Patients with ulcerative colitis, a form of inflammatory bowel disease, face an increased risk of developing colorectal cancer. Although advances have been made in the therapeutic management of this disease, much less attention has been given to the development of cancer preventive strategies for this high-risk population. Ulcerative colitis patients often develop folate deficiencies that require supplementation with folic acid (FA), a synthetic form of folate. The effect of FA on risk for colorectal cancer remains unclear and the recent suggestion that FA supplements may be useful in preventing colitis-associated colorectal cancer is of great concern. Preliminary studies from this group provide the first evidence that FA supplementation (8 mg/kg diet) causes a dose-dependent increase in the formation of colorectal tumors in mice with AOM/DSS-induced colitis. Results from associated RNASeq and in vitro analyses suggest that FA promotes tumorigenesis by activating ERK and inducing NF-κB signaling in colonic epithelial cells with dysfunctional p53, the gatekeeping event in the development of colitis-associated cancers. The hypothesis of the proposed studies is that FA supplementation promotes UC-associated cancer in cells with dysfunctional/mutant p53, but not in cells with wild type p53, via an inflammatory pathway mediated by ERK and NF-κB. The mechanism by which FA induces tumor formation will be investigated in Aim 1 using CRISPR engineered isogenic human RKO colon carcinoma cells with varying p53 status (p53+/+, p53-/- and p53+/Mut). The impact of FA and dysfunctional p53 on activation of ERK, NF-κB signaling, as well as cell cycle progression will be evaluated. In vitro findings will be validated in Aim 2, where complimentary in vivo analyses will examine the combined effect of FA and mutant p53 on DSS-induced colitis-associated tumorigenesis and associated biomarkers in p53+/+ and p53+/515A (mutant) mice. In addition, the impact of high dose FA given prior to the induction of colitis will be examined. Tumor incidence and multiplicity, as well as degree of intestinal inflammation, will serve as primary endpoints of these studies. Based on the important contribution of intestinal microbiota to colitis and their ability to synthesize folate de novo, the impact of FA administration on the diversity and relative abundance of fecal and adherent bacteria within the intestine of p53+/515A mice with DSS-induced colitis will be examined in Aim 3. Mice will be treated with 5-aminosalicylic acid (5-ASA), a common maintenance therapy for ulcerative colitis, for the duration of FA exposure to recapitulate the clinical therapy of a patient following a diagnosis of colitis. The composition of the fecal and colon-adherent microbiomes, determined from 16S rRNA and metagenomic sequencing data, will be correlated with colonic inflammation, barrier function and tumor incidence/multiplicity. The results are expected to provide significant insight into the impact of FA supplementation on colitis-associated tumorigenesis and inform the first guidelines for the use of FA supplements by patients with ulcerative colitis.