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

Abstract

Inactivation of the tumor suppressor genes tumor protein p53 ( TP53 ) and cyclin-dependent kinase inhibitor 2A ( CDKN2A ) occurs early during gastroesophageal junction (GEJ) tumorigenesis. However, because of a paucity of GEJ-specific disease models, cancer-promoting consequences of TP53 and CDKN2A 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 TP53 and CDKN2A knockout ( TP53/CDKN2A 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 TP53/CDKN2A 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, TP53/CDKN2A dual inactivation disrupted both the transcriptome and the DNA methylome, likely mediated by key transcription factors, particularly forkhead box M1 (FOXM1). FOXM1 activated PTAFR transcription by binding to the PTAFR 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 TP53/CDKN2A inactivation in early GEJ neoplasia, which may facilitate early diagnosis and prevention of GEJ neoplasms.

Cancer Spheroids and Organoids as Novel Tools for Research and Therapy: State of the Art and Challenges to Guide Precision Medicine

“…A novel model of CRSIPR-Cas9-engineered TP53-CDKN2A dual KO human normal gastroesophageal junction (GEJ) organoids was generated for the first time. TP53-CDKN2A KO in GEJ induced morphological dysplasia as well as pro-neoplastic features in vitro and in vivo [ 81 ]. Takeda Haruna et al used genetically defined benign tumor-derived organoids carrying two frequent gene mutations (APC and KRAS mutations; AK organoids), which mainly contribute to the disease progress in the early stage of colorectal cancer (CRC) [ 82 ].…”

Advanced strategies for CRISPR/Cas9 delivery and applications in gene editing, therapy, and cancer detection using nanoparticles and nanocarriers

“…This investigation unveiled the significant involvement of TP53/CDKN2A inactivation in the initial stages of GEJ neoplasia, potentially enhancing the ability to diagnose and prevent the disease at an early stage. 168 The efficacy and straightforward nature of CRISPR-Cas9 as a technique for creating disease models has considerably advanced the understandings of disease pathologies. 169…”

Modelling esophageal adenocarcinoma and Barrett’s esophagus with patient-derived organoids

“…In another recent study, Zhao et al, 2022 investigated the effect of various gene knockouts on normal gastroesophageal junction (GEJ) organoids. GEJ cancers and those of the gastric cardia are thought to be related to EAC ( Nowicki-Osuch et al, 2021 ).…”