Comparison of ATP-binding cassette transporter interactions with the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib

Abstract

Although the development of tyrosine kinase inhibitors (TKIs) to control the unregulated activity of BCR-ABL revolutionized the therapy of chronic myeloid leukemia, resistance to TKIs is a clinical reality. Among the postulated mechanisms of resistance is the overexpression of ATP-binding cassette (ABC) transporters, such as P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2), which mediate reduced intracellular drug accumulation. We compared the interactions of the TKIs imatinib, nilotinib, and dasatinib with ABCB1 and ABCG2 in ex vivo and in vitro systems. The TKIs inhibited rhodamine 123 and Hoechst 33342 efflux mediated by endogenous expression of the transporters in murine and human hematopoietic stem cells with potency order nilotinib >> imatinib >> dasatinib. Studies with ABCB1-, ABCG2-, and ABCC1-transfected human embryonic kidney 293 cells verified that nilotinib was the most potent inhibitor of ABCB1 and ABCG2. Cytotoxicity assays in stably transduced K562-ABCG2 and K562-ABCB1 cells confirmed that the TKIs were also substrates for the two transporters. Like imatinib, both nilotinib and dasatinib decreased ABCG2 surface expression in K562-ABCG2 cells. Finally, we found that all TKIs were able to compete labeling of ABCB1 and ABCG2 by the photo-cross-linkable prazosin analog [(125)I]iodoarylazidoprazosin, suggesting interaction at the prazosin-binding site of both proteins. Our experiments support the hypothesis that all three TKIs are substrates of ABC transporters and that, at higher concentrations, TKIs overcome transporter function. Taken together, the results suggest that therapeutic doses of imatinib and nilotinib may diminish the potential of ABCB1 and ABCG2 to limit oral absorption or confer resistance. Clinical data are required to definitively answer the latter question.

Fig. 1.

Inhibition of Abcb1- and Abcg2-mediated transport in murine KSL cells by the TKIs imatinib, nilotinib, and dasatinib. A, KSL cells in murine bone marrow were labeled and incubated with 0.2 μg/ml rhodamine 123 for 30 min at 37°C and then with 2.5 μg/ml Hoechst 33342 in the absence or presence of known inhibitors (B) or varying concentrations of imatinib (C), nilotinib (D), or dasatinib (E and F) as outlined under Materials and Methods. For plots B to F, the y-axis is Hoechst fluorescence and the x-axis is rhodamine fluorescence. Tariquidar (1 μM) served as a positive control for inhibition of ABCB1 and ABCG2, FTC (10 μM) for inhibition of ABCG2, and verapamil (15 μM) for inhibition of ABCB1.

Fig. 2.

Inhibition of ABCB1 in human ex vivo systems. A, mononuclear cells were isolated from human femora as described under Materials and Methods and incubated with 0.5 μg/ml rhodamine 123 for 30 min at 37°C in the absence or presence of the desired concentration of imatinib, nilotinib, dasatinib, or verapamil. Cells were then washed and allowed to incubate in rhodamine-free medium for 60 min continuing without or with inhibitor. Cells were subsequently stained with APC-Cy7-conjugated lineage markers to isolate the CD38⁻ and CD34⁺ population. Verapamil (black line) at a concentration of 50 μM served as a positive control for ABCB1 inhibition. B, peripheral blood mononuclear cells were separated by density gradient and incubated with rhodamine 123 with or without inhibitors as in A above. Subsequent to the 60-min efflux period, cells were stained with anti-CD56 antibody. Valspodar (3 μg/ml) served as a positive control for ABCB1 inhibition.

Fig. 3.

Interaction between TKIs and multidrug resistance proteins. Trypsinized HEK293 cells transfected with ABCG2, ABCB1, or ABCC1 were incubated with the desired fluorescent substrate (1 μM pheophorbide a or 200 nM BODIPY-prazosin for ABCG2, 0.5 μg/ml rhodamine 123 or 200 nM BODIPY-prazosin for ABCB1, or 200 nM calcein AM for ABCC1) in the presence or absence of the desired inhibitor for 30 min at 37°C. Cells were subsequently washed and allowed to incubate in substrate-free medium for 60 min continuing in the absence or presence of inhibitor. FTC (10 μM) served as a positive control for inhibition of ABCG2, valspodar (3 μg/ml) for ABCB1, and MK571 (100 μM) for ABCC1.

Fig. 4.

TKIs compete for photolabeling of ABCB1 and ABCG2 by [¹²⁵I]IAAP. We incubated crude membranes from High Five insect cells expressing ABCB1 (A) or ABCG2-expressing MCF-7 FLV1000 cells (B) with [¹²⁵I]IAAP and increasing doses of TKIs. The samples were then cross-linked by UV illumination, subjected to electrophoresis, and analyzed as outlined under Materials and Methods. Autoradiograms from one of at least three independent experiments are shown. ATPase activity was determined in crude membranes from High Five insect cells expressing ABCB1 (C) or ABCG2 (D) incubated in the presence of varying concentrations of dasatinib as outlined under Materials and Methods. Results compiled from at least three independent experiments are shown in C and D.

Fig. 5.

ABC transporters prevent TKI-mediated dephosphorylation of pCRKL in K562 cells. A, total cell lysates were obtained from K562 parental, K562-ABCG2, and K562-ABCB1 cells incubated with various concentrations of imatinib or dasatinib for 12 h or nilotinib for 24 h. Lysates were subjected to SDS-polyacrylamide gel electrophoresis, and protein was transferred to nitrocellulose membranes. The membranes were incubated overnight with anti-pCRKL antibody and subsequently incubated with secondary antibody that was detected with the Odyssey infrared imaging system. B, expression of pCRKL obtained from at least two independent experiments described in part A was normalized to GAPDH expression. Expression of pCRKL is reported based on assigning control cells a value of 100. Error bars represent S.D. Pgp, ABCB1.

Fig. 6.

TKIs decrease ABCG2 surface expression. A, ABCG2-transfected K562 cells were incubated for 14 h with 1 μM imatinib, nilotinib, or dasatinib, after which time cells were incubated with anti-ABCG2 antibody (denoted by solid line) or a negative control antibody (denoted by black histogram). Fluorescence was then determined with a flow cytometer. Histograms from one of at least three independent experiments are shown. Untreated cells are represented by the gray histogram. B, effect of varying concentrations of imatinib, nilotinib, or dasatinib on ABCG2 surface expression as measured by 5D3 antibody staining of K562 cells. Results are relative to ABCG2 surface expression levels found on untreated cells and assigned a value of 100.