Osimertinib (AZD9291)

Published OnlineFirst May 18, 2016; DOI: 10.1158/1535-7163.MCT-15-0939

Small Molecule Therapeutics Molecular Cancer Therapeutics Osimertinib (AZD9291) Enhanced the Efﬁcacy of Chemotherapeutic Agents in ABCB1- and ABCG2- Overexpressing Cells In Vitro, In Vivo, and Ex Vivo Zhen Chen1,2, Yifan Chen1, Meng Xu1, Likun Chen1, Xu Zhang1, Kenneth Kin Wah To3, Hongyun Zhao1, Fang Wang1,2, Zhongjun Xia1, Xiaoqin Chen1, and Liwu Fu1,2

Abstract

The overexpression of ATP-binding cassette (ABC) transporters overexpressing cells but not in their parental sensitive cells. has been proved to be a major trigger for multidrug resistance Furthermore, osimertinib stimulated the ATPase activity of both (MDR) in certain types of cancer. In our study, we investigated ABCB1 and ABCG2 and competed with the [125I] iodoarylazido- whether osimertinib (AZD9291), a third-generation irreversible prazosin photolabeling bound to ABCB1 or ABCG2, but did tyrosine kinase inhibitor of both activating EGFR mutations and not alter the localization and expression of ABCB1 or ABCG2 resistance-associated T790M point mutation, could reverse MDR in mRNA and protein levels nor the phosphorylations of induced by ABCB1 and ABCG2 in vitro, in vivo, and ex vivo. Our EGFR, AKT, and ERK. Importantly, osimertinib also enhanced results showed that osimertinib significantly increased the sensi- the cytotoxicity of DOX and intracellular accumulation of Rho 123 tivity of ABCB1- and ABCG2-overexpressing cells to their substrate in ABCB1-overexpressing primary leukemia cells. Overall, these chemotherapeutic agents in vitro and in the model of ABCB1- findings suggest osimertinib reverses ABCB1- and ABCG2-medi- overexpressing KBv200 cell xenograft in nude mice. Mechanisti- ated MDR via inhibiting ABCB1 and ABCG2 from pumping cally, osimertinib increased the intracellular accumulations of out chemotherapeutic agents and provide possibility for doxorubicin (DOX) and Rhodamine 123 (Rho 123) by inhibiting cancer combinational therapy with osimertinib in the clinic. the efflux function of the transporters in ABCB1- or ABCG2- Mol Cancer Ther; 15(8); 1845–58. 2016 AACR.

Introduction effluxing out of the cells against a concentration gradient coupled to the energy of ATP hydrolysis (9). Oneofthemajorobstacletosuccessful treatment of cancer is ABCB1, coded by ABCB1 gene, is composed of two homolo- the development of multidrug resistance (MDR), which means gous halves, each containing six transmembrane domains and cancer cells become resistant to structurally and mechanistically an ATP binding/utilization domain, separated by a flexible poly- distinct classes of anticancer drugs (1–4). Among several factors peptide linker (10). ABCB1 renders MDR in cancer cells by responsible for development of MDR, the overexpression of transporting a wide spectrum of compounds including the vinca ATP-binding cassette (ABC) transporters is a major concern alkaloids [vinblastine and vincristine (VCR)], the anthracyclines (5, 6). Currently, 49 members of the ABC transporter family [doxorubicin (DOX) and daunorubicin], epipodophyllotoxins, have been identified and are dividing into seven subfamilies taxanes, and some tyrosine kinase inhibitors (TKI) out of the cells (ABCA through ABCG) based on the sequence and structure (11, 12). The overexpression of ABCB1 could predict poor prog- similarities (7, 8). The major members of the ABC transporters nosis of cancer patients (13–15). leading to MDR in cancer cells include ABCB1 (P-glycoprotein, ABCG2, encoded by the ABCG2 gene, is known as a half P-gp) and ABCG2 (breast cancer resistance protein, BCRP, transporter that effluxes agents with amphiphilic characteristics mitoxantrone resistance protein, MXR, and placenta-specific (16, 17). ABCG2 has been reported to mediate MDR by effluxing ABC transporter, ABCP), which mediate the substrate agents numerous agents, including mitoxantrone (MX), topotecan, iri- notecan, DOX, nilotinib, and imatinib (16, 18–20). Indeed, 1Collaborative Innovation Center for Cancer Medicine, State Key Lab- upregulated expression of ABCG2 is observed in a variety of oratory of Oncology in South China, Sun Yat-Sen University Cancer cancer stem cells, leading to the speculation that ABCG2 provides Center, Guangzhou, China. 2Guangdong Esophageal Cancer Institute, cellular protection against cytotoxic agents for cancer stem cells, Guangzhou, China. 3School of Pharmacy, the Chinese University of Hong Kong, Hong Kong, China. which are inherently present in the cancer cell populations (21, 22). Note: Supplementary data for this article are available at Molecular Cancer Based on the discoveries of ABC transporters, identifying inhi- Therapeutics Online (http://mct.aacrjournals.org/). bitors that can either regulate their expression or inhibit their Z. Chen, Y. Chen, and M. Xu contributed equally to this article. function may represent a promising strategy to circumvent MDR. Corresponding Author: Liwu Fu, State Key Laboratory of Oncology in South Numerous studies over the past three decades have been devoted China, Sun Yat-Sen University Cancer Center, East, 651 Dongfeng Road, Guangz- to developing ABC transporter inhibitors (23, 24). However, hou, 510060 Guangdong, China. Phone: 86-20-873-431-63; Fax: 86-20-873-431- many of these inhibitors failed to be utilized in clinical applica- 70; E-mail: [email protected] tion due to the unacceptable toxicity as well as problematic doi: 10.1158/1535-7163.MCT-15-0939 pharmacokinetic interactions (4). But recently, we have reported 2016 American Association for Cancer Research. that several TKIs, including lapatinib, erlotinib, apatinib, and

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afatinib, can overcome MDR mediated by ABC transporters (25– Cell cytotoxicity assay 30). And the preliminary results with inhibitors showed no The MTT assay was used to assess the cell sensitivity to che- appreciable impacts on cytochrome P4503A4 drug metabolism motherapeutic agents as described previously (38). The concen- and no significant drug interactions with common chemothera- tration required to inhibit cell growth by 50% (IC50) was calcu- peutic agents, offering new hope for combination therapy in lated from survival curves using the Bliss method (39). The fold of cancer patients. resistance was calculated by dividing the IC50 for the MDR cells by Osimertinib (AZD9291), a mono-anilino-pyrimidine com- that for the parental sensitive cells. The degree of reversal of MDR pound, is an irreversible TKI selective for activating EGFR muta- (fold reversal) was calculated by dividing the IC50 for cells with the tions (EGFRdel19 and EGFRL858R) and resistance mutation chemotherapeutic agents in the absence of osimertinib by that (EGFRT790M) with structure different from the first- and sec- obtained in the presence of osimertinib. ond-generation EGFR-TKIs (31–33). Because osimertinib acts at the ATP binding site of the tyrosine kinase domains of EGFR, it is Animal experiments conceivable that osimertinib may inhibit functions of ABC trans- The KBv200 cell xenograft model was established as porters by binding to their ATP-binding sites, which is similar to described previously (40). All experiments were performed in the mechanism on reversing MDR by several TKIs mentioned accordance with the guidelines for the use of laboratory ani- above. These have spurred on efforts to investigate whether mals of the Sun Yat-Sen University Institutional Animal Care osimertinib can enhance the efficacy of conventional chemother- and Use Committee. Briefly, KBv200 cells (2.0 106)were apeutic agents via interaction with ABCB1 or ABCG2 in vitro, subcutaneously injected into the right armpit of athymic nude in vivo, and ex vivo. mice (BALB/C-nu/nu, female, 5 to 6 weeks of age, 18 to 24 g of weight, purchased from Hunan SJA Laboratory Animal Co., Materials and Methods Ltd). When the tumor reached a mean diameter of 5 mm, the Chemicals and reagents mice were randomized into four groups and received various Osimertinib was purchased from KareBay Biochem. The anti- treatments: (a) saline (q3d); (b) paclitaxel (20 mg/kg, intra- bodies against ABCB1/p-gp (sc-13131), ABCG2 (sc-377176), peritoneal injection/i.p., q3d); (c) osimertinib (10 mg/kg, AKT (sc-8312), p-AKT (sc-7985-R), ERK (sc-514302), p-ERK (sc- gavage, q3d); (d) osimertinib (10 mg/kg, gavage, q3d, given 7383), and EGFR (sc-514995) were purchased from Santa Cruz 1 hour before paclitaxel administration) plus paclitaxel (20 Biotechnology. Antibody against p-EGFR (#3777) was pur- mg/kg, i.p., q3d). The body weights of the animals and the two chased from Cell Signaling Technology. Flow cytometry anti- perpendicular tumor diameters (A and B) were measured every bodies against ABCB1 (#557002) and IgG2a (#559532) were 2 days, and the tumor volume (V) was calculated according to ¼ p þ 3 purchased from BD Biosciences. Glyceraldehyde-3-phosphate the following formula: V ( /6)[(A B)/2] . At the end of the dehydrogenase (GAPDH) antibody was purchased from Kang- observation period, the mice were euthanized and the xeno- cheng Co. SYBR Green qPCR Master Mix was purchased from grafts were excised and weighed. The curves of tumor growth ExCell Bio. DMEM and RPMI 1640 medium were purchased and body weight were drawn according to tumor volume and from Life Technologies. Rhodamine 123 (Rho 123), 1-(4, 5- time of treatment. The ratio of growth inhibition (IR) was dimethylthiazol-2-yl)-3, 5-diphenylformazan (MTT), G418, estimated according to the following formula: paclitaxel, DOX, VCR, verapamil (VRP), topotecan, MX, cisplat- 1 Mean tumor weight of experimental group in, fumitremorgin C (FTC), and other chemicals were purchased IRð%Þ¼ 100 from Sigma Aldrich. Mean tumor weight of control group

