Osimertinib (AZD9291) Attenuates the Function of Multidrug

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Osimertinib (AZD9291) attenuates the function of multidrug resistance-linked ATP-binding cassette transporter ABCB1 in vitro Sung-Han Hsiao, Yu-Jen Lu, Yan-Qing Li, Yang-Hui Huang, Chia-Hung Hsieh, and Chung-Pu Wu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00249 • Publication Date (Web): 12 May 2016 Downloaded from http://pubs.acs.org on May 18, 2016

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Molecular Pharmaceutics

Osimertinib (AZD9291) attenuates the function of multidrug resistance-linked ATP-binding cassette transporter ABCB1 in vitro

Sung-Han Hsiao †, Yu-Jen Lu ‡, Yan-Qing Li , Yang-Hui Huang , Chia-Hung ＃

Hsieh

＆,$

, Chung-Pu Wu †,‡,＃,

※,

※

*

Authors' Affiliations: †

Graduate Institute of Biomedical Sciences, ‡ Department of Neurosurgery, Chang

Gung Memorial Hospital,

＃

Department of Physiology and Pharmacology, and

※

Molecular Medicine Research Center, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan.

＆

Graduate Institute of Basic Medical Science, and $ Department

of Medical Research, China Medical University Hospital, Taichung, Taiwan.

* Corresponding author at: 259 Wen-Hwa 1st Road, Kwei-Shan, Tao-Yuan 333, Taiwan. Phone: +886-3-2118800, ext. 3754. Fax: +886-3-2118700. E-mail address: [email protected]

Keywords: Multidrug resistance; ABCB1; Osimertinib; AZD9291; EGFR.

Running Title: Osimertinib inhibits ABCB1-mediated transport.

Abbreviations: MDR, multidrug resistance; ABC, ATP-binding cassette; EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer; TKIs, tyrosine kinase inhibitors; FDA, Food and Drug Administration; FCS, fetal calf serum; CCK-8, Cell Counting Kit-8; IMDM, Iscove's Modified Dulbecco's Medium; BBB, blood–brain barrier; RF, resistance factor. 1

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ABSTRACT

The effectiveness of cancer chemotherapy is often circumvented by multidrug resistance (MDR) caused by the overexpression of ATP-binding cassette (ABC) drug transporter ABCB1 (MDR1, P-glycoprotein). Several epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have been shown previously capable of modulating the function of ABCB1 and reversing ABCB1-mediated MDR in human cancer cells. Furthermore, some TKIs are transported by ABCB1, resulting in low oral bioavailability, reduced distribution and the development of acquired resistance to these TKIs. In this study, we investigated the interaction between ABCB1 and osimertinib, a novel selective, irreversible third-generation EGFR TKI that has recently been approved by the U.S. Food and Drug Administration (FDA). We also evaluated the potential impact of ABCB1 on the efficacy of osimertinib in cancer cells, which can present a therapeutic challenge to clinicians in the future. We revealed that although osimertinib stimulates the ATPase activity of ABCB1, overexpression of ABCB1 does not confer resistance to osimertinib. Our results suggest that it is unlikely that the overexpression of ABCB1 can be a major contributor to the development of osimertinib resistance in cancer patients. More significantly, we revealed an additional action of osimertinib that directly inhibits the function of ABCB1without affecting the expression level of ABCB1, enhances drug-induced apoptosis and reverses the MDR phenotype in ABCB1-overexpressing cancer cells. Considering that osimertinib is a clinically approved third-generation EGFR TKI, our findings suggest that a combination therapy with osimertinib and conventional anticancer drugs may be beneficial to patients with MDR tumors.

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INTRODUCTION

The inevitable emergence of multidrug resistance (MDR) caused by the overexpression of ATP-binding cassette (ABC) drug transporters in cancer cells often leads to failure in chemotherapy.1, 2ABCB1 (MDR1; P-glycoprotein) is the first mammalian ABC protein discovered capable of utilizing energy derived from ATP hydrolysis to efflux and confer resistance to a large variety of chemotherapeutic agents, resulting in the development of MDR phenotype, cancer relapse and death.2, 3 Moreover, ABCB1 is highly expressed at barrier sites such as the intestinal walls and the blood-brain barrier (BBB), affecting the oral bioavailability and distribution of most drugs, and thus is considered as a crucial endogenous protective mechanism against xenobiotics.4, 5 Therefore, modulating the function or expression of ABCB1 has great clinical significance.

Currently, inhibition of the function of ABCB1 remains as one of the most effective ways to restore chemosensitivity in ABCB1-overexpressing MDR cancer cells.6 Although tremendous efforts have been invested in discovering and synthesizing novel modulators of ABCB1, drug repositioning (also referred as drug repurposing) of approved drugs with known pharmacological and toxicological properties is a promising and practical approach to overcome MDR in cancer patients.6-9 Numerous clinically active tyrosine kinase inhibitors (TKIs), including epidermal growth factor receptor (EGFR) TKIs, have previously been shown to interact with ABCB1.10-15 Some TKIs are transported substrates of ABCB1, which leads to problems such as reduced oral bioavailability, distribution and the development of acquired resistance.10, 11, 15 On the other hand, some TKIs are capable of inhibiting ABCB1-mediated drug efflux in a competitive manner, which 3



