SUMMARY: Inflammatory bowel disease (IBD) afflicts over three million people in the USA, is increasing worldwide, and leads to colitis-associated carcinogenesis (CAC). We are seeking new adjunctive IBD therapies that are safe, effective, and inexpensive that could also reduce risk for CAC. Spermidine (Spd) is a polyamine generated from putrescine and from back-conversion of spermine by spermine oxidase (SMOX). Recent high-impact studies have shown that Spd supplementation improves cardiovascular health, longevity and quality of life in aging, and does not increase the risk for cancer. Spd has been used successfully in Phase I clinical trials. We have developed a sensitive, accurate mass spectrometry-based assay for polyamine detection, which has enhanced our capabilities for this project. Our recent discoveries support the use of Spd as a new strategy for colitis treatment and CAC prevention. The long-term goal is to further elucidate the scope and mechanisms underlying the protective effect of Spd to gain insights needed for future human clinical trials in IBD. Our proposed studies are supported by our data indicating that: 1) Expression of SMOX, a key source of Spd, is reduced in patients with ulcerative colitis (UC) and associated dysplasia. 2) Mice with Smox deletion have reduced Spd in the colon and exacerbation of both dextran sulfate sodium (DSS) colitis, a model with features of UC, and CAC in the azoxymethane (AOM)-DSS model. 3) Spd supplementation restores colon Spd levels and protects against colitis and CAC. 4) Spd is the substrate for generation of hypusine, an amino acid produced by deoxyhypusine synthase (DHPS), which is required for a highly specific form of protein translation, hypusination, involving activation of eukaryotic translation initiation factor 5A (EIF5A) and formation of hypusinated EIF5A (EIF5AHyp). DHPS levels are low in UC patients. EIF5AHyp is reduced by Smox deletion and restored by Spd during DSS colitis and AOM- DSS-induced CAC, regulates macrophage function, and enhances colonic epithelial restitution. 5) Electrophiles, such as levuglandins (LGs), are damaging products of lipid peroxidation that can lead to immune dysfunction and neoplastic risk by forming adducts with proteins and DNA; we found increased adducts in human UC and CAC, and a specific scavenger of electrophiles reduced adduct formation and carcinogenesis in the AOM-DSS model. Importantly, we demonstrate that Spd can scavenge LGs. We hypothesize that potential benefits of Spd in colitis and carcinogenesis are due to effects on immune responses, epithelial function, hypusination, and electrophile scavenging. Our Aims are: 1) To determine the molecular and cellular mechanisms of protection by Spd in acute and chronic colitis models and CAC, including effects on the transcriptome/metabolome, autophagy, and immune cell versus epithelial cell function using transgenic mice with cell-specific human SMOX expression. 2) To determine if Spd acts through hy...