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

Abstract

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 μm) followed by ponatinib > bosutinib > dasatinib > imatinib. Imatinib inhibited hCNT2 with an IC50 value of 2.3 μm Ponatinib inhibited all five hNTs with the greatest effect seen for hENT1 (IC50 value, 9 μm). TKIs inhibited [(3)H]uridine uptake in a competitive manner. Studies in yeast with mutants at two amino acid residues of hENT1 (L442I, 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 [(3)H]cytarabine, [(3)H]cladribine, or [(3)H]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-ABL inhibitors and reduced interaction with M33A mutant hENT1.

Keywords: Saccharomyces cerevisiae; drug resistance; drug transport; inhibition mechanism; membrane transport; nucleoside/nucleotide transport; receptor tyrosine kinase.

FIGURE 1.

Structures of BCR-ABL TKIs. Structures of bosutinib, dasatinib, imatinib, nilotinib, and ponatinib.

FIGURE 2.

Effects of BCR-ABL TKIs on [³H]uridine uptake in yeast and CEM cells. Yeast cells were incubated with 1 μm [³H]uridine for 10 min in the absence or presence of increasing concentrations of imatinib (●) (0–300 μm), dasatinib (○) (0–300 μm), or nilotinib (□) (0–30 μm) (panel A). Data presented are averages of three experiments each conducted with six replicates per concentration and are expressed as mean ± S.E. Uptake values represent % uridine uptake in the presence of BCR-ABL TKIs relative to that in its absence (control). Error bars are not shown where S.E. values are smaller than the size of the symbol. Panel B shows effects of increasing concentrations (0–30 μm) of imatinib (●), dasatinib (○), or nilotinib (□) on [³H]uridine uptake in CEM cells. Values with mean ± S.E. are shown in each panel. Each experiment was repeated three times with four replicates per condition.

FIGURE 3.

Inhibition of hENT1-mediated uridine uptake by BCR-ABL TKIs. Effects of fixed concentrations of bosutinib, dasatinib, imatinib, ponatinib (0 (○), 10 (▴), 25 (●), or 50 (□) μm) and nilotinib 0 (○), 1 (▴), 2.5 (●), or 5 (□) μm on varying concentrations of [³H]uridine uptake in hENT1-producing yeast cells. Panels A–E show Lineweaver-Burk transformations of the data and panels F–J show Dixon slope replots. Values with mean ± S.E. are shown in each panel. Each experiment was repeated three times with four replicates for each experiment.

FIGURE 4.

Effect of BCR-ABL TKIs on uptake and accumulation of cytotoxic nucleoside drugs. Uptake (1 min) and accumulation (60 min) of 1 μm [³H]cytarabine, [³H]cladribine, or [³H]fludarabine in CEM cells was examined in the absence or presence 10 μm bosutinib, dasatinib, imatinib, ponatinib, or dipyridamole or 2 μm nilotinib. Panels A–C show 1-min uptake and panels D–F show 60-min accumulation. The symbols (●, ▴, and ■) represent experiments done on different days. Values plotted are % control values obtained in the absence of BCL-ABL TKIs and values from two to three experiments each conducted with four replicates are shown in each panel as scatter plots.

FIGURE 5.

BCR-ABL TKIs decrease hENT1 surface expression. CEM cells were incubated overnight with 1 μm bosutinib, dasatinib, imatinib, nilotinib, or ponatinib after which cells were washed and incubated with 100 nm SAHENTA-FITC for 1 h. Fluorescence was then determined with a flow cytometer. Panel A shows histograms from ponatinib-treated cells from one of three independent experiments. Stained untreated cells are shown in light gray, stained ponatinib-treated cells in white, and unstained cells in dark gray histograms. Panel B shows the results plotted from such experiments with all five TKIs as % Control of untreated cells. The symbols (●, ▴, and ■) represent experiments done on different days. Values with mean ± S.D. are shown as scatter plot.

FIGURE 6.

Effects of different modes of administration of [³H]uridine with BCR-ABL TKIs on accumulation of [³H]uridine in A549 cells. Panels A–E show [³H]uridine (1 μm) uptake in A549 that were either treated without or with 10 μm of each of the BCR-ABL TKIs (bosutinib, dasatinib, imatinib, or ponatinib) or 5 μm nilotinib for 15 min before, during, or after exposure to [³H]uridine for 15 min as described under ”Experimental Procedures.“ The symbols (●, ▴, and ■) represent experiments done on different days. Values plotted are % control values obtained in the absence of additives and values from three experiments each conducted with three replicates are shown in as scatter plots in each panel.