Cell lines and cell culture DOX and Rho 123 accumulation assay The following cell lines were cultured in essential medium Flow cytometry assay was performed to measure the effect of containing 10% FBS at 37 C in the presence of 5% CO2: the osimertinib on the intracellular accumulation of DOX and Rho human oral epidermoid carcinoma cell line KB and its VCR- 123 in cancer cells as previously described (35). Briefly, the cells selected ABCB1-overexpressing cell line KBv200 were generous were cultured in the 6-well plates and grew overnight. Then the gifts from Dr. Xu-Yi Liu (Cancer Hospital of Beijing, Beijing, cells were treated with different concentrations of osimertinib or China). We used the human non–small cell lung cancer cell vehicle at 37 C for 3 hours. Following the osimertinib treatment, line H460 and its MX-selected ABCG2-overexpressing cell line 10 mmol/L DOX or 5 mmol/L Rho 123 was added and incubation H460/MX20, the human colon carcinoma cell line S1 and its MX- was continued for additional 3 hours or 0.5 hours, respectively. selected ABCG2-overexpressing cell line S1-MI-80, the human The cells were then harvested, washed 3 times with chilled PBS, breast carcinoma cell line MCF-7 and its DOX-selected ABCB1- and analyzed by flow cytometry analysis (Beckman Coulter; overexpressing cell line MCF-7/adr. Stable-transfected HEK293/ CytomicsFC500). VRP was used as a control inhibitor in pcDNA3.1, HEK293/ABCB1, and HEK293/ABCG2 (wild-type) ABCB1-overexpressing cells, whereas FTC was used in ABCG2- cells were established by selection with G418 after transfecting overexpressing cells. HEK293 with empty pcDNA3.1 vector or pcDNA3.1 vector containing full-length ABCB1 or pcDNA3.1 vector containing full- Intracellular Hoechst 33342 accumulation assay length ABCG2 coding arginine (R) at amino acid 482 position, The intracellular accumulation assay of Hoechst 33342 was respectively (34–37), were generous gifts from Dr. Susan Bates performed as described by Goodell and colleagues with some (NCI, NIH, Bethesda, MD). Cell lines used in this study were modifications (41). In brief, cells were incubated in 6-well plates thawed from early passage stocks and were passaged for less than 6 with exposure to 0.05, 0.1, and 0.2 mmol/L osimertinib for 3 months. hours. Then Hoechst 33342 dye (0.5 mg/mL) was added and

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further incubated for 1.5. hours. Finally, cells were washed with designated time points. Then the proteins were extracted and the ice-cold PBS 3 times and resuspended in PBS for flow cytometry concentrations were determined using the BCA Protein Assay analysis. (Thermo Fisher Scientific). Equal amounts of proteins were separated by SDS-PAGE gel and transferred to polyvinylidene difluor- fl Rho 123 ef ux assay ide membranes followed by blocking with 5% non-fat milk. fl Rho 123 ef ux assay was performed as described previously Membranes were sequentially incubated with the primary and (42). S1, S1-MI-80, KB, and KBv200 cells were treated with secondary antibodies. The signal was detected using the enhanced m 5 mol/L Rho 123 at 37 C for 30 minutes. Then the cells were chemiluminescence solutions (#1705061; Bio-Rad) and exposed washed 3 times with PBS and were subsequently incubated at to a Kodak medical x-ray processor (Kodak). 37C with fresh media in the presence or absence of 0.2 (S1, S1- m MI-80) or 0.4 (KB, KBv200) mol/L osimertinib for 0, 15, 30, 60, PCR and real-time quantitative PCR and 90 minutes. The cells were collected at the designed time ABCB1 and ABCG2 expressions in mRNA level were assayed as points and washed three times with chilled PBS. Finally, cells were previously described (28). After a range of different concentrations fl resuspended in chilled PBS buffer for ow cytometric analysis and designed time points of osimertinib treatment, total cellular immediately. RNA was isolated by trizol reagent RNA extraction kit (#15596026; Thermo Fisher Scientific Inc). The first strand cDNA was synthe- ABCB1 and ABCG2 ATPase activity assay sized by cDNA reverse transcription kit (Promega Corp). The PCR A colorimetric ATPase assay was performed as previously 0 0 primers were 5 -CAGGCTTGCTGTAATTACCCA-3 (forward) and described with minor modification (43). Briefly, crude mem- 0 0 0 5 -TCAAAGAAACAACGGTTCGG-3 (reverse) for ABCB1; 5 -TGG- branes isolated from High Five insect cells expressing either 0 0 CTGTCATGGCTTCAGTA-3 (forward) and 5 -GCCACGTGATTCT- ABCB1 or ABCG2 (100 mg protein/mL) were incubated at 37 C 0 0 TCCACAA-3 (reverse) for ABCG2; 5 -GAGTCAAGGATTTGG- with a range of different concentrations of osimertinib in 0 0 0 TCGT-3 (forward) and 5 -GATCTCGCTCCTGGAAGATG-3 the presence or absence of sodium orthovanadate (0.3 mmol/L (reverse) for GAPDH, respectively. After 35 cycles of amplification, for ABCB1 and 1.2 mmol/L for ABCG2) in ATPase assay buffer products were separated by 1.0% agarose gel electrophoresis. Real- (50 mmol/L KCl, 5 mmol/L sodium azide, 2 mmol/L EDTA, time PCR was performed using SYBR GREEN Mix. Relative quan- 10 mmol/L MgCl , 1 mmol/L DTT, pH 6.8) for 5 minutes. ATP DD 2 tification of ABCB1 or ABCG2 was analyzed using the 2 Ct hydrolysis reaction was then started by the addition of 5 mmol/L method as a ratio relative to the GAPDH expression level in each Mg-ATP (concentration in a final volume of 60 mL) and incubated sample, respectively (44). for 20 minutes (for ABCB1) or 10 minutes (for ABCG2). SDS solution (30 mL of 10% SDS) was added to terminate the reaction. Cell surface expression of ABCB1 and ABCG2 Absorbance was subsequently measured at 750 nm after the examined by flow cytometry addition of a detection reagent (35 mmol/L ammonium molyb- Flow cytometry analysis was performed as described previously date, 15 mmol/L zinc acetate, 10% ascorbic acid) and incubation (45). Cell surface expression of ABCB1 or ABCG2 in KBv200 or S1- at 37C for 20 minutes. The amount of inorganic phosphate MI-80 cells was measured by flow cytometry after being treated released was quantified by reading from a standard curve. Specific with designed concentrations of osimertinib for 48 hours, respec- osimertinib-stimulated ABCB1 and ABCG2 ATPase activity (i.e., tively. Single-cell suspensions were prepared and washed 3 times vanadate-sensitive) was determined as the difference between the with ice-cold PBS (supplemented with 0.5% BSA). Then, 10 mLof amounts of inorganic phosphate released from ATP in the absence FITC-conjugated ABCB1 or FITC-conjugated ABCG2 antibody and presence of sodium orthovanadate. was mixed with 25 mL of cells (4 106 cells/mL) and incubated Photoaffinity labeling of ABCB1 and ABCG2 with [125I]-IAAP in the dark for 45 minutes at 4 C, respectively. The cells were Crude membrane from High Five insect cells expressing ABCB1 washed twice with chilled PBS (supplemented with 0.5% BSA) m fl or ABCG2 (50 mg protein) was incubated with 0 to 12.5 mmol/L and were resuspended in 400 L of PBS for ow cytometric osimertinib for 5 minutes at room temperature in 50 mmol/L Tris- analysis. Isotype control samples were treated in an identical a HCl (pH 7.5). [125I]-Iodoarylazidoprazosin (IAAP; 2,200 Ci/ manner with FITC-conjugated IgG2 for ABCB1 or ABCG2. nmole, 3 nmol/L) was added, and incubation was continued for a further 5 minutes under subdued light. The samples were then Patient samples cross-linked by UV illumination (365 nm) on ice. The labeled Bone marrow samples from diagnosed acute myeloid leukemia – – ABCB1 and ABCG2 were immunoprecipitated using the corre- (AML) patients according to the French American British (FAB) fi sponding antibody [ABCB1 (UIC2) from Abcam (cat#ab166957) classi cation were obtained after their informed consent (46). and ABCG2 (BXP-21) from Santa Cruz Biotechnology (cat#sc- This study was approved by the Ethics Review Committee of 58222)], respectively. The samples were then subjected to SDS- Sun Yat-Sen University. Leukemic blasts were isolated using – PAGE using a 7% Tris-acetate NuPAGE gel, dried, and exposed to Ficoll Hypaque density gradient by centrifugation and then Bio-Max MR film (Eastman Kodak Co.) at 80C for overnight. cultured in RPMI 1640 medium. Expressions of ABCB1 and fl The radioactivity incorporated into the transporter protein was Rho123 intracellular accumulation were detected by ow cyto- quantified using the Storm 860 PhosphorImager system (Molec- metry as described above. ular Dynamics; ref. 43). Statistical analysis Western blot analysis Results were shown as mean SD. All experiments were Western blot analysis was performed as described earlier (28). repeated at least three times, and the differences were determined For Western blot analysis, the cells were treated with a range of by using the Student t test. The statistical significance was deter- different concentrations of osimertinib and then were collected at mined to be "" P < 0.05 and "" P < 0.01.

