The novel BCR-ABL and FLT3 inhibitor ponatinib is a potent inhibitor of the MDR-associated ATP-binding cassette transporter ABCG2

Abstract

Ponatinib is a novel tyrosine kinase inhibitor with potent activity against BCR-ABL with mutations, including T315I, and also against fms-like tyrosine kinase 3. We tested interactions between ponatinib at pharmacologically relevant concentrations of 50 to 200 nmol/L and the MDR-associated ATP-binding cassette (ABC) proteins ABCB1, ABCC1, and ABCG2. Ponatinib enhanced uptake of substrates of ABCG2 and ABCB1, but not ABCC1, in cells overexpressing these proteins, with a greater effect on ABCG2 than on ABCB1. Ponatinib potently inhibited [(125)I]-IAAP binding to ABCG2 and ABCB1, indicating binding to their drug substrate sites, with IC(50) values of 0.04 and 0.63 μmol/L, respectively. Ponatinib stimulated ABCG2 ATPase activity in a concentration-dependent manner and stimulated ABCB1 ATPase activity at low concentrations, consistent with it being a substrate of both proteins at pharmacologically relevant concentrations. The ponatinib IC(50) values of BCR-ABL-expressing K562 cells transfected with ABCB1 and ABCG2 were approximately the same as and 2-fold higher than that of K562, respectively, consistent with ponatinib being a substrate of both proteins, but inhibiting its own transport, and resistance was also attenuated to a small degree by ponatinib-induced downregulation of ABCB1 and ABCG2 cell-surface expression on resistant K562 cells. Ponatinib at pharmacologically relevant concentrations produced synergistic cytotoxicity with ABCB1 and ABCG2 substrate chemotherapy drugs and enhanced apoptosis induced by these drugs, including daunorubicin, mitoxantrone, topotecan, and flavopiridol, in cells overexpressing these transport proteins. Combinations of ponatinib and chemotherapy drugs warrant further testing.

Figure 1. Ponatinib enhances uptake of substrates of ABCG2 and ABCB1, but not ABCC1, in cells overexpressing these proteins

Ponatinib effect on transport mediated by ABCB1 (A), ABCG2 (B) and ABCC1 (C) was measured by comparing cellular fluorescence after uptake of their fluorescent substrates DiOC₂(3), pheophorbide A (PhA) and rhodamine 123 (RH 123), respectively, in the presence and absence of ponatinib in relevant cell lines, with specific modulators 2.5 μM PSC-833, 10 μM fumitremorgin C (FTC) and 1 mM probenecid (proben) as positive controls. Each bar represents the mean ± SEM of three individual experiments. D-value is the Kolmogorov-Smirnov statistic. The chemical structure of ponatinib is shown in D.

Figure 2

A. Ponatinib decreases [¹²⁵I]-IAAP photolabeling of ABCB1 and ABCG2 Crude membranes from High-Five cells expressing ABCB1 and MCF-7 FLV1000 cells expressing ABCG2 were incubated with 0–10 μM ponatinib and [¹²⁵I]-IAAP. Representative autoradiograms are shown in the upper panel. In the lower panel, incorporation of [¹²⁵I]-IAAP into the ABCB1 and ABCG2 bands was plotted as a function of ponatinib concentration. Ponatinib inhibits [¹²⁵I]-IAAP binding to ABCG2 and ABCB1 with IC₅₀’s of 0.04 μM and 0.63 μM, respectively. Values are mean ± SD from three independent experiments. B. Ponatinib increases ABCB1 and ABCG2 ATPase activity. Crude membrane protein from High-Five cells expressing ABCB1 or ABCG2 was incubated with ponatinib at a range of concentrations in the presence or absence of sodium orthovanadate or beryllium sulfate and sodium fluoride, respectively. Average ABCB1 ATPase activity (upper panel), ABCG2 ATPase activity at the same ponatinib concentrations as for ABCB1 (middle panel) and ABCG2 ATPase activity at low ponatinib concentrations (lower panel) from independent duplicate experiments are shown, with standard errors.

Figure 3

A. Ponatinib cytotoxicity in K562/ABCB1 and K562/ABCG2 cells. K562, K562/ABCB1 and K562/ABCG2 cells were incubated with ponatinib (0–10 nM) for 96 hours and cell viability was measured using the WST-1 assay. Each data point represents the mean ± SEM of at least three experiments in triplicate. IC₅₀s were 0.46 nM, 0.51 nM and 0.92 nM for K562, K562/ABCB1 and K562/ABCG2 cells, respectively. B. Ponatinib decreases ABCG2 and ABCB1 surface expression on K562/ABCG2 and K562/ABCB1 cells. K562/ABCB1 and K562/ABCG2 cells were treated with ponatinib (0, 0.5 and 1 nM) for 48 hours, then stained with MRK16 and 5D3 antibodies, respectively. Each bar represents the mean ± SEM of fluorescence intensity in at least two experiments in duplicate. C. Ponatinib does not decrease ABCB1 and ABCG2 surface expression on HL60/VCR and 8226/MR20 cells. HL60/VCR and 8226/MR20 cells were treated with ponatinib (0, 50 and 100 nM) for 48 hours, then stained as above.

Figure 4

A. Ponatinib cytotoxicity in HL60/VCR, 8226/MR20 and MCF7/AdrVp cells 8226/MR20, HL60/VCR and MCF7/AdrVp cells were incubated with ponatinib at a range of concentrations for 96 hours and cell viability was measured using the WST-1 assay. Ponatinib was cytotoxic to HL60/VCR and 8226/MR20, but not MCF7/AdrVP, cells at pharmacologically relevant concentrations of 50 to 200 nM. B. Ponatinib and mitoxantrone or topotecan interactions in 8226/MR20 cells. Upper panels, response surfaces of ponatinib with mitoxantrone and with topotecan in 8226/MR20 cells, and lower panels, corresponding contour plots of the interaction index surfaces. Dashed lines indicate 95% confidence surface for additive action (interaction index =1) as described in Results. C. Ponatinib and daunorubicin interactions in HL60/VCR cells. Upper panel, response surface of ponatinib with daunorubicin in HL60/VCR cells, and lower panel, corresponding contour plot of the interaction index surface. D. Mitoxantrone was studied with ponatinib at 0, 50, and 100 nM in MCF7/AdrVP cells, yielding IC₅₀s of 44, 28 and 18 μM, respectively (left panel), while the IC₅₀ of mitoxantrone in parental MCF7 cells was 3.2 nM (right panel).

Figure 5. Ponatinib enhances apoptosis in combination with ABCG2 and ABCB1 substrate chemotherapy drugs

(A) ABCG2-overexpressing 8226/MR20 cells were treated with the ABCG2 substrates mitoxantrone, topotecan and flavopiridol, and ABCB1-overexpressing HL60/VCR (B) and 8226/Dox6 (C) cells were treated with the ABCB1 substrate daunorubicin at fixed concentrations, alone and in combination with 50, 100 and 200 nM ponatinib for 48 hours. Percentages of apoptotic cells are shown. Each bar represents the mean ± SEM of at least three experiments. Mitoxantrone, topotecan, flavopiridol and daunorubicin chemical structures are shown in D.

Figure 6. Effect of ponatinib on cell cycle distribution

HL60/VCR, 8226/MR20, K562 and MV4–11 cells treated with ponatinib at the indicated concentrations for the indicated times were harvested and fixed in 70% ethanol, then stained with propidium iodide and analyzed by flow cytometry.