Abstract
In patients with ALL the most common NT5C2 mutation class disrupted its switch-off mechanism.
Major finding: In patients with ALL the most common NT5C2 mutation class disrupted its switch-off mechanism.
Concept: Other mutations induce constitutive NT5C2 activation or allosteric regulation to confer chemoresistance.
Impact: Uncovering mechanisms by which NT5C2 confers chemoresistance may guide development of inhibitors.
NT5C2 is a ubiquitously expressed cytosolic nucleotidase that facilitates export of excess purine nucleotides out of the cell to maintain intracellular nucleotide pool homeostasis. NT5C2 can also dephosphorylate and inactivate cytotoxic thiopurine monophosphate nucleotides, including two generated by the thiopurine nucleoside analogs used to treat acute lymphoblastic leukemia (ALL): 6-thioguanine (6-TG) and 6-mercaptopurine (6-MP). Thus, somatic gain-of-function mutations in NT5C2 with increased nucleotidase activity can promote resistance to chemotherapy with 6-MP and 6-TG. The helix A segment of NT5C2 exists in a disordered inactive form and adopts a helical configuration that facilitates substrate binding and catalysis upon allosteric activation. A mutation in this region strongly activates NT5C2, and the role of helix A in activation is supported by modeling studies. However, the majority of NT5C2 mutations associated with ALL relapse occur outside of this region, prompting Dieck, Tzoneva, Forouhar, and colleagues to investigate additional regulatory elements may control NT5C2 activity. Analysis of allelic data from 643 patients with 6-MP–treated relapsed ALL revealed 32 independent NT5C2 mutations distributed throughout the gene, including 27 substitutions, 4 in-frame indel mutations, and a C-terminal truncating mutation. Determination of the crystal structure of wild-type and mutant NT5C2 homotetramers revealed 3 classes of mutations with different mechanisms of action. Class I mutations locked the activating helix A in a constitutively active configuration. Class II mutations were the most common and resulted in loss of the NT5C2 switch-off mechanism. Class III mutations promoted allosteric activation of NT5C2 via loss of a C-terminal acidic tail that functioned as a brake to restrain NT5C2 activation. Collectively, these findings reveal three distinct mechanisms by which NT5C2 mutations can confer resistance to thiopurines and may guide the development of NT5C2 inhibitors aimed at overcoming resistance in patients with ALL.
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