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A N

O NH N N

N NN H O Osimertinib B C 120 KB 120 S1 100 KBv200 100 S1-MI-80

80 80 60 60 40 40 20 20 Survival (% of control)

0 Survival (% of control) 0 0.01 0.1 1 10 100 0.01 0.1 1 10 100 µ Concentration of osimertinib ( mol/L) Concentration of osimertinib (µmol/L) Figure 1. D E The structure of osimertinib, cytotoxicity of osimertinib in the 120 120 MCF7 H460 sensitive parental and drug-resistant cell lines, and potentiation 100 MCF7/adr 100 H460/MX20 of the anticancer effect of paclitaxel by osimertinib in the

80 80 KBv200 cell xenograft nude mice model. A, the structure of osimertinib. MTT cytotoxicity assay was used to assess the cell 60 60 survival in KB and ABCB1-overexpressing KBv200 cells (B), 40 40 S1 and ABCG2-overexpressing S1-MI-80 cells (C), MCF-7 and 20 20 ABCB1-overexpressing MCF-7/adr cells (D), H460 and Survival (% of control) 0 Survival (% of control) 0 ABCG2-overexpressing H460/MX20 cells (E), HEK293/Vector 0.01 0.1 1 10 100 0.01 0.1 1 10 100 Concentration of osimertinib (µmol/L) Concentration of osimertinib (µmol/L) and stable ABCB1-transfected HEK293/ABCB1 cells (F), and HEK293/Vector and stable ABCG2-transfected HEK293/ABCG2 cells (G). All cells were treated with different FG concentrations of osimertinib for 72 hours. Data points 120 120 HEK293/Vector HEK293/Vector represent the mean SD of at least three independent HEK293/ABCG2 100 HEK293/ABCB1 100 experiments performed in quadruple. H, the tumor growth 80 80 curve was drawn according to the tumor volume and time after 60 60 implantation. Data shown are mean SD of tumor volume for 40 40 each group (n ¼ 9). I, the tumor tissues were excised and the

20 20 tumor size was measured on the 21st day after implantation. Survival (% of control)

Survival (% of control) J, animals' body weights were measured every 2 days, and the 0 0 0.01 0.1 1 10 100 0.01 0.1 1 10 100 average percentage change was calculated after treatment. Concentration of osimertinib (µmol/L) Concentration of osimertinib (µmol/L) K, the average tumor weight of each group was calculated after the tumors excised from the mice. The four treatment HI groups were: (a) saline (q3d); (b) paclitaxel Control (20 mg/kg, intraperitoneal injection/i.p., q3d); (c) osimertinib Paclitaxel Control Osimertinib (10 mg/kg, gavage, q3d); (d) osimertinib (10 mg/kg, gavage, 3,000

) Osimertinib+paclitaxel q3d, given 1 hour before paclitaxel administration) plus 3 2,500 Paclitaxel paclitaxel (20 mg/kg, i.p., q3d). Data shown are mean SD for 2,000 each group; , P < 0.01. Osimertinib 1,500 1,000 Osimertinib + 500 paclitaxel Tumor volume (m volume Tumor 0 7 9 11 13 15 17 19 21 Days after implantation J K Control Paclitaxel 50 2.5 Osimertinib 40 Osimertinib+paclitaxel 30 2.0 20 10 1.5 0 –10 1.0 –20 –30 0.5 –40 (g) weight Tumor –50 7 9 11 13 15 17 19 21 0.0 Average % body weight change % body weight Average Days after implantation Control + Paclitaxel Osimertinib Osimertinib paclitaxel

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Table 1A. Effect of osimertinib on reversing ABCB1- and ABCG2-mediated MDR in drug-selected cell lines

IC50 SD (mmol/L; fold-reversal) Compounds KB KBv200 (ABCB1) VCR 0.0020 0.0003 (1.00) 0.5651 0.0325 (1.00) þ 0.1 mmol/L osimertinib 0.0019 0.0002 (1.05) 0.1176 0.0121 (4.81) þ 0.2 mmol/L osimertinib 0.0018 0.0004 (1.11) 0.0905 0.0029 (6.24) þ 0.4 mmol/L osimertinib 0.0018 0.0005 (1.11) 0.0534 0.0041 (10.58) þ 10 mmol/L VRP 0.0017 0.0003 (1.18) 0.0497 0.0023 (11.37) DOX 0.0311 0.0021 (1.00) 1.3346 0.1267 (1.00) þ 0.1 mmol/L osimertinib 0.0292 0.0022 (1.07) 0.4527 0.0312 (2.95) þ 0.2 mmol/L osimertinib 0.0307 0.0018 (1.01) 0.2782 0.0216 (4.80) þ 0.4 mmol/L osimertinib 0.0279 0.0030 (1.11) 0.1875 0.0129 (7.12) þ 10 mmol/L VRP 0.0294 0.0035 (1.06) 0.1241 0.0112 (10.75) Paclitaxel 0.0021 0.0005 (1.00) 0.4912 0.0274 (1.00) þ 0.1 mmol/L osimertinib 0.0020 0.0006 (1.05) 0.1695 0.0136 (2.90) þ 0.2 mmol/L osimertinib 0.0019 0.0002 (1.11) 0.1074 0.0095 (4.57) þ 0.4 mmol/L osimertinib 0.0017 0.0008 (1.24) 0.0563 0.0053 (8.72) þ 10 mmol/L VRP 0.0019 0.0004 (1.11) 0.0367 0.0031 (13.38) Cisplatin 0.6688 0.0715 (1.00) 1.4710 0.0543 (1.00) þ 0.4 mmol/L osimertinib 0.6727 0.0673 (0.99) 1.4860 0.0472 (0.99)

