Abstract Colorectal cancer (CRC) is the leading cause of cancer-related death in the United States. Sprouty2 (Spry2) is an endogenous suppressor of receptor tyrosine kinase (RTK)/Ras/Mitogen Activated Protein Kinase (MAPK) signaling pathways. The objectives of this study are: 1. To assess requirement of Spry2 in CRC mouse models. 2. To generate and analyze multi-omics data set from mouse tumors and patient organoids. 3. To assess whether differential 5mC and 5hmC hotspot loci in Spry2 may contain acquired differentially methylated/hydroxymethlyated CpGs that can distinguish between adenomas, adenocarcinomas, and adjacent control mucosa. 4. Furthermore, increased Spry2 mRNA expression and differential promoter methylation within regions susceptible to 5mC changes associated with Spry2 expression may correlate with worse prognosis in advanced rectal cancer patients undergoing preoperative chemoradiotherapy (PCRT) and surgery. Therefore, Spry2 expression and promoter 5mC status together could serve as prognostic epimarkers in advanced rectal cancer patients. 5. As Spry2 is regulated in CRC by DNA-methylation within the promoter and can therefore be modulated by CRISPR-dCas9 tool to affect overall Spry2 expression and tumor growth, it could be considered as a future treatment strategy in patients. In previous studies, Spry2 accentuates cancer phenotype in CRC. It is hypothesized that deletion of Spry2 would suppress tumorigenesis in intestinal specific mouse models. To test this hypothesis, in Aim 1a, Sprouty2flox/flox-Cdx2-P-Cre-ERT2, and Sprouty2flox/flox-Cdx2-P-Cre-ERT2/KrasLSL-G12D double mutant mouse models are established and effect of Spry2 deletion on azoxymethane-induced colonic tumorigenesis is studied. Further, Spry2 deletion in mouse models is complemented with shRNA mediated suppression of Spry2 in Kras wild-type and KrasG12D mutant organoids established from adenomas of CRC patients (Aim 1b). Studies are extended to generate multi-omics dataset from mouse adenomas and patients’ organoids using omics methodologies to assess signaling pathways regulated by Spry2 (Aim 1c). Perturbing epigenetic regulation of Spry2 in patients could modify disease progression. In Aim 2a, we will identify differentially methylated 5mC and 5hmC hotspot regions of Spry2 in CRC patients to distinguish between adenomas, adenocarcinomas, and adjacent control mucosa. In Aim 2b, we will assess Spry2 mRNA expression and promoter methylation status in pathology collection of patients who had undergone PCRT followed by surgery (a retrospective study) to differentiate between PCRT responders vs non- responders by comparing disease free survival. Studies will be extended to test whether 5mC is an epigenetic target using CRISPR-dCas9 editing in CRC. In Aim 2c, by utilizing different CRC cell lines and immunocompromised mice, we will dissect the relationship between Spry2 methylation levels in the promoter region with Spry2 expression and tumor growth. The propose...