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 [125I]-IAAP binding to ABCG2 and ABCB1, indicating binding to their drug substrate sites, with IC50 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 IC50 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. Mol Cancer Ther; 11(9); 2033–44. ©2012 AACR.

Cancer cell resistance to structurally unrelated drugs, termed MDR, is implicated in chemotherapy failure. The ATP-binding cassette (ABC) transport proteins ABCB1 [P-glycoprotein (Pgp), MDR1], ABCC1 [MDR protein-1 (MRP1)], and ABCG2 [breast cancer resistance protein (BCRP), mitoxantrone resistance protein (MXR)] are strongly implicated in MDR (1). These proteins are expressed on leukemia cells of all subtypes and transport structurally and functionally diverse drugs used to treat leukemias (1).

Diverse novel tyrosine kinase inhibitors (TKI) used to treat leukemias are ABCB1, ABCC1, and/or ABCG2 substrates and/or inhibitors. These include the BCR-ABL inhibitors imatinib mesylate, nilotinib, and dasatinib (2–8) used to treat chronic myelogenous leukemia (CML) and Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL), and the fms-like tyrosine kinase 3 (FLT3) inhibitors midostaurin (9), tandutinib (10), sorafenib (11), and sunitinib (12), in clinical trials in acute myeloid leukemia (AML) with FLT3 internal tandem duplication (ITD), present in 30% of cases and associated with adverse treatment outcome (13). TKI interactions with ABC proteins should be considered in design of treatment regimens, as they may cause resistance to TKIs, sensitization to chemotherapy drugs, and/or significant drug interactions.

Ponatinib (AP24534) is a novel TKI, currently in clinical trials, with potent activity in cells with BCR-ABL mutations including T315I, which confers resistance to the approved and available BCR-ABL inhibitors imatinib mesylate, nilotinib, and dasatinib (14). Ponatinib inhibits BCR-ABL at concentrations above 40 nmol/L (15), which are achieved with doses of 30 mg and greater (16), and shows promising clinical activity (16, 17). Ponatinib also potently inhibits FLT3 and thus may also have a role in AML therapy (18). It also inhibits fibroblast growth factor receptors, VEGF receptors (VEGFR), and angiopoietin (Tie2; ref. 14), promising targets in solid tumor therapy (19).

Given that other BCR-ABL inhibitors interact with MDR proteins and given the potential role of ponatinib in treating AML and solid tumors, in addition to CML and Ph+ ALL, we studied interaction of ponatinib with MDR-associated ABC proteins.

Cell lines

HL60, K562, and MV4-11 leukemia cells were obtained from the American Type Culture Collection; vincristine-selected HL60/VCR cells overexpressing ABCB1 (20) from Dr. Ahmad R. Safa, Indiana University, Indianapolis, IN; and doxorubicin-selected HL60/ADR cells overexpressing ABCC1 (21) from Dr. Kapil Bhalla, University of Kansas Cancer Center, Kansas City, KS. Parental 8226 myeloma cells and doxorubicin-selected 8226/Dox6 and mitoxantrone-selected 8226/MR20 cells, overexpressing ABCB1 or wild-type (R482) ABCG2, respectively (22), were obtained from Dr. William Dalton, Moffitt Cancer Center, Tampa, FL. Transfected K562/ABCB1 and K562/ABCG2 cells, stably overexpressing ABCB1 (23) or wild-type ABCG2 (24), were gifts from Dr. Michael Gottesman, National Cancer Institute, Bethesda, MD and Dr. Yoshikazu Sugimoto, Kyoritsu University of Pharmacy, Tokyo, Japan, respectively. Doxorubicin- and verapamil-selected MCF7/AdrVp breast carcinoma cells, overexpressing ABCG2 with the R482T mutation (25), were obtained from Dr. Douglas Ross, University of Maryland Greenebaum Cancer Center, Baltimore, MD, and flavopiridol-selected MCF-7/Flv1000 cells (26), overexpressing wild-type ABCG2, from Dr. Susan Bates, National Cancer Institute. All cells were cultured in RPMI 1640, pH 7.4, with 10% FBS at 37°C in a humidified atmosphere containing 5% CO2. No authentication of cell lines was carried out by the authors.

Reagents

Ponatinib (AP24534) was purchased from ChemieTek and was stocked at 10 mmol/L in dimethyl sulfoxide at −20°C. The fluorescent ABCB1 and ABCC1 substrates 3,3′-diethyloxacarbocyanine iodide [DiOC2(3)] and rhodamine 123 (RH 123; ref. 27) were purchased from Sigma-Aldrich and the fluorescent ABCG2 substrate pheophorbide A (PhA; ref. 28) from Frontier Scientific. The ABCB1 inhibitor PSC-833 was obtained from Novartis Pharmaceutical Corporation. The ABCC1 and ABCG2 inhibitors p-[dipropylsulfamoyl] benzoic acid (probenecid) and fumitremorgin C (FTC), respectively, were purchased from Sigma-Aldrich (29). Daunorubicin, mitoxantrone, and topotecan were purchased from Sigma-Aldrich and flavopiridol from Enzo Life Sciences. MRK16 antibody to an ABCB1 cell-surface epitope was purchased from Alexis Biochemicals, allophycocyanin conjugate (APC)-tagged 5D3 antibody to an ABCG2 cell-surface epitope from BD Biosciences, and BXP-21 ABCG2 antibody from Signet Laboratories. Fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI) were purchased from Trevigen, APC Annexin V from BD Biosciences, and LIVE/DEAD fixable near-IR dead cell stain from Invitrogen. Cell proliferation reagent WST-1 was purchased from Roche Diagnostics and [125I]Iodoarylazidoprazosin (IAAP; 2,200 Ci/mmol) from PerkinElmer Life and Analytical Sciences.