MCF-7 MCF-7/adr (ABCB1) DOX 0.0917 0.0031 (1.00) 4.3203 0.2543 (1.00) þ 0.1 mmol/L osimertinib 0.0923 0.0027 (0.99) 2.3475 0.1542 (1.84) þ 0.2 mmol/L osimertinib 0.0861 0.0048 (1.07) 0.7412 0.0436 (5.83) þ 0.4 mmol/L osimertinib 0.0889 0.0065 (1.03) 0.4959 0.0312 (8.71) þ 10 mmol/L VRP 0.0934 0.0076 (0.98) 0.2667 0.0722 (16.20) Paclitaxel 0.0089 0.0009 (1.00) 0.6967 0.0543 (1.00) þ 0.1 mmol/L osimertinib 0.0087 0.0008 (1.02) 0.2916 0.0275 (2.39) þ 0.2 mmol/L osimertinib 0.0099 0.0009 (0.90) 0.1594 0.0114 (4.37) þ 0.4 mmol/L osimertinib 0.0083 0.0007 (1.07) 0.0815 0.0078 (8.55) þ 10 mmol/L VRP 0.0085 0.0006 (1.05) 0.0443 0.0042 (15.73) Cisplatin 1.0585 0.2354 (1.00) 5.3143 0.2987 (1.00) þ 0.4 mmol/L osimertinib 1.0960 0.2574 (0.97) 5.4766 0.6573 (0.97)

S1 S1-MI-80 (ABCG2) MX 0.1293 0.0982 (1.00) 19.5305 0.7642 (1.00) þ 0.05 mmol/L osimertinib 0.1187 0.0875 (1.09) 7.5039 0.5428 (2.60) þ 0.1 mmol/L osimertinib 0.1098 0.0542 (1.18) 3.1861 0.6539 (6.13) þ 0.2 mmol/L osimertinib 0.1152 0.0362 (1.12) 1.1542 0.3214 (16.92) þ 2.5 mmol/L FTC 0.1360 0.0487 (0.95) 0.8689 0.3573 (22.48) Topotecan 0.2677 0.0125 (1.00) 14.4553 0.8625 (1.00) þ 0.05 mmol/L osimertinib 0.2549 0.0265 (1.05) 4.5483 0.6722 (3.18) þ 0.1 mmol/L osimertinib 0.2763 0.0627 (0.97) 3.4653 0.2986 (4.17) þ 0.2 mmol/L osimertinib 0.2451 0.0362 (1.09) 1.9404 0.3497 (7.05) þ 2.5 mmol/L FTC 0.2634 0.0543 (1.02) 0.7717 0.2574 (18.73) Cisplatin 17.6519 0.4527 (1.00) 18.4539 0.7862 (1.00) þ 0.2 mmol/L osimertinib 16.3288 0.6793 (1.08) 17.6709 0.8095 (1.04)

H460 H460/MX20 (ABCG2) MX 0.0033 0.0004 (1.00) 0.4017 0.0763 (1.00) þ 0.1 mmol/L osimertinib 0.0028 0.0007 (1.18) 0.1480 0.0659 (2.71) þ 0.2 mmol/L osimertinib 0.0030 0.0009 (1.10) 0.0903 0.0864 (4.45) þ 0.4 mmol/L osimertinib 0.0027 0.0008 (1.22) 0.0519 0.0402 (7.74) þ 2.5 mmol/L FTC 0.0025 0.0004 (1.32) 0.0397 0.0675 (10.12) Cisplatin 3.1759 0.7622 (1.00) 7.7430 0.5439 (1.00) þ 1.5 mmol/L osimertinib 2.9254 0.5421 (1.09) 7.2012 0.4328 (1.08) NOTE: MTT assay was performed to analyze the cell survival after treatment with anticancer agents in the absence or presence of osimertinib. VRP (speciﬁc inhibitor of ABCB1) and FTC (speciﬁc inhibitor of ABCG2) were used as the positive control. The fold reversal of MDR (values given in parentheses) was calculated by dividing the IC50 value for cells with the anticancer drug in the absence of osimertinib by that obtained in the presence of osimertinib. Data represent the mean SD of at least three independent experiments. , P < 0.05; , P < 0.01.

Results assay. As shown in Fig. 1B–G, osimertinib displayed moderate fi Osimertinib enhanced the ef cacy of substrate cytotoxic effect on both parental and resistant cells. The IC50 chemotherapeutic agents in ABCB1- and ABCG2- values of osimertinib alone were 4.194 0.332, 6.330 0.736, overexpressing cells in vitro 5.092 0.837, 4.983 0.372, 1.434 0.276,1.884 0.125, The structure of osimertinib is shown in Fig. 1A. To find out 6.787 0.725, 6.648 0.575, 3.451 0.498, 8.027 0.975, whether osimertinib could reverse MDR in vitro,wefirst examined 6.536 0.736 mmol/L for KB, KBv200, MCF-7, MCF-7/adr, S1, the cytotoxic effect of osimertinib on different cell lines by MTT S1-MI-80, H460, H460/MX20, HEK293/Vector, HEK293/ABCB1,

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Table 1B. Effect of osimertinib on reversing ABCB1- and ABCG2-mediated MDR in stable-transfected cells

IC50 SD (mmol/L; fold-reversal) Compounds HEK293/Vector HEK293/ABCB1 DOX 0.0686 0.0054 (1.00) 1.1795 0.1722 (1.00) þ 0.1 mmol/L osimertinib 0.0665 0.0072 (1.03) 0.3512 0.0237 (3.36) þ 0.2 mmol/L osimertinib 0.0646 0.0083 (1.06) 0.2719 0.0254 (4.34) þ 0.4 mmol/L osimertinib 0.0677 0.0056 (1.01) 0.1204 0.0108 (9.80) þ 10 mmol/L VRP 0.0567 0.0077 (1.21) 0.0598 0.0068 (19.72) Paclitaxel 0.0452 0.0021 (1.00) 2.5177 0.2376 (1.00) þ 0.1 mmol/L osimertinib 0.0422 0.0043 (1.07) 0.9275 0.0386 (2.71) þ 0.2 mmol/L osimertinib 0.0465 0.0032 (0.97) 0.6254 0.0452 (4.03) þ 0.4 mmol/L osimertinib 0.0443 0.0026 (1.02) 0.3271 0.0311 (7.70) þ 10 mmol/L VRP 0.0476 0.0054 (0.95) 0.2652 0.0264 (9.49) Cisplatin 4.3137 0.3763 (1.00) 4.3944 0.2752 (1.00) þ 0.4 mmol/L osimertinib 4.2425 0.2543 (1.02) 4.2390 0.8463 (1.04)

HEK293/Vector HEK293/ABCG2 MX 0.0601 0.0062 (1.00) 0.4522 0.0762 (1.00) þ 0.1 mmol/L osimertinib 0.0565 0.0036 (1.06) 0.1484 0.0121 (3.05) þ 0.2 mmol/L osimertinib 0.0579 0.0075 (1.04) 0.0937 0.0072 (4.83) þ 0.4 mmol/L osimertinib 0.0570 0.0082 (1.05) 0.0738 0.0063 (6.13) þ 2.5 mmol/L FTC 0.0597 0.0043 (1.01) 0.0648 0.0021 (7.00) Topotecan 0.0633 0.0072 (1.00) 2.7392 0.3213 (1.00) þ 0.1 mmol/L osimertinib 0.0603 0.0035 (1.05) 1.1723 0.2123 (2.34) þ 0.2 mmol/L osimertinib 0.0617 0.0062 (1.03) 0.6331 0.0435 (4.33) þ 0.4 mmol/L osimertinib 0.0625 0.0034 (1.01) 0.2910 0.0108 (9.41) þ 2.5 mmol/L FTC 0.0623 0.0083 (1.02) 0.2435 0.0483 (11.25) Cisplatin 4.3539 0.6552 (1.00) 4.1673 0.5241 (1.00) þ 0.4 mmol/L osimertinib 4.1473 0.2651 (1.05) 4.2504 0.1252 (0.98) NOTE: MTT assay was performed to analyze the cell survival after treatment with anticancer agents in the absence or presence of osimertinib. VRP (speciﬁc inhibitor of ABCB1) and FTC (speciﬁc inhibitor of ABCG2) were used as the positive control. The fold reversal of MDR (values given in parentheses) was calculated by dividing

the IC50 value for cells with the anticancer drug in the absence of osimertinib by that obtained in the presence of osimertinib. Data represent the mean SD of at least three independent experiments. , P < 0.05; , P < 0.01.

