Project Summary Colorectal cancer (CRC) ranks as the 2nd most common cause of cancer mortality. Nearly all patients suffering from Lynch Syndrome as well as 15-20% of patients with sporadic early CRC have microsatellite instability (MSI) due to DNA mismatch repair (MMR) deficiency. Notably, defective MMR by itself is not sufficient to drive cell transformation and tumorigenesis, but microsatellite mutations in a limited number of target genes might be positively selected during tumor development, underlying MSI-associated pathogenesis. Unfortunately, relatively little is known about the molecular underpinnings of MSI target genes and their mechanism of action in MSI-associated disease penetrance. This project will fill this gap, capitalizing on our recent discovery of a strong correlation between the MSI phenotype and recurrent frameshift (FS) mutation in the autophagy tumor suppressor UVRAG. The protein product of this FS mutation functions as an oncogene and a bona fide trigger of centrosome amplification (CA) in CRC. We now bring within this proposal a collaboration of leaders in CRC genetics and molecular biology along with clinicians and pathologists to understand the molecular mechanism by which CA augments the expressivity of MMR mutations, contributing to human colonic carcinogenesis. To achieve this goal, we propose three Specific Aims, including (1) identifying the molecular mechanism of CA in MSI CRC associated with UVRAGFS expression; (2) examining the impact of CA on the differentiation and metastatic capacity of MSI patient-derived colonic organoids; and (3) investigating the in vivo role of MSI-derived UVRAGFS in disease penetrance and cancer progression using targeted mutant mouse models. These aims will be addressed using multidisciplinary innovative approaches that integrate state-of-the-art genetic, biochemistry, live- cell imaging, and physiological assays in cells and in mice with targeted mutations in genes related to MMR deficiency and centrosome deregulation. We have access to the right cohort of patients and our use of patient-derived colonic organoids will maximize the relevance of our findings for eventual translation to cancer patients in the clinic. Together, we anticipate that our studies will delineate a novel mechanism underlying MSI-associated disease penetrance and provide compelling in vivo validation of CA as a novel prognostic and predictive biomarker and a therapeutic target for personalized treatment of MSI CRC including Lynch Syndrome.