Uptake of fluorescent ABC protein substrates

To measure ponatinib effect on uptake of fluorescent ABC protein substrates, HL60/VCR, 8226/Dox6, and K562/ABCB1 cells (1 × 106) were incubated for 30 minutes at 37°C with DiOC2(3) (0.6 ng/mL) and ponatinib (0–200 nmol/L) or PSC-883 (2.5 μmol/L) as a control, HL60/ADR cells with RH 123 (0.5 μg/mL) and ponatinib (0–200 nmol/L) or probenecid (1 mmol/L) as a control and 8226/MR20, K562/ABCG2, and MCF7/AdrVp cells with PhA (1 μmol/L) and ponatinib (0–200 nmol/L) or FTC (10 μmol/L) as a control. The cells were then washed twice, resuspended in PBS, then acquired on a FACSCanto II flow cytometer (BD Biosciences) and analyzed using FlowJo software (Tree Star, Inc.). Substrate content after uptake with and without modulator was compared by the Kolmogorov–Smirnov statistic, expressed as a D value ranging from 0 (no difference) to 1 (no overlap), with D values 0.2 or greater indicating significant modulation (27).

Photoaffinity labeling of ABCB1 and ABCG2 with [125I]IAAP

High-Five insect cell membrane vesicles expressing ABCB1 and crude membranes from MCF-7/Flv1000 cells (30 μg) expressing ABCG2 were incubated with 0 to 10 μmol/L ponatinib for 5 minutes at 21°C to 23°C in 50 mmol/L Tris-HCl, pH 7.5. [125I]-IAAP (2200 Ci/mmol), 3 to 6 nmol/L, was added and photoaffinity labeling of ABCB1 and ABCG2 by [125I]-IAAP was measured as previously described (30, 31).

ABCB1 and ABCG2 ATPase assay

Crude membrane protein (100 μg protein/mL) from ABCB1- and ABCG2-expressing High-Five insect cells was incubated at 37°C with ponatinib in varying concentrations, with and without 0.3 mmol/L sodium orthovanadate, for ABCB1 or BeFx (0.2 mmol/L beryllium sulfate and 2.5 mmol/L sodium fluoride), for ABCG2, and the amount of inorganic phosphate released and the Vi- or BeFx-sensitive ATPase activity were measured as previously described (32).

MDR protein cell surface expression

To detect ABCG2 cell-surface expression, cells were incubated with APC-conjugated 5D3 antibody at room temperature for 30 minutes, then washed twice with PBS. To detect ABCB1 cell-surface expression, cells were incubated with MRK16 antibody for 1 hour, washed twice with PBS, then incubated with phycoerythrin (PE)-conjugated anti-human antibody for 30 minutes. Cells were acquired on a FACSCanto II (BD Biosciences) and analyzed with FlowJo. Replicate measurements of mean fluorescence intensity under different conditions were compared using the Student t test.

Cell viability assay

A total of 1 × 104 log-phase cells were seeded per well in 96-well tissue culture plates and incubated with ponatinib (0–10 μmol/L) or chemotherapy drugs at a range of concentrations at 37°C in 5% CO2 for 96 hours. Viable drug-treated cells were quantified using the WST-1 assay (29). Experiments were carried out in triplicate at least 3 times.

Drug interactions and statistical analysis

For cell lines for which ponatinib was cytotoxic at pharmacologically relevant concentrations in cell viability assays, ponatinib effects on chemotherapy drug cytotoxicity were evaluated in drug combination studies. Combination experiments were designed by the maximal power design (33, 34), using SynStat version 1.beta software (35). The method maximizes the power of the F-test to detect departures from the additive action of drugs. It does not assume a constant relative potency of the 2 drugs. With information from single-agent experiments, SynStat derives mixtures of 2 drugs and replicates of each mixture based on the pooled variations in single-agent experiments, which have 80% statistical power to detect at least a 15% difference in viability between the predicted additive values and the observed values at a significance level of 5%. Cells are then exposed to these multiple mixtures, and the cytotoxicity of these combinations is determined. Upon completion of the experiments, the F-statistic (33) is used to test the hypothesis of the additive action of 2 drugs and calculate the P value of the F-test. If the P value is greater than 0.05, the hypothesis of additive action is accepted. Otherwise, we calculate the interaction index (τ) (36) as

formula

in which, for a given cytotoxic effect, xA and xB are the concentrations of drugs A and B in the combination, and XA and XB are the concentrations of drugs A and B that achieve the same cytotoxic effect when given alone. A τ value of 1 indicates additivity, τ less than 1 indicates synergy, and τ greater than 1 indicates antagonism. The combination index surface is then fitted using the 2-dimensional B-spline method (34), and the contour plot shows the dose–mixture areas of additive action, synergy, and antagonism for the joint action of the 2 drugs.

Curve shift assay

MCF7/AdrVP cells, for which ponatinib was not cytotoxic at pharmacologically relevant concentrations in cell viability assays, were plated with mitoxantrone at a range of concentrations in a cell viability assay in the presence and absence of ponatinib at several concentrations, with analysis by the WST-1 colorimetric assay as described above.

Measurement of apoptosis

8226/MR20 cells, overexpressing ABCG2, were incubated with mitoxantrone, topotecan, or flavopiridol for 48 hours in the presence and absence of ponatinib, and apoptosis and necrosis were measured by staining with Annexin V–FITC and PI. HL60/VCR and 8226/Dox6 cells, overexpressing ABCB1, were incubated with daunorubicin for 48 hours in the presence and absence of ponatinib, and apoptosis and necrosis were measured using APC Annexin V and LIVE/DEAD fixable near-IR dead cell stain, to avoid spectral overlap with daunorubicin. After treatment, cells (2 × 105–3 × 105) were washed with PBS, resuspended in Annexin V binding buffer (1x), stained with Annexin V–FITC (1 μL) and PI (2 μL) or APC Annexin V (2.5 μL) and LIVE/DEAD fixable near-IR dead cell stain (0.5 μL), incubated at room temperature in the dark, then washed and acquired on a FACSCanto II and analyzed with FlowJo.

Flow cytometric cell-cycle analysis

A total of 1 × 105 HL60/VCR, 8226/MR20, K562, and MV4-11 cells were treated with 0, 1, 5, 50, and 100 nmol/L ponatinib for 24 and 48 hours, fixed in chilled ethanol (70%), washed with PBS, then treated with DNase-free RNase (200 μg/mL) for 1 hour at 37°C, stained with PI (40 μg/mL) and kept in the dark for 15 minutes at 20°C to 25°C. Staining was measured on a FACScan, and percentages of cells in different cell-cycle phases were determined using FlowJo.