and HEK293/ABCG2 cells, respectively. Thus, we chose 0.2 mmol/ enhances the efficacy of substrate conventional chemothera- L and 0.4 mmol/L osimertinib as the maximum concentration peutic agent in ABCB1-overexpressing cell xenografts in vivo. for further reversal assay in ABCG2- and ABCB1-overexpressing Importantly, we did not find any death or decrease of body cells with more than 90% of cells surviving. As expected, KBv200 weight in the four groups (Fig. 1J), suggesting that the combi- cells or MCF-7/adr cells overexpressing ABCB1 and S1-MI-80 cells nation regimen did not result in increased toxicity. or H460/MX20 cells overexpressing ABCG2 were extremely resistant to substrate chemotherapeutic agents (such as VCR, DOX, Osimertinib increased the intracellular accumulation of DOX paclitaxel, topotecan) when compared with their parental cells and Rho 123/Hoechst 33342 in ABCB1- and ABCG2- (Tables 1A and 1B). But with osimertinib in a designed concen- overexpressing cells tration-dependent manner, the IC50 of resistant cell lines signif- The results above indicated that osimertinib could signifi- icantly decreased while their parental sensitive cells showed no cantly enhance the sensitivity of MDR cancer cells to substrate difference. Besides, osimertinib did not increase the cytotoxicity of conventional chemotherapeutic agents in vitro and in vivo. cisplatin which is not a substrate of ABCB1 or ABCG2 in the Mechanistically, we detected the intracellular accumulation of ABCB1- or ABCG2-overexpressing MDR cells. These results sug- DOX and Rho 123 in the presence or absence of osimertinib in gested that osimertinib potentiated the sensitivity of ABCB1- and the KBv200 and S1-MI-80 cells by flow cytometric analysis. Our ABCG2-overexpressing cells to conventional chemotherapeutic data showed that the intracellular accumulations of DOX and agents such as VCR, DOX, paclitaxel, and topotecan in vitro. Rho 123 were significantly lower in the resistant cells than that in their parental sensitive cells (Fig. 2A–D). In the presence of Osimertinib potentiated the anticancer efficacy of paclitaxel in osimertinib, the intracellular accumulations of DOX and Rho model of ABCB1-overexpressing KBv200 cell xenografts in vivo 123 significantly increased in KBv200 and S1-MI-80 cells while To investigate whether osimertinib could enhance the effi- not in the sensitive KB and S1 cells. In addition, the similar cacy of chemotherapeutic agents in vivo, we established the results were observed in H460/MX20 and HEK293/ABCG2 ABCB1-overexpressing multidrug-resistant KBv200 cell xeno- cells both of which express wild-type ABCG2 which do not graft model in nude mice. There was no significant difference in transport DOX or Rho 123. Osimertinib increased the intra- tumor size between animals treated with saline, osimertinib, or cellular accumulation of Hoechst 33342 in resistant H460/ paclitaxel alone (Fig. 1H). The combination of osimertinib and MX20 and HEK293/ABCG2 cells rather than in sensitive paclitaxel produced a greater inhibitory effect on xenograft H460 and HEK293/Vector cells (Fig. 2E and F). Furthermore, growth compared with animals treated with only saline, pac- the increased effect produced by osimertinib at 0.2 mmol/L or litaxel, or osimertinib (P < 0.05; Fig. 1H, I, and K), and the 0.4 mmol/L was comparable with that of FTC at 2.5 mmol/L or inhibition rate was 52.7%. These indicated that osimertinib VRP at 10 mmol/L (Fig. 2).

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In order to confirm whether the increased intracellular accu- up to the concentration of 2.0 mmol/L (in S1-MI-80 cells) and 4.0 mulations of DOX and Rho 123 were due to the blockage of mmol/L (in KBv200 cells). In addition, we performed the flow transporter-mediated efflux, a time course study was performed to cytometric assay to determine the effect of osimertinib on the assess the effect of osimertinib on the efflux of Rho 123. In the membrane-bound levels of ABCB1 and ABCG2. As shown absence of osimertinib, the intracellular Rho 123 was much less in in Fig. 4E and F, osimertinib did not influence the membrane the KBv200 and S1-MI-80 cells than in their parental KB and S1 location of ABCB1 and ABCG2. These results indicated that neither cells (Fig. 3A and B) because of the active ABCB1 and ABCG2 the expression nor membrane location of ABCB1 and ABCG2 was efflux. While in the presence of osimertinib, the retention of Rho altered by osimertinib at the designed concentrations. The reversal 123 in KBv200 and S1-MI-80 cells significantly increased at the of MDR by osimertinib was possible through inhibiting the trans- designed time points comparing to the group without osimertinib port function of ABCB1 and ABCG2, but not by downregulating treatment. These results indicated that osimertinib could inhibit their expression or altering membrane location. the efflux activity of ABCB1 and ABCG2. Osimertinib did not block the phosphorylations of EGFR, AKT, Osimertinib stimulated the ATPase activity of ABCB1 and and ERK at the concentration of MDR reversal ABCG2 Previous studies have reported that the inhibition of AKT A crucial role of ABC transporters in the development of MDR is and ERK1/2 pathways could block the resistance to chemother- achieved by pumping out drugs from the cells, and this process is apeutic agents in cancer cells (47, 48). Because osimertinib is a coupled to the energy of ATP hydrolysis on the ATPase domain of potent inhibitor of EGFR and regulates two important down- ABC transporters, which is stimulated in the presence of transport stream AKT and ERK, we conducted Western blots to investigate substrates. Thus, ATP consumption reflects ATPase activity. Based whether osimertinib alters the phosphorylation level of EGFR, on this, we evaluated the vanadate-sensitive ABCB1 or ABCG2 AKT, or ERK in the concentration of MDR reversal. As shown ATPase activity in the presence of indicated concentrations of in Fig. 4G–J, osimertinib did not alter the phosphorylation nor osimertinib. As shown in Fig. 3C and D, ABCB1 ATPase activity total levels of EGFR, AKT, and ERK in KBv200 and S1-MI-80 cells was stimulated by osimertinib until it reached a plateau close to both of which expressed wild-type EGFR (Supplementary Fig. S1) 40 nmol/min/mg protein, and it remained steady at osimertinib in the concentration of reversing MDR. These results indicated concentration >1.3 mmol/L. On the other hand, a maximum that the blockade of EGFR, AKT, and ERK activity was unlikely to ABCG2 ATPase activity up to 60 nmol/min/mg protein was play a significant role in the sensitizing effect of osimertinib on the attained in the presence of 0.5 mmol/L osimertinib. It seems that ABCB1- and ABCG2-overexpressing cells. osimertinib stimulated the ATPase activity by interacting with the drug-substrate-binding sites on the transporters, possibly similar Osimertinib reversed MDR in ex vivo to other substrates. To explore whether osimertinib could reverse ABCB1-mediated MDR in ex vivo, bone marrow samples were collected from drug- Osimertinib inhibited the photolabeling of ABCB1 and resistant patients who were diagnosed as AML. Four patients were ABCG2 with [125I]-IAAP detected with high ABCB1 expression (Fig. 5A). We then exam- The photoaffinity analogue of prazosin, [125I]-IAAP, which is ined the effect of osimertinib on intracellular accumulation of transported by both ABCB1 and ABCG2, has been widely used Rho 123 in these high ABCB1-expressing primary leukemia blasts to determine the binding regions of ABCB1 and ABCG2 that using flow cytometric analysis. Our results showed that osimerti- interact with substrates and inhibitors (30). Therefore, to nib could increase the intracellular accumulation of Rho 123 investigate the interaction of osimertinib with the substrate- (Fig. 5B and C). In addition, we used the MTT assay to assess the binding sites of ABCB1 and ABCG2, crude membranes from sensitization effect of osimertinib in the clinical samples. As High Five insect cells expressing ABCB1 or ABCG2 were incu- shown in Fig. 5D, osimertinib at 0.4 mmol/L significantly sensi- bated with [125I]-IAAP and increasing concentration (0–12.5 tized all four samples to DOX treatment when comparing with the mmol/L) of osimertinib. The autoradiogram showed that osi- DOX-alone group (P < 0.05). These results suggested that osimertinib strongly inhibited the [125I]-IAAP photolabeling of mertinib was able to sensitize high ABCB1-expressing leukemia both ABCB1 and ABCG2 in a concentration-dependent manner cells to conventional chemotherapeutic agents, and it may be and displayed higher affinity to ABCG2 than to ABCB1 (Fig. 3E useful in combination regimen to combat ABCB1-mediated MDR and F). The concentration of osimertinib required for 50% in clinic. inhibition of photolabeling of ABCB1 and ABCG2 with [125I]- IAAP was approximately 12 mmol/L and 3 mmol/L, respectively. These led to a speculation that osimertinib, like [125I]-IAAP, Discussion might complete with substrates and incorporate into the sub- This study sought to utilize the combination of osimertinib strate-binding sites of ABCB1 and ABCG2. with conventional anticancer drugs to reverse ABCB1- or ABCG2-mediated MDR. It has been well known that ABC Osimertinib did not alter the expression levels or membrane- transporters–mediated MDR is a major obstacle to the success bound levels of ABCB1 and ABCG2 of chemotherapy. And the most widely studied ABCB1 and The reversal of ABCB1- and ABCG2-mediated MDR could be ABCG2 lead to MDR through pumping out substrate chemo- achieved by either inhibiting their function or downregulating their therapeutic agents with the energy from ATP hydrolysis. Luck- expression level. Therefore, the effect of osimertinib on the expres- ily, several TKIs, such as lapatinib, erlotinib, apatinib, and sion of ABCB1 and ABCG2 was detected by Western blot and RT- afatinib, were reported to inhibit the function of ABC trans- PCR assay, respectively (Fig. 4A–D). It turned out that osimertinib porters, and these discoveries of TKIs represent a new strategy to did not alter the protein or mRNA level of both transporters, even circumvent MDR (25–30). Osimertinib (AZD9291), a third-

