Project Summary Significant hurdles to developing new cancer therapeutics include the discovery of new targets and of novel protein pockets and interfaces amenable to therapeutic development. The gene methylthioadenosine phosphorylase (MTAP) is frequently co-deleted with the tumor suppressor gene CDKN2A in 25-50% of pancreas cancer, glioblastoma and mesothelioma, where there is a desperate need for new therapeutics. We found that MTAP deletion leads to accumulation of its substrate methylthioadenosine (MTA), and that MTA acts as an endogenous SAM-competitive inhibitor of the methyltransferase PRMT5. This leads to ?synthetic lethal? dependence of MTAP deleted tumors on PRMT5. Unfortunately, due to their SAM-cooperative mechanism, current PRMT5 inhibitors have no selectivity for MTAP deleted tumors and instead are broadly anti-proliferative. PRMT5 methylates substrates in complex with 3 adaptor proteins RIOK1, pICln and COPR5. Notably, RIOK1 and pICln were also synthetic lethal interactors with MTAP deletion. These data suggest that PRMT5 activity AND its interaction with the adaptors are required for the viability of MTAP deleted cancers. We have discovered a short peptide sequence, present in each adaptor, which mediates binding to PRMT5 (the PRMT5 binding motif or PBM). We solved the structure of the PBM bound to PRMT5 at a site we term the PBM groove. Since the adaptors are required for viability in MTAP deleted cells this interaction site may provide a new route to distinct PRMT5 therapeutics. Hence, we will test the hypothesis that the PBM groove and the PBM are necessary for PRMT5 methyltransferase function and maintenance of cell viability. The aims are: Aim 1: To test whether the PBM and the PBM-binding site on PRMT5 are necessary for the methylation of PRMT5 substrates and for the maintenance of cell viability in MTAP-deficient cells. Aim 2: To test the therapeutic validity of targeting the PBM groove of PRMT5. New preliminary data suggests that the mechanism of adaptor protein recruitment that we have identified mediates the methylation of certain but not all PRMT5 substrates. With the genetic tools and reagents that we have developed we can now determine which substrates are recruited through the PBM-mediate mechanism and/or whether there are substrates that are recruited independently of the PBM. A prime candidate for a substrate potentially recruited through an independent mechanism is the androgen receptor. Therefore in Aim 3 we will use a mass spectrometry proteomic approach: Aim 3: To determine the extent to which the PBM groove mediates PRMT5 substrate methylation.