Ponatinib increases substrate uptake in cells overexpressing ABCB1 and ABCG2

Ponatinib produced a significant concentration-dependent increase in uptake of the ABCB1 substrate DiOC2(3) in ABCB1-overexpressing HL60/VCR, K562/ABCB1, and 8226/Dox6 cells, and of the ABCG2 substrate PhA in ABCG2-overexpressing 8226/MR20, K562/ABCG2, and MCF7/AdrVP cells, with greater inhibition of ABCG2 than of ABCB1 (Fig. 1). The effect in MCF7/AdrVp was less than in 8226/MR20 and K562/ABCG2, likely because of a greater degree of resistance in the solid tumor in relation to hematopoietic cell lines, rather than to the presence of the R482T mutation in MCF7/AdrVp, though the latter is also possible. Because the R482T ABCG2 mutation is not clinically relevant, we did not pursue this distinction. Ponatinib had no effect on RH 123 uptake in ABCC1-overexpressing HL60/ADR cells.

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), PhA, and rhodamine 123 (RH 123), respectively, in the presence and absence of ponatinib in relevant cell lines, with specific modulators 2.5 μmol/L PSC-833, 10 μmol/L FTC and 1 mmol/L probenecid (proben) as positive controls. Each bar represents the mean ± SEM of 3 individual experiments. D value is the Kolmogorov–Smirnov statistic. The chemical structure of ponatinib is shown in D.

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), PhA, and rhodamine 123 (RH 123), respectively, in the presence and absence of ponatinib in relevant cell lines, with specific modulators 2.5 μmol/L PSC-833, 10 μmol/L FTC and 1 mmol/L probenecid (proben) as positive controls. Each bar represents the mean ± SEM of 3 individual experiments. D value is the Kolmogorov–Smirnov statistic. The chemical structure of ponatinib is shown in D.

Close modal

Ponatinib inhibits [125I]-IAAP photolabeling of ABCB1 and ABCG2

Given that ponatinib inhibited transport by ABCB1 and ABCG2, we studied its binding to their drug substrate sites by measuring its effect on their photolabeling with [125I]-IAAP. Crude membranes from High-Five cells expressing ABCB1 and MCF-7 FLV1000 cells expressing ABCG2 were photo-cross-linked with 3 to 6 nmol/L [125I]-IAAP (2,200 Ci/mmole) in the presence of 0 to 10 μmol/L ponatinib. Ponatinib inhibited [125I]-IAAP binding to ABCG2 and ABCB1 with IC50 values of 0.04 and 0.63 μmol/L, respectively (Fig. 2A), indicating strong and weaker binding, respectively, to the ABCG2 and ABCB1 drug substrate sites.

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 to 10 μmol/L ponatinib and [125I]-IAAP. Representative autoradiograms are shown in the top panel. In the bottom 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 values of 0.04 and 0.63 μmol/L, respectively. Values are mean ± SD from 3 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 (top), ABCG2 ATPase activity at the same ponatinib concentrations as for ABCB1 (middle) and ABCG2 ATPase activity at low ponatinib concentrations (bottom) from independent duplicate experiments are shown, with SEs.

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 to 10 μmol/L ponatinib and [125I]-IAAP. Representative autoradiograms are shown in the top panel. In the bottom 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 values of 0.04 and 0.63 μmol/L, respectively. Values are mean ± SD from 3 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 (top), ABCG2 ATPase activity at the same ponatinib concentrations as for ABCB1 (middle) and ABCG2 ATPase activity at low ponatinib concentrations (bottom) from independent duplicate experiments are shown, with SEs.

Close modal

Ponatinib stimulates ABCB1 and ABCG2 ATPase activity

Given that ponatinib exhibited binding to the ABCG2 and ABCB1 drug substrate sites, we determined its effect on their ATPase activity, as drug-stimulated ATPase activity is a useful measure of substrate interaction at the drug-binding sites of these transporters (37). Ponatinib stimulated ABCG2 ATPase activity in a concentration-dependent manner (Fig. 2B) and stimulated ABCB1 ATPase activity at low, pharmacologically relevant, concentrations, but not at higher concentrations (Fig. 2B). Thus, ponatinib significantly increased ABCG2 and ABCB1 ATPase activity at lower concentrations, indicating direct interaction at these transporters' drug substrate binding sites, similarly to other TKIs (38).

Ponatinib cytotoxicty in K562, K562/ABCB1, and K562/ABCG2 cells

K562, K562/ABCB1, and K562/ABCG2 cells, with the BCR-ABL translocation, were incubated with ponatinib in cell viability assays, yielding IC50 values of 0.46, 0.5, and 0.92 nmol/L, respectively (Fig. 3A). Thus ABCG2 confers resistance, albeit low-level, to ponatinib, attributable to efflux of this drug by this transporter, but the resistance was modest and was likely attenuated by inhibition of ABCG2-mediated ponatinib transport by ponatinib itself. ABCB1-mediated ponatinib transport was also likely attenuated by ponatinib. An additional possible mechanism of attenuation of resistance is ponatinib-induced decreased cell surface expression of ABCG2 and ABCB1.

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 nmol/L) for 96 hours and cell viability was measured using the WST-1 assay. Each data point represents the mean ± SEM of at least 3 experiments in triplicate. IC50 values were 0.46, 0.51, and 0.92 nmol/L 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 nmol/L) for 48 hours, then stained with MRK16 and 5D3 antibodies, respectively. Each bar represents the mean ± SEM of fluorescence intensity in at least 2 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 nmol/L) for 48 hours, then stained as above.

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 nmol/L) for 96 hours and cell viability was measured using the WST-1 assay. Each data point represents the mean ± SEM of at least 3 experiments in triplicate. IC50 values were 0.46, 0.51, and 0.92 nmol/L 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 nmol/L) for 48 hours, then stained with MRK16 and 5D3 antibodies, respectively. Each bar represents the mean ± SEM of fluorescence intensity in at least 2 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 nmol/L) for 48 hours, then stained as above.