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A KB KBv200

Blank Blank Vehicle 128 Vehicle 128 Vehicle 10 0.1 µmol/L Osimertinib 0.1 µmol/L Osimertinib 0.1 µmol/L Osimertinib 0.2 µmol/L Osimertinib 0.2 µmol/L Osimertinib 0.2 µmol/L Osimertinib µ µ µ 0.4 mol/L Osimertinib 0.4 mol/L Osimertinib 0.4 mol/L Osimertinib 8 µ 10 µmol/L Verapamil 10 µmol/L Verapamil 10 mol/L Verapamil

6 Events Events 4

Relative value Relative 2

0 0 0 100 101 102 103 104 105 100 101 102 103 104 105 KB KBv200

B DOX DOX Blank Blank Vehicle 128 Vehicle 128 Vehicle 20 0.1 µmol/L Osimertinib 0.1 µmol/L Osimertinib 0.1 µmol/L Osimertinib 0.2 µmol/L Osimertinib 0.2 µmol/L Osimertinib 0.2 µmol/L Osimertinib µ µ µ 0.4 mol/L Osimertinib 0.4 mol/L Osimertinib 0.4 mol/L Osimertinib µ 10 µmol/L Verapamil 10 µmol/L Verapamil 15 10 mol/L Verapamil

10 Events Events

Relative value Relative 5

0 0 0 100 101 102 103 104 105 100 101 102 103 104 105 KB KBv200 Rho 123 Rho 123

C S1 S1-MI-80 Vehicle 128 Blank 128 Blank 15 0.05 µmol/L Osimertinib Vehicle Vehicle µ 0.05 µmol/L Osimertinib 0.05 µmol/L Osimertinib 0.1 mol/L Osimertinib 0.1 µmol/L Osimertinib 0.1 µmol/L Osimertinib 0.2 µmol/L Osimertinib 0.2 µmol/L Osimertinib 0.2 µmol/L Osimertinib 2.5 µmol/L FTC µ µ 2.5 mol/L FTC 2.5 mol/L FTC 10 Events Events 5 Relative values Relative

0 0 0 100 101 102 103 104 105 100 101 102 103 104 105 S1 S1-MI-80

D DOX DOX Vehicle Blank Blank µ 128 Vehicle 128 Vehicle 30 0.05 mol/L Osimertinib 0.05 µmol/L Osimertinib 0.05 µmol/L Osimertinib 0.1 µmol/L Osimertinib 0.1 µmol/L Osimertinib 0.1 µmol/L Osimertinib 0.2 µmol/L Osimertinib 0.2 µmol/L Osimertinib 0.2 µmol/L Osimertinib 2.5 µmol/L FTC 2.5 µmol/L FTC 2.5 µmol/L FTC 20 Events Events 10 Relative values Relative

0 0 0 100 101 102 103 104 105 100 101 102 103 104 105 S1 S1-MI-80 Rho 123 Rho 123

Figure 2. Effect of osimertinib on the intracellular accumulation of DOX, Rho 123, and Hoechst 33342 in MDR cells and their parental sensitive cells. The intracellular accumulation of DOX (A and C) and Rho 123 (B and D) in KBv200, S1-MI-80 cells, and their parental sensitive KB, S1 cells was measured by ﬂow cytometric analysis, respectively. (Continued on the following page.)

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E H460 H460/MX20 Blank 426 Blank Vehicle 519 Vehicle Vehicle µ 0.05 µmol/L Osimertinib 0.05 µmol/L Osimertinib 2,500 0.05 mol/L Osimertinib 0.1 µmol/L Osimertinib 0.1 µmol/L Osimertinib 0.1 µmol/L Osimertinib 0.2 µmol/L Osimertinib 0.2 µmol/L Osimertinib 0.2 µmol/L Osimertinib 2.5 µmol/L FTC 2.5 µmol/L FTC 2,000 2.5 µmol/L FTC 1,500

1,000 Events Events

Relative value Relative 500

0 0 0 H460 H460/MX20 100 101 102 103 104 100 101 102 103 104 Hoechst 33342 Hoechst 33342

HEK293/Vector HEK293/ABCG2 F 487 Vehicle Blank Blank 0.05 µmol/L Osimertinib 391 Vehicle Vehicle 0.1 µmol/L Osimertinib µ 0.05 µmol/L Osimertinib 0.05 mol/L Osimertinib 0.2 µmol/L Osimertinib µ 0.1 µmol/L Osimertinib 0.1 mol/L Osimertinib 3,000 2.5 µmol/L FTC 0.2 µmol/L Osimertinib 0.2 µmol/L Osimertinib 2.5 µmol/L FTC 2.5 µmol/L FTC 2,000

Events Events 1,000 Relative value Relative

0 0 0 100 101 102 103 104 100 101 102 103 104 HEK293/Vector HEK293/ABCG2 Hoechst 33342 Hoechst 33342

