Pharmacogenetic characterization of naturally occurring germline NT5C1A variants to chemotherapeutic nucleoside analogs

Abstract

Background Mutations or alteration in expression of the 5’ nucleotidase gene family can confer altered responses to treatment with nucleoside analogs. While investigating leukemia susceptibility genes, we discovered a very rare p.L254P NT5C1A missense variant in the substrate recognition motif. Given the paucity of cellular drug response data from NT5C1A germline variation, we characterized p.L254P and eight rare variants of NT5C1A from genomic databases. Methods Through lentiviral infection, we created HEK293 cell lines that stably overexpress wildtype NT5C1A, p.L254P, or eight NT5C1A variants reported in the NHLBI Exome Variant server (one truncating and seven missense). IC50 values were determined by cytotoxicity assays after exposure to chemotherapeutic nucleoside analogs (Cladribine, Gemcitabine, 5-Fluorouracil). In addition, we used structure-based homology modeling to generate a 3D model for the C-terminal region of NT5C1A. Results The p.R180X (truncating), p.A214T, and p.L254P missense changes were the only variants that significantly impaired protein function across all nucleotide analogs tested (>5-fold difference versus WT; p<.05). Several of the remaining variants individually displayed differential effects (both more and less resistant) across the analogs tested. The homology model provided a structural framework to understand the impact of NT5C1A mutants on catalysis and drug processing. The model predicted active site residues within NT5C1A motif III and we experimentally confirmed that p.K314 (not p.K320) is required for NT5C1A activity. Conclusion We characterized germline variation and predicted protein structures of NT5C1A. Individual missense changes showed substantial variation in response to the different nucleoside analogs tested, which may impact patients’ responses to treatment.

Cytosolic 5′-nucleotidase 1A is overexpressed in pancreatic cancer and mediates gemcitabine resistance by reducing intracellular gemcitabine metabolites

“…Under physiological conditions, NT5C1A regulates the pool of adenosine monophosphate (AMP), which is, in turn, responsible for allosterically stimulating AMPK activity [ 18 , 19 ]. In addition, NT5C1A is a previously unrecognized gemcitabine inactivating enzyme that dephosphorylates gemcitabine monophosphate (dFdCMP) to the prodrug dFdC, thus potentially limiting the cytotoxicity of gemcitabine by decreasing the formation of dFdCTP [ [20] , [21] , [22] ].…”