Abstract 1964: Age-related <i>p16</i> epimutation is a targetable driver of <i>Kras</i> -mutant lung cancer.

Abstract Background: Lung cancer is a leading cause of cancer death worldwide; however, current targeted therapies have limited efficacy for most patients. p16 epimutation, characterized by epigenetic silencing of p16 by promoter DNA hypermethylation, is common in lung cancer. Elucidating how p16 epimutation drives lung tumorigenesis may uncover new therapeutic opportunities. Methods: We developed a mouse model combining the conditional Cre-inducible KrasG12D-mutation with epigenetically engineered p16 epimutation and conducted time-course study to analyze the tumor phenotype and survival. Organoids derived from normal and tumorous lung tissue were used to assess the role of p16 epimutation in tumor initiation and maintenance. Moreover, spatial transcriptomic analysis was used to characterize tumor heterogeneity and define distinct cell populations. To test the therapeutic potential of reversing p16 epimutation, we evaluated the efficacy of hypomethylating agents 5-Aza-2’-Deoxycytidine (DAC) and GSK3685032 on p16 reactivation in organoid and in vivo. Furthermore, we developed mice enabling conditional, inducible and site-specific DNA demethylation using a CRISPR-dCas9-Tet1 based epigenetic editing system. We assessed the effect of targeted p16 promoter demethylation on tumor progression in vivo. Results: Mice with combined Kras-mutation and p16 epimutation developed malignant tumors more rapidly and had significantly shorter survival than mice with Kras-mutation only (Median survival: 161 days vs. 223 days, n=50, p=0.0003). Histopathological analyses confirmed the tumor progression to adenocarcinoma with distinct papillary and intraluminal growth patterns in combined mice, but not in Kras-mutation only mice. Spatial transcriptomic analysis confirmed that tumors with this growth patterns were enriched in transitional airway progenitors co-expressing Sox2, Nkx2-1, and ciliated markers. Moreover, normal and tumor organoids derived from the combined mice proliferated robustly whereas organoids from Kras-mutation only mice stopped growing shortly after establishment. While global hypomethylation agents significantly reactivated p16 and inhibited proliferation in tumor organoids, these agents failed to block tumor development in vivo (Fold change of tumor burden to control: DAC 1.41 ± 0.11, GSK3685032 1.33 ± 0.18, n=5, p=0.07). In contrast, CRISPR-mediated targeted p16 promoter demethylation significantly reduced both tumor number and size in vivo compared with control mice (Tumor number: 16.17 vs. 6.33 per mouse, n=6, p=0.04). Conclusion: Our work identifies p16 as a bona fide epigenetic driver and therapeutic target for Lung cancer. Importantly, this study provides proof-of-concept that precise, locus-specific DNA demethylation can restore tumor suppressor function, highlighting a promising strategy for targeted epigenetic therapy in lung cancer patients. Citation Format: Xiaomin Chen, Li Yang, Eduardo Lopez, Lili Ma, Chao Cheng, Lanjing Zhang, Lanlan Shen. Age-related p16 epimutation is a targetable driver of Kras-mutant lung cancer [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 1964.