Figure 2. (Continued. ) Meanwhile, the intracellular accumulation of Hoechst 33342 in H460 and ABCG2-overexpressing H460/MX20 cells (E) and HEK293/Vector and stable ABCG2-transfected HEK293/ABCG2 (wild-type) cells (F) also was measured by flow cytometric analysis. The results were quantified as fold change in fluorescence intensity compared with the paired MDR cells. They were calculated by dividing the fluorescence intensity of each sample with that of MDR cells treated with DOX, Rho 123, or Hoechst 33342 alone. Data represent the mean SD of at least three independent experiments. , P < 0.05; , P < 0.01 generation EGFR TKI, is potent for patients with non–small cell ters make use of the energy released from ATP hydrolysis by lung cancer who failed prior treatment with first- and second- their ATPase to pump their substrates out of cells. Therefore, the generation EGFR TKIs due to the resistance mutation rate of ATP hydrolysis is directly proportional to the transport EGFRT790M (49). Based on these, we hypothesized that osimer- activity of the transporters. Osimertinib stimulated the ATPase tinib might interact with ABC transporters and overcome MDR activity of ABCB1 and ABCG2 at low concentration, supporting as well as other TKIs. that osimertinib may be similar to the substrates and be a As expected, osimertinib significantly potentiated the efficiency competitive inhibitor of these transporters. Furthermore, the of the known ABCB1 and ABCG2 substrate chemotherapeutic interactions between osimertinib and ABCB1 and ABCG2 were agents in ABCB1- or ABCG2-overexpressing cells under the more confirmed by its competition with the [125I]-IAAP photoaffinity than 90% cell survival concentration of osimertinib. Meanwhile, labeling of the transporters. All of these are consistent with our osimertinib did not alter the sensitivity of the parental sensitive earlier findings with other TKIs (25–30). KB, MCF-7, S1, H460, or HEK293/Vector cells to chemothera- Because osimertinib could inhibit the transport function of peutic agents. Moreover, osimertinib also did not enhance the ABCB1 and ABCG2, we continue to verify whether osimertinib cytotoxicity of cisplatin (nonsubstrate of ABCB1 and ABCG2) in would influence their expression level. Osimertinib did not ABCB1- or ABCG2-overexpressing cells. These results ascertained alter the protein or mRNA level of ABCB1 and ABCG2 trans- the sensitization of the MDR cells by osimertinib in vitro.To porters under the more than 90% cell survival concentration. further investigate whether osimertinib could enhance the efficacy Moreover, osimertinib did not alter the membrane bound of of chemotherapeutic agents in vivo, we assessed this action with both ABCB1 and ABCG2 in KBv200andS1-MI-80cells.These KBv200 cell xenograft nude mice model. Osimertinib or paclitaxel indicated that neither the expression nor membrane location of alone did not significantly inhibit the growth of KBv200 cell ABCB1andABCG2wasalteredbyosimertinibatthedesigned xenografts compared with saline control, but osimertinib remark- concentrations. As an EGFR inhibitor, it has been reported that ably potentiated the antitumor activity of paclitaxel without osimertinib inhibits phosphorylation of EGFR, AKT, and ERK increasing toxicity. across cell lines harboring sensitizing or T790M resistance The reversal of ABCB1- and ABCG2-mediated drug resistance mutations (31). In addition, activation of PI3K/AKT and/or could be achieved by either antagonizing their function or ERK pathways is related to cancer cells acquired resistance to down regulating their expression level. And osimertinib was chemotherapy (9). In our study, osimertinib did not inhibit the found to increase the intracellular accumulation of DOX and phosphorylation of EGFR, AKT, or ERK in S1-MI-80 and Rho 123 in resistant cell lines by inhibiting the efflux function KBv200 cells by osimertinib under the more than 90% cell of ABCB1 and ABCG2 transporters. In addition, ABC transpor- survival concentration, indicating that the blockade of EGFR,

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A KB B S1 KB+0.4 µmol/L Osimertinib S1+0.2 µmol/L Osimertinib KBv200 S1-MI-80 120 KBv200+0.4 µmol/L Osimertinib 120 S1-MI-80+0.2 µmol/L Osimertinib

100 100

80 80

60 60

40 40

20 20 Rho 123 (% of initial) Rho 123 (% of initial) 0 0 0 15 30 45 60 75 90 0 15 30 45 60 75 90 Time/min Time/min

C D 60 80

60 40

ATPase activity ATPase 20 ATPase activity ATPase (nmol/min/mg protein) (nmol/min/mg protein) 0 ABCG2 Vanadate-sensitive 0 Vanadate-sensitive ABCB1 Vanadate-sensitive 0 1 2 3 4 5 0 1 2 3 4 5 Concentration of osimertinib (µmol/L) Concentration of osimertinib (µmol/L)

EFµmol/L µmol/L 3.13 6.25 12.5 0 0.39 1.56 3.13 6.25 12.5 0 0.100.20 0.39 0.78 1.56 120 0.10 0.20 0.78 120 kDa kDa 170 170 130 ABCB1 100 100 130 100 70 (170 kDa) 100 55 80 70 ABCG2 80 40 35 55 (72 kDa) 25 60 40 60 35 40 25 40 (% control value)

(% control value) 20 20 [125I]-IAAP incorporated [125I]-IAAP incorporated 0 0 0 20 40 60 0 20 40 60 [Osimertinib] µmol/L [Osimertinib] µmol/L

Figure 3. Effect of osimertinib on the efflux of Rho 123, the ATPase activity, and the [125I]-IAAP photoaffinity labeling of ABCB1 and ABCG2. A and B, the Rho 123 retention was measured by flow cytometry in KB, KBv200, S1, and S1-MI-80 cells, respectively. The cells were preincubated with 5 mmol/L of Rho 123 at 37C for 30 minutes and then incubated with osimertinib for indicated time points in Rho 123–free media. C and D, the vanadate-sensitive ABCB1 or ABCG2 ATPase activity was evaluated in the presence of the indicated concentrations of osimertinib. E and F, osimertinib competed for photolabeling of ABCB1 or ABCG2 by [125I]-IAAP. A representative autoradiogram was shown from three independent experiments. The relative amount of [125I]-IAAP incorporated was plotted against the concentration of osimertinib present. 100% incorporation refers to the absence of osimertinib. Data points represent the mean SD of at least three independent experiments. , P < 0.01.

AKT, and ERK activity was unlikely to play a significant role in Treatment failure in AML is frequently due to resistance to the sensitizing effect of osimertinib on the ABCB1- and ABCG2- chemotherapy by drug efflux via ABC proteins, specifically overexpressing cells. ABCB1. We found four of eleven samples collected from

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A 48 h 0.4 µmol/L 48 h 0.4 µmol/L Osimertinib (µmol/L)0 0.1 0.2 0.4 4.0 0 24 48 72 (h) (µmol/L) 0 0.1 0.2 0.4 4.0 0 24 48 72 (h) ABCB1 170 kDa ABCB1 265 bp 36 kDa GAPDH GAPDH 475 bp KBv200 KBv200

B 48 h 0.4 µmol/L 48 h 0.2 µmol/L Osimertinib (µmol/L) 0 0.05 0.1 0.2 2.0 0 24 48 72 (h) (µmol/L) 0 0.05 0.1 0.2 2.0 0 24 48 72 (h)

ABCG2 70 kDa ABCG2 235 bp

GAPDH 36 kDa GAPDH 475 bp S1-MI-80 S1-MI-80 C D 1.2 1.2

1.0 1.0

0.8 0.8

0.6 0.6

0.4 0.4 (relative values) (relative 0.2 values) (relative 0.2 ABCB1 mRNA level ABCG2 mRNA level 0.0 0.0 Figure 4. Osimertinib 0 0.1 0.2 0.4 4.0 (µmol/L) 0 24 48 72 (h) Osimertinib 0 0.05 0.1 0.2 2.0 (µmol/L) 0 24 48 72 (h) Effect of osimertinib on the expression of 48 h 0.4 µmol/L 48 h 0.2 µmol/L KBv200 S1-MI-80 ABCB1, ABCG2, and total levels or phosphorylation inhibition of EGFR, AKT, and IgG Control IgG F µ Control E Control Control 0.2 mol/L Osimertinib ERK. A and B, the protein and mRNA levels of µ 128 1.2 0.4 µmol/L Osimertinib 128 0.4 mol/L Osimertinib 1.2 0.4 µmol/L Osimertinib ABCB1 and ABCG2 were detected by Western 1.0 1.0 0.8 blot and RT-PCR assay, respectively. C and D, 0.8 0.6 real-time PCR assay was performed to quantify 0.6 Events

Events 0.4 the mRNA levels of ABCB1 and ABCG2 in the 0.4 expression level expression Relative ABCG2 Relative 0.2 expression level expression KBv200 or S1-MI-80 cells. E and F, the ABCB1 Relative 0.2 0 0.0 membrane-bound levels of ABCB1 or ABCG2 in 0 0.0 100 101 102 103 10 4 105 100 101 102 103 10 4 105 KBv200 or S1-MI-80 cells were detected by KBv200 KBv200 S1-MI-80 S1-MI-80 ﬂow cytometer, respectively. KB (G), KBv200 (H), S1 (I), and S1-MI-80 (J) cells were G 48 h 0.4 µmol/L H 48 h 0.4 µmol/L treated with osimertinib at indicated Osimertinib (µmol/L) 0 0.1 0.2 0.4 4.0 0 24 48 72 (h) (µmol/L) 0 0.1 0.2 0.4 4.0 0 24 48 72 (h) concentrations and designed time points. 170 kDa 4.0 mmol/L (in KB and KBv200 cells) or p-EGFR m 2.0 mol/L (in S1 and S1-MI-80 cells) of EGFR 170 kDa osimertinib was used as positive control for the blockage of EGFR, AKT, and ERK p-AKT 60 kDa phosphorylation. Representative results are shown from three independent experiments in AKT 60 kDa each panel. p-ERK 42/44 kDa

