Project Summary/Abstract Despite intensive chemotherapy, 20% of pediatric and over 50% of adult acute lymphoblastic leukemia (ALL) ALL patients fail to achieve a complete remission or relapse after intensified chemotherapy, making relapse and resistance to therapy the most significant challenge in the treatment of this disease. This project seeks to dissect the structure and function of NT5C2, a cytosolic nucleotidase activated by gain of function mutations in about 45% of early relapse B-precursor ALL and 35% of early relapse T-ALL cases. NT5C2 mutations are characteristically associated with early relapse and progression under therapy and confer resistance to 6- mercaptopurine chemotherapy in vitro and in vivo. Our central hypothesis is that activating mutations in NT5C2 lock the NT5C2 protein in an active state either by forcing a constitutively active configuration similar to that induced by allosteric activators or via disruption of intramolecular switch off mechanisms responsible for returning the enzyme to its resting inactive state after activation. In this context, we will perform detailed structure-function analysis of NT5C2 mutant proteins to guide the development of new therapies for the treatment of relapse and refractory ALL.