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leads to resensitization of MDR cancer cells to anticancer agents. 12-14, 16

Gefitinib and erlotinib are first-generation EGFR TKIs that have been shown capable of modulating the function of ABCB1 and reversing chemotherapy resistance in ABCB1-expressing MDR cancer cells.17-20 These EGFR TKIs were effective in treating patients with advanced non-small cell lung cancer (NSCLC) harboring activating EGFR mutations.21-23 However, like most chemotherapeutic agents, many patients with activating EGFR mutations eventually develop acquired resistance to these EGFR TKIs through the acquisition of a second T790M mutation in EGFR.24-27 Recently, Wang et al. demonstrated that afatinib, a second-generation EGFR TKI, has potent inhibitory activity against ABCB1 and reverses MDR in ABCB1-overexpressing A2780T ovarian cancer cells.28 Moreover, it was also reported that NSCLC with acquired resistance to afatinib is associated with the overexpression of ABCB1.29 Together, these studies revealed a strong interaction between EGFR TKIs and ABCB1, which led us to investigate the interaction between osimertinib and ABCB1. Recently, the U.S. Food and Drug Administration (FDA) approved osimertinib (AZD9291), a novel and selective third-generation irreversible EGFR TKI, for the treatment of patients with metastatic EGFR T790M mutation-positive NSCLC.30, 31, 32 Recent studies indicate that acquired resistance to osimertinib is linked to acquired EGFR C797S mutation in NSCLC33 or associated with increased dependence on RAS signaling.34 However, the potential impact of ABCB1 on the efficacy of osimertinib in cancer cells has not been examined.

In the present study, we demonstrated that osimertinib inhibits the function of ABCB1 without affecting the expression of ABCB1. More importantly, osimertinib is capable of enhancing drug-induced apoptosis and reversing MDR in 4


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ABCB1-overexpressing cancer cells. Although osimertinib stimulates the ATPase activity of ABCB1, which indicates that osimertinib behaves as a high-affinity substrate of ABCB1, we discovered that ABCB1-overexpressing cells were not resistant to osimertinib. Taken together, our data suggest that ABCB1 overexpression is unlikely to contribute to the development of resistance to osimertinib in cancer cells, and patients with MDR tumors may benefit from a combination therapy of anticancer agents and osimertinib.

EXPERIMENTAL SECTION

Chemicals. Phosphate-buffered saline (PBS), fetal calf serum (FCS), Dulbecco's Modified Eagle Medium (DMEM), RPMI-1640, Iscove's modified Dulbecco's medium (IMDM), trypsin-EDTA, penicillin and streptomycin were purchased from Gibco, Invitrogen (Carlsbad, CA, USA). Calcein-AM, Cell Counting Kit-8 (CCK-8), MTT dye, colchicine and other chemicals were purchased from Sigma (St. Louis, MO, USA), unless stated otherwise. Tariquidar was a generous gift from Dr. Susan Bates (National Cancer Institute, NIH, Bethesda, MD, USA). Osimertinib and afatinib were purchased from Selleckchem (Houston, TX, USA). Annexin V : FITC Apoptosis Detection Kit was purchased from BD Pharmingen (San Diego, CA, USA).

Cell lines and culture conditions. The human epidermal tumor line KB-3-1 and its ABCB1-expressing variant KB-V-1, human ovarian tumor line OVCAR-8 and its ABCB1-expressing variant NCI-ADR-RES, mouse fibroblast NIH3T3 and NIH3T3-G185 (transfected with human ABCB1) cells, as well as 5



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pcDNA3.1-HEK293 and MDR19-HEK293 (HEK293 cells transfected with human ABCB1) cells were cultured in DMEM, supplemented with 10% FCS, 2 mM L-glutamine and 100 units of penicillin/streptomycin/mL. KB-V-1 cells were cultured in media containing 1 mg/mL vinblastine,35 whereas NIH3T3-G185 cells were cultured in the presence of 60 ng/mL colchicine.36 pcDNA3.1-HEK293 and MDR19-HEK293 were cultured in the presence of 2 mg/mL G418.37 Human myelogenous leukemia K562 and K562/i-S9 (ABCB1-expressing ) cells were maintained in RPMI, supplemented with 10% FCS, 2 mM L-glutamine and 100 units of penicillin/streptomycin/mL.38 Cell lines were generous gifts from Dr. Suresh V. Ambudkar (National Cancer Institute, NIH, Bethesda, MD, USA), all maintained at 37 °C in 5% CO2 humidified air and placed in drug-free medium 7 days prior to assay.