ERK 42/44 kDa

GAPDH 36 kDa

KB KBv200

IJ48 h 0.2 µmol/L 48 h 0.2 µmol/L Osimertinib (µmol/L) 0 0.05 0.1 0.2 2.0 0 24 48 72 (h) (µmol/L) 0 0.05 0.1 0.2 2.0 0 24 48 72 (h)

p-EGFR 170 kDa

EGFR 170 kDa

p-AKT 60 kDa

AKT 60 kDa

p-ERK 42/44 kDa

ERK 42/44 kDa

GAPDH 36 kDa

S1 S1-MI-80 drug-resistant patients who were diagnosed as acute leukemia patients, so there is slight difference in response to osimertinib with high ABCB1 expression. The expression of ABCB1 was sensitization. We found thatosimertinibcouldsigniﬁcantly slightly different in four bone marrow samples collected from increase the accumulation of Rho 123 and enhance the

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ABCD Blank Control 128 IgG 128 Control 0.4 µmol/L Osimertinib Anti-ABCB1 0.4 µmol/L Osimertinib 10 µmol/L Verapamil 120 DOX 10 µmol/L Verapamil 12 0.4 µmol/L Osimertinib 100 53.1% 10 10 µmol/L Verapamil 8 80

6 60 Events Events 4 40 Patient 1 Patient Relative values Relative 2 20 Survival (% of control) 0 0 0 0 100 101 102 103 10 4 105 100 101 102 103 10 4 105 0.01 0.1 1 10 Expression of ABCB1 Rho 123 accumulation Rho 123 accumulation Concentration of DOX (µmol/L)

Control Blank 0.4 µmol/L Osimertinib 128 128 Control IgG µ 10 µmol/L Verapamil Anti-ABCB1 0.4 mol/L Osimertinib 120 DOX 10 µmol/L Verapamil 6 0.4 µmol/L Osimertinib 100 26.1% 10 µmol/L Verapamil 4 80 60 Events Events 2 40 Patient 2 Patient

Relative values Relative 20 Survival (% of control) 0 0 0 0 0 1 2 3 4 5 0 1 2 3 4 5 10 10 10 10 10 10 10 10 10 10 10 10 0.01 0.1 1 10 Expression of ABCB1 Rho 123 accumulation Rho 123 accumulation Concentration of DOX (µmol/L)

Blank Control 128 IgG 128 Control µ 0.4 µmol/L Osimertinib 0.4 mol/L Osimertinib 120 DOX Anti-ABCB1 10 µmol/L Verapamil 12 10 µmol/L Verapamil 0.4 µmol/L Osimertinib 100 49.0% 10 10 µmol/L Verapamil 8 80

6 60 Events Events

Patient 3 Patient 4 40

Relative values Relative 2 20 Survival (% of control) 0 0 0 0 0 1 2 3 4 5 100 101 102 103 10 4 105 10 10 10 10 10 10 0.01 0.1 1 10 Expression of ABCB1 Rho 123 accumulation Rho 123 accumulation Concentration of DOX (µmol/L)

Blank 128 IgG 128 Control Control µ 0.4 µmol/L Osimertinib Anti-ABCB1 0.4 mol/L Osimertinib 12 120 DOX 10 µmol/L Verapamil 10 µmol/L Verapamil 0.4 µmol/L Osimertinib 100 47.4% 10 10 µmol/L Verapamil 8 80

6 60 Events Events

Patient 4 Patient 4 40 Relative values Relative 2 20 Survival (% of control) 0 0 0 0 0 1 2 3 4 5 0 1 2 3 4 5 10 10 10 10 10 10 10 10 10 10 10 10 0.01 0.1 1 10 Expression of ABCB1 Rho 123 accumulation Rho 123 accumulation Concentration of DOX (µmol/L)

Figure 5. Osimertinib increased the accumulation of Rho 123 and enhanced the cytotoxicity of DOX in ABCB1 high–expressing primary leukemia blasts. Bone marrow was collected from four newly diagnosed AML patients. A, the mononuclear cells were isolated and their expression levels of ABCB1 were determined by flow cytometry. B, intracellular accumulation of Rho 123 in primary leukemia blasts with or without osimertinib treatment was analyzed by flow cytometry. VRP, an ABCB1 inhibitor, was used as positive control. C, the results were calculated by dividing the fluorescence intensity of each sample with that of mononuclear cells treated with Rho 123 alone. D, enhanced cytotoxicity of DOX in ABCB1–high expressing primary leukemia blasts by osimertinib. Data represent the mean SD of at least three independent experiments. Representative results are shown in A and B. , P < 0.05; , P < 0.01.

cytotoxicity of DOX in these four high ABCB1-expressing pri- ventional chemotherapeutic agents in ABCB1- and ABCG2- mary leukemia blast cells. These results suggested that osimer- overexpressing cells. The reversal of MDR by osimertinib was tinib was able to sensitize high ABCB1-expressing leukemia mediated by inhibition of ABCB1/ABCG2 transport function, cells to conventional chemotherapeutic agents, and it may be resulting in an increase of intracellular accumulation substrate useful in combination regimen to combat ABCB1-mediated chemotherapeutic agents but not by downregulating their MDR in clinic. expression or altering membrane location. In addition, the In conclusion, we provide the ﬁrst in vitro, in vivo,andex vivo reversal of MDR by osimertinib was not associated with the evidence that osimertinib could enhance the efﬁcacy of con- blockage of the phosphorylation of EGFR, AKT, and ERK. Our

1856 Mol Cancer Ther; 15(8) August 2016 Molecular Cancer Therapeutics

Reversal of MDR by Osimertinib

results suggested that osimertinib could be used in combina- Administrative, technical, or material support (i.e., reporting or organizing tion therapy with conventional ABCB1 or ABCG2 substrate data, constructing databases): Y. Chen, F. Wang, X. Chen, L. Fu drugs to overcome MDR mediated by ABCB1 or ABCG2 in Study supervision: X. Zhang clinical practice. Acknowledgments The authors thank Dr. Susan Bates (NCI, NIH, Bethesda, MD) for the ABCG2- Disclosure of Potential Conﬂicts of Interest overexpressing cell lines. No potential conﬂicts of interest were disclosed. Grant Support Authors' Contributions All authors received grants from the Natural Science Foundation of China Conception and design: K.K.W. To, L. Fu (No. 81473233 and No. 81402503) and the Ph.D. Programs Foundation for Development of methodology: Y. Chen, X. Zhang, K.K.W. To, L. Fu young teachers of Ministry of Education of China (No. 20130171120108). Acquisition of data (provided animals, acquired and managed patients, The costs of publication of this article were defrayed in part by the provided facilities, etc.): Z. Chen, Y. Chen, M. Xu, L. Chen, K.K.W. To, payment of page charges. This article must therefore be hereby marked advertisement H. Zhao, Z. Xia, L. Fu in accordance with 18 U.S.C. Section 1734 solely to indicate Analysis and interpretation of data (e.g., statistical analysis, biostatistics, this fact. computational analysis): Z. Chen, Y. Chen, M. Xu, X. Zhang, K.K.W. To, L. Fu Writing, review, and/or revision of the manuscript: Z. Chen, M. Xu, Received December 3, 2015; revised March 30, 2016; accepted May 2, 2016; K.K.W. To, L. Fu published OnlineFirst May 18, 2016.

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1858 Mol Cancer Ther; 15(8) August 2016 Molecular Cancer Therapeutics

Osimertinib (AZD9291) Enhanced the Efficacy of Chemotherapeutic Agents in ABCB1- and ABCG2-Overexpressing Cells In Vitro, In Vivo , and Ex Vivo

Zhen Chen, Yifan Chen, Meng Xu, et al.

Mol Cancer Ther 2016;15:1845-1858. Published OnlineFirst May 18, 2016.

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