Mechanisms of -Mediated Thiopurine Resistance in Acute Lymphoblastic Leukemia.

Relapse remains a formidable challenge for acute lymphoblastic leukemia (ALL). Recently, recurrent mutations in were identified as a common genomic lesion unique in relapsed ALL and were linked to acquired thiopurine resistance. However, molecular mechanisms by which regulates thiopurine cytotoxicity were incompletely understood. To this end, we sought to comprehensively characterize the biochemical and cellular effects of 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 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 mutations alter thiopurine metabolism and cellular disposition, but also influence endogenous nucleotide homeostasis and thiopurine-induced metabolomic response. These complex mechanisms contributed to 2-mediated drug resistance in ALL and pointed to potential opportunities for therapeutic targeting in relapsed ALL.