Fluorescent drug accumulation assay. A FACSort flow cytometer equipped with CellQuest software was used to performed ABCB1-mediated efflux assays. Briefly, cells were harvested after trypsinization by centrifugation at 500 g and then resuspended in IMDM supplemented with 5% FCS. Calcein-AM was added to 3 × 105 cells in 4 mL of IMDM in the presence or absence of tested drugs. The effect of osimertinib, afatinib or tariquidar on calcein-AM efflux mediated by ABCB1 was measured and analyzed according to the method described by Gribar et al.39

Immunoblotting. Primary C219 antibody (1:1000) was used to detect ABCB1, whereas anti-α-tubulin (1:2000) was used to detect positive control tubulin for Western blotting. The secondary antibody used was the horseradish peroxidase-conjugated goat anti-mouse IgG (1:10,000). Signals were detected as described previously.40 6


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Cytotoxicity assay. MTT assays were performed to determine the sensitivity of attached cancer cell lines to the tested compounds. In contrast, CCK-8 assays were used to determine the cytotoxicity of tested compounds in HEK293, MDR19-HEK293, K562 and K562/i-S9 cells according to the method described by Ishiyama et al.41 Briefly, cells were plated into 96-well plates at a density of 5,000 cells per well in 100 µL of culture medium and maintained at 37 °C for 24 h before adding drugs to make a final volume of 200 µL. Cells were incubated for an additional 72 h with various concentrations of drugs before development with MTT or CCK-8 reagent. For the reversal of cytotoxicity assays, a non-toxic concentration of osimertinib or tariquidar was added to the cytotoxicity assay, and the extent of reversal was then calculated based on the relative resistance values as described previously.13

Apoptosis assay. Apoptosis assays were carried out to determine the percentage of apoptotic cells induced by indicated regimens. Cells were treated with colchicine in the absence or presence of osimertinib or tariquidar for 48 h before harvested, centrifuged and resuspended in FACS buffer containing 1.25 µg/ml annexin V–FITC (PharMingen) and 0.1 mg/ml propidium iodide (PI), and cells were incubated at room temperature for 15 min. The labeled cells (10,000 per sample) were then analyzed by FACScan (BD Biosciences) using the CellQuest software. Cells in the lower right dot-plot quadrant were counted as apoptotic and have intact plasma membranes (phosphatidylserine PS-positive and PI-negative), whereas cells in the upper right dot-plot quadrant have leaky membranes (PS-positive and PI-positive) and considered as either necrotic or late apoptotic.

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ATPase assay of ABCB1. The osimertinib-stimulated vanadate (Vi)-sensitive ABCB1-specific ATPase activity was recorded by using the Pgp-Glo assay system (Promega, WI, USA) according to the manufacturer's instructions and determined based on endpoint Pi assay as described previously.42, 43, 44

Statistical analysis. GraphPad Prism (La Jolla, CA, USA ) and KaleidaGraph (Reading, PA, USA ) software were used to plot the curves and perform statistical analysis. Data are presented as mean ± SEM, and IC50 values were calculated as mean ± SD from at least three independent experiments. Differences between any mean values were analyzed by two-sided Student’s t-test and results were considered statistically significant at P < 0.05.

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RESULTS

Osimertinib inhibits ABCB1-mediated efflux of calcein-AM. In order to determine the potential interaction between osimertinib and ABCB1, we examined the effect of osimertinib on the function and the protein expression of ABCB1. We first evaluated the capability of osimertinib to inhibit ABCB1-mediated drug efflux in drug accumulation assays as described in Materials and methods. We discovered that without affecting the drug-sensitive parental cells (left panels, Figures 1 A and 1 B), osimertinib (10 µM) significantly increased the accumulation of fluorescent calcein, an established substrate of ABCB145 in ABCB1-overexpressing KB-V-1 cells (right panel, Figure 1 A) and ABCB1-transfected MDR19-HEK293 cells (right panel, Figure 1 B) to the same extent as tariquidar, a known inhibitor of ABCB1 (dotted lines). Moreover, we demonstrated that osimertinib inhibited the transport function of ABCB1 in KB-V-1 (Figure 1 C) and MDR19-HEK293 (Figure 1 D) cells in a concentration-dependent manner, with calculated IC50 values of approximately 5 µM and 3 µM, respectively. The second-generation EGFR TKI afatinib was tested here for comparison purposes as it has recently been shown to significantly inhibit ABCB1-mediated efflux of drugs.28 As expected, afatinib also inhibited the transport of calcein-AM in a concentration-dependent manner (Figures 1 C and 1 D).

Given that several ABCB1-interacting compounds, including EGFR TKIs, have been reported to down-regulate the protein expression of ABCB1 in cancer cell lines,28, 46-48 we examined the effect of osimertinib on ABCB1 protein expression in drug-resistant, ABCB1-overexpressing human KB-V-1 epidermal (Figure 2 A) and NCI-ADR-RES ovarian cancer cell lines (Figure 2 B). Cells were maintained in the absence or presence of increasing concentrations (0.1 - 1 µM) of osimertinib for 72 h, 9



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harvested and processed for immunoblotting as described in Materials and methods. Our results show that osimertinib does not significantly alter the expression of ABCB1 at the protein level in these cancer cell lines (Figure 2C).

Osimertinib reverses ABCB1-mediated multidrug resistance in ABCB1-overexpressing cells. Previously, several first-generation and second-generation EGFR TKIs were found capable of inhibiting the function of ABCB1 and reversing MDR in ABCB1-overexpressing cancer cells in a competitive manner.17-19, 28, 49 Therefore, we examined the reversal effect of osimertinib on ABCB1-mediated MDR in ABCB1-overexpressing human cancer cells (Figure 3, right panels). Multiple non-toxic concentrations of osimertinib (more than 90% of viable cells) were tested in the experiments (Figure 3, left panels), and the reversal effect of osimertinib on ABCB1-mediated drug resistance is summarized in Table 1. The relative resistance (RR) value represents the relative resistance of ABCB1-overexpressing cells to a particular anticancer drug in the presence or absence of a reversing agent, which was calculated as described previously.40 We discovered that osimertinib was able to re-sensitize ABCB1-overexpressing KB-V-1 and NCI-ADR-RES cancer cells to ABCB1 substrates50 colchicine, vincristine and paclitaxel in a concentration-dependent manner. Our data suggest that at low, non-toxic concentrations (0.1 - 0.8 µM), osimertinib can be used to restore the chemosensitivity of ABCB1-overexpressing MDR cells to anticancer agents.

