the molecular and immunological mechanisms, and to develop an improved CTX/IACS combination therapy to facilitate clinical translation. To achieve these goals, we will elucidate the mechanisms by which IACS is synergistic with CTX against OSCC by inducing NLRP3 inflammasome-dependent pyroptosis (Aim 1) and define the immunomodulatory mechanisms and effects associated with OSCC cell pyroptosis induced by the combination of CTX/IACS (Aim 2). We will also evaluate an improved CTX/IACS combination therapy designed with tumor-targeting nanotechnology to maximize efficacy while minimizing toxicity to facilitate clinical translation (Aim 3). Results of this project should provide us with preclinical data for the translational development of a Phase 1 clinical trial using this novel combination strategy to preferentially target EGFR-overexpressing OSCC.

PROJECT NARRATIVE Effective control of advanced oral squamous cell carcinoma (OSCC) remains a clinical challenge. The therapeutic efficacy of cetuximab (CTX)-based anti-epidermal growth factor receptor (EGFR) therapy is unsatisfactory in limiting OSCC, which can be significantly improved in combination with IACS-010759 (IACS), an orally bioavailable mitochondrial complex I inhibitor, by inducing pyroptosis-mediated anti-tumor immunity. Successful completion of this project will provide a comprehensive understanding of the molecular and immunological mechanisms of CTX/IACS combination, leading to an improved therapy that maximizes anti- tumor efficacy while minimizing toxicity.