PROJECT SUMMARY In recent years, adenosine has been identified as an important therapeutic target due to its observed immunosuppressive effects in the tumor microenvironment. By agonism to adenosine receptors, adenosine is a broad suppressor of immune function, decreased cytotoxic activity of T cells and NK cells, and increased differentiation of T cells to T regulatory cells. In the tumor microenvironment adenosine are elevated due to the overwhelming release of adenosine triphosphate and nicotinamide adenine dinucleotide, both of which are degraded to adenosine in the extracellular environment during cancer-associated stress conditions. Structurally similar 5’methylthioadenosine, and adenosine’s extracellular degradation product inosine are also observed to stimulate adenosine receptors. Independent of immunomodulatory mechanisms, tumor- expressed adenosine receptors have been observed to contribute to growth, metastasis, and proliferation of cancer cells. This effect is less defined compared to the immunosuppressive activities, but has significance in both solid tumors and post-chemotherapy or radiation models where adenosine and inosine are often greatly elevated due to surrounding dead or dying cells. Individual cancer cell lines commonly delete the gene encoding methylthioadenosine phosphorylase, responsible for depleting 5’methylthioadenosine, and are separately observed to modulate the expression of enzymes responsible for producing or degrading adenosine, or expression of adenosine receptors. The varied and redundant pathways resulting in adenosine receptor stimulation limits the effectiveness of single receptor agonists or enzymatic inhibitors. For this reason, in Aim 1 I will engineer a stable, high affinity human methylthioadenosine phosphorylase which substantially degrades both adenosine and 5’methylthioadenosine. Engineering an enzyme with favorable kinetic parameters, selectivity, and stability will allow for therapeutic characterization. In Aim 2, I will evaluate the in vitro efficacy of the enzyme and immune-independent mechanism with human cancer cell lines. Finally, in Aim 3 I will perform in vivo analysis of therapeutic potential. Following toxicology and pharmacologic studies, I will define the therapeutic effect on a the CT26 colon carcinoma syngeneic murine cancer model. I will use CD4 and CD8 T cell depletion and tumor immunophenotyping to gain insight into the immunomodulatory mechanism, specifically the balance of effector versus regulatory lymphocytes.