Osimertinib enhances colchicine-induced apoptosis in ABCB1-overexpressing cancer cells. Next, we examined the potentiating effect of osimertinib on drug-induced apoptosis in ABCB1-overexpressing cancer cells. As shown in Figure 10


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4, we measured the level of apoptosis induced by colchicine in human KB cancer cell lines in the presence or absence of osimertinib or tariquidar. In the absence of colchicine, the level of apoptosis in drug-sensitive parental KB-3-1 and drug-resistant ABCB1-overexpressing KB-V-1 cell lines was similar and unaffected by osimertinib or tariquidar (Figure 4 A). When cells were treated with colchicine (500 nM), the percentage of apoptosis increased significantly from approximately 6% to 42% in drug-sensitive KB-3-1 cell line (Figure 4 B, upper left panel). In contrast, the apoptotic effect of colchicine, a known substrate of ABCB1,51 was considerably less in ABCB1-overexpressing KB-V-1 cells (Figure 4 B, lower left panel). As shown in Figure 4B, osimertinib (lower middle panel) and tariquidar (lower right panel) increased the level of apoptosis induced by colchicine in KB-V-1 cells from approximately 12% to 38% and 62%, respectively. Our results show that osimertinib enhanced sensitivity of anticancer drug to ABCB1-overexpressing cells.

Osimertinib stimulates ABCB1 ATPase activity. ABCB1-mediated substrate transport is coupled to ATP hydrolysis by ABCB1.43 Therefore, we examined the effect of osimertinib on Vi-sensitive ATPase activity of ABCB1 as described in Materials and methods. As shown in Figure 5, osimertinib stimulated Vi-sensitive ABCB1 ATP hydrolysis in a concentration-dependent manner, producing a maximum stimulation of approximately 4-fold (basal, 55.5 ± 3.1 nmole Pi/min/mg protein) and a concentration of approximately 80 nM required for 50% stimulation. Our results suggest a high affinity interaction between osimertinib and ABCB1, which is in agreement with results from the drug efflux assays (Figure 1).

Overexpression of ABCB1 does not confer resistance to osimertinib in cancer cells. Next, we examined the cytotoxicity of osimertinib in multiple drug-sensitive 11



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and ABCB1-overexpressing MDR cancer cell lines, and the calculated IC50 values are summarized in Table 2. The resistance factor (RF) value represents the extent of cellular resistance to osimertinib caused by the overexpression of ABCB1, which was calculated by dividing the IC50 value of ABCB1-overexpressing MDR subline by the IC50 value of the respective parental line. No significant differences were found when we compared the RF values in ABCB1-overexpressing MDR cancer cell lines in respective to the drug-sensitive cancer cell lines. To further confirm our findings, we compared the cytotoxicity of osimertinib in NIH3T3 mouse embryonic fibroblast cells and NIH3T3 cells transfected with human ABCB1 (NIH3T3-G185), as well as in HEK293 cells and ABCB1-transfected MDR19-HEK293 cells. We found that these cell lines were equally sensitive to osimertinib treatment (Table 2).

DISCUSSION

Multidrug resistance to chemotherapeutic agents is a major challenge to the effective treatment of cancer. The MDR phenomenon in cancer is mostly contributed by the overexpression of ABCB1 drug transporter in cancer cells.1, 2 Instead of developing new reversing agents, we and other groups have investigated the probability of repositioning approved drugs to overcome ABCB1-mediated MDR in cancer cells.6 In the present study, we investigated the effect of recently FDA approved EGFR TKI osimertinib on the function and expression of ABCB1, and the potential impact of ABCB1 overexpression on the cytotoxicity of osimertinib in cancer cells. First, we discovered that osimertinib significantly increased the accumulation of ABCB1 substrate in ABCB1-overexpressing cells, and that osimertinib appeared to be more effective in modulating the function of ABCB1 than afatinib, a second-generation EGFR TKI that was reported to inhibit the 12


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function of ABCB128 (Figure 1). However, in contrast to afatinib,28 osimertinib failed to attenuate the expression of ABCB1 in human cancer cells (Figure 2). Next, we demonstrated that osimertinib is capable of resensitizing ABCB1-overexpressing cancer cells to multiple conventional anticancer agents (Figure 3) in the same manner as other EGFR TKIs, such as gefitinib,14, 17, 18, 52 erlotinib19, 20, 52 and afatinib.28 Moreover, treating ABCB1-overexpressing KB-V-1 cancer cells with a non-toxic concentration of osimertinib resulted in a significant potentiation of colchicine-induced apoptosis (Figure 4). These results further support the notion that osimertinib reverses ABCB1-mediated MDR by inhibiting the function of ABCB1 without altering the expression level of ABCB1. However, to our surprise, we discovered that osimertinib is effective in reversing ABCB1-mediated resistance to colchicine, vincristine and paclitaxel (Table 1), but not to doxorubicin (data not shown). Our findings were similar to those reported by Noguchi et al. that erlotinib suppresses ABCB1-mediated resistance to vincristine and paclitaxel, but does not suppress resistance to doxorubicin,20 suggesting the possibility that osimertinib also modulates ABCB1-mediated drug resistance in a substrate-dependent manner.

