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AXL degradation in combination with EGFR-TKI can delay and overcome acquired resistance in human non-small cell lung cancer cells

AXL degradation in combination with EGFR-TKI can delay and overcome acquired resistance in human... Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) has been a major obstacle in the treatment of non-small cell lung cancer (NSCLC) patients. AXL has been reported to mediate EGFR-TKIs. Recently, third generation EGFR-TKI osimertinib has been approved and yet its acquired resistance mechanism is not clearly understood. We found that AXL is involved in both gefitinib and osimertinib resistance using in vitro and in vivo model. In addition, AXL overexpression was correlated with extended protein degradation rate. We demonstrate targeting AXL degradation is an alternative route to restore EGFR-TKIs sensitivity. We confirmed that the combination effect of YD, an AXL degrader, and EGFR-TKIs can delay or overcome EGFR-TKIs-driven resistance in EGFR-mutant NSCLC cells, xenograft tumors, and patient-derived xenograft (PDX) models. Therefore, combination of EGFR-TKI and AXL degrader is a potentially effective treatment strategy for overcoming and delaying acquired resistance in NSCLC. Introduction amplification . Although a number of agents have been Epidermal growth factor receptor (EGFR) mutation is suggested for development to target the L858R/T790M/ one of the major driver oncogenes in non-small cell lung C797S triple mutation of EGFR, alternative approaches to cancer (NSCLC) and most frequently found in Asian control resistance are needed and the use of drug com- 1–3 patients . Although the first generation of EGFR tyr- binations may benefit patients who do not respond to 7,8 osine kinase inhibitors (TKIs), such as gefitinib and current treatment . erlotinib, have led to improved prognoses for NSCLC AXL is a receptor tyrosine kinase that belongs to the patients, their long-term efficacy is questionable due to TAM family which consists of three members: Tyro3, the emergence of acquired resistance within a year of MERTK, and AXL . Dysregulation of TAM signaling has 4,5 treatment . Recently, a third generation EGFR-TKIs been reported to be associated with cancer, chronic osimertinib, a specific inhibitor of mutant EGFR, has been inflammation, and autoimmune disease .Among three approved for clinical use. However, several resistance TAM member, AXL, both growth arrest-specificgene6 mechanisms were subsequently identified from patients’ (GAS6)-dependent and (GAS6)-independent, can promote samples including C797S and L718Q EGFR mutations, many downstream signaling pathways and transcription SCLC transformation, HER2 amplification, and MET factors regulating cell survival, growth, EMT, metastasis, 11–13 and tumor microenvironment in cancer cells . Recently, AXL has been reported to play a role in drug resistance Correspondence: Sang Kook Lee ([email protected]) mechanisms for many anti-cancer drugs, as well as in College of Pharmacy, Natural Products Research Institute, Seoul National 14–17 ionizing radiation therapy for multiple cancers .AXL University, Seoul 08826, Korea Edited by S. Tait © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Kim et al. Cell Death and Disease (2019) 10:361 Page 2 of 12 receptor kinase inhibitors have shown profound effects in treated with indicated concentrations of gefitinib, osi- overcoming the acquired resistance to EGFR-TKIs in mertinib, or YD alone or in combination for 72 h. IC mesenchymal cancer cells, but their anti-proliferative effects values were calculated via non-linear regression analysis as a singleagent areverylimited . Since AXL is considered using TableCurve 2D v5.01 (Systat Software Inc., San Jose, as an attractive target to overcome the resistance to CA, USA). The combination effect was evaluated by the EGFR-TKIs, several AXL kinase inhibitors, antibody drug value of the combination index (CI) values which were conjugates and decoy receptors are currently under calculated as follows: CI = D /(D ) +D /(D ) .D and D 1 x 1 2 x 2 1 2 19–21 investigation in clinical trials for cancer treatment . are the concentrations of the combined test compounds In this study, we determined that activation of the AXL that achieve the expected effect, and (D ) and (D ) are x 1 x 2 in EGFR-TKIs resistant cells is associated with extended the concentrations that achieve similar effects when the protein degradation of AXL. We further demonstrated the test compounds are used alone. The CI values were combining YD, an AXL degrader, and EGFR-TKI resulted compared with the reference values reported by Chou . in overcoming resistance in EGFR-TKIs resistant NSCLC cells also delaying the emergence of resistance in EGFR- Gene knockdown using siRNA transfection TKI sensitive NSCLC cells using tumor xenograft, and siRNAs were purchased from Invitrogen. Negative PDX model. control siRNA from the same company was used for control. After seeding, cells were transfected with Materials and methods 100 pmol siRNA duplex for 24 h using Lipofectamine Chemicals and reagents RNAiMAX (Invitrogen, CA, USA) according to the Gefitinib (CAS No. HY-50895) and Osimertinib (AZD- manufacturer’s instructions. The coding strand for AXL 9291, CAS No. HY-15772) were purchased from Med- was as follows: sense CCA GCA CCU GUG GUC AUC ChemExpress (NJ, USA). Cycloheximide (CAS No. 66-81- UUA CCU U and antisense AAG GUA AGA UGA CCA 9) was purchased from A.G. Scientific (CA, USA). Yuan- CAG GUG CUG G. huadine (YD; purity >98.5%) was isolated from a CHCl - soluble fraction of the flowers of Daphne genkwa,as Western blotting analysis described previously . All chemicals were dissolved in The cells were lysed in 2× sample loading buffer DMSO for in vitro experiments. (250 mM Tris-HCl pH 6.8, 4% SDS, 10% glycerol, 0.006% Antibodies against C-terminal AXL (sc-1096), EGFR bromophenol blue, 2% β-mercaptoethanol, 50 mM (sc-03), p-ERK (sc-7383), ERK (sc-94), MET (sc-10), sodium fluoride, and 5 mM sodium orthovanadate). β-actin (sc-47778) were obtained from Santa Cruz Bio- Tumor tissues were collected in RIPA buffer (Thermo- technology (Santa Cruz, CA, USA). p-AXL (#5724), p- fisher, Rockford, IL, USA), and then further lysed with 2x EGFR (#2234), p-MET (#3077), p-Akt (#9271), Akt laemmli sample buffer with 2% β-mercaptoethanol (#9272), p-p70S6 Kinase (#9205), p70S6 Kinase (#9202), (Biorad). The collected samples were subjected to 6-12% p-SAPK/JNK (#9091), SAPK/JNK (#9252), and snail SDS-PAGE gel and transferred onto PVDF membranes (#3879) were obtained from Cell Signaling Technology (Millipore, Bedford, MA, USA). The membranes were (Danvers, MA, USA). blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature, and Cell culture then incubated with primary antibodies in 2.5% BSA in Human non-small lung cancer cells HCC827, HCC827- TBST overnight at 4 °C on a shaker. The membranes were gef, PC9, PC9-gef cells were a kind gift of Dr. Jae Cheol washed three times with TBST and incubated with the Lee and Dr. Jin Kyung Rho (Asan Medical Center, Seoul, secondary antibodies (HRP) (Younginfrontier, Seoul, Korea). HCC827-gef and PC9-gef cells were subcultured Korea) diluted in TBST for 2 h at room temperature. After in the presence of 1 µM gefitinib. Resistant cell line washing with TBST, the membranes were exposed to HCC827-osi was generated in vitro by culturing HCC827 enhanced chemiluminescence (ECL) solution (Intron, cells with escalating doses (0.001–0.5 μM) of osimertinib. Daejon, Korea). The chemiluminescence signals were All the cells were maintained in RPMI 1640 media sup- captured using LAS-4000 (Fuji Film