PROJECT SUMMARY/ABSTRACT Classifying cancers based on molecular signatures, as opposed to tissue of origin, has allowed for the development of molecularly targeted therapeutics which maximize efficacy while minimizing toxicity for each patient. The vast majority of these therapies are inhibitors of oncogenic kinases, thus limiting the potential application of these therapies to specific cancer subtypes. Given their mechanism of action, resistance to kinase based therapies almost inevitably develops. Alternatively, the therapeutic potential of activating endogenous tumor suppressors has not been thoroughly explored. Unlike oncogene based therapies, re- activation of tumor suppressors holds the potential to target multiple molecular drivers, enhancing the applicability of such a therapy. One major class of tumor suppressor enzymes, Protein Phosphatase 2A (PP2A), is functionally inactivated in the majority of human cancers, rendering it a potential therapeutic target. The complex heterotrimeric structure of PP2A, consisting of an obligate scaffold and enzymatic subunit coupled to one of four classes of regulatory subunits, allows for broad substrate specificity and tumor suppressive functions. PP2A inactivation occurs through several mechanisms including increased expression of endogenous inhibitors, epigenetic silencing, and post-translational modifications to various subunits. Our lab has engineered a novel class of small molecule activators of PP2A (SMAPs) which direct conformational changes on the core dimer, re-activating PP2A, resulting in pre-clinical efficacy in xenograft, patient derived, and genetically engineered mouse models of cancer. Our preliminary studies have identified SMAP induced alterations to key regulators of PP2A, including the PP2A inhibitor, PME-1, an oncogenic methyl-esterase. Importantly, PME-1 is overexpressed in an array of human cancers, and has been shown to directly enhance tumorigenesis in cellular systems. Select cancer derived mutations in the scaffold subunit of PP2A demonstrate resistance to SMAP therapy, while demonstrating enhanced binding to PME-1. We seek to outline the mechanism by which PME-1 abrogates the tumor suppressive functions of PP2A as well as the mechanism by which SMAPs regulate PP2A:PME-1 complex formation to promote tumor suppression. This knowledge will provide critical insight into key regulatory elements responsible for directing PP2A substrate specificity and overall function. Understanding the SMAP induced mechanism of tumor suppression will lay the foundation for the development of next generation compounds.