IDDF2024-ABS-0085 Mutational order and epistasis regulate the transcriptional consequences of FBXW7 mutations during early colorectal cancer

Background

Somatic mutations, in genes such as FBXW7, have been discovered in phenotypically normal colonic tissue before the acquisition of cancer driver mutations such as APC, or TP53. Yet, their role in oncogenesis remains unknown. Here, we uncover the effects of FBXW7 mutations on future cancer driver mutations.

Methods

Wildtype (W) colon organoids were generated using patient-derived normal tissue. To model pre-cancer and cancer, APC^-/- (A) and APC^-/-/TP53^-/- (AP) double mutant organoids were generated. FBXW7^-/- (F) mutation was then introduced on W, A and AP backgrounds to generate F, AF and APF organoids respectively (IDDF2024-ABS-0085 Figure 1). In another model, we interrogated the effect of changing the order of mutation. We first generated A and F organoids separately. On A organoids, FBXW7^-/- mutation was introduced to generate AF organoids. On F organoids, APC^-/- mutation was introduced to generate FA organoids (IDDF2024-ABS-0085 Figure 2).

Results

RNAseq showed an FBXW7^-/- on different mutational backgrounds, resulting in increasing transcriptional heterogeneity. F organoids were transcriptionally quiescent and resembled W organoids. AF vs A showed more transcriptional variation, though the greatest variation was observed in APF vs AP (IDDF2024-ABS-0085 Figure 3). When the order of mutations was switched, morphologically, FA organoids resembled F organoids, while AF organoids resembled A organoids, although both FA and AF organoids have the same mutations (IDDF2024-ABS-0085 Figure 4). Principle component analysis (PCA) showed AF organoids were transcriptionally similar to A organoids, while FA organoids clustered with W and F organoids (IDDF2024-ABS-0085 Figure 5). There was a significant overlap between DE genes in FA and AF organoids. Among these overlap genes, the magnitude of perturbation was consistently muted in FA versus AF. Further analysis with scRNAseq showed a significant upregulation of the YAP/TAZ regenerative signature, and downregulation of the intestinal stem cell signature (IDDF2024-ABS-0085 Figure 6). ATAC-seq of W, AF and FA organoids showed reduced chromatin accessibility in FA organoids, suggesting that an early FBXW7 mutation abrogates the chromatin effects of an APC mutation.

Conclusions

These findings point towards a protective role of FBXW7 mutations in normal colonic tissue. Both the order of mutations and mutational background, therefore, play a hitherto undescribed role in colorectal cancer evolution.