ABSTRACT: Colorectal adenocarcinoma (CRC) is a leading cause of cancer deaths globally. CRC tumorigenesis proceeds through a model of stepwise transformation of the colonic epithelium from benign adenoma to invasive carcinoma. This genetic process generally initiates from loss-of-function mutations in APC, which hyperactivate the Wnt signaling pathway, and requires eventual mutations in p53 for tumor progression and invasion. A majority of CRC cases is also characterized by chromosomal instability (CIN), which arises early and increases through tumor progression and which enables mutations that drive carcinogenesis. Despite enablement of transformation by CIN, the mechanisms by which CIN arises are not completely understood. A strong candidate contributor to CIN in early CRC is telomere dysfunction, which occurs when chromosome ends become shortened and uncapped and activate a DNA damage response (DDR) that leads to chromosome end-fusions. Telomere erosion and fusions have been observed in adenoma and carcinoma biopsies and have been shown to correlate with CIN, consistent with the possibility that telomeres may function as a driver of carcinogenesis. Given these observations, a deeper understanding of the mechanisms that regulate telomere capping and the consequences of telomere uncapping during CRC progression is needed. Our laboratory previously uncovered a novel feedback loop between telomere capping and Wnt signaling that contributes to homeostatic intestinal maintenance in mice and humans. Through this loop, loss of telomere capping leads to a broad suppression of Wnt pathway activity in intestinal crypt epithelia and underlying stroma. We determined that in mice this regulation involves p53- mediated expression miR-34a, a p53-activated microRNA which targets Wnt pathway components. Moreover, activation of the Wnt pathway reinforces telomere capping through upregulation of the shelterin protein TRF2. Thus, telomere capping and Wnt signaling in the gut are mutually supportive, and dysregulation of this feedback loop in CRC may be permissive for cells harboring dysfunctional telomeres. The study proposed herein aims to understand how oncogenic CRC mutations that might compromise Wnt-telomere feedback impact telomere status during CRC progression, as well as the extent to which perturbed telomere capping promotes tumor growth. My central hypothesis is that the regulatory loop between Wnt signaling and telomere capping is disrupted in CRC such that dysfunctional telomeres enhance, rather than inhibit, tumorigenesis. In Aim 1, I will use a human colonic organoid model to determine how APC and p53 mutations disrupt Wnt-telomere feedback. In Aim 2, I will characterize the relationship between these mutations and Wnt signaling/TRF2 expression in human CRC samples and determine whether induced telomere dysfunction in the setting of APC and p53 loss enhances organoid growth in murine xenografts. Taken together, these studies will provide unprecedent...