Corp., Tokyo, Japan). plemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics-antimycotics (AA) (PSF; 100 units/mL peni- Real-time PCR analysis cillin G sodium, 100 μg/mL streptomycin, and 250 ng/mL The total RNA of the cells was isolated with TRI reagent amphotericin B). (Invitrogen, Grand Island, NY, USA). The isolated RNA (1 µg) was reverse-transcribed using ReverTra Ace qPCR Cell viability assay RT Master Mix (TOYOBO, Osaka, Japan) according to Cell viability was assayed by MTT method. Cells were the manufacturer’s instructions. Using synthesized cDNA, TM ® seeded in 96-well plates. On the next day, cells were Real-time PCR was conducted using iQ SYBR Green Official journal of the Cell Death Differentiation Association Kim et al. Cell Death and Disease (2019) 10:361 Page 3 of 12 Supermix (Bio-Rad, Hercules, CA, USA), according to the was approved by the institutional review board of Sever- manufacturer’s instructions. The comparative C method ance Hospital (4-2013-0526), and all patients provided was used to determine the relative expression normalized written informed consent. Tumors and paired peripheral by β-actin. The sequences of the primers are listed below. blood samples were consecutively collected for PDX AXL establishment and further genetic analysis. PDXs were (F) 5′-CGTAACCTCCACCTGGTCTC-3′; created using 6–8-week-old female severe combined immunodeficient (NOG) and nude (nu/nu) mice (R) 5′-TCCCATCGTCTGACAGCA-3′ GAS6 (OrientBio, Seoul, Korea). The tumors and related PDXs (F) 5′-CATCAACAAGTATGGGTCTCCGT-3′; were assigned Yonsei Human In Mouse (YHIM) identi- (R) 5′-GTTCTCCTGGCTGCATTCGTTGA-3′ fiers that corresponded to the original patient-derived β-actin tumors. Tumor dimensions were measured twice a week (F) 5′-AGCACAATGAAGATCAAGAT-3′; with a digital caliper and tumor volume was calculated as (R) 5′-TGTAACGCAACTAAGTCATA-3′ follows: (length × width )/2. Establishment of acquired gefitinib-resistant PDX tumors (YHIM-1009) and drug Immunocytochemistry administration was performed in Yonsei Cancer Center The cells were grown on a confocal dish pre-coated with and carried out as described previously . 0.2% gelatin. The cells were fixed with 4% paraformalde- hyde (in PBS) for 15 min and were blocked in 1% BSA (in Immunohistochemistry staining PBS containing 0.1% Triton X-100) for 30 min at room The tumors after the end of xenograft experiment were temperature. Cells were incubated with primary antibody excised, fixed in 4% paraformaldehyde (in PBS), and (AXL, 1:50) at 4 °C overnight and further incubated with embedded in paraffin. The embedded specimens were secondary antibody (anti-mouse Alexa 647, 1:250) for 2 h sectioned carefully, serially deparaffinized, rehydrated, at room temperature. The nuclei were stained with DAPI and subjected to antigen retrieval. Immunohistochemical (0.5 µg/ml). The images were detected using a confocal analysis of the tumor tissues was carried out as described microscope (Leica, TCS SP8). previously with the indicated antibodies . Tumor xenograft study Statistical analysis Balb/c-nu mouse (male, 4-weeks-old; OrientBio, Seoul, The data are presented as the means ± SD for the Korea) were allowed one-week acclimation prior to the minimum three independently performed experiments. 6 6 experiment. HCC827 (2 × 10 cells), HCC827-gef (4 × 10 The statistical significance (*P< 0.05, **P< 0.01, ***P< cells), or HCC827-osi (4 × 10 cells) cells were prepared in 0.005) were determined by ANOVA using Dunnett’s test. 100 µl PBS and mixed with the equal amount of Matrigel All statistical tests were two-sided. (Corning, Bedford, MA, USA) right before injecting sub- cutaneously into the flanks of the mice. When the tumor Results 3 3 volume reached 50 mm (HCC827) and 100 mm Differential sensitivity to osimertinib is observed in EGFR- (HCC827-Gef, HCC827-osi) on average, the mice were mutant acquired gefitinib resistant NSCLC cells randomized into the vehicle control and treatment groups To investigate the molecular mechanism of EGFR-TKIs (n = 5). Drugs were mixed with vehicle (EtOH:Tween80: resistance, we primarily evaluated the effects of gefitinib Saline solution 1:1:98). Each drug was administrated orally and osimertinib, the first and third generation of EGFR- once a day and 6 times per week for 22 days (HCC827-gef, TKIs, respectively, on growth of EGFR-mutant NSCLC HCC827-osi) and 90 days (HCC827). The body weight cell lines after 72 h of treatment (PC9, PC9-gef, HCC827, and tumor size were measured every 3–7 days. The tumor HCC827-gef, HCC827-osi) (Fig. 1a). While both HCC827 size was measured using a digital slide caliper and and PC9 cells harbor the EGFR Exon 19 deletion muta- volumes (mm ) were calculated as follows: (width × tion, HCC827 has been reported to have higher MET length × height) × π/6. The normalized tumor volume as expression while PC9 has higher EGFR and FGFR3 follows: (TV /TV ), where TV is the initial expression among NSCLC cell lines harboring mutant j,treated i,control i tumor volume of first administration, and TV is the EGFR status . Resistant cells were established through tumor volume of day j. Animals were sacrificed after the continuous exposure to gefitinib or osimertinib using a final drug administration and tumors were collected for dose-escalation procedure, and finally they exhibited at ex vivo analysis. least 100-fold greater IC than did the parental cells. We found that PC9 and HCC827 cells were initially very Patient-derived xenograft study sensitive to both gefitinib and osimertinib (Fig. 1b). Patient-derived tumor specimens were collected at Interestingly, two gefitinib-resistant cells showed different Yonsei University Severance Hospital. The study protocol sensitivity to osimertinib. Osimertinib effectively inhibited Official journal of the Cell Death Differentiation Association Kim et al. Cell Death and Disease (2019) 10:361 Page 4 of 12 A B C Fig. 1 Overexpression of AXL is observed in EGFR-TKI resistant NSCLC cells. NSCLC cells (PC9, PC9-gef, HCC827, HCC827-gef, and HCC827-osi) were treated with gefitinib or osimertinib for 72 h. Cell viability was measured by MTT assay as described in Methods (a). IC values and subculture conditions were stated (b). The protein expressions of major RTKs, p-AKT, and p-ERK were determined by Western blotting (c). Cells were incubated on the confocal dish for 24 h, fixed, and stained with C-term AXL (red) and DAPI (blue) for visualization of cellular localization (d). Data are presented as the mean fold changes ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test the growth of PC9-gef cells, but not HCC827-gef cells. compensating for EGFR inactivation. In addition, the Moreover, acquired osimertinib resistant cells developed levels of phosphorylated Akt and ERK were elevated from HCC827 also became intrinsically resistant to gefi- compared with those of HCC827 cells (Fig. 1c). The tinib. This suggests acquired resistant cells established expressions of AXL in HCC827, HCC827-gef and from HCC827 cells develop EGFR-independent resistance HCC827-osi cells were also evaluated by immunocy- mechanism. tochemistry staining of C-term AXL (Fig. 1d). AXL was clearly overexpressed in the resistant cells and mainly AXL is a major bypass pathway in osimertinib resistant localized in the cytosol. cells To examine the role of AXL in the resistant cells, the In order to investigate the mechanisms of acquired effects of AXL knockdown on the drug sensitivity of resistance of HCC827-gef and HCC827-osi cells, the EGFR-TKIs in HCC827-gef and HCC827-osi cells were expression levels of major receptor tyrosine kinases evaluated. The efficiency of siRNA was confirmed prior to (RTKs) and major downstream targets were evaluated by the experiments (Fig. 2a). AXL inhibition significantly Western blotting. The activation of phosphorylated Akt restored the sensitivity of gefitinib and osimertinib in and ERK has been reported to drive resistance and both HCC827-gef and HCC827-osi cells (Fig. 2b, c). 