Abstract 7226: Development of syngeneic tobacco associated oral cancer models using <i>Trp53</i> and <i>Cdkn2a</i> mutant mice

Abstract Background: Immunotherapy has revolutionized cancer treatment by leveraging therapeutic antibodies to block immune checkpoint inhibitors (ICIs). However, despite numerous clinical trials, success remains limited, likely due to tumor models lacking the appropriate genetic mutations or being studied in non-orthotopic sites factors critical to tumor response. To address these limitations, we developed syngeneic oral cancer models featuring defined mutations in Trp53 and Cdkn2a, the most frequently altered genes in human papillomavirus-negative head and neck squamous cell carcinoma (HNSCC). This mutation combination correlates with aggressive disease and poor survival outcomes in HNSCC patients. Using a carcinogenesis approach in single- and double-mutant Trp53/Cdkn2a mice, we established a preclinical platform to study HNSCC, a malignancy with limited therapeutic options. Methods: Krt14-Cre transgenic mice carrying Trp53-LOF, Trp53R172H, Trp53R245W, and Cdkn2a-LOF mutations were backcrossed onto a C57BL/6 genetic background. Oral carcinogenesis was induced via 16 weeks of low-dose 4-nitroquinoline 1-oxide in drinking water. Tumors from the tongues of treated mice underwent histopathological analysis and tissue culture immortalization. Orthotopic injections of immortalized tumor cells into C57BL/6J mice enabled validation of tumorigenic cell lines. RNA profiling quantified ICIs, cytokines, chemokines, and immune cell markers via qPCR. Sensitivity of the resulting genetically defined ROC tongue tumors to cisplatin, gemcitabine, and anti-PD-1 immunotherapy was assessed. Results: Mutations in Trp53 and Cdkn2a significantly accelerated tongue tumor development. Mice harboring double germline mutations exhibited more aggressive tumor progression compared to single-mutation or wild-type controls. From these tumors, we established eight syngeneic ROC cell lines, each harboring distinct Trp53/Cdkn2a co-mutations confirmed through sequencing. These cell lines, free of mycoplasma and verified to have unique mouse-origin STR profiles, were tumorigenic in C57BL/6J mice. The cell lines displayed variable expression levels of ICIs, cytokines, chemokines, and immune cell markers, and exhibited differential responses to chemotherapy and anti-PD-1 immunotherapy. Importantly, the models demonstrated immune "hot" and "cold" tumor microenvironments, including immune-excluded phenotypes. Conclusions: Our study establishes syngeneic oral cancer models with Trp53 and Cdkn2a mutations as robust platforms for investigating oral cancer immune evasion and optimizing immune-based therapies. These models provide a complementary tool for human clinical trials by facilitating hypothesis testing and advancing therapeutic strategies targeting the tumor microenvironment and immune system. Citation Format: Roberto Rangel, Roberto Rangel Jr, Alanis E. Rodriguez-Rosario, Nicholas Balbin, Liping Dong, Arnoldo Corona, Ahmed Tajmim Noor, Guillermina Lozano, Andrew G. Sikora, Jeffrey N. Myers. Development of syngeneic tobacco associated oral cancer models using Trp53 and Cdkn2a mutant mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7226.