Next, we found that osimertinib stimulated ABCB1-mediated ATP hydrolysis in a concentration-dependent manner (Figure 5). The chemical event of ATP hydrolysis mediated by ABCB1 is closely linked to ABCB1-mediated substrate transport.53 Our results demonstrate that osimertinib behaves in the same manner as other well-established substrates of ABCB1,13, 42-44, 54, 55 and that it is most likely that osimertinib inhibits the efflux function of ABCB1 by competing with the binding of another drug at the substrate binding site(s) of ABCB1. Of note, the calculated Ki value for osimertinib (Ki ≈ 80 nM) is considerably lower than that for afatinib (Ki ≈ 2.5 µM),28 indicating that ABCB1 displays a high-affinity interaction with 13



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osimertinib, which is consistent with the results obtained from the fluorescent drug accumulation assay (Figure 1). However, it is important to note that favorable MDR reversal results from in vitro or in vivo studies do not necessarily translate into successful clinical outcomes.6 For instance, the expression level of ABCB1 varies in different individuals and may lead to high variability in the response rate in patients receiving ABCB1 modulators. Moreover, modulators may exhibit unfavorable pharmacokinetic interactions with co-administered anticancer drugs, causing enhanced toxicity in patients. In view of the pharmacological properties of osimertinib30-32, it remains to be determined whether osimertinib interferes with the clearance or metabolism of other anticancer drugs.

The development of acquired resistance to TKIs has become a significant therapeutic challenge.56 The overexpression of ABCB1 has been implicated in contributing to reduced therapeutic efficacy and/or conferring resistance to several EGFR TKIs,13, 29, 57 we thus decided to compare the cytotoxicity of osimertinib in multiple drug-sensitive cell lines and respective ABCB1-overexpressing MDR sublines. We found that ABCB1-overexpressing human epidermal KB-V-1 cells, human ovarian NCI-ADR-RES cells and leukemia K562-is9 cells were all equally sensitive to osimertinib as their respective parental cells. Moreover, ABCB1-transfected NIH3T3-G185 and MDR19-HEK293 cells were also equally sensitive to osimertinib as their parental cells (Table 2). Therefore, despite the fact that osimertinib stimulates the ATPase activity of ABCB1 (Figure 5), ABCB1 does not appear to confer resistance to osimertinib in cancer cells.

In conclusion, this study demonstrated that osimertinib can reverse MDR by attenuating the transport activity of ABCB1. Moreover, we found that ABCB1 14


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overexpression in cancer cells does not lead to resistance to osimertinib. Given that osimertinib has recently been approved by the U.S. FDA, our study suggests that the advantage of combination therapy with osimertinib and other anticancer agents should be further evaluated in clinical practice.

CONFLICT OF INTEREST. None.

ACKNOWLEDGMENTS. The authors thank Dr. Suresh V. Ambudkar (National Cancer Institute, NIH) for generously providing cell lines. This work was supported by funds from the Chang Gung Medical Research Program (CMRPD1D0153), the Ministry of Science and Technology of Taiwan (MOST-104-2320-B-182-039), and a grant to Chang Gung University from the Ministry of Education (EMRPD1F0191).