Close modal

Ponatinib decreases ABCG2 and ABCB1 cell-surface expression

To determine whether ponatinib might also decrease ABCG2 and ABCB1 cell-surface expression on K562/ABCG2 and K562/ABCB1 cells, respectively, cells were incubated for 48 hours with ponatinib at 0, 0.5, and 1.0 nmol/L. Surface ABCG2 (mean ± SEM, 1,612 ± 2 vs. 2,103 ± 21; P = 0.0017) and ABCB1 (mean ± SEM, 596 ± 16 vs. 759 ± 58; P = 0.05) expression decreased on K562/ABCG2 and K562/ABCB1 cells incubated with 1 nmol/L ponatinib, in relation to control (Fig. 3B). This effect might contribute to sensitization of K562/ABCG2 and K562/ABCB1 cells to ponatinib, albeit minimally, as decreases in expression were small. In contrast, ABCG2 (mean ± SEM, 2,650 ± 70 vs. 3,210 ± 197; P = 0.056) and ABCB1 (mean ± SEM, 31,224 ± 1,865 vs. 36,030 ± 2,898; P = 0.2) cell-surface expression did not decrease on 8226/MR20 and HL60/VCR cells, which do not express BCR-ABL, incubated for 48 hours with versus without ponatinib (Fig. 3C). ABCG2 and ABCB1 cell-surface expression on cells expressing BCR-ABL may decrease because of inhibition of Akt downstream of BCR-ABL (39). Alternatively, 5D3 binding is altered by ABCG2 conformational changes induced by effects on function (40), though this does not explain different effects in K562/ABCG2 and 8226/MR20.

Ponatinib and chemotherapy drug interactions

Because ponatinib was cytotoxic to 8226/MR20 and HL60/VCR cells at pharmacologically relevant concentrations, with IC50 values of 160 and 180 nmol/L, respectively (Fig. 4A, left and middle panels), ABCG2 and ABCB1 substrate chemotherapy drug cytotoxicity in the presence of ponatinib was analyzed in drug interaction studies in these cells.

Figure 4.

A, ponatinib cytotoxicity in HL60/VCR, 8226/MR20, and MCF7/AdrVp cells. The 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 nmol/L. B, ponatinib and mitoxantrone or topotecan interactions in 8226/MR20 cells. Top, response surfaces of ponatinib with mitoxantrone and with topotecan in 8226/MR20 cells; bottom, 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. Top, response surface of ponatinib with daunorubicin in HL60/VCR cells; bottom, corresponding contour plot of the interaction index surface. D, mitoxantrone was studied with ponatinib at 0, 50, and 100 nmol/L in MCF7/AdrVP cells, yielding IC50 values of 44, 28, and 18 μmol/L, respectively (left), whereas the IC50 value of mitoxantrone in parental MCF7 cells was 3.2 nmol/L (right).

Figure 4.

A, ponatinib cytotoxicity in HL60/VCR, 8226/MR20, and MCF7/AdrVp cells. The 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 nmol/L. B, ponatinib and mitoxantrone or topotecan interactions in 8226/MR20 cells. Top, response surfaces of ponatinib with mitoxantrone and with topotecan in 8226/MR20 cells; bottom, 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. Top, response surface of ponatinib with daunorubicin in HL60/VCR cells; bottom, corresponding contour plot of the interaction index surface. D, mitoxantrone was studied with ponatinib at 0, 50, and 100 nmol/L in MCF7/AdrVP cells, yielding IC50 values of 44, 28, and 18 μmol/L, respectively (left), whereas the IC50 value of mitoxantrone in parental MCF7 cells was 3.2 nmol/L (right).

Close modal

In the ponatinib and mitoxantrone combination experiments in 8226/MR20 cells, mitoxantrone concentrations were 0.09 to 0.12 μmol/L and ponatinib concentrations 0.001 to 0.25 μmol/L. On the basis of maximal power design, 18 mixtures were chosen, with 5 replicates of each mixture, yielding 108 total observations. The maximum and minimum viabilities (% of control) were 55% and 1.28%, mean 12.77% and SD 13.111. Figure 4B, top left, shows the response surface of the combination of ponatinib and mitoxantrone in 8226/MR20 cells. With the observations from the combination experiments, the F-test (34) shows that we cannot accept that ponatinib and mitoxantrone have additive activity [F(16, 90) = 40.20, P < 0.0001]. The contour plot of the interaction index surface (Fig. 4B, bottom left) shows that the combination of ponatinib and mitoxantrone in 8226/MR20 cells is antagonistic or additive at lower ponatinib concentrations (less than 40 nmol/L, approximately), synergistic at ponatinib concentrations above approximately 40 nmol/L, and additive at the highest concentrations of both drugs studied.

In the ponatinib and topotecan combination experiments in 8226/MR20 cells, topotecan concentrations were 0.045 to 0.175 μmol/L, and ponatinib concentrations were 0.05 to 0.25 μmol/L. On the basis of maximal power design, 16 mixtures were chosen, with 5 replicates of each mixture yielding 96 total observations. Maximum and minimum viabilities were 100% and 15%, mean 50.85%, and SD 23.00. Figure 4B, top right, shows the response surface of the combination of ponatinib and topotecan against 8226/MR20 cells. With the observations from the combination experiments, the F-test (34) shows that we cannot accept that topotecan and ponatinib have additive action [F(14, 78) = 14.55, P < 0.0001]. The contour plot of the interaction index surface (Fig. 4B, bottom right) shows that the combination of topotecan with ponatinib is antagonistic in 8226/MR20 cells at lower concentrations of both drugs and synergistic at the higher concentrations tested.

Ponatinib and daunorubicin combination experiments were carried out in HL60/VCR cells, overexpressing ABCB1. Daunorubicin concentrations were 0.04 to 2.20 μmol/L, and ponatinib concentrations were 0.001 to 0.25 μmol/L. On the basis of the maximal power design, 19 mixtures were chosen, with 4 replicates of each mixture, for 95 total observations. Maximum and minimum viabilities were 100% and 0.1%, mean 53.60% and SD 40.027. Figure 4C, top panel, shows the response surface of the combination of ponatinib and daunorubicin in HL60/VCR cells. With the observations from the combination experiments, the F-test (34) showed that we cannot accept that ponatinib and daunorubicin have additive effects in HL60/VCR cells [F(17, 76) = 85.70, P < 0.0001]. The contour plot of the interaction index surface (Fig. 4C, bottom panel) showed that the combination of ponatinib and daunorubicin is antagonistic in HL60/VCR cells for daunorubicin concentrations less than approximately 0.25 μmol/L for all concentrations of ponatinib studied, but that daunorubicin and ponatinib are synergistic at daunorubicin concentrations of 0.25 to 1.1 μmol/L and ponatinib concentrations greater than approximately 0.18 μmol/L.

