CDKN2A-p16 deletion and activated KRAS G12D drive Barrett's-like gland hyperplasia-metaplasia and synergize in the development of dysplasia pre-cancer lesions.

BACKGROUND & AIMS: Barrett's esophagus (BE) is the precursor of esophageal dysplasia and adenocarcinoma (EAC). CDKN2A-p16 deletions were reported in 34-74% of BE patients who progressed to dysplasia and EAC, suggesting that p16 loss may drive neoplastic progression. KRAS activation frequently occurs in EAC and pre-cancer lesions. LGR5+ stem cells in the squamocolumnar-junction (SCJ) of mouse stomach, contribute as BE progenitors. We aimed to determine the functional effects of p16 loss and KRAS activation in Barrett's-like metaplasia and dysplasia development.

METHODS: We established mouse models with conditional knockout of CDKN2A-p16 (p16KO) and/or activated KRASG12D expression in interleukin-1b transgenic mice targeting SCJ LGR5+ cells and characterized histologic alterations (mucous-gland hyperplasia/metaplasia, inflammation, and dysplasia) in mouse SCJ. Gene expression was determined by microarray, RNAseq, and immunohistochemistry of SCJ tissues and cultured 3D organoids.

RESULTS: p16KO mice exhibited increased mucous-gland hyperplasia/metaplasia vs. controls (p=.0051). Combined p16KO+KRASG12D resulted in more frequent dysplasia and higher dysplasia scores with 82% of p16KO+KRASG12D mice developing high-grade dysplasia (p=0.0036). SCJ transcriptome analysis showed several activated pathways in p16KO vs. control mice (apoptosis, TNF-alpha/NFkB, proteasome degradation, p53 signaling, MAPK, KRAS, and G1-to-S transition).

CONCLUSIONS: p16 deletion in LGR5+ cell precursors triggers increased SCJ mucous-gland hyperplasia/metaplasia. KRASG12D synergizes with p16 deletion resulting in higher grades of SCJ glandular dysplasia, mimicking Barrett's high-grade dysplasia. These genetically modified mouse models establish a functional role of p16 and activated KRAS in the progression of Barrett's like lesions to dysplasia in mice, representing an in vivo model of esophageal pre-cancer. Derived 3D organoid models further provide in vitro modeling opportunities of esophageal pre-cancer stages.