Proposal Abstract Methionine adenosyltransferase 2 alpha (MAT2A) and protein arginine methyltransferase 5 (PRMT5) are cancer targets that are synthetically lethal with MTAP deletions and have several drug candidates in clinical trials targeting MTAP-/- cancers. MTAP is deleted in ~15% of human cancers and encodes the metabolic enzyme 5’-methylthioadenosine phosphorylase, the sole enzyme in humans responsible for recycling of methylthioadenosine (MTA) to methionine. MAT2A synthesizes S-adenosyl methionine (SAM), the methyl donor substrate for methyltransferase reactions. PRMT5 utilizes SAM as a substrate and is inhibited by MTA, and MTAP-/- cells in culture demonstrate elevated MTA levels. In vivo observations of glioblastoma tumors suggest however, that MTAP-/- does not always lead to increased tumoral MTA levels due to MTA efflux into matrix MTAP-competent cells. Additionally, MTAP deletions are a rare (~2%) occurrence in colorectal cancers (CRCs), precluding MAT2A and PRMT5 inhibitors’ use for most CRCs. The Schramm laboratory has previously solved the transition state (TS) structure of MTAP and synthesized a potent small molecule inhibitor methylthio-DADMe-immucillin-A (MTDIA) that recapitulates the in vitro effects of MTA accumulation within tissues. MTDIA has been shown to inhibit tumor growth in several cancer models, including CRC, and is linked to a decrease in PRMT5 activity through elevation of MTA levels. We propose that MTDIA be used in combination with MAT2A inhibitor AG-270, currently in Phase I clinical trials, to harness their synthetic lethality by targeting PRMT5. We will test the safety, target engagement, and anti-cancer efficacy of MTDIA in combination with AG-270 in ApcMin/+ and CRC patient-derived xenograft (PDX) mice. To determine mechanisms of anti-cancer effects, we will probe the upstream and downstream effects related to PRMT5 activity. We will perform tumor metabolomic quantification of relevant metabolites and histone and protein- arginine methylation characterization using immunohistochemistry and proteomic techniques. We will also profile the gene expression changes using single-cell RNA sequencing to determine how combination therapy alters tumor architecture and growth. Finally, we will solve the transition state structure of PRMT5 with the goal of laying the foundations for development of novel transition state analogue inhibitors. This work will expand upon the use of MAT2A and PRMT5 inhibitors beyond the ~15% of MTAP-deleted cancers and provide avenues for MTDIA to be used in clinical trials.