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Activating mutations in the

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Watanabe, M.; Maki, Y.; Soh, J.; Asano, H.; Tsukuda, K.; Miyoshi, S.; Toyooka, S. Acquisition of cancer stem cell-like properties in non-small cell lung cancer with acquired resistance to afatinib. Cancer Sci 2015, 106, (10), 1377-84. 30. Finlay, M. R.; Anderton, M.; Ashton, S.; Ballard, P.; Bethel, P. A.; Box, M. R.; Bradbury, R. H.; Brown, S. J.; Butterworth, S.; Campbell, A.; Chorley, C.; Colclough, N.; Cross, D. A.; Currie, G. S.; Grist, M.; Hassall, L.; Hill, G. B.; James, D.; James, M.; Kemmitt, P.; Klinowska, T.; Lamont, G.; Lamont, S. G.; Martin, N.; McFarland, H. L.; Mellor, M. J.; Orme, J. P.; Perkins, D.; Perkins, P.; Richmond, G.; Smith, P.; Ward, R. A.; Waring, M. J.; Whittaker, D.; Wells, S.; Wrigley, G. L. Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor. J Med Chem 2014, 57, (20), 8249-67. 31. Cross, D. A.; Ashton, S. E.; Ghiorghiu, S.; Eberlein, C.; Nebhan, C. A.; Spitzler, P. J.; Orme, J. P.; Finlay, M. R.; Ward, R. A.; Mellor, M. J.; Hughes, G.; Rahi, A.; Jacobs, V. N.; Red Brewer, M.; Ichihara, E.; Sun, J.; Jin, H.; Ballard, P.; Al-Kadhimi, K.; Rowlinson, R.; Klinowska, T.; Richmond, G. H.; Cantarini, M.; Kim, D. W.; Ranson, M. R.; Pao, W. AZD9291, an irreversible EGFR TKI, overcomes T790M-mediated resistance to EGFR inhibitors in lung cancer. Cancer Discov 2014, 4, (9), 1046-61. 32. Janne, P. A.; Yang, J. C.; Kim, D. W.; Planchard, D.; Ohe, Y.; Ramalingam, S. S.; Ahn, M. J.; Kim, S. W.; Su, W. C.; Horn, L.; Haggstrom, D.; Felip, E.; Kim, J. H.; Frewer, P.; Cantarini, M.; Brown, K. H.; Dickinson, P. A.; Ghiorghiu, S.; Ranson, M. AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer. N Engl J Med 2015, 372, (18), 1689-99. 33. Thress, K. S.; Paweletz, C. P.; Felip, E.; Cho, B. C.; Stetson, D.; Dougherty, B.; Lai, Z.; Markovets, A.; Vivancos, A.; Kuang, Y.; Ercan, D.; Matthews, S. E.; Cantarini, M.; Barrett, J. C.; Janne, P. A.; Oxnard, G. R. Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small cell lung cancer harboring EGFR T790M. Nat Med 2015, 21, (6), 560-2. 34. Eberlein, C. A.; Stetson, D.; Markovets, A. A.; Al-Kadhimi, K. J.; Lai, Z.; Fisher, P. R.; Meador, C. B.; Spitzler, P.; Ichihara, E.; Ross, S. J.; Ahdesmaki, M. J.; Ahmed, A.; Ratcliffe, L. E.; O'Brien, E. L.; Barnes, C. H.; Brown, H.; Smith, P. D.; Dry, J. R.; Beran, G.; Thress, K. S.; Dougherty, B.; Pao, W.; Cross, D. A. Acquired Resistance to the Mutant-Selective EGFR Inhibitor AZD9291 Is Associated with Increased Dependence on RAS Signaling in Preclinical Models. Cancer Res 2015, 75, (12), 2489-500. 35. Shen, D. W.; Fojo, A.; Chin, J. E.; Roninson, I. B.; Richert, N.; Pastan, I.; Gottesman, M. M. Human multidrug-resistant cell lines: increased mdr1 19



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Overexpression of human ABCB1 in cancer cells leads to reduced

activity of GSK461364, a specific inhibitor of polo-like kinase 1. Mol Pharm 2014, 11, (10), 3727-36. 43. Ambudkar, S. V. Drug-stimulatable ATPase activity in crude membranes of human MDR1-transfected mammalian cells. Methods Enzymol 1998, 292, 504-514. 44. Wu, C. P.; Hsieh, C. H.; Hsiao, S. H.; Luo, S. Y.; Su, C. Y.; Li, Y. Q.; Huang, Y. H.; Huang, C. W.; Hsu, S. C. Human ATP-Binding Cassette Transporter ABCB1 Confers Resistance to Volasertib (BI 6727), a Selective Inhibitor of Polo-like Kinase 1. Mol Pharm 2015, 12, (11), 3885-95. 45. Hollo, Z.; Homolya, L.; Davis, C. W.; Sarkadi, B.

Calcein accumulation as a

fluorometric functional assay of the multidrug transporter. Biochim Biophys Acta 1994, 1191, (2), 384-8. 46. Ludwig, J. A.; Szakacs, G.; Martin, S. E.; Chu, B. F.; Cardarelli, C.; Sauna, Z. 20


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E.; Caplen, N. J.; Fales, H. M.; Ambudkar, S. V.; Weinstein, J. N.; Gottesman, M. M. Selective toxicity of NSC73306 in MDR1-positive cells as a new strategy to circumvent multidrug resistance in cancer. Cancer Res 2006, 66, (9), 4808-4815. 47. Cuestas, M. L.; Castillo, A. I.; Sosnik, A.; Mathet, V. L. Downregulation of mdr1 and abcg2 genes is a mechanism of inhibition of efflux pumps mediated by polymeric amphiphiles. Bioorg Med Chem Lett 2012, 22, (21), 6577-9. 48. Natarajan, K.; Bhullar, J.; Shukla, S.; Burcu, M.; Chen, Z. S.; Ambudkar, S. V.; Baer, M. R.

The Pim kinase inhibitor SGI-1776 decreases cell surface expression

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Restricted brain penetration of the tyrosine kinase inhibitor erlotinib

due to the drug transporters P-gp and BCRP. Invest New Drugs 2012, 30, (2), 443-9.

22


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FIGURE LEGENDS

Figure 1. Osimertinib inhibits ABCB1-mediated drug efflux. The accumulation of fluorescent calcein in (A) drug-sensitive human epidermal KB-3-1 (left panel) or its ABCB1-overexpressing variant KB-V-1 (right panel) cancer cell line, or in (B) drug-sensitive parental human HEK293 (left panel) or its ABCB1-transfected MDR19-HEK293 (right panel) cell line, was measured in the absence (solid lines) or presence of 10 µM osimertinib (shaded, solid lines) or 3 µM of known ABCB1 inhibitor tariquidar (dotted lines) and analyzed immediately by flow cytometry as described in Materials and methods. Representative histograms of three independent are shown. The concentration-dependent inhibition of ABCB1-mediated calcein-AM efflux by osimertinib (open circles) or afatinib (filled circles) in (C) KB-V-1 or (D) MDR19-HEK293 cells. Values are presented as mean ± SEM calculated from at least three independent experiments. The respective IC50 values were calculated as the concentrations required to inhibit the efflux of calcein-AM to 50% of the control values.

