Predicting colorectal cancer risk in FAP patients using patient-specific organoids.

Colorectal cancer (CRC), a prevalent global cancer, is mostly sporadic. Familial adenomatous polyposis (FAP), arises from APC germline mutations. We established FAP-human embryonic stem cell lines (FAP1,2,3) with distinct APC mutations and differentiated them into colon organoids to study cancer development. While normal expressing APC lines and FAP3 formed complex organoids, FAP1,2 failed to differentiate. By utilizing CRISPR editing to correct APC mutations in FAP1,2, we succeeded in restoring their ability to form complex organoids expressing colon gene (CDX2). To elucidate the truncated APC proteins' mechanism of action, we used AlphaFold2 algorithm to model their secondary structures. Structural analysis of the normal phenotype organoids (normal and FAP3) revealed 5-6 salt bridges only at the N-terminal oligomerization domain. In contrast, analysis of disease organoids-phenotype (FAP1,2) revealed a production of novel salt bridges, likely act in a dominant-negative manner on full-length APC, disrupting APC function and promoting tumorigenesis. Our study underscores the critical role of germline APC mutations in colon cancer initiation, revealing how specific mutations influence disease severity. By deciphering APC structure-function relationships, we illuminate potential therapies and the molecular underpinnings of APC mutations that precede clinical presentation.