Multi-omic study of genome-edited human colonoid models of colorectal cancer reveal genotype-specific patterns of microRNA regulation

Combinations of oncogenic mutations drive inter-tumor heterogeneity in colorectal cancer (CRC), which promotes distinct phenotypes and affects therapeutic efficacy. We recently demonstrated that combinations of mutations in mouse small intestinal organoids lead to unique changes in microRNA (miRNA) expression profiles. However, it remains unknown how different mutational backgrounds shape miRNA profiles in the human colon. We leveraged human colonic organoid models, termed colonoids, with gene edits targeting genes commonly mutated in CRC to profile genotype-specific changes in miRNA expression. By small RNA-sequencing we characterized genotype-specific miRNA profiles. We identified one group of miRNAs, including mir-34a-5p and mir-10a-5p, that is strongly downregulated in APC/KRAS/TP53 mutant (AKP-mutant) colonoids. Using chromatin run-on sequencing, we showed that most miRNA alterations in AKP-mutant colonoids are concordant with transcriptional changes. Transcription factor (TF) motif enrichment analysis using transcriptional regulatory elements with increased activity in AKP-mutant colonoids revealed an enrichment of binding sites for multiple oncogenic TFs. Several of these harbor predicted binding sites for mir-10a-5p and/or mir-34a-5p, suggesting these miRNAs may play a role in regulating transcriptional programs in AKP-mutant contexts. Ultimately, our study offers a glimpse into regulatory mechanisms that drive inter-tumor heterogeneity, and we highlight candidate therapeutic targets for the advancement of precision medicine.