Post-Translational Mechanisms of NT5C2 Activation As Non-Genetic Drivers of Chemotherapy Resistance in Relapsed Acute Lymphoblastic Leukemia

Despite intensive chemotherapy, 20% of pediatric acute lymphoblastic leukemia (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. We have shown that NT5C2 mutations resulting in deregulated enzymatic activity are present in 10% of relapsed B-precursor ALL and 20% of relapsed T-ALL cases. These genetic alterations abrogate the activity of an intramolecular switch-off mechanism resulting in increased nucleotidase activity. NT5C2 metabolizes and clears the activated forms of 6-mercaptopurine (6-MP), a critical drug in the treatment of ALL. As a result, activating mutations in the NT5C2 cytosolic nucleotidase gene are characteristically associated with early relapsed leukemia and progression under therapy and confer resistance to 6-mercaptopurine chemotherapy.

A saturated mutagenesis positive selection screen for all possible NT5C2 variants (10640 alleles) conferring resistance to 6-MP recovers mutational hotspots involving recurrent relapse-associated mutations and identifies new residues and protein domains involved in NT5C2 regulation whose inactivation drives resistance to 6-MP. Moreover, a genome wide CRISPR screen analysis of 6-MP-gene interactions in NT5C2 wild type REH ALL cells identifies NT5C2 as the most prominent hit whose inactivation increases sensitivity to 6-MP. Taken together, these results point to heretofore uncharacterized mechanisms of NT5C2 regulation and support a role for wild type NT5C2 in 6-MP metabolism and inactivation. Thus post-translational modifications enhancing NT5C2 activity could attenuate the therapeutic activity of 6-MP in NT5C2 wild type cells as non-mutational mechanism of 6-MP resistance.

Mass spectrometry analysis of NT5C2 protein revealed the presence of two NT5C2 acetylations and two NT5C2 phosphorylation sites. Functional analyses of these modifications revealed increased nucleotidase activity for the NT5C2 S502D phospho-mimic mutation. Consistently, expression of NT5C2 S502D in leukemia lymphoblasts induced resistance to 6-MP. Importantly, western blot analysis revealed increased NT5C2 S502 phosphorylation in relapsed leukemia xenograft samples compared with matched diagnosis derived ALL xenografts. Crystal structure analysis of NT5C2 recombinant protein supports that S502 engages D229 in the partner protomer of the NT5C2 dimer. Structure-function analysis of S502D, S502A and D229A mutants targeting this interaction supports the disruption of S502-D229 interaction via S502 phosphorylation as mechanism of NT5C2 activation.

In all, these results highlight a prominent role for wild type NT5C2 activity in the detoxification of 6-MP in ALL lymphoblasts, identifies NT5C2 S502 phosphorylation as a prevalent non-genetic mechanism of NT5C2 activation at relapse and provides novel insight on the mechanisms of NT5C2 regulation of relevance for the therapeutic targeting of NT5C2 in relapsed ALL.