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

<h3>Background</h3> Somatic mutations, in genes such as <i>FBXW7</i>, have been discovered in phenotypically normal colonic tissue before the acquisition of cancer driver mutations such as <i>APC</i>, or <i>TP53</i>. Yet, their role in oncogenesis remains unknown. Here, we uncover the effects of <i>FBXW7</i> mutations on future cancer driver mutations. <h3>Methods</h3> Wildtype (W) colon organoids were generated using patient-derived normal tissue. To model pre-cancer and cancer, <i>APC^-/-</i> (A) and <i>APC^-/-/TP53^-/-</i> (AP) double mutant organoids were generated. <i>FBXW7^-/-</i> (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, <i>FBXW7^-/-</i> mutation was introduced to generate AF organoids. On F organoids, <i>APC^-/-</i> mutation was introduced to generate FA organoids (IDDF2024-ABS-0085 Figure 2). <h3>Results</h3> RNAseq showed an <i>FBXW7^-/-</i> 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 <i>FBXW7</i> mutation abrogates the chromatin effects of an <i>APC</i> mutation. <h3>Conclusions</h3> These findings point towards a protective role of <i>FBXW7</i> mutations in normal colonic tissue. Both the order of mutations and mutational background, therefore, play a hitherto undescribed role in colorectal cancer evolution.