Abstract Somatic driver mutations, in genes such as FBXW7, have been discovered in phenotypically normal colonic tissue, however their role in cancer initiation remains elusive. Here, using patient-derived human colon organoids as models of early tumour evolution we investigate the consequences of FBXW7 mutations in normal and gene-edited organoids. We observed that FBXW7 mutations exert an epistatic effect where the transcriptional consequences of the mutation are dependent on the background mutational makeup of the cell. Specifically, we found the timing of acquiring an FBXW7 mutation relative to APC mutation, led to profound differences. When FBXW7 was mutated before APC , repression of the APC transcriptional response and maintenance of near-normal cell state was seen. However, when APC was mutated before FBXW7 , cells acquired classic cancer-stem cell features. Single-cell RNA sequencing revealed that FBXW7 mutations in normal tissue also function by subtly reordering stem cell hierarchies and priming a fetal/regenerative phenotype through upregulation of YAP/TAZ signaling. Further analysis using transposase-accessible chromatin sequencing found this cellular plasticity was driven by changes in the chromatin accessibility of 36 transcriptional start site regions associated with TEAD1/TEAD2 motifs, which in turn upregulated YAP. Taken together, we demonstrate a critical role of FBXW7 mutations in preventing the initiation of colorectal cancer, and provide exemplar evidence for the importance of epistasis and mutational order in cancer biology.

Mutational order and epistasis determine the consequences ofFBXW7mutations during colorectal cancer evolution