Abstract 6010: Targeting p16 epimutation during lung cancer progression for anticancer therapy
Authors: Chen Xiaomin , Lawrence Emily Brisnna , Farmer Leah , Yang Li , Perry Robert Craig , Sequeira David Joel , Zhang Lanjing , Shen Lanlan 
Published: April 2023

Abstract
Background: Whole-genome DNA methylation profiling revealed that epimutation (gene silencing associated with alteration in DNA methylation) is common in human lung cancer, but it remains unclear whether the aberrant DNA methylation is a cause or a consequence of tumorigenesis. Furthermore, different from genetic mutations, which are irreversible, epigenetic mutation is reversible and could be restored to normal status. The potential reversibility of epimutation presents new therapeutic opportunities for cancer treatment.
Methods and Results: We utilized an epigenetically engineered mouse cancer model in which the expression of p16 (also known as Cyclin-dependent kinase inhibitor 2A) is silenced by accumulating DNA methylation at the promoter region (p16meth). To investigate whether p16 epimutation functionally contributes to the lung cancer initiation and progression, we obtained Cre recombinase inducible K-ras+/LSL-G12D mice from the Jackson Laboratory and crossed them with p16meth mice to produce K-ras+/LSL-G12D; p16meth/meth mice (with combined K-ras mutation and p16 epimutation). These mice have been maintained on the C57BL/6J background. We administered 5 × 107 pfu of adenovirus-Cre (Ad5-CMV-Cre) to mice at 20 weeks (wk) of age through intranasal instillation to activate oncogenic K-ras. We monitored the lung tumor development at 6, 8, 12, and 16 wk post-infection. Remarkably, as early as 6 wk post-infection, we found substantially increased tumor numbers and sizes in the K-ras+/LSL-G12D; p16meth/meth mice compared with the K-ras+/LSL-G12D mice (n=4 per group). Histopathological analyses confirmed the tumor progression to adenocarcinoma. Interestingly, the K-ras+/LSL-G12D; p16meth/meth lung tumors exhibited distinct papillary and intraluminal growth patterns, which were not observed in mice with K-ras mutation alone. In addition, to investigate the therapeutic effects of reversing p16 epimutation, we generated MEFs from these mice and compared the efficiency of p16 reactivation by either a standard hypomethylating agent 5-Aza-2’-Deoxycytidine (DAC) or a first-in-class reversible DNMT1-selective inhibitor GSK3685032. We found that GSK3685032 induced p16 demethylation more effectively than DAC. Treatment of GSK3685032 at 1.5µM for 6 days resulted in robust p16 hypomethylation (58% loss) and three-fold increase in gene expression.
Conclusion: Our study validates that p16 epimutation functionally contributes to lung cancer initiation and provides an ideal in vivo preclinical platform for testing epigenetic therapeutic strategies
Citation Format: Xiaomin Chen, Emily Brisnna Lawrence, Leah Farmer, Li Yang, Robert Craig Perry, David Joel Sequeira, Lanjing Zhang, Lanlan Shen. Targeting p16 epimutation during lung cancer progression for anticancer therapy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6010.