Figure 2. Effect of osimertinib on ABCB1 protein expression in human cancer cells. Immunoblot detection of human ABCB1 protein expression in (A) human KB-3-1 and KB-V-1 epidermal cancer cells or (B) human ovarian OVCAR-8 and NCI-ADR-RES cancer cells and (C) quantification of total lysate protein (10 µg) from cells treated with increasing concentrations of osimertinib (0-1 µM) for 72 h as described previously.42 α-tubulin was used as an internal control for equal loading. Values are presented as mean ± SEM calculated from three independent experiments.

23



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Page 24 of 35

Figure 3. Osimertinib reverses ABCB1-mediated drug resistance. Parental KB-3-1 cells (left panels) and ABCB1-overexpressing KB-V-1 cells (right panels) were treated with (A) colchicine, (B) paclitaxel or (C) vincristine in the absence (open circles) or presence of osimertinib at 0.1 µM (open squares), 0.2 µM (filled squares), 0.5 µM (open triangles) or 0.8 µM (filled triangles) as described in Materials and Methods. Points, means from at least three independent experiments; bars; SEM.

Figure 4. Osimertinib enhances colchicine-induced apoptosis in ABCB1-overexpressing cancer cells. Parental KB-3-1 (top panels) and ABCB1-overexpressing KB-V-1 (lower panels) cells were isolated and analyzed 48 h after treatment with either (A) DMSO (left panels), 3 µM osimertinib (middle panels), 1 µM tariquidar (right panels) or (B) 0.5 µM colchicine (left panels), 0.5 µM colchicine and 3 µM osimertinib (middle panels), or 0.5 µM colchicine and 1 µM tariquidar (right panels). Apoptotic cells were quantified by flow cytometry as described previously.44 Representative histograms and the mean values of at least three independent are shown.

Figure 5. Osimertinib stimulates the vanadate (Vi)-sensitive ATPase activity of ABCB1. The effect of 0 - 10 µM and 0 - 0.5 µM (inset) of osimertinib on the Vi-sensitive ABCB1 ATP hydrolysis was determined as described previously.55 Points indicate the mean of at least three independent experiments; bars indicate the SEM.

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Table 1. Chemosensitization effect of osimertinib on ABCB1-mediated drug resistance in ABCB1-overexpressing human cancer cells. R.R

Concentration

KB-3-1

KB-V-1

(µM)

(parental)

(resistant)

IC50 (nM)

IC50 (µM)

-

9.47 ± 3.24

2.16 ± 0.33

228

+ Osimertinib

0.1

10.46 ± 4.27

0.94 ± 0.11**

90

+ Osimertinib

0.2

10.44 ± 4.29

0.70 ± 0.07**

67

+ Osimertinib

0.5

8.78 ± 3.12

0.41 ± 0.03***

47

+ Osimertinib

0.8

10.95 ± 3.97

0.19 ± 0.02***

17

+ Tariquidar

1.0

8.57 ± 2.87

10.45 ± 3.39*** (nM)

1

-

2.19 ± 0.65

4.92 ± 0.83

2247

+ Osimertinib

0.1

2.02 ± 0.50

3.10 ± 0.38*

1535

+ Osimertinib

0.2

2.06 ± 0.53

2.14 ± 0.20**

1039

+ Osimertinib

0.5

2.02 ± 0.45

0.66 ± 0.07***

327

+ Osimertinib

0.8

2.30 ± 0.54

0.34 ± 0.04***

148

+ Tariquidar

1.0

2.16 ± 0.60

1.85 ± 0.44*** (nM)

1

-

1.43 ± 0.31

2.53 ± 0.41

1769

+ Osimertinib

0.1

1.69 ± 0.41

0.99 ± 0.10**

586

+ Osimertinib

0.2

1.72 ± 0.44

0.68 ± 0.07**

395

+ Osimertinib

0.5

1.28 ± 0.31

0.24 ± 0.04***

188

+ Osimertinib

0.8

1.33 ± 0.29

0.12 ± 0.02***

90

+ Tariquidar

1.0

1.26 ± 0.29

1.93 ± 0.47*** (nM)

2

Concentration

OVCAR-8

NCI-ADR-RES

R.R

(µM)

(parental)

(resistant)

Treatment

Colchicine

Paclitaxel

Vincristine

Treatment

25



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IC50 (nM)

IC50 (µM)

-

23.10 ± 9.22

1.45 ± 0.28

63

+ Osimertinib

0.1

24.31 ± 10.28

0.98 ± 0.23

40

+ Osimertinib

0.2

24.13 ± 9.89

0.93 ± 0.24

39

+ Osimertinib

0.5

21.97 ± 9.29

0.49 ± 0.14**

22

+ Osimertinib

0.8

20.39 ± 8.67

0.30 ± 0.09**

15

+ Tariquidar

1.0

22.76 ± 9.35

23.83 ± 10.48*** (nM)