27,28 growth in tumor cells . In HCC827-gef and HCC827- Although AXL was overexpressed in PC9-gef cells, osi cells, both AXL and MET were activated in knockdown of AXL was less effective in restoring the Official journal of the Cell Death Differentiation Association Kim et al. Cell Death and Disease (2019) 10:361 Page 5 of 12 HCC827-gef AB 120 120 siNC siNC siAXL siAXL ** 100 100 *** HCC827-osi HCC827-gef *** *** *** *** *** 80 80 HCC827-osi HCC827-gef 1.2 60 60 1.2 ** 1.0 1.0 40 40 0.8 0.8 20 20 0.6 *** 0.6 0 0 0 0.04 0.2 1 5 0 0.04 0.2 1 5 0.4 0.4 Gefitinib (μM) Osimertinib (μM) *** 0.2 0.2 0.0 0.0 HCC827-osi siCTL siAxl siCTL siAxl siNC siAXL siNC siAXL 120 siNC kDa siNC kDa AXL 140 siAXL siAXL AXL 140 100 *** *** *** *** *** *** *** β-actin 42 β-actin 42 ** 0 0.04 0.2 1 5 0 0.04 0.2 1 5 Gefitinib (μM) Osimertinib (μM) HCC827 DE HCC827-gef Time (hr) 01248 16 20 24 Time (hr) 01248 16 20 24 CHX - + ++++++ CHX - + ++++++ kDa kDa AXL AXL 16 140 140 HCC827 HCC827 β-actin 42 β-actin 42 H HCC827-gef CC827-gef HCC827-osi HCC827-osi HCC827-osi Half-life of AXL after Cell lines CHX treatment (hr) * Time (hr) 01248 16 20 24 ** HCC827 1.8 CHX - +++++++ * kDa 2 AXL HCC827-gef > 24 0 AXL AXL GAS6 β-actin HCC827-osi > 24 Fig. 2 The total AXL protein degradation rate is extended in EGFR-TKI resistant cells. The efficiency of siAXL was confirmed using real-time PCR and Western blotting (a). The effects of gefitinib or osimertinib on the cell viability were evaluated after cells were transfected with siNC or siAXL compared with control (no transfection) in HCC827-gef cells (b) and HCC827-osi cells (c). Cycloheximide (CHX) was treated to stop the protein production and the protein expression of AXL (140 kda) was detected by Western blotting over the indicated time points. The expressions of AXL and β-actin were quantified by densitometry using ImageJ. AXL expressions were normalized to β-actin and further compared with 0 h expressions (d). mRNA expression of AXL and GAS6 were detected by real-time PCR using β-actin as a reference gene (e). Data are presented as the mean fold changes ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test gefitinib sensitivity (Supplementary Fig. S1a–c). Together, AXL than that of HCC827 parent cells (Fig. 2d). Western these results suggest AXL is the major bypass pathway blotting bands of AXL expression was normalized by and plays a significant role in HCC827-gef and HCC827- β-actin expression to quantify half-life. The half-life of osi cells which are resistant to both gefitinib and AXL protein was calculated to be 1.8 h in HCC827 cells, osimertinib. while it was longer than 24 h in the resistant cells. A ligand of AXL, GAS6 binds and activates AXL receptor Overexpression of AXL is associated with extended protein signaling thereby stimulating the cell proliferation .We degradation rate found that the gene copy number of AXL and its ligand To further assess the status of AXL in the resistant cells genes, GAS6, were increased in the resistant cells (Fig. 2e). compared with the parent cells, we measured the AXL AXL was found to be overexpressed in PC9-gef cells, so protein degradation rate using Western blotting analysis. we also compared the half-life of AXL in PC9 and PC9-gef The protein degradation rate of full-length AXL was cells. The overexpression of AXL was also correlated with measured after blocking new protein synthesis using extended degradation rate in PC9-gef cells (Supplemen- cycloheximide (CHX). Both HCC827-gef and HCC827- tary Fig. S1d). These results indicated that resistant cells osi cells displayed extended protein degradation rates of exhibit higher GAS6 and AXL expression and AXL Official journal of the Cell Death Differentiation Association Normalized mRNA Normalized mRNA expression of Axl expression of AXL Normalized mRNA Normalized mRNA expression of AXL expression of Axl Cell Viability (% of control) Cell Viability (% of control) Normalized mRNA expression Cell Viability (% of control) Cell Viability (% of control) Kim et al. Cell Death and Disease (2019) 10:361 Page 6 of 12 overexpression is closely associated with extended protein pathway were further examined in both sensitive and degradation rates. resistant cells. YD concentration-dependently inhibited full-length AXL and its downstream targets including YD inhibits cell proliferation and by AXL degradation in phosphorylated Akt and ERK without affecting the total resistant cells protein expression (Fig. 3d). Interestingly, YD suppressed YD, derived from the flowers of Daphne genkwa, inhi- the levels of phosphorylated MET and total MET expressions. Collectively, YD efficiently regulates AXL bits AXL expression by targeting protein degradation (Fig. 3a) . To access the effects of AXL degradation on degradation, MET, and their downstream signaling in cell viability, we examined the effects of YD in HCC827, both EGFR-TKI-sensitive and EGFR-TKI-resistant cells. HCC827-gef, and HCC827-osi cells. The treatment of YD for 72 h reduced cell viability of HCC827, HCC827-gef, YD resensitizes HCC827-gef and HCC827-osi cells to EGFR- and HCC827-osi with IC value of 7.06 nM, 2.92 nM, and TKIs 9.2 nM, respectively (Fig. 3b). Moreover, YD treatment AXL kinase inhibitors are reported to resensitize EGFR- accelerated the degradation of AXL protein of resistant TKIs resistant cells to EGFR-TKIs . We further evaluated cells. The calculated half-life of AXL protein after YD whether AXL degrader, YD, could resensitize resistant treatment was 1.2 h in HCC827-gef cells and 2.7 h in cells to gefitinib and osimertinib. Treatment of YD with HCC827-osi cells which became similar to that of parent gefitinib or osimertinib for 72 h inhibited cell growth cells (Fig. 3c). The effects of YD on the AXL signaling more than any single drug treatment (Fig. 4a left and B Fig. 3 YD can regulate the degradation of AXL and effectively inhibit the growth of both EGFR-TKI sensitive and resistant cells. Chemical structure of yuanhuadine (YD) (a). Indicated cell lines were treated with YD for 72 h and viability was measured. (b). The protein expression of AXL (140 kda) was detected after co-treatment of CHX and YD by Western blotting analysis over the indicated time points to test the effects of YD on the protein degradation (c). The expressions of AXL and β-actin were quantified by densitometry using ImageJ. AXL expressions were normalized to β-actin and further compared with 0 h expression (d). Cells were treated with indicated concentrations of YD for 24 h (e). Data are presented as the mean fold changes ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test Official journal of the Cell Death Differentiation Association Kim et al. Cell Death and Disease (2019) 10:361 Page 7 of 12 YD 0 nM YD 1 nM HCC827-gef YD (nM) YD 5 nM YD 10 nM 510 HCC827-gef HCC827-osi Gefitinib (μM) CI YD -+ -+ YD - + - + 1 0.81 0.87 Gefitinib - - + + kDa Osimertinib - - + + 5 0.67 0.46 p-AXL p-AXL 10 0.41 0.34 (Y702) (Y702) Gefitinib (µM) AXL AXL 140 YD 0 nM p-MET p-MET YD 1 nM (Y1234/1235) HCC827-osi YD (nM) (Y1234/1235) YD 5 nM YD 10 nM MET MET 145 Osimertinib p-Akt p-Akt (μM) CI (S473) (S473) 0.5 0.69 0.33 Akt Akt 1 0.67 0.46 5 0.54 0.50 p-p70S6 p-p70S6 70 Osimertinib (µM) p70S6 p70S6 70 p-ERK1/2 p-ERK1/2 HCC827-gef or HCC827-osi ERK1/2 ERK1/2 21 days (administration 6 days per week) 54 p-JNK p-JNK JNK JNK Vehicle YD 46 Vehicle YD Snail 29 Snail Gefitinib Combination Osimertinib Combination β-actin 42 β-actin 800 p-Akt/Akt p-Akt/Akt HCC827-gef HCC827-osi p-ERK/ERK p-ERK/ERK xenograft 700 xenograft p-p70S6/p70S6 p-p70S6/p70S6 1.4 1.4 p-JNK/JNK p-JNK/JNK 400 600 1.2 1.2 1.0 1.0 400 0.8 0.8 0.6 0.6 200 300 0.4 0.4 0.2 0.2 0.0 0.0 CTL YD Osi Combi CTL YD Gef Combi 0 7 14 21 0 7 14 21 Days of administration Days of administration Fig. 4 YD and EGFR-TKIs synergistically inhibit the growth of gefitinib and osimertinib cross-resistant cells in in vitro and in vivo. Cell viability was measured after combined treatment of YD and gefitinib or osimertinib for 72 h in HCC827-gef (a) and HCC827-osi (b), respectively. Based on the cell viability results, CI values were calculated to demonstrate the combination effects in each cell line. The effects of combination treatment compared with single treatment were accessed using western blot. Cells were treated with YD (10 nM) and/or gefitinib (1 μM) in HCC827-gef cells, and YD (10 nM) and/or osimertinib (500 nM) in HCC827-osi cells (c). HCC827-gef cells were subcutaneously implanted into the flanks of Balb/c-nude mice (n = 5). Mice were orally administered with compounds 6 times per week for 21 days and doses indicated are 1 mg/kg for YD, 10 mg/kg for gefitinib. HCC827-osi cells were subcutaneously implanted into the flanks of Balb/c-nude mice (n = 5). Mice were orally administered with compounds 6 times per week for 21 days and doses indicated are 0.5 mg/kg for YD, 5 mg/kg for osimertinib (d). Data are presented as the mean fold changes ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test left). These synergistic effects were evaluated by combi- strongly inhibited the expressions of p-Akt, p-ERK, p- nation index (CI) analysis using the Chou-Talalay p70S6, and p-JNK which regulate cell proliferation and method, which is based on the median-effect equation survival (Fig. 4c). Moreover, YD and combined treatment 23,32 and also provide algorithms for computer simulation inhibited snail expression, which is one of the major EMT (CI < 1: synergism, CI = 1: additive; CI > 1: antagonism). target genes regulated by AXL signaling . In search of The formula can be applied for any dose-effect analysis potential application of YD in a combination therapy, we and even suitable for small-scale experiments. Various also investigated the combined effects of YD and other doses of compounds were tested to accurately determine kinase inhibitors such as PHA-665752 (c-MET inhibitor), the effects of drug combinations. The calculated CI values SP600125 (JNK inhibitor), and LY294002 (PI3K inhibitor) were smaller than 1, which indicates synergism (Fig. 4a which were showed limited efficacy as a single agent in the right and b right). resistant cells. Combined treatment with YD resulted in Treatment with either gefitinib or osimertinib alone did significant suppression of cell viability and showed not have significant effects on the expression of AXL, synergism with all of three kinase inhibitors (Supple- MET, and downstream signaling in resistant cells. On the mentary Fig. S2). These results indicate targeting AXL other hand, the combination of YD and EGFR-TKIs degradation by YD can resensitize resistant cells not only Official journal of the Cell Death Differentiation Association Tumor volume (mm3) Cell Viability (% of control) Cell Viability (% of control) Tumor volume (mm ) Fold change compared to control cells Fold change compared to control cells Kim et al. Cell Death and Disease (2019) 10:361 Page 8 of 12 Acquired gefitinib resistant patient derived tumors (YHIM-1009) 31 days (administration 7 days per week) PDX model Vehicle YD Gefitinib Combination 500 24 4 8 12 16 20 24 28 31 4 8 12 16 20 24 28 31 Days of administration Days of administration Fig. 5 YD and EGFR-TKIs synergistically inhibit the growth in acquired gefitinib-resistant PDX models. When tumor volumes reached 200 to 250 mm3, mice engrafted with YHIM-1009 tumors were segregated into appropriate treatment groups (three to five mice per group). Mice were orally administered with compounds every day for 31 days and doses indicated are 1 mg/kg for YD, 10 mg/kg for gefitinib (a). *P < 0.05, **P < 0.01, ***P < 0.005 by t-test to EGFR-TKIs, but also potentially to c-MET inhibitor, activity of single agent administration and combination JNK inhibitor, or PI3K inhibitor. administration in acquired gefitinib-resistant patient derived xenograft (PDX) model. The third mouse gen- Combined administration of YD with EGFR-TKIs eration of YHIM-1009 (EGFR 19del mutation/ PIK3CA synergistically inhibits tumor growth in xenograft model E542K) tumor model, an acquired-gefitinib resistant bearing HCC827-gef and HCC827-osi cells tumor, was used for drug efficacy testing. The previous To further confirm the synergistic activity of YD and study demonstrated that YHIM-1009 did not show EGFR-TKIs using in vivo model, we examined their effi- response to both gefitinib (25 mg/kg) and osimertinib cacy in a xenograft model engrafted with HCC827-gef and (6.25 mg/kg) administration . When tumor volumes HCC827-osi cells. The drugs were orally administered reached 200 to 250 mm , animals were administrated with once a day, 6 days per week, for 3 weeks. Resistant cells vehicle, YD (1 mg/kg), gefitinib (10 mg/kg), or YD and developed in vitro were implanted in the right flank of gefitinib every day for 31 days. Combined administration nude mice. After one week, mice were randomly assigned of YD and gefitinib notably inhibited the tumor growth to each group. Consistent with in vitro data, the combi- derived from acquired gefitinib-resistant patient tumor nation administration effectively inhibited the tumor more efficiently than a single treatment (Fig. 5a). Body- group in both HCC827-gef and HCC827-osi xenograft weight was measured to monitor the toxicity, and there tumor model (Fig. 4d). YD as a single treatment group was no significant change in bodyweight for all groups. resulted in tumor-regression after 14 days in HCC827-gef Unlike the results of HCC827-gef xenograft model, YD tumor model and 17 days in HCC827-osi tumor model. single administration resulted in only slight inhibition of Collectively, the combination of YD and EGFR-TKIs tumor growth. These results indicate that YD potentially shows potential for both inhibitions of in vitro cell pro- more useful for combination therapy than as a single liferation and in vivo tumor growth via AXL degradation. agent. Combined administration of YD with gefitinib Combined administration of YD with gefitinib delays the synergistically inhibits tumor growth of gefitinib-resistant emergence of resistance in long-term xenograft model PDX model bearing HCC827 cells In order to assess the combination effects of YD and Because AXL is regarded as an acquired resistance gefitinib in more clinically relevant setting, we compared mechanism to EGFR-TKIs, we hypothesized that AXL Official journal of the Cell Death Differentiation Association Tumor volume (mm ) Body weight (g) Kim et al. Cell Death and Disease (2019) 10:361 Page 9 of 12 Vehicle YD A B Vehicle YD Gefitinib Combination Gefitinib Combination 2400 600 2000 500 1600 400 1200 300 1000 8 800 200 *** 11 46 60 90 *** 400 100 Days of administration *** Combi- Day Vehicle YD Gefitinib nation 0 10 20 30 40 50 60 0 15 30 45 60 75 90 11 7.3 4.0 1.3 0.9 Days of administration 46 27.6 9.0 2.8 1.0 60 32.6 9.5 5.3 1.6 YD -+ -+ 90 42.4 10.6 12.5 8.1 Gefinib -- + + Vehicle YD AXL Gefitinib Combination Fig. 6 The combined administration of YD and gefitinib can delay the emergence of resistance and inhibit AXL overexpression in a long- term in vivo xenograft model. HCC827-Luc cells were subcutaneously implanted into the flanks of Balb/c-nude mice (five mice per group). Drugs were orally administered 6 times per week for 90 days and doses indicated are 0.5 mg/kg for YD, 10 mg/kg for gefitinib (a). The graph represents tumor volumes of indicated days which were normalized to the initial tumor volume for comparison (b). Bioluminescence images of mice at the final day before sacrifice were measured and mice were injected with Firefly D-luciferin before imaging (c). Immunohistochemical analysis of AXL was performed in tumor tissue (d). The schematic diagram illustrating the significance of combined YD with EGFR-TKI treatment in NSCLC (e). *P < 0.05, **P < 0.01, ***P < 0.005 by t-test inhibition with EGFR-TKIs from the beginning of treat- the initial size and started to grow rapidly after 35 days of ment would prevent the emergence of acquired resistance, drug administration (Fig. 6a right). The tumor volumes on enhance the therapeutic efficacy, and extend drug day 11, 46, 60, and 90 were normalized to the initial tumor response time. Therefore, we performed a long-term volume before the administration to quantify the effects (90 days) xenograft experiment using a xenograft model (Fig. 6b). Tumors in the combination group later started implanted with EGFR-TKI-sensitive HCC827 cells (Fig. 6a to grow after day 60, but their final volumes were still left). Not every vehicle group survived throughout the smaller than those of the YD group or gefitinib group long-term experiment due to the tumor burden, so (Fig. 6c). Although tumor size in the vehicle-administered remaining mice were still used to record tumor size and group was much larger, AXL expression was undetected body weights. We observed no change in body weights in in immunohistochemistry staining. These data confirm combination group and gefitinib group, while the body that AXL increases in response to the long-term admin- weight was only slightly reduced in YD group (data not istration of gefitinib regardless of tumor size (Fig. 6d). shown). Combination of YD and gefitinib both almost Combined treatment with YD and gefitinib was not completely prevented the tumor growth for 60 days, while resulted in the AXL expression in the tumors. However, the tumors of the gefitinib administration group exceeded the tumor sizes of the combination treatment group also Official journal of the Cell Death Differentiation Association Tumor Volume ( Tumor Volume ( Normalized tumor volume Kim et al. Cell Death and Disease (2019) 10:361 Page 10 of 12 increased at later points in time, suggesting that tumors without toxicity . Therefore, N-terminal fragment gen- may have acquired resistance by additional mechanisms. erated by AXL may prevent binding of GAS6 and AXL Further studies are needed to investigate the mechanism receptor thereby enhancing the AXL blockade activity. involved with resistance to the combination treatment of YD inhibited AXL, as well as MET protein expression YD and gefitinib. Collectively, these results suggest that which resulted in more sustained inhibition of down- using a combined treatment as a first-line therapy may stream signaling pathway. Previous study also reported delay the emergence of acquired-resistance and as a that tumor suppressor OPCML inactivates AXL- second-line therapy may overcome resistance (Fig. 6e). dependent oncogenic signaling in ovarian cancer and also prevent transactivation of other RTKs such as EGFR Discussion and cMET . Although the mutation and activation of EGFR are It is well reported that AXL inhibition using kinase recognized as important oncogenic drivers in NSCLC inhibitors can sensitize or synergistically work with sev- patients, targeting EGFR using currently existing EGFR- eral drugs such as antimitotic drugs, erlotinib, and ima- 8,17,19 TKIs has shown limited success due to the emergence of tinib . In addition, several studies suggested that acquired resistance . Osimertinib, a third-generation there are crosstalks between AXL and other RTKs; (1) TKIs, has been developed to treat mutant EGFR or/and AXL clusters together with other cell surface RTKs in the T790M-positive NSCLC patients and also has been mesenchymal cells which leads to crosstalk-amplified reported to have superior efficacy as first-line treatment . signaling network and a limited response to single TKIs A recent study showed the activation of AXL as an treatment . (2) When head and neck and esophageal adaptive response to osimertinib, and SPRY4 as a poten- squamous cell carcinomas become resistant to PI3Kα tial negative regulator of AXL signaling in lung cancer . inhibition, AXL dimerizes with EGFR and activates However, little has been studied about the roles of over- EGFR/PKC/mTOR pathway . (3) AXL not only forms a expressed AXL in acquired resistance mechanisms against complex with EGFR but also may promote nuclear 47,48 osimertinib. Our study report that extended degradation translocation of the EGFR . (4) MGCD265, which is a rate of AXL is pivotal in the development of resistance to multi-targeted TKIs targeting MET, AXL, and PDGFR, gefitinib, osimertinib; thus, we investigated therapeutic completely inhibited the growth in only tumors with high approaches using drug combination to overcome resis- MET gene copy gains, and combination of MGCD265 and tance by targeting AXL degradation. erlotinib effectively inhibited the tumor growth in most of Targeting protein degradation may have an advantage triple breast cancer cells and tumors . (5) AXL kinase over kinase inhibition in the regulation of cancer cell inhibitor (R428) suppressed EMT-induced metastasis and growth; thus the efficacy of drugs with proteolysis- combined treatment enhanced the effects of lapatinib or targeting chimera (PROTAC) technology is under inves- trastuzumab in HER2-overexpressed breast cancer PDX 37,38 50 tigation . Several studies with EGFR, HER2, and c- model . These studies support combination treatment MET inhibitors have confirmed that targeted protein ought to be superior than single drug treatment. degradation may have an advantage because it yields more In this study, we confirmed that AXL protein degrada- sustained loss of functions and prevents kinome re- tion with EGFR-TKIs synergistically suppresses the wiring . Along these lines, we utilized the natural anti- growth of EGFR mutant cells and tumors. The effects of tumor compound YD to target AXL degradation which combined administration were very significant in PDX exhibits anti-proliferative activities against various human model. The tumor model used in PDX harbors EGFR lung cancer cells by regulating AXL, SerpinB2, NNMT, mutation, as well as PI3KA mutation, which may explain 22,39–42 and BMP4 . YD exhibits nanomolar IC in NSCLC results of inefficient YD single administration group. cell lines which is much lower than currently existing Nonetheless, the combined administration showed very AXL kinase inhibitors based on their reported IC potent tumor growth inhibition. As previously reported, value . tumor tissues obtained from gefitinib-administrated We previously found that YD cleaves AXL independent group in a long-term xenograft study with HCC827 cells of kinase, metalloprotease, proteasome and lysosome showed significantly higher AXL expression from the 30 17 activities . Treatment of YD can release N-terminal into immunohistochemistry results . Although tumor size in the culture medium and sequentially generate intracel- the vehicle-administered group was much bigger in long- lular domain thereby degrading the full-length AXL. term xenograft, the AXL expression was negligible. These When N-terminal of AXL is cleaved, it may work as a findings suggest that AXL is upregulated in response to decoy receptor to inhibit the binding of GAS6 and AXL the long-term administration of gefitinib, regardless of receptor. For example, an engineered AXL decoy recep- tumor size, and affect sensitivity of tumors to EGFR-TKIs. tor, which antagonizes the GAS6/AXL system by cap- When administrated in combination of YD and gefitinib, turing GAS6, can inhibit cancer cells and tumor growth AXL was not detected in the tumors from the Official journal of the Cell Death Differentiation Association Kim et al. Cell Death and Disease (2019) 10:361 Page 11 of 12 immunohistochemistry results. These results indicate that 9. Axelrod, H. & Pienta, K. J. Axl as a mediator of cellular growth and survival. Oncotarget 5,1–35 (2014). YD completely suppressed the AXL activation and was 10. Lemke, G. Biology of the TAM receptors. CSH Perspect. Biol. 5, a009076 (2013). able to delay the tumor regrowth. 11. Ammoun, S. et al. Axl/Gas6/NFkappaB signalling in schwannoma pathological In conclusion, we have elucidated AXL as a driver of proliferation, adhesion and survival. Oncogene 33,336–346 (2014). 12. Antony, J., Huang,R.Y.J.&AXL-Driven,E.M.T.State as a targetableconduit in cross-resistance to gefitinib and osimertinib and demon- cancer. Cancer Res. 77, 3725–3732 (2017). strated the efficacy of combining YD with gefitinib and/or 13. Antony, J. et al. The GAS6-AXL signaling network is a mesenchymal (Mes) molecular subtype-specific therapeutic target for ovarian cancer. Sci. Signal. 9, osimertinib in overcoming EGFR-TKI resistance in cells, ra97 (2016). resistant tumor xenograft and PDX model, and in delaying 14. Aguilera, T. A. et al. Reprogramming the immunological microenvironment the emergence of acquired resistance. These results sug- through radiation and targeting Axl. Nat. Commun. 7, 13898 (2016). gest combination use of YD and EGFR-TKIs may repre- 15. Brand, T. M. et al. AXL mediates resistance to cetuximab therapy. Cancer Res. 74,5152–5164 (2014). sent a potential therapeutic strategy for NSCLC patients. 16. Muller, J. et al. Low MITF/AXL ratio predicts early resistance to multiple tar- geted drugs in melanoma. Nat. Commun. 5, 5712 (2014). Acknowledgements 17. Zhang,Z.F.etal. Activation ofthe AXL kinase causes resistance to EGFR- The authors thank Byoung Chul Cho (JE-UK Institute for Cancer Research, JEUK targeted therapy in lung cancer. Nat. Genet. 44, 852 (2012). Co. Ltd., Gumi-City, Kyungbuk, Republic of Korea /Division of Medical 18. Wilson, C. et al. AXL inhibition sensitizes mesenchymal cancer cells to anti- Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei mitotic drugs. Cancer Res. 74,5878–5890 (2014). University College of Medicine, Seoul, Republic of Korea) for carrying out the 19. Ben-Batalla, I. et al. Axl blockade by BGB324 inhibits BCR-ABL tyrosine kinase PDX study. This research was supported by a National Research Foundation of inhibitor-sensitive and -resistant chronic myeloid leukemia. Clinical Cancer Res. Korea (NRF) grant funded by the Korean Government (MSIP) 23,2289–2300 (2017). (2016R1C1B1008540 and 2016M3A9B6903499). 20. Boshuizen, J. et al. Cooperative targeting of melanoma heterogeneity with an AXL antibody-drug conjugate and BRAF/MEK inhibitors. Nat. Med. 24,203 Data availability and materials (2018). Please contact authors for data request. 21. Kariolis, M. S. et al. An engineered Axl ‘decoy receptor’ effectively silences the Gas6-Axl signaling axis. Nat. Chem. Biol. 10,977–983 (2014). Conflict of interest 22. Hong, J. Y., Nam,J.W., Seo, E. K. &Lee,S.K.Daphnanediterpene esters with The authors declare that they have no competing interest. anti-proliferative activities against human lung cancer cells from Daphne genkwa. Chem. Pharm. Bull. 58,234–237 (2010). Ethics approval and consent to participate 23. Chou, T. C. Theoretical basis, experimental design, and computerized simu- In vivo tumor xenograft experiments followed the guidelines of and approved lation of synergism and antagonism in drug combination studies. Pharmacol. by Institute of Laboratory Animal Resources of Seoul National University (SNU- Rev. 58,621–681 (2006). 161108-2). 24. Kang, H. N. et al. Establishment of a platform of non-small-cell lung cancer patient-derived xenografts with clinical and genomic annotation. Lung Cancer 124,168–178 (2018). 25. Kim, W. K. et al. Esculetin suppresses tumor growth and metastasis by tar- Publisher’s note geting Axin2/E-cadherin axis in colorectal cancer. Biochem. Pharmacol. 152, Springer Nature remains neutral with regard to jurisdictional claims in 71–83 (2018). published maps and institutional affiliations. 26. Karachaliou, N. et al. Common co-activation of AXL and CDCP1 in EGFR- mutation-positive non-smallcell lung cancer associated with poor prognosis. Supplementary Information accompanies this paper at (https://doi.org/ Ebiomedicine 29,112–127 (2018). 10.1038/s41419-019-1601-6). 27. Jacobsen, K. et al. Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer. Nat. Commun. 8, 410 (2017). Received: 13 February 2019 Revised: 25 March 2019 Accepted: 17 April 28. Normanno, N. et al. The MEK/MAPK pathway is involved in the resistance of breast cancer cells to the EGFR tyrosine kinase inhibitor gefitinib. J. Cell Physiol. 207,420–427 (2006). 29. Varnum, B. C. et al. Axl receptor tyrosine kinase stimulated by the vitamin K- dependent protein encoded by growth-arrest-specificgene6. Nature 373, 623–626 (1995). References 30. Bae,S.Y., Hong,J.Y., Lee, H. J.,Park, H. J. &Lee,S.K.Targeting thedegradation 1. Kim, T. Y.,Han,S.W.&Bang,Y.J.Chasing targetsfor EGFR tyrosine kinase of AXL receptor tyrosine kinase to overcome resistance in gefitinib-resistant inhibitors in non-small-cell lung cancer: Asian perspectives. Exp. Rev. Mol. non-small cell lung cancer. Oncotarget 6,10146–10160 (2015). Diagnost. 7,821–836 (2007). 31. Giles, K. M. et al. Axl mediates acquired resistance of head and neck cancer 2. Liam, C. K., Pang, Y. K. & Poh, M. E. EGFR mutations in Asian Patients with cells to the epidermal growth factor receptor inhibitor erlotinib. Mol. Cancer advanced lung adenocarcinoma. J. Thorac. Oncol. 9,E70–E71 (2014). Ther. 12, 2541–2558 (2013). 3. Min, H. Y. & Lee, H. Y. Oncogene-driven metabolic alterations in cancer. Biomol. 32. Chou, T. C. Drug combination studies and their synergy quantification using Ther. 26,45–56 (2018). the Chou-Talalay method. Cancer Res. 70,440–446 (2010). 4. Soh, J. et al.EGFRmutationstatusin pleural fluid predicts tumor respon- 33. Asiedu, M. K. et al. AXL induces epithelial-to-mesenchymal transition and siveness and resistance to gefitinib. Lung Cancer 56,445–448 (2007). regulates the function of breast cancer stem cells. Oncogene 33,1316–1324 5. Uramoto, H. et al. Resistance to gefitinib. Int. J. Clin. Oncol. 11,487–491 (2006). (2014). 6. Yang, Z. et al. Investigating novel resistance mechanisms to third-generation 34. Wu, S. G. & Shih, J. Y. Management of acquired resistance to EGFR TKI-targeted EGFR tyrosine kinase inhibitor osimertinib in non-small cell lung cancer therapy in advanced non-small cell lung cancer. Mol. Cancer 17,38 (2018). patients. Clin. Cancer Res. 24,3097–3107 (2018). 35. Passiglia, F., Raez,L.E.& Rolfo, C. Moving osimertinibto first-line: the right 7. Zhang, H. et al. Discovery of 2,4,6-trisubstitued pyrido[3,4-d]pyrimidine deri- “strategy” in the chessboard of epidermal growth factor receptor-mutated vativesasnew EGFR-TKIs. Eur. J. Med. Chem. 148,221–237 (2018). non-small cell lung cancer? J. Thorac Dis. 10, S1076–S1080 (2018). 8. Gunther, M. et al. Trisubstituted pyridinylimidazoles as potent inhibitors of the 36. Taniguchi, H. et al. AXL confers intrinsic resistance to osimertinib and advances clinically resistant L858R/T790M/C797S EGFR mutant: targeting of both the emergence of tolerant cells. Nat. Commun. 10, 259 (2019). hydrophobic regions and the phosphate binding site. J. Med. Chem. 60, 37. Burslem, G. M. et al. The advantages of targeted protein degradation over 5613–5637 (2017). inhibition: an RTK case study. Cell Chem. Biol. 25,67–77 e63 (2018). Official journal of the Cell Death Differentiation Association Kim et al. Cell Death and Disease (2019) 10:361 Page 12 of 12 38. Raina,K.etal. PROTAC-induced BET protein degradation as a therapy for 44. Antony, J. et al. The tumour suppressor OPCML promotes AXL inactivation by castration-resistant prostate cancer. Proc. Natl Acad. Sci. USA 113, 7124–7129 the phosphatase PTPRG in ovarian cancer. EMBO Rep. 19, e45670 (2018). (2016). 45. Halmos, B. & Haura, E. B. New twists in the AXL(e) of tumor progression. Sci. 39. Bach, D. H. et al. Targeting nicotinamide N-Methyltransferase and miR-449a in Signal. 9,fs14 (2016). EGFR-TKI-resistant non-small-cell lung cancer cells. Mol. Ther. Nucleic Acids 11, 46. Elkabets, M. et al. AXL mediates resistance to PI3Kalpha inhibition by activating 455–467 (2018). the EGFR/PKC/mTOR axis in head and neck and esophageal squamous cell 40. Bae, S.Y., Park,H.J., Hong,J.Y., Lee, H. J. &Lee,S.K. Down-regulation of carcinomas. Cancer Cell 27,533–546 (2015). SerpinB2 is associated with gefitinib resistance in non-small cell lung cancer 47. Meyer, A. S., Miller, M. A., Gertler, F. B. & Lauffenburger, D. A. The receptor AXL and enhances invadopodia-like structure protrusions. Sci. Rep-Uk 6, 32258 diversifies EGFR signaling and limits the response to EGFR-targeted inhibitors (2016). in triple-negative breast cancer cells. Sci. Signal. 6, ra66 (2013). 41. Bach, D. H. et al. BMP4 upregulation is associated with acquired drug resis- 48. Brand, T. M. et al. The receptor tyrosine kinase AXL mediates nuclear trans- tance and fatty acid metabolism in EGFR-mutant non-small-cell lung cancer location of the epidermal growth factor receptor. Sci. Signal. 10,eaag1064 cells. Mol. Ther. Nucleic Acids 12,817–828 (2018). (2017). 42. Jo, S. K.,Hong, J. Y., Park, H. J. &Lee,S.K.Anticanceractivityofnovel daphnane 49. Linklater, E. S. et al. Targeting MET and EGFR crosstalk signaling in triple- diterpenoids from Daphne genkwa through cell-cycle arrest and suppression negative breast cancers. Oncotarget 7, 69903–69915 (2016). of Akt/STAT/Src signalings in human lung cancer cells. Biomol. Ther. 20, 50. Goyette,M.A.etal. Thereceptortyrosine kinase AXL is required at multiple 513–519 (2012). steps of the metastatic cascade during HER2-positive breast cancer progres- 43. Kariolis, M. S. et al. Inhibition of the GAS6/AXL pathway augments the efficacy sion. Cell Rep. 23,1476–1490 (2018). of chemotherapies. J. Clin. Invest. 127,183–198 (2017). Official journal of the Cell Death Differentiation Association http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Death & Disease Springer Journals

AXL degradation in combination with EGFR-TKI can delay and overcome acquired resistance in human non-small cell lung cancer cells

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Life Sciences; Life Sciences, general; Biochemistry, general; Cell Biology; Immunology; Cell Culture; Antibodies
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Abstract

Acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) has been a major obstacle in the treatment of non-small cell lung cancer (NSCLC) patients. AXL has been reported to mediate EGFR-TKIs. Recently, third generation EGFR-TKI osimertinib has been approved and yet its acquired resistance mechanism is not clearly understood. We found that AXL is involved in both gefitinib and osimertinib resistance using in vitro and in vivo model. In addition, AXL overexpression was correlated with extended protein degradation rate. We demonstrate targeting AXL degradation is an alternative route to restore EGFR-TKIs sensitivity. We confirmed that the combination effect of YD, an AXL degrader, and EGFR-TKIs can delay or overcome EGFR-TKIs-driven resistance in EGFR-mutant NSCLC cells, xenograft tumors, and patient-derived xenograft (PDX) models. Therefore, combination of EGFR-TKI and AXL degrader is a potentially effective treatment strategy for overcoming and delaying acquired resistance in NSCLC. Introduction amplification . Although a number of agents have been Epidermal growth factor receptor (EGFR) mutation is suggested for development to target the L858R/T790M/ one of the major driver oncogenes in non-small cell lung C797S triple mutation of EGFR, alternative approaches to cancer (NSCLC) and most frequently found in Asian control resistance are needed and the use of drug com- 1–3 patients . Although the first generation of EGFR tyr- binations may benefit patients who do not respond to 7,8 osine kinase inhibitors (TKIs), such as gefitinib and current treatment . erlotinib, have led to improved prognoses for NSCLC AXL is a receptor tyrosine kinase that belongs to the patients, their long-term efficacy is questionable due to TAM family which consists of three members: Tyro3, the emergence of acquired resistance within a year of MERTK, and AXL . Dysregulation of TAM signaling has 4,5 treatment . Recently, a third generation EGFR-TKIs been reported to be associated with cancer, chronic osimertinib, a specific inhibitor of mutant EGFR, has been inflammation, and autoimmune disease .Among three approved for clinical use. However, several resistance TAM member, AXL, both growth arrest-specificgene6 mechanisms were subsequently identified from patients’ (GAS6)-dependent and (GAS6)-independent, can promote samples including C797S and L718Q EGFR mutations, many downstream signaling pathways and transcription SCLC transformation, HER2 amplification, and MET factors regulating cell survival, growth, EMT, metastasis, 11–13 and tumor microenvironment in cancer cells . Recently, AXL has been reported to play a role in drug resistance Correspondence: Sang Kook Lee ([email protected]) mechanisms for many anti-cancer drugs, as well as in College of Pharmacy, Natural Products Research Institute, Seoul National 14–17 ionizing radiation therapy for multiple cancers .AXL University, Seoul 08826, Korea Edited by S. Tait © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to theCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Kim et al. Cell Death and Disease (2019) 10:361 Page 2 of 12 receptor kinase inhibitors have shown profound effects in treated with indicated concentrations of gefitinib, osi- overcoming the acquired resistance to EGFR-TKIs in mertinib, or YD alone or in combination for 72 h. IC mesenchymal cancer cells, but their anti-proliferative effects values were calculated via non-linear regression analysis as a singleagent areverylimited . Since AXL is considered using TableCurve 2D v5.01 (Systat Software Inc., San Jose, as an attractive target to overcome the resistance to CA, USA). The combination effect was evaluated by the EGFR-TKIs, several AXL kinase inhibitors, antibody drug value of the combination index (CI) values which were conjugates and decoy receptors are currently under calculated as follows: CI = D /(D ) +D /(D ) .D and D 1 x 1 2 x 2 1 2 19–21 investigation in clinical trials for cancer treatment . are the concentrations of the combined test compounds In this study, we determined that activation of the AXL that achieve the expected effect, and (D ) and (D ) are x 1 x 2 in EGFR-TKIs resistant cells is associated with extended the concentrations that achieve similar effects when the protein degradation of AXL. We further demonstrated the test compounds are used alone. The CI values were combining YD, an AXL degrader, and EGFR-TKI resulted compared with the reference values reported by Chou . in overcoming resistance in EGFR-TKIs resistant NSCLC cells also delaying the emergence of resistance in EGFR- Gene knockdown using siRNA transfection TKI sensitive NSCLC cells using tumor xenograft, and siRNAs were purchased from Invitrogen. Negative PDX model. control siRNA from the same company was used for control. After seeding, cells were transfected with Materials and methods 100 pmol siRNA duplex for 24 h using Lipofectamine Chemicals and reagents RNAiMAX (Invitrogen, CA, USA) according to the Gefitinib (CAS No. HY-50895) and Osimertinib (AZD- manufacturer’s instructions. The coding strand for AXL 9291, CAS No. HY-15772) were purchased from Med- was as follows: sense CCA GCA CCU GUG GUC AUC ChemExpress (NJ, USA). Cycloheximide (CAS No. 66-81- UUA CCU U and antisense AAG GUA AGA UGA CCA 9) was purchased from A.G. Scientific (CA, USA). Yuan- CAG GUG CUG G. huadine (YD; purity >98.5%) was isolated from a CHCl - soluble fraction of the flowers of Daphne genkwa,as Western blotting analysis described previously . All chemicals were dissolved in The cells were lysed in 2× sample loading buffer DMSO for in vitro experiments. (250 mM Tris-HCl pH 6.8, 4% SDS, 10% glycerol, 0.006% Antibodies against C-terminal AXL (sc-1096), EGFR bromophenol blue, 2% β-mercaptoethanol, 50 mM (sc-03), p-ERK (sc-7383), ERK (sc-94), MET (sc-10), sodium fluoride, and 5 mM sodium orthovanadate). β-actin (sc-47778) were obtained from Santa Cruz Bio- Tumor tissues were collected in RIPA buffer (Thermo- technology (Santa Cruz, CA, USA). p-AXL (#5724), p- fisher, Rockford, IL, USA), and then further lysed with 2x EGFR (#2234), p-MET (#3077), p-Akt (#9271), Akt laemmli sample buffer with 2% β-mercaptoethanol (#9272), p-p70S6 Kinase (#9205), p70S6 Kinase (#9202), (Biorad). The collected samples were subjected to 6-12% p-SAPK/JNK (#9091), SAPK/JNK (#9252), and snail SDS-PAGE gel and transferred onto PVDF membranes (#3879) were obtained from Cell Signaling Technology (Millipore, Bedford, MA, USA). The membranes were (Danvers, MA, USA). blocked with 5% BSA in Tris-buffered saline containing 0.1% Tween-20 (TBST) for 1 h at room temperature, and Cell culture then incubated with primary antibodies in 2.5% BSA in Human non-small lung cancer cells HCC827, HCC827- TBST overnight at 4 °C on a shaker. The membranes were gef, PC9, PC9-gef cells were a kind gift of Dr. Jae Cheol washed three times with TBST and incubated with the Lee and Dr. Jin Kyung Rho (Asan Medical Center, Seoul, secondary antibodies (HRP) (Younginfrontier, Seoul, Korea). HCC827-gef and PC9-gef cells were subcultured Korea) diluted in TBST for 2 h at room temperature. After in the presence of 1 µM gefitinib. Resistant cell line washing with TBST, the membranes were exposed to HCC827-osi was generated in vitro by culturing HCC827 enhanced chemiluminescence (ECL) solution (Intron, cells with escalating doses (0.001–0.5 μM) of osimertinib. Daejon, Korea). The chemiluminescence signals were All the cells were maintained in RPMI 1640 media sup- captured using LAS-4000 (Fuji Film Corp., Tokyo, Japan). plemented with 10% Fetal Bovine Serum (FBS) and 1% antibiotics-antimycotics (AA) (PSF; 100 units/mL peni- Real-time PCR analysis cillin G sodium, 100 μg/mL streptomycin, and 250 ng/mL The total RNA of the cells was isolated with TRI reagent amphotericin B). (Invitrogen, Grand Island, NY, USA). The isolated RNA (1 µg) was reverse-transcribed using ReverTra Ace qPCR Cell viability assay RT Master Mix (TOYOBO, Osaka, Japan) according to Cell viability was assayed by MTT method. Cells were the manufacturer’s instructions. Using synthesized cDNA, TM ® seeded in 96-well plates. On the next day, cells were Real-time PCR was conducted using iQ SYBR Green Official journal of the Cell Death Differentiation Association Kim et al. Cell Death and Disease (2019) 10:361 Page 3 of 12 Supermix (Bio-Rad, Hercules, CA, USA), according to the was approved by the institutional review board of Sever- manufacturer’s instructions. The comparative C method ance Hospital (4-2013-0526), and all patients provided was used to determine the relative expression normalized written informed consent. Tumors and paired peripheral by β-actin. The sequences of the primers are listed below. blood samples were consecutively collected for PDX AXL establishment and further genetic analysis. PDXs were (F) 5′-CGTAACCTCCACCTGGTCTC-3′; created using 6–8-week-old female severe combined immunodeficient (NOG) and nude (nu/nu) mice (R) 5′-TCCCATCGTCTGACAGCA-3′ GAS6 (OrientBio, Seoul, Korea). The tumors and related PDXs (F) 5′-CATCAACAAGTATGGGTCTCCGT-3′; were assigned Yonsei Human In Mouse (YHIM) identi- (R) 5′-GTTCTCCTGGCTGCATTCGTTGA-3′ fiers that corresponded to the original patient-derived β-actin tumors. Tumor dimensions were measured twice a week (F) 5′-AGCACAATGAAGATCAAGAT-3′; with a digital caliper and tumor volume was calculated as (R) 5′-TGTAACGCAACTAAGTCATA-3′ follows: (length × width )/2. Establishment of acquired gefitinib-resistant PDX tumors (YHIM-1009) and drug Immunocytochemistry administration was performed in Yonsei Cancer Center The cells were grown on a confocal dish pre-coated with and carried out as described previously . 0.2% gelatin. The cells were fixed with 4% paraformalde- hyde (in PBS) for 15 min and were blocked in 1% BSA (in Immunohistochemistry staining PBS containing 0.1% Triton X-100) for 30 min at room The tumors after the end of xenograft experiment were temperature. Cells were incubated with primary antibody excised, fixed in 4% paraformaldehyde (in PBS), and (AXL, 1:50) at 4 °C overnight and further incubated with embedded in paraffin. The embedded specimens were secondary antibody (anti-mouse Alexa 647, 1:250) for 2 h sectioned carefully, serially deparaffinized, rehydrated, at room temperature. The nuclei were stained with DAPI and subjected to antigen retrieval. Immunohistochemical (0.5 µg/ml). The images were detected using a confocal analysis of the tumor tissues was carried out as described microscope (Leica, TCS SP8). previously with the indicated antibodies . Tumor xenograft study Statistical analysis Balb/c-nu mouse (male, 4-weeks-old; OrientBio, Seoul, The data are presented as the means ± SD for the Korea) were allowed one-week acclimation prior to the minimum three independently performed experiments. 6 6 experiment. HCC827 (2 × 10 cells), HCC827-gef (4 × 10 The statistical significance (*P< 0.05, **P< 0.01, ***P< cells), or HCC827-osi (4 × 10 cells) cells were prepared in 0.005) were determined by ANOVA using Dunnett’s test. 100 µl PBS and mixed with the equal amount of Matrigel All statistical tests were two-sided. (Corning, Bedford, MA, USA) right before injecting sub- cutaneously into the flanks of the mice. When the tumor Results 3 3 volume reached 50 mm (HCC827) and 100 mm Differential sensitivity to osimertinib is observed in EGFR- (HCC827-Gef, HCC827-osi) on average, the mice were mutant acquired gefitinib resistant NSCLC cells randomized into the vehicle control and treatment groups To investigate the molecular mechanism of EGFR-TKIs (n = 5). Drugs were mixed with vehicle (EtOH:Tween80: resistance, we primarily evaluated the effects of gefitinib Saline solution 1:1:98). Each drug was administrated orally and osimertinib, the first and third generation of EGFR- once a day and 6 times per week for 22 days (HCC827-gef, TKIs, respectively, on growth of EGFR-mutant NSCLC HCC827-osi) and 90 days (HCC827). The body weight cell lines after 72 h of treatment (PC9, PC9-gef, HCC827, and tumor size were measured every 3–7 days. The tumor HCC827-gef, HCC827-osi) (Fig. 1a). While both HCC827 size was measured using a digital slide caliper and and PC9 cells harbor the EGFR Exon 19 deletion muta- volumes (mm ) