Functional characterization of CNOT3 variants identified in familial adenomatous polyposis adenomas

Germline mutations in the tumor suppressor Adenomatous Polyposis Coli (APC) define Familial Adenomatous Polyposis (FAP), the genetic predisposition to developing adenomatous polyps. Recent sequencing of FAP adenomas have challenged established dogma that APC mutations alone represent the adenoma mutational landscape because recurrent somatic mutations in non-WNT pathway genes were also discovered. In particular, one of these novel genes, CNOT3, presented E20K and E70K mutations that are predicted to be deleterious in silico. We utilized zebrafish embryos to determine if these mutations affect CNOT3 function and perform novel biology in an APC-dependent pathway in vivo. Human CNOT3 (hCNOT3) and E20K mRNA injection rescued zebrafish cnot3a knockdown lordosis phenotype while E70K did not. In the FAP apc(mcr) zebrafish model, we show that ctbp1, but not retinoic acid, regulates cnot3a expression. Injection of hCNOT3 and E20K, but not E70K, to homozygous apc(mcr) zebrafish initiated gut differentiation while cnot3a knockdown in wildtype embryos led to decreased intestinal development and differentiation. Finally, targeted sequencing of 37 additional FAP adenomas revealed CNOT3 mutations in 20% of these samples. Overall, our work supports a mechanism where CTBP1 regulates CNOT3 and that overall CNOT3 perturbation could work in concert with germline APC mutations in advancing adenomas to a more transformed state prior to progression to adenocarcinoma.