Selective Inhibition of Human Equilibrative and Concentrative Nucleoside Transporters by BCR-ABL Kinase Inhibitors IDENTIFICATION OF KEY hENT1 AMINO ACID RESIDUES FOR INTERACTION WITH BCR-ABL KINASE INHIBITORS

Human nucleoside transporters (hNTs) mediate cellular influx of anticancer nucleoside drugs, including cytarabine, cladribine, and fludarabine. BCR-ABL tyrosine kinase inhibitors (TKIs) imatinib and dasatinib inhibit fludarabine and cytarabine uptake. We assessed interactions of bosutinib, dasatinib, imatinib, nilotinib, and ponatinib with recombinant hNTs (hENT1, 2; hCNT1, -2, and-3) produced individually in yeast Saccharomyces cerevisiae. Nilotinib inhibited hENT1-mediated uridine transport most potently (IC50 value, 0.7 mu m) followed by ponatinib > bosutinib > dasatinib > imatinib. Imatinib inhibited hCNI2 with an IC50 value of 2.3 mu m. Ponatinib inhibited all five hNTs with the greatest effect seen for hENT1 (IC50 value, 9 gm). TKIs inhibited [H-3]uridine uptake in a competitive manner. Studies in yeast with mutants at two amino acid residues of hENT1 (L4421, L442T, M33A, M33A/L442I) previously shown to be involved in uridine and dipyridamole binding, suggested that BCR-ABL TKIs interacted with Met(33) (TM1) and Leu(442) (TM11) residues of hENT1. In cultured human CEM lymphoblastoid cells, which possess a single hNT type (hENT1), accumulation of [H-3]cytarabine, [H-3]cladribine, or [H-3]fludarabine was reduced by each of the five TKIs, and also caused a reduction in cell surface expression of hENT1 protein. In conclusion, BCR-ABL TKIs variously inhibit five different hNTs, cause a decrease in cell surface hENT1 expression, and decrease uridine accumulation when presented together with uridine or when given before uridine. In experiments with mutant hENT1, we showed for the first time interaction of Met(33) (involved in dipyridamole binding) with BCR-ABI, inhibitors and reduced interaction with M33A mutant hENT1.