BRAFV600E augments WNT signaling in colorectal cancer via aberrant DNA methylation

The BRAF^V600E mutation drives an aggressive subtype of colorectal cancer (CRC). Although WNT signaling activation is a hallmark of CRC, APC mutations are uncommon in BRAF-V600E mutant CRC, and RNF43 mutations are instead suspected to drive WNT pathway activation. Here, we investigated WNT pathway activation in BRAF-V600E mutant CRC using CRISPR-LbCpf1-corrected <i>BRAF</i> (V600E) and <i>RNF43</i> (P441fs) organoids. BRAF^E600V organoids regained dependency on EGF receptor signaling, and lost tumorigenic potential. Under identical growth conditions, correction of BRAF^V600E, rather than RNF43^P441fs, suppressed WNT target genes and upregulated epithelial differentiation genes and WNT antagonist genes. DNA methylation analysis revealed promoter hypermethylation of WNT antagonist genes and gene body hypermethylation -associated with transcriptional upregulation- of key WNT effectors (LGR5, EPHB2, and TCF4) in BRAF^V600E organoids. Demethylation treatment resulted in upregulation of WNT antagonists and reduced WNT target gene expression in BRAF^V600E organoids. Our results demonstrate that BRAF^V600E enhances WNT pathway activation through modulation of DNA methylation patterns.