Sensitization to chemotherapy drug

As the IC50 of ponatinib in ABCG2-overexpressing MCF7/AdrVP cells was 1.2 μmol/L and ponatinib was not cytotoxic at the pharmacologically relevant concentrations of 50 and 100 nmol/L (Fig. 4A, right panel), its effect on sensitivity of these cells to the ABCG2 substrate chemotherapy drug mitoxantrone was studied in a curve shift assay. Mitoxantrone IC50 values in the presence of 0, 50, and 100 nmol/L ponatinib were 44, 28, and 18 μmol/L (Fig. 4D, left panel), showing concentration-dependent ponatinib sensitization of MCF7/AdrVP cells to mitoxantrone. In contrast, mitoxantrone was cytotoxic to parental MCF7 cells in nanomolar range, with anIC50 of 3.2 nmol/L (Fig. 4D, right panel).

Ponatinib enhances apoptosis induction by ABCG2 or ABCB1 substrate chemotherapy drugs in cell lines overexpressing these proteins

Ponatinib at pharmacologically relevant concentrations of 50, 100, and 200 nmol/L sensitized 8226/MR20 cells, overexpressing ABCG2, to apoptosis induction by 100 nmol/L mitoxantrone, 300 nmol/L topotecan, and 200 nmol/L flavopiridol (Fig. 5A) and also increased apoptosis induced by daunorubucin (250 nmol/L) in HL60/VCR (Fig. 5B) and 8226/Dox6 (Fig. 5C) cells, overexpressing ABCB1, in a concentration-dependent manner.

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 nmol/L ponatinib for 48 hours. Percentages of apoptotic cells are shown. Each bar represents the mean ± SEM of at least 3 experiments. Mitoxantrone, topotecan, flavopiridol, and daunorubicin chemical structures are shown in D.

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 nmol/L ponatinib for 48 hours. Percentages of apoptotic cells are shown. Each bar represents the mean ± SEM of at least 3 experiments. Mitoxantrone, topotecan, flavopiridol, and daunorubicin chemical structures are shown in D.

Close modal

Cell-cycle effects of ponatinib

Finally, TKIs can cause cell-cycle arrest, which can result in kinetic resistance when they are combined with chemotherapy drugs. We therefore studied the effect of ponatinib on cell cycle in HL60/VCR and 8226/MR20 cells. Ponatinib had no effect on cell-cycle parameters in these drug-resistant cells without BCR-ABL rearrangement or FLT3-ITD (Fig. 6). In contrast, as expected, it caused cell-cycle arrest and apoptosis in K562 cells, with BCR-ABL rearrangement, and in MV4-11 cells, with FLT3-ITD (Fig. 6).

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.

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.

Close modal

We have shown that the novel BCR-ABL and FLT3 inhibitor ponatinib is a potent inhibitor of drug transport by ABCG2 at pharmacologically relevant concentrations and synergizes with ABCG2 substrate chemotherapy drugs in inducing cytotoxicity and apoptosis in cells overexpressing ABCG2. Ponatinib also inhibits drug transport by ABCB1, albeit less potently, and synergizes with ABCB1 substrate drugs.

Ponatinib inhibition of drug transport by ABCG2 and ABCB1 seems to occur by direct interaction with these transporters. The results of the [125I]-IAAP photolabeling assay indicated strong binding of ponatinib to the drug substrate site of ABCG2 and weaker binding to that of ABCB1. In addition, ponatinib stimulated ABCG2 ATPase activity in a concentration-dependent manner and stimulated ABCB1 ATPase activity at low, pharmacologically relevant, concentrations. Thus ponatinib directly interacts at the substrate-binding sites of ABCG2 and ABCB1 at pharmacologically relevant low concentrations. Ambudkar and colleagues (41) defined 3 classes of ABCB1 inhibitors based on their effects on ABCB1 ATPase activity. Class I agents stimulate ATPase activity at low concentrations but inhibit it at high concentrations, whereas Class II compounds stimulate ATPase activity in a concentration-dependent manner without any inhibition, and Class III compounds inhibit ATPase activity. We found ponatinib to be a Class I ABCB1 inhibitor.

All currently available BCR-ABL inhibitors are ABCB1 and ABCG2 substrates and/or inhibitors (2–8), and the relative order of potency was recently shown to be nilotinib, then imatinib, then dasatinib (8). Two BCR-ABL inhibitors in current development have also been studied. Bosutinib was found not to be a substrate of ABCB1 or ABCG2 (42), whereas danusertib was found to be susceptible to resistance mediated by ABCG2 (43). Of note, it is important that drugs be studied at pharmacologically relevant concentrations.

Mechanism and concentration dependence of inhibition of transport have been most extensively studied for imatinib. Using [125I]-IAAP photolabeling and ATPase assays, Shukla and colleagues showed that imatinib mesylate is a substrate of both ABCB1 and ABCG2, and that it interacts with both transporters at low micromolar concentrations, indicating relatively high affinity (5). Imatinib seemed not to be transported at higher concentrations, likely because it inhibits its own transport at those concentrations (5), an observation that resolved seemingly contradictory earlier findings (3, 4). Nakanishi and colleagues also showed that imatinib decreased cell surface expression of ABCG2 on K562/BCRP-MX10 cells, expressing BCR-ABL, but not on ABCG2-overexpressing cells without BCR-ABL rearrangement, likely by inhibition of Akt downstream of BCR-ABL (39). We found that ponatinib also decreases surface ABCB1 and ABCG2 expression in cells with, but not without, BCR-ABL rearrangement.

Whereas BCR-ABL inhibitors are used as single agents to treat CML, in the treatment of Ph+ ALL, they are administered in combination with chemotherapy drugs, including the ABCB1 substrates doxorubicin and vincristine and the ABCG2 substrates 6-mercaptopurine (44) and methotrexate (45). ABCB1 and ABCG2 inhibition by BCR-ABL inhibitors might, therefore, be beneficial in combination regimens, and might also cause clinically significant drug interactions.

