Founder Wnt alterations guide the evolutionary trajectories of colorectal tumorigenesis

Colorectal cancer (CRC) is classically divided into hypermutated (HM) and non-hypermutated (NHM) subtypes based on mutation burden. Although NHM tumors account for ~80% of CRC, no widely accepted genetic framework is available to understand CRC heterogeneity. In this study, we analyzed a total of 4,437 CRCs using targeted-capture sequencing across 169 driver genes, along with RNA sequencing of 2,833 tumors. Based on the bimodal distribution of mutation burden, 3,986 were classified as NHM, while HM cases were further resolved into MSI- and POLE -mutated CRC. Notably, within NHM CRCs, APC mutations, CTNNB1 alterations affecting exon 3, biallelic RNF43 mutations, and RSPO2/3 fusions were almost completely mutually exclusive, a pattern that also held in hypermutated tumors. Leveraging this mutual exclusivity, we classified NHM CRCs into five non-overlapping subgroups: APC -mutant ( APC mut ; 90%), CTNNB1 exon 3-altered ( CTNNB1 Exon3 ; 2.4%), biallelic RNF43 -mutant ( RNF43 biallelic ; 1.7%), RSPO2/3 fusion-positive ( RSPO2/3 fusion ; 1.7%), and Wnt-negative (Wnt WT ; ~1.5%). Accounting for ~10% of NHM CRCs, the APC -wild-type subtypes- CTNNB1 Exon3 , RNF43 biallelic , RSPO2/3 fusion , and Wnt WT -showed distinct genomic and transcriptomic features and consistently poorer outcomes than APC mut CRCs. Among them, the Wnt WT subtype exhibited attenuated Wnt signaling with prominent inflammatory and immune-response signatures, representing a highly inflamed subset distinct from Wnt-pathway-altered CRCs. To further dissect the heterogeneity of APC mut CRCs, we quantified the selection pressure acting on different APC mutations by calculating amino-acid-level dN/dS values. This analysis revealed five segments within the APC coding region with distinct profiles. Mutations within three segments were mutually exclusive even after accounting for biallelic APC inactivation. Leveraging this mutually exclusive architecture, we classified APC mut tumors into four subgroups (APC1-4). As expected, these CRC subgroups were characterized by different Wnt activation levels as assessed by AXIN2 expression or GSVA analysis of Wnt-pathway gene sets. Subgroups with lower-Wnt activation (APC3-4) were enriched for AMER1 or CTNNB1 minor-hotspot mutations, whereas those with higher-Wnt activation (APC1-2) frequently harbored TCF7L2/TCF7 mutations. These mutational patterns indicate that colorectal tumors avoid excessively high or low Wnt activity, instead maintaining signaling within a relatively restricted range. The APC mut subgroups also differed in their co-mutation profiles, chromosomal instability, tumor location, and clinical outcomes, suggesting that founder APC mutations dictate evolutionary trajectories and clinical behavior.In conclusion, we provide an integrated view of how founder Wnt alterations and the positional effects of APC truncations shape the molecular and evolutionary landscape of CRC.