Abstract 4273: Oncogenic BRAFV600E drives stem cell niche factors-independent growth and tumorigenic transformation in colon organoids

Herein, we report a model for how BRAF mutations, in contrast to KRAS mutations, specifically associate with and induce, in mouse colon organoids without a microenvironment, the phenotype of human sporadic right-sided colon adenocarcinomas (COAD). The human BRAF mutant COAD are particularly distinguished by having an increased incidence of promoter CpG island methylation, termed CpG island methylator phenotype (CIMP)-High. This latter phenotype contrasts with the majority of the COAD which are classified in the CIMP-low to intermediate groups and have mostly KRAS mutations. To understand how these mutations influence tumor evolution and the methylation landscape, we modeled the early carcinogenesis of colorectal cancer by inducing BRAF V600E and KRAS G12D mutations individually in 3D organoids prepared from mouse proximal colon. The induction of BRAF V600E mutation, but not KRAS G12D mutation, showed various features of progressive transformation. In vitro induction of BRAF V600E drove the organoids to derive cystic changes in morphology and promoted adoption of a stem cell niche independency characterized by growth in the absence of added stem cell niche factors including Wnt3a, R-Spondin, and Noggin. This independency is not due to increased secretion of niche factors by the Paneth-like cells in BRAF V600E mutant organoids. In addition, BRAF V600E organoids showed dysplastic changes such as high nuclear to cytoplasmic ratio and abnormal budding. The most exciting finding is that induction of BRAF V600E mutation, but not KRAS G12D mutation, induced complete transformation of the organoids forming xenograft tumors in NOD/SCID mice. The tumors exhibit histological characteristics of human mucinous adenocarcinoma, which is the tumor type highly associated with BRAF V600E mutation in human COAD. Gene expression analyses revealed up-regulation of intestinal stem cell signature genes and down-regulation of genes related to intestinal differentiation in BRAF V600E organoids. In addition, BRAF V600E organoids showed increased expression levels of Wnt pathway target genes indicating an enhanced and sustained Wnt-signaling. Analyses of CpG-island methylation in a panel of genes showed increased CpG-island methylation only in the BRAF V600E mutant organoids. In conclusion, in mouse colon organoids, BRAF V600E drives a human right-sided COAD phenotype with adoption of a stem cell niche independency, activation of the Wnt pathway, and induction of CpG island methylation. Citation Format: Byunghak Kang, Julie In, Nicholas Zachos, David Huso, Shinji Maegawa, Jean-Pierre Issa, Hariharan Easwaran, Stephen B. Baylin. Oncogenic BRAFV600E drives stem cell niche factors-independent growth and tumorigenic transformation in colon organoids. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4273.