Ponatinib also potently inhibits FLT3 and may thus be applicable in AML therapy (18). FLT3 inhibitors are being combined with chemotherapy to treat AML with FLT3 mutations (9, 46–50) and also have activity in AML with wild-type FLT3 (46). Initial FLT3 inhibitors tested included the staurosporine derivatives lestaurtinib and midostaurin, but their use is complicated by high plasma protein binding, cell-cycle inhibition (47), and multikinase inhibition potentially causing off-target effects and toxicities. Lestaurtinib was not efficacious following chemotherapy in patients with AML with FLT3 mutations in first relapse in a randomized trial (48), and results of a randomized trial of midostaurin in newly diagnosed AML patients with FLT3 mutations are awaited. Sorafenib has been combined with idarubicin and infusional high-dose cytarabine (49). The selective FLT3 inhibitor AC220 (50) has not yet been combined with chemotherapy. Ponatinib has favorable features, including low plasma protein binding (15), good tolerability (16, 17) and, as shown here, no induction of cell-cycle arrest in cells with wild-type FLT3. Inhibition of ABCB1 and ABCG2 makes it attractive for further testing in combination with chemotherapy in AML.

No potential conflicts of interest were disclosed.

Conception and design: R. Sen, M.R. Baer

Development of methodology: R. Sen, Z.-S. Chen, M.R. Baer

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): R. Sen, S. Shukla, S.V. Ambudkar, M.R. Baer

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): R. Sen, K. Natarajan, J. Bhullar, S. Shukla, H.-B. Fang, L. Cai, S.V. Ambudkar, M.R. Baer

Writing, review, and/or revision of the manuscript: R. Sen, K. Natarajan, J. Bhullar, S. Shukla, H.-B. Fang, Z.-S. Chen, S.V. Ambudkar, M.R. Baer

