PROJECT ABSTRACT The two greatest fears expressed by patients with ulcerative colitis (UC) are developing colon cancer and losing their colon. Although colitis-associated cancer is less common, dysplasia is more common and often results in colectomy. Our proposal leverages the work of the previous funding period and advances in the field to take a translational approach to unraveling dysplasia in colitis. The focus of our studies has been the link between innate immune signaling and intestinal bacteria leading to colitis-associated neoplasia. Our results have led us to focus on the role of dual oxidase 2 (DUOX2) in dysplasia. DUOX2 is a NADPH oxidase that catalyzes the conversion of oxygen into hydrogen peroxide (H2O2) upon interaction with the maturation factor DUOXA2. It is consistently upregulated in biopsies from IBD patients and DUOX2/DUOXA2 expression is further increased in patients who have had dysplasia. Our group has shown that both inflammatory and microbial signals induce the expression and activity (H2O2 production) of DUOX2 in colonic epithelial cells. We have proven that chronic activation of DUOX2 leads to the formation of tumors, which is almost totally abrogated by inactivating DUOX2 in the epithelium. In the current proposal, we hypothesize that IBD-associated dysbiosis activates Duox2 and local production of H2O2 leading to epithelial barrier dysfunction, recruitment of tumor-promoting myeloid derived suppressor cells (MDSCs), and generation of a tumorigenic microbiome in a feed forward loop. This is pursued in the following specific aims: 1) Determine the dependence of inflammatory colonic dysplasia on epithelial Duox2 signaling. Here we will investigate epithelial barrier dysfunction and DNA damage pathways caused by epithelial Duox2 activation using colonoid models (human and murine). Using humanized germ-free mice, we will determine if the UC-dysplasia microbiome is sufficient to cause DUOX2-mediated permeability defects and DNA damage. 2) Di