Generation and multiomic profiling of a<i>TP53/CDKN2A</i>double-knockout gastroesophageal junction organoid model

Abstract

Inactivation of the tumor suppressor genes tumor protein p53 (<i>TP53</i>) and cyclin-dependent kinase inhibitor 2A (<i>CDKN2A</i>) occurs early during gastroesophageal junction (GEJ) tumorigenesis. However, because of a paucity of GEJ-specific disease models, cancer-promoting consequences of <i>TP53</i> and <i>CDKN2A</i> inactivation at the GEJ have not been characterized. Here, we report the development of a wild-type primary human GEJ organoid model and a CRISPR-edited transformed GEJ organoid model. CRISPR-Cas9-mediated <i>TP53</i> and <i>CDKN2A</i> knockout (<i>TP53/CDKN2A</i>^KO) in GEJ organoids induced morphologic dysplasia and proneoplastic features in vitro and tumor formation in vivo. Lipidomic profiling identified several platelet-activating factors (PTAFs) among the most up-regulated lipids in CRISPR-edited organoids. PTAF/PTAF receptor (PTAFR) abrogation by siRNA knockdown or a pharmacologic inhibitor (WEB2086) reduced proliferation and other proneoplastic features of <i>TP53/CDKN2A</i>^KO GEJ organoids in vitro and tumor formation in vivo. In addition, murine xenografts of Eso26, an established human esophageal adenocarcinoma cell line, were suppressed by WEB2086. Mechanistically, <i>TP53/CDKN2A</i> dual inactivation disrupted both the transcriptome and the DNA methylome, likely mediated by key transcription factors, particularly forkhead box M1 (FOXM1). FOXM1 activated <i>PTAFR</i> transcription by binding to the <i>PTAFR</i> promoter, further amplifying the PTAF-PTAFR pathway. Together, these studies established a robust model system for investigating early GEJ neoplastic events, identified crucial metabolic and epigenomic changes occurring during GEJ model tumorigenesis, and revealed a potential cancer therapeutic strategy. This work provides insights into proneoplastic mechanisms associated with <i>TP53/CDKN2A</i> inactivation in early GEJ neoplasia, which may facilitate early diagnosis and prevention of GEJ neoplasms.