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M.R. Baer

Study supervision: S.V. Ambudkar, M.R. Baer

The study was supported by a Leukemia and Lymphoma Society Translational Research Award (M.R. Baer), University of Maryland, Baltimore UMMG Cancer Research Grant #CH 649 CRF, State of Maryland Department of Health and Mental Hygiene (DHMH) under the Cigarette Restitution Fund Program (M.R. Baer), NCI Cancer Center Support Grant P30 CA134274 (UMGCC), and NIH Intramural Research Program, National Cancer Institute, Center for Cancer Research (S. Shukla, S.V. Ambudkar).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Szakács
G
,
Paterson
JK
,
Ludwig
JA
,
Booth-Genthe
C
,
Gottesman
MM
. 
Targeting multidrug resistance in cancer
.
Nat Rev Drug Discov
2006
;
5
:
219
34
.
2.
Illmer
T
,
Schaich
M
,
Platzbecker
U
,
Freiberg-Richter
J
,
Oelschlägel
U
,
von Bonin
M
, et al
P-glycoprotein-mediated drug efflux is a resistance mechanism of chronic myelogenous leukemia cells to treatment with imatinib mesylate
.
Leukemia
2004
;
18
:
401
8
.
3.
Burger
H
,
van Tol
H
,
Boersma
AW
,
Brok
M
,
Wiemer
EA
,
Stoter
G
, et al
Imatinib mesylate (STI571) is a substrate for the breast cancer resistance protein (BCRP)/ABCG2 drug pump
.
Blood
2004
;
104
:
2940
2
.
4.
Houghton
PJ
,
Germain
GS
,
Harwood
FC
,
Schuetz
JD
,
Stewart
CF
,
Buchdunger
E
, et al
Imatinib mesylate is a potent inhibitor of the ABCG2 (BCRP) transporter and reverses resistance to topotecan and SN-38 in vitro
.
Cancer Res
2004
;
64
:
2333
7
.
5.
Shukla
S
,
Sauna
ZE
,
Ambudkar
SV
. 
Evidence for the interaction of imatinib at the transport-substrate site(s) of the multidrug-resistance-linked ABC drug transporters ABCB1 (P-glycoprotein) and ABCG2
.
Leukemia
2008
;
22
:
445
7
.
6.
Tiwari
AK
,
Sodani
K
,
Wang
SR
,
Kuang
YH
,
Ashby
CR
 Jr
,
Chen
X
, et al
Nilotinib (AMN107, Tasigna) reverses multidrug resistance by inhibiting the activity of the ABCB1/Pgp and ABCG2/BCRP/MXR transporters
.
Biochem Pharmacol
2009
;
78
:
153
61
.
7.
Lagas
JS
,
van Waterschoot
RA
,
van Tilburg
VA
,
Hillebrand
MJ
,
Lankheet
N
,
Rosing
H
, et al
Brain accumulation of dasatinib is restricted by P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) and can be enhanced by elacridar treatment
.
Clin Cancer Res
2009
;
15
:
2344
51
.
8.
Dohse
M
,
Scharenberg
C
,
Shukla
S
,
Robey
RW
,
Volkmann
T
,
Deeken
JF
, et al
Comparison of ATP-binding cassette transporter interactions with the tyrosine kinase inhibitors imatinib, nilotinib, and dasatinib
.
Drug Metab Dispos
2010
;
38
:
1371
80
.
9.
Hunter
HM
,
Pallis
M
,
Seedhouse
CH
,
Grundy
M
,
Gray
C
,
Russell
NH
. 
The expression of P-glycoprotein in AML cells with FLT3 internal tandem duplications is associated with reduced apoptosis in response to FLT3 inhibitors
.
Br J Haematol
2004
;
127
:
26
33
.
10.
Yang
JJ
,
Milton
MN
,
Yu
S
,
Liao
M
,
Liu
N
,
Wu
JT
, et al
P-glycoprotein and breast cancer resistance protein affect disposition of tandutinib, a tyrosine kinase inhibitor
.
Drug Metab Lett
2010
;
4
:
201
12
.
11.
Lagas
JS
,
van Waterschoot
RA
,
Sparidans
RW
,
Wagenaar
E
,
Beijnen
JH
,
Schinkel
AH
. 
Breast cancer resistance protein and P-glycoprotein limit sorafenib brain accumulation
.
Mol Cancer Ther
2010
;
9
:
319
26
.
12.
Shukla
S
,
Robey
RW
,
Bates
SE
,
Ambudkar
SV
. 
Sunitinib (Sutent, SU11248), a small-molecule receptor tyrosine kinase inhibitor, blocks function of the ATP-binding cassette (ABC) transporters P-glycoprotein (ABCB1) and ABCG2
.
Drug Metab Dispos
2009
;
37
:
359
65
.
13.
Schlenk
RF
,
Döhner
K
,
Krauter
J
,
Fröhling
S
,
Corbacioglu
A
,
Bullinger
L
, et al
Mutations and treatment outcome in cytogenetically normal acute myeloid leukemia
.
N Engl J Med
2008
;
358
:
1909
18
.
14.
O'Hare
T
,
Shakespeare
WC
,
Zhu
X
,
Eide
CA
,
Rivera
VM
,
Wang
F
, et al
AP24534, a pan-BCR-ABL inhibitor for chronic myeloid leukemia, potently inhibits the T315I mutant and overcomes mutation-based resistance
.
Cancer Cell
2009
;
16
:
401
12
.
15.
Huang
WS
,
Metcalf
CA
,
Sundaramoorthi
R
,
Wang
Y
,
Zou
D
,
Thomas
RM
, et al
Discovery of 3-[2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl-N-{4-[(4-methylpiperazin-1yl)methyl]-3-(trifluoromethyl)phenyl}benzamide (AP24534), a potent, orally active pan-inhibitor of breakpoint cluster region-abelson (BCR-ABL) kinase including the T315I gatekeeper mutant
.
J Med Chem
2010
;
53
:
4701
19
.
16.
Cortes
J
,
Talpaz
M
,
Bixby
D
,
Deininger
M
,
Shah
N
,
Flinn
IW
, et al
A phase 1 trial of oral ponatinib (AP24534) in patients with refractory chronic myelogenous leukemia (CML) and other hematologic malignancies: Emerging safety and clinical response findings
.
Blood
2010
;
116
:
210
.
17.
Cortes
JE
,
Kim
D-W
,
Pinilla-Ibarz
J
,
Le Coutre
PD
,
Chuah
C
,
Nicolini
FE
, et al
Initial findings from the PACE trial: A pivotal phase 2 study of ponatinib in patients with CML and Ph+ ALL resistant or intolerant to dasatinib or nilotinib, or with the T315I mutation
.
Blood
2011
;
118
:
109
.
18.
Gozgit
JM
,
Wong
MJ
,
Wardwell
S
,
Tyner
JW
,
Loriaux
MM
,
Mohemmad
QK
, et al
Potent activity of Ponatinib (AP24534) in models of FLT3-driven acute myeloid leukemia and other hematologic malignancies
.
Mol Cancer Ther
2011
;
10
:
1028
35
.
19.
Gozgit
JM
,
Wong
MJ
,
Moran
L
,
Wardwell
S
,
Mohemmad
QK
,
Narasimhan
NI
, et al
Ponatinib (AP24534), a multi-targeted pan-FGFR inhibitor with activity in multiple FGFR-amplified or mutated cancer models
.
Mol Cancer Ther
2012
;
11
:
690
9
.
20.
Ogretmen
B
,
Safa
AR
. 
Identification and characterization of the MDR1 promoter-enhancing factor 1 (MEF1) in the multidrug resistant HL60/VCR human acute myeloid leukemia cell line
.
Biochemistry
2000
;
39
:
194
204
.
21.
Marsh
W
,
Sicheri
D
,
Center
MS
. 
Isolation and characterization of adriamycin-resistant HL60 cells which are not defective in the initial accumulation of the drug
.
Cancer Res
1986
;
46
:
4053
7
.
22.
Hazlehurst
LA
,
Foley
NE
,
Gleason-Guzman
MC
,
Hacker
MP
,
Cress
AE
,
Greenberger
LW
, et al
Multiple mechanisms confer drug resistance to mitoxantrone in the human 8226 myeloma cell line
.
Cancer Res
1999
;
59
:
1021
8
.
23.
Hafkemeyer
P
,
Licht
T
,
Pastan
I
,
Gottesman
MM
. 
