Abstract 1250: Genetically-defined organoid models reveal early mechanisms of squamous neoplastic progression and therapeutic vulnerabilities in squamous cell carcinoma

Abstract Upper aerodigestive squamous cell carcinoma (UASCC) represents an aggressive and often fatal group of malignancies. Mechanistically, the early steps of neoplastic transformation remain poorly defined. Translationally, despite extensive genomic characterization of UASCC, the application of these findings to clinical practice remains highly limited. We have identified a crucial gap in the field - the lack of viable and valid human models that can: i) faithfully recapitulate the stepwise progression from precursor lesions to tumors, and ii) rigorously model genotype-phenotype relationships. Here, we developed two complementary, cross-species, genetically-defined organoid systems: (1) a genome-sequenced, patient-derived organoid platform comprising 46 models spanning normal tissue, precursor lesions, and tumors along the oral-esophageal neoplastic continuum; and (2) a CRISPR/Cas9-engineered organoid platform of 16 models, targeting key UASCC genomic drivers to recreate the full spectrum of malignant transformation (hyperplasia → dysplasia → carcinoma). Using these models, we uncovered critical insights into early squamous neoplastic evolution. For example, single-cell RNA sequencing of TP53/CDKN2A double-knockout (DKO) organoids revealed an expanded population of quiescent basal and proliferative squamous cells, alongside a reduction in differentiated squamous cells. This differentiation imbalance was exacerbated by the introduction of additional drivers, such as PIK3CAE545K mutation (DKOP). Furthermore, a senescence-like program, identified as a hallmark of normal squamous epithelium, was markedly attenuated in DKO organoids and further diminished in DKOP organoids, with ANXA1 emerging as a master regulator. We identified a novel ANXA1-SMAD3-p27KIP1 pathway as a critical driver of this senescence-like program, suppressing neoplastic phenotypes in squamous organoid models. Leveraging these genetically-defined organoid systems, we further performed high-throughput drug screening at single-organoid resolution, utilizing bioprinting technology coupled with machine learning. As anticipated, PIK3CA mutations conferred a survival advantage against most therapies tested. However, intriguingly, genetically-engineered organoids with PIK3CA mutations demonstrated heightened sensitivity to mitomycin C and onalespib. These findings were further validated using independent patient-derived HNSCC organoids harboring PIK3CA mutations. In summary, this study provides critical insights into the mechanisms of early squamous neoplastic progression and underscores the utility of genetically-defined organoid platforms for forward genetic studies in cancer. These rigorously validated models offer a powerful platform for identifying genome-guided therapies and advancing precision oncology. Citation Format: Hua Zhao, Young Min Park, Yueyuan Zheng, Qiong Mao, Luda Lin, Casey Collet, Stephanie Wong, Boyan Hu, Uttam K. Sinha, Alice Soragni, Dechen Lin. Genetically-defined organoid models reveal early mechanisms of squamous neoplastic progression and therapeutic vulnerabilities in squamous cell carcinoma [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 1250.