Disentangling BRAF-V600E signalling in colorectal cancer

Abstract

Colorectal cancer is one of the most common malignancies worldwide and arises when normal intestinal cells accumulate genetic and epigenetic alterations. These changes disrupt critical signaling pathways that regulate cell proliferation, survival, and differentiation, ultimately driving uncontrolled growth. Among the most frequently deregulated pathways are WNT/β-catenin and MAPK signaling. Tumors develop through distinct molecular routes, most prominently the adenoma–carcinoma sequence and the serrated pathway. While adenoma–carcinoma progression is often initiated by APC mutations, the serrated pathway is strongly associated with the activating BRAF-V600E mutation. Patients whose tumors carry this mutation typically face poor prognosis and more aggressive disease. This dissertation focuses on understanding why BRAF-V600E colorectal cancers behave so aggressively, and how their molecular features might be exploited therapeutically. Using CRISPR-based gene editing in patient-derived organoids, we corrected the BRAF-V600E mutation back to wild-type and, conversely, introduced the mutation into healthy tissue. These complementary approaches revealed profound molecular consequences: loss of tumorigenicity in xenografts, restored dependence on growth factors, and widespread transcriptional and epigenetic reprogramming. The results suggest that BRAF-V600E directly drives both MAPK hyperactivation and WNT/β-catenin pathway deregulation, not only through canonical signaling but also via epigenetic repression of pathway antagonists and activation of WNT enhancers. Importantly, the BRAF-V600E mutation proved essential for maintaining the CpG Island Methylator Phenotype (CIMP), a hallmark epigenetic program characterized by widespread promoter hypermethylation. This phenotype silences tumor suppressors and DNA repair genes, thereby reinforcing malignant behavior. Correcting BRAF abolished CIMP features, demonstrating a causal role for the mutation in sustaining this epigenetic state. Another gene frequently altered in BRAF-mutant colorectal cancer is RNF43, a negative regulator of WNT signaling. By editing RNF43 back to wild-type, we observed striking effects: organoids lost their ability to metastasize when transplanted into mice. Mechanistic analysis revealed that RNF43 mutations foster the emergence of niche cells that secrete growth factors, supporting tumor progression. When RNF43 is restored, this tumor–microenvironment interaction collapses, and metastatic capacity is lost. A final part of this work addresses treatment resistance. Clinically, patients with BRAF-mutant metastatic colorectal cancer are treated with combined BRAF and EGFR inhibition, but resistance invariably develops. To model this, we established long-term regrowth assays in organoids, uncovering that tumors rapidly escape via compensatory activation of insulin and IGF1 receptor signaling. Adding the IR/IGF1R inhibitor linsitinib to the current therapeutic backbone effectively suppressed regrowth and sustained pathway inhibition, pointing toward a rational triple-therapy approach. In summary, this dissertation provides new mechanistic insights into how BRAF-V600E mutations shape colorectal cancer biology through both genetic and epigenetic mechanisms, how RNF43 cooperates in metastatic progression, and how feedback activation of alternative receptor tyrosine kinases undermines current therapies. These findings highlight the need for tailored therapeutic strategies that simultaneously target the unique vulnerabilities of BRAF-mutant tumors and suggest new avenues for improving patient outcomes.