Chemoprotection of hematopoietic cells by a mutant P-glycoprotein resistant to a potent chemosensitizer of multidrug-resistant cancers
.
Hum Gene Ther
2000
;
11
:
555
65
.
24.
Yanase
K
,
Tsukahara
S
,
Asada
S
,
Ishikawa
E
,
Imai
Y
,
Sugimoto
Y
. 
Gefitinib reverses breast cancer resistance protein-mediated drug resistance
.
Mol Cancer Ther
2004
;
3
:
1119
25
.
25.
Chen
YN
,
Mickley
LA
,
Schwartz
AM
,
Acton
EM
,
Hwang
JL
,
Fojo
AT
. 
Characterization of adriamycin-resistant human breast cancer cells which display overexpression of a novel resistance-related membrane protein
.
J Biol Chem
1990
;
265
:
10073
80
.
26.
Robey
RW
,
Medina-Pérez
WY
,
Nishiyama
K
,
Lahusen
T
,
Miyake
K
,
Litman
T
, et al
Overexpression of the ATP-binding cassette half-transporter, ABCG2 (Mxr/BCrp/ABCP1), in flavopiridol-resistant human breast cancer cells
.
Clin Cancer Res
2001
;
7
:
145
52
.
27.
Minderman
H
,
Suvannasankha
A
,
O'Loughlin
KL
,
Scheffer
GL
,
Scheper
RJ
,
Robey
RW
, et al
Flow cytometric analysis of breast cancer resistance protein expression and function
.
Cytometry
2002
;
48
:
59
65
.
28.
Robey
RW
,
Steadman
K
,
Polgar
O
,
Morisaki
K
,
Blayney
M
,
Mistry
P
, et al
Pheophorbide a is a specific probe for ABCG2 function and inhibition
.
Cancer Res
2004
;
64
:
1242
6
.
29.
Minderman
H
,
O'Loughlin
KL
,
Pendyala
L
,
Baer
MR
. 
VX-710 (biricodar) increases drug retention and enhances chemosensitivity in resistant cells overexpressing P-glycoprotein, multidrug resistance protein, and breast cancer resistance protein
.
Clin Cancer Res
2004
;
10
:
1826
34
.
30.
Sauna
ZE
,
Ambudkar
SV
. 
Evidence for a requirement for ATP hydrolysis at two distinct steps during a single turnover of the catalytic cycle of human P-glycoprotein
.
Proc Natl Acad Sci U S A
2000
;
97
:
2515
20
.
31.
Shukla
S
,
Robey
RW
,
Bates
SE
,
Ambudkar
SV
. 
The calcium channel blockers, 1,4-dihydropyridines, are substrates of the multidrug resistance-linked ABC drug transporter, ABCG2
.
Biochemistry
2006
;
45
:
8940
51
.
32.
Ambudkar
SV
. 
Drug-stimulatable ATPase activity in crude membranes of human MDR1-transfected mammalian cells
.
Methods Enzymol
1998
;
292
:
504
14
.
33.
Tan
M
,
Fang
HB
,
Tian
GL
,
Houghton
PJ
. 
Experimental design and sample size determination for testing synergism in drug combination studies based on uniform measures
.
Stat Med
2003
;
22
:
2091
100
.
34.
Fang
HB
,
Ross
DD
,
Sausville
E
,
Tan
M
. 
Experimental design and interaction analysis of combination studies of drugs with log-linear dose responses
.
Stat Med
2008
;
27
:
3071
83
.
36.
Berenbaum
MC
. 
Synergy, additivism and antagonism in immunosuppression. A critical review
.
Clin Exp Immunol
1977
;
28
:
1
18
.
37.
Evans
GL
,
Ni
B
,
Hrycyna
CA
,
Chen
D
,
Ambudkar
SV
,
Pastan
I
, et al
Heterologous expression systems for P-glycoprotein: E. coli, yeast and baculovirus
.
J Bioenerg and Biomembr
1995
;
27
:
43
52
.
38.
Brózik
A
,
Hegedüs
C
,
Erdei
Z
,
Hegedus
T
,
Özvegy-Laczka
C
,
Szakács
G
, et al
Tyrosine kinase inhibitors as modulators of ATP binding cassette multidrug transporters: substrates, chemosensitizers or inducers of acquired multidrug resistance?
Expert Opin Drug Metab Toxicol
2011
;
7
:
623
42
.
39.
Nakanishi
T
,
Shiozawa
K
,
Hassel
BA
,
Ross
DD
. 
Complex interaction of BCRP/ABCG2 and imatinib in BCR-ABL-expressing cells: BCRP-mediated resistance to imatinib is attenuated by imatinib-induced reduction of BCRP expression
.
Blood
2006
;
108
:
678
84
.
40.
Ozvegy-Laczka
C
,
Várady
G
,
Köblös
G
,
Ujhelly
O
,
Cervenak
J
,
Schuetz
JD
, et al
Function-dependent conformational changes of the ABCG2 multidrug transporter modify its interaction with a monoclonal antibody on the cell surface
.
J Biol Chem
2005
;
280
:
4219
27
.
41.
Ambudkar
SV
,
Dey
S
,
Hrycyna
CA
,
Ramachandra
M
,
Pastan
I
,
Gottesman
MM
. 
Biochemical, cellular, and pharmacological aspects of the multidrug transporter
.
Annu Rev Pharmacol Toxicol
1999
;
39
:
361
98
.
42.
Hegedus
C
,
Ozvegy-Laczka
C
,
Apati
A
,
Magocsi
M
,
Nemet
K
,
Orfi
L
, et al
Interaction of nilotinib, dasatinib and bosutinib with ABCB1 and ABCG2: implications for altered anti-cancer effects and pharmacological properties
.
Br J Pharmacol
2009
;
158
:
1153
64
.
43.
Balabanov
S
,
Gontarewicz
A
,
Keller
G
,
Raddrizzani
L
,
Braig
M
,
Bosotti
R
, et al
Abcg2 overexpression represents a novel mechanism for acquired resistance to the multi-kinase inhibitor danusertib in BCR-ABL-positive cells in vitro
.
PLoS One
2011
;
6
:
e19164
.
44.
de Wolf
C
,
Jansen
R
,
Yamaguchi
H
,
de Haas
M
,
van de Wetering
K
,
Wijnholds
J
, et al
Contribution of the drug transporter ABCG2 (breast cancer resistance protein) to resistance against anticancer nucleosides
.
Mol Cancer Ther
2008
;
7
:
3092
102
.
45.
Volk
EL
,
Farley
KM
,
Wu
Y
,
Li
F
,
Robey
RW
,
Schneider
E
. 
Overexpression of wild-type breast cancer resistance protein mediates methotrexate resistance
.
Cancer Res
2002
;
62
:
5035
40
.
46.
Fischer
T
,
Stone
RM
,
Deangelo
DJ
,
Galinsky
I
,
Estey
E
,
Lanza
C
, et al
Phase IIB trial of oral midostaurin (PKC412), the FMS-like tyrosine kinase 3 receptor (FLT3) and multi-targeted kinase inhibitor, in patients with acute myeloid leukemia and high-risk myelodysplastic syndrome with either wild-type or mutated FLT3
.
J Clin Oncol
2010
;
28
:
4339
45
.
47.
Levis
M
,
Pham
R
,
Smith
BD
,
Small
D
. 
In vitro studies of a FLT3 inhibitor combined with chemotherapy: sequence of administration is important to achieve synergistic cytotoxic effects
.
Blood
2004
;
104
:
1145
50
.
48.
Levis
M
,
Ravandi
F
,
Wang
ES
,
Baer
MR
,
Perl
A
,
Coutre
S
, et al
Results from a randomized trial of salvage chemotherapy followed by lestaurtinib for patients with FLT3 mutant AML in first relapse
.
Blood
2011
;
117
:
3294
301
.
49.
Ravandi
F
,
Cortes
JE
,
Jones
D
,
Faderl
S
,
Garcia-Manero
G
,
Konopleva
MY
, et al
Phase I/II study of combination therapy with sorafenib, idarubicin, and cytarabine in younger patients with acute myeloid leukemia
.
J Clin Oncol
2010
;
28
:
1856
62
.
50.
Zarrinkar
PP
,
Gunawardane
RN
,
Cramer
MD
,
Gardner
MF
,
Brigham
D
,
Belli
B
, et al
AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML)
.
Blood
2009
;
114
:
2984
92
.