Relapse remains a formidable challenge for acute lymphoblastic leukemia (ALL). Recently, recurrent mutations in NT5C2 were identified as a common genomic lesion unique in relapsed ALL and were linked to acquired thiopurine resistance. However, molecular mechanisms by which NT5C2 regulates thiopurine cytotoxicity were incompletely understood. To this end, we sought to comprehensively characterize the biochemical and cellular effects of NT5C2 mutations. Compared with wild-type NT5C2, mutant proteins showed elevated 5'-nucleotidase activity with a stark preference of thiopurine metabolites over endogenous purine nucleotides, suggesting neomorphic effects specific to thiopurine metabolism. Expression of mutant NT5C2 mutations also significantly reduced thiopurine uptake in vitro with concomitant increase in efflux of 6-mercaptopurine (MP) metabolites, plausibly via indirect effects on drug transporter pathways. Finally, intracellular metabolomic profiling revealed significant shifts in nucleotide homeostasis induced by mutant NT5C2 at baseline; MP treatment also resulted in global changes in metabolomic profiles with completely divergent effects in cells with mutant versus wild-type NT5C2. Collectively, our data indicated that NT5C2 mutations alter thiopurine metabolism and cellular disposition, but also influence endogenous nucleotide homeostasis and thiopurine-induced metabolomic response. These complex mechanisms contributed to NT5C2-mediated drug resistance in ALL and pointed to potential opportunities for therapeutic targeting in relapsed ALL.

Department of Oncology Pharmacology Core Karmanos Cancer Institute Wayne State University School of Medicine Detroit Michigan

Department of Oncology St Jude Children's Research Hospital Memphis Tennessee

Department of Pathology New York University Langone Medical Center New York New York

Department of Pediatrics Benioff Children's Hospital University of California San Francisco California

Department of Pediatrics New York University Langone Medical Center New York New York

Department of Pharmaceutical Sciences St Jude Children's Research Hospital Memphis Tennessee

Hematology Oncology Center Beijing Children's Hospital Capital Medical University Beijing China

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Prague Czech Republic

New York University Cancer Institute New York University Langone Medical Center New York New York

Protein Production Center St Jude Children's Hospital Memphis Tennessee

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