Abstract 7524: Genetically defined organoid systems uncover PIK3CA-mediated suppression of an oral-immune program during early squamous neoplastic evolution

Abstract Head and neck squamous cell carcinoma (HNSCC) is an aggressive and lethal neoplasm, yet its early neoplastic transformation mechanisms remain poorly defined. Despite extensive genomic characterization, translation of these molecular insights into clinical practice has been limited. A critical barrier has been the absence of physiologically relevant human models capable of faithfully recapitulating the stepwise progression from precursor lesions to invasive tumors while enabling rigorous interrogation of genotype-phenotype relationships. Here, we developed two complementary, cross-species, genetically defined organoid systems—a genome-sequenced patient-derived platform spanning normal tissue, precursor lesions, and tumors, and a CRISPR/Cas9-engineered human and mouse organoid platform targeting key HNSCC drivers (TP53, CDKN2A, PIK3CA) to reconstruct the continuum of squamous malignant transformation. Using these models, we uncovered critical insights into early neoplastic evolution. TP53/CDKN2A double-knockout (DKO) organoids exhibited morphological dysplasia, hyperproliferation, loss of squamous differentiation, and tumorigenicity—phenotypes further exacerbated by introducing mutant PIK3CAE545K (DKOP). Single-cell RNA sequencing of DKO and DKOP organoids revealed expansion of quiescent basal and proliferative squamous populations and depletion of differentiated cells. Notably, an Oral-Immune transcriptional program characteristic of normal squamous epithelium was attenuated in DKO organoids and further diminished in DKOP. The Oral-Immune program was strongly correlated with intratumoral T-cell infiltration in HNSCC, marked by enrichment of cytotoxic and proliferative CD8+ subsets and upregulation of IFN-pathway and cytotoxic effector genes. Strikingly, across 46 ICB-treated cohorts, its strongest association with therapeutic response occurred in an HNSCC cohort, where it outperformed 17 established immune gene-expression signatures, including IFNG and PD-L1. Both CD8+ T-cell scores and the Oral-Immune score were inversely correlated with PIK3CA mutation status. Mutant PIK3CAE545K suppressed Oral-Immune program genes in HNSCC, and this effect was reversible with PI3K inhibition. Functionally, PIK3CA mutations reduced CD8+ T-cell infiltration in both in vitro CD8+ T cell-organoid coculture assays and in vivo orthotopic allograft models. Together, these findings identify the Oral-Immune program as a key determinant of immune-inflamed tumor states, reveal PIK3CA mutations as drivers of immune evasion and diminished immunotherapy responsiveness through suppression of this program, and underscore the power of genetically defined organoid models for dissecting early cancer evolution. Citation Format: Hua Zhao, Young Min Park, Yueyuan Zheng, Qiong Mao, Hao Wu, Fanyi Mo, Uttam K. Sinha, Parish Sedghizadeh, De-Chen Lin. Genetically defined organoid systems uncover PIK3CA-mediated suppression of an oral-immune program during early squamous neoplastic evolution abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7524.