In colon cancer, the BRAF^V600E mutation is strongly associated with the CpG island methylator phenotype (CIMP). Here, we characterized the contribution of BRAF^V600E to maintenance of aberrant DNA methylation using CRISPR-LbCpf1-corrected BRAF (V600E) organoids. DNA methylation analyses identified 5,187 differentially methylated CpGs within CpG islands—82% hypermethylated in BRAF^V600E organoids—including CIMP-associated genes and polycomb repressor complex 2 (PRC2) target genes. RNA sequencing showed concordant repression of these genes. Furthermore, BRAF^V600E organoids demonstrated high expression of PRC2 core components (EZH2, SUZ12, and EED), showed PRC2-induced H3K27 trimethylation in promoter regions, and maintained a PRC2-associated embryonic phenotype. This phenotype was lost following mutation correction or DNA methylation inhibition. These findings show that BRAF^V600E maintains aberrant DNA and histone methylation patterns in advanced colon cancer, likely preserving the transformed phenotype. Silencing of PRC2 target genes may contribute to this phenomenon. Epigenetic therapies may have value in the treatment of BRAF^V600E-mutant colon cancer.

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 BRAF (V600E) and RNF43 (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.

Summary: In colorectal cancer (CRC), RNF43 mutations are linked to BRAF V600E-initiated serrated adenomas that advance into mucinous adenocarcinomas with poor prognosis upon metastasis. How RNF43 mutations facilitate a metastasis-prone growth state remains unknown. Here, we addressed this issue by repairing mutant RNF43 in patient-derived BRAF-mutant CRC organoids using gene editing. Upon RNF43 correction, CRC organoids exhibit strongly decreased mucus production and, moreover, display loss of niche factor independence and metastatic capacity upon orthotopic transplantation in mice. Mechanistically, we show that mutant RNF43 promotes cancer cell lineage specification towards a non-dividing niche population that secretes essential growth factors, providing a state of self-sufficiency to the cancer epithelium. We show that phenotypic diversification into tumour-intrinsic niche cells (TINCs) and proliferative cancer stem cells depends on tuneable WNT levels enabled by mutations in RNF43 , but not APC . In patient samples, enhanced TINC profiles correlate with RNF43 -mutant CRC, mucinous histology and metastatic disease, thus representing a general cellular mechanism by which tumours acquire a self-sufficient, pro-metastatic growth state.

The current treatment for refractory BRAF-V600E mutant metastatic colorectal cancer (mCRC) involves combined inhibition of BRAF and the epidermal growth factor receptor (EGFR). However, tumour responses are often short-lived due to a rebound in mitogen-activated protein kinase (MAPK) activity. In this study, we combined short-term cell viability assays with long-term regrowth assays following drug removal over a period of three weeks. This allowed assessment of regrowth after therapy discontinuation. We tested the effect of combined BRAF inhibition (encorafenib) and EGFR inhibition (afatinib) on BRAF-V600E mutant CRC patient-derived organoids (PDOs). Combined EGFR/BRAF inhibition initially caused a major reduction in PDO growth capacity in BRAF-V600E mutant PDOs. This was followed by rapid regrowth after drug removal, mirroring clinical outcomes. EGFR inhibition in BRAF-V600E mutant PDOs led to activation of the insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R). The IGF1R/IR inhibitor linsitinib prevented the rebound in MAPK activity following removal of afatinib and encorafenib, prevented regrowth of CRC PDOs, and improved the anti-tumour response in an in vivo model. PDO regrowth assays allow the identification of pathways driving tumour recurrence. IR/IGF1R-inhibition prevents regrowth following golden standard MAPK pathway-targeted therapy and provides a strategy to improve the treatment of BRAF-V600E mutant CRC