Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancerrelated death in the world. Somatic mutations drive colon tumor development by altering the activity of gene regulatory mechanisms that promote tumor growth. Different combinations of mutations can have unique effects on gene regulatory pathways, thereby promoting inter-tumor heterogeneity and affecting treatment outcomes. Individual oncogenic mutations can alter expression of oncogenes and tumor suppressors by changing the activity of regulatory elements known as enhancers. Aberrant enhancer activity can drive cancer phenotypes. Very few studies have systematically investigated how different combinations of mutations affect enhancer profiles and the downstream effects on other gene regulatory mechanisms. I hypothesize that different combinations of somatic CRC driver mutations will lead to unique changes in enhancer profiles. Understanding how mutation status affects transcriptional networks and key oncogenic pathways of colon tumor development will be important for the advancement of precision medicine by identifying candidate therapeutic targets for different mutational contexts of CRC.

The proposed experiments will leverage cutting-edge human, colonic organoid (colonoid) models that are genetically altered (CRISPR/Cas9 technology) to harbor combinations of mutations that frequently occur in CRC (APC, KRAS, P53). Utilizing chromatin run-on sequencing (ChRO-seq) data from these models, I will evaluate mutation-specific patterns of nascent transcription that can be used to assess enhancer activity across the genome. Using the underlying sequences of enhancers with mutation-specific patterns, I will perform transcription factor motif enrichment analysis to identify candidate transcription factors that contribute to mutation-driven tumor heterogeneity. Furthermore, I will characterize the effects of aberrant transcriptional programs on downstream gene regulatory mechanisms, with a focus on miRNAs. MiRNAs are small, noncoding RNAs that have been shown to be important negative regulators of gene expression in CRC and can be regulated transcriptionally. I will utilize ChRO- and small RNA-seq data from our colonoid models to identify transcriptionally regulated miRNAs and the transcription factors that regulate them. Successful completion of the proposed aims will have important implications for enhancer, miRNA, and colon biology while also identifying novel therapeutic targets for CRC.