1

-

10.28 ± 2.15

5.45 ± 1.41

530

+ Osimertinib

0.1

7.89 ± 2.20

2.98 ± 0.66

378

+ Osimertinib

0.2

7.51 ± 2.08

2.77 ± 0.33*

369

+ Osimertinib

0.5

7.09 ± 1.90

1.53 ± 0.21**

216

+ Osimertinib

0.8

5.65 ± 1.34*

0.89 ± 0.08**

158

+ Tariquidar

1.0

6.63 ± 1.26

7.01 ± 1.57*** (nM)

1

-

12.38 ± 3.26

2.41 ± 0.65

195

+ Osimertinib

0.1

10.67 ± 3.45

1.27 ± 0.30

119

+ Osimertinib

0.2

9.86 ± 3.26

0.88 ± 0.18*

89

+ Osimertinib

0.5

8.21 ± 2.86

0.33 ± 0.08**

40

+ Osimertinib

0.8

7.11 ± 2.45

0.23 ± 0.04**

32

+ Tariquidar

1.0

8.81 ± 1.96

10.03 ± 2.78*** (nM)

1

Colchicine

Paclitaxel

Vincristine

Abbreviation: R. R, relative resistance. †

IC50 values are mean ± SD calculated from dose-response curves obtained from at

least three independent experiments. ‡ R.R values were calculated by dividing IC50 values of ABCB1 overexpressing cells by IC50 values of respective parental cells. *P < 0.05; **P < 0.01; ***P < 0.001.

26


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Table 2. Sensitivity of drug-sensitive and ABCB1-overexpressing drug-resistant cell lines to osimertinib. IC50 (µM)a

R.Fb

-

4.80 ± 1.25

-

epidermal

ABCB1

5.22 ± 1.34

1

OVCAR-8

ovarian

-

3.13 ± 0.73

-

NCI-ADR-RES

ovarian

ABCB1

3.22 ± 0.69

1

K562

leukemic

-

2.75 ± 1.09

-

K562/i-S9

leukemic

ABCB1

2.23 ± 0.72

1

NIH3T3

-

-

3.98 ± 0.81

-

NIH3T3-G185

-

ABCB1

4.75 ± 1.48

1

pcDNA-HEK293

-

-

4.05 ± 0.93

-

MDR19-HEK293

-

ABCB1

3.68 ± 0.77

1

Cell line

Cancer

Transporter

origin

expressed

KB-3-1

epidermal

KB-V-1

Abbreviation: R.F, resistance factor. a

IC50 values are mean ± SD calculated from dose-response curves obtained from

three independent experiments using cytotoxicity assay as described in Materials and methods. b RF values were calculated by dividing IC50 values of ABCB1 overexpressing cells by IC50 values of respective parental cells. * P < 0.05 ; ** P < 0.01 ; *** P < 0.001.

27



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Page 28 of 35

For Table of Contents Only

28


Page 29 of 35


Wu et al., Figure 1

A KB-3-1 Cell Number

KB-V-1

Control + Osimertinib + Tariquidar

Fluorescence Intensity


pcDNA-HEK293

MDR19-HEK293

B

Cell Number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

Control + Osimertinib + Tariquidar





Page 30 of 35

Wu et al., Figure 1 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

C KB-V-1 ○ + Osimertinib ● + Afatinib

D

MDR19-HEK293 ○ + Osimertinib ● + Afatinib


Page 31 of 35


Wu et al., Figure 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

A

KB-V-1

KB-3-1 0 0.1 0.2 0.5 0.8 1.0

0 0.1 0.2 0.5 0.8 1.0

Osimertinib [μM]

ABCB1 Tubulin

B

NCI-ADR-RES

OVCAR8 0 0.1 0.2 0.5 0.8 1.0

0 0.1 0.2 0.5 0.8 1.0

Osimertinib [μM]

ABCB1 Tubulin

C



Wu et al., Figure 3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

A

KB-3-1

KB-V-1

KB-3-1

KB-V-1

KB-3-1

KB-V-1

B

C


Page 32 of 35

Page 33 of 35 Wu

Propidium iodide

KB-V-1

KB-3-1

A


Control

+ Osimertinib

+ Tariquidar

0.3%

2.3%

0.2%

1.6%

0.2%

3.1%

93.8%

3.6%

95.1%

3.1%

93.9%

2.8%

0.4%

4.4%

0.3%

3.3%

0.7%

4.6%

91.1%

4.1%

92.4%

4.0%

91.3%

3.4%

Annexin V-FITC + Colchicine + Osimertinib

+ Colchicine

+ Colchicine + Tariquidar

2.1%

25.0%

2.4%

24.2%

2.7%

25.5%

55.1%

17.8%

52.5%

20.9%

49.8%

22.0%

0.5%

5.2%

1.1%

17.1%

3.7%

39.8%

87.3%

7.0%

60.6%

21.2%

34.1%

22.4%

Propidium iodide

KB-3-1

B

KB-V-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

et al., Figure 4

Annexin V-FITC ACS Paragon Plus Environment

Wu et al., Figure 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58



Page 34 of 35

Page 35 of 35 Molecular Pharmaceutics

1 2 3 4 5 6 7


Osimertinib (AZD9291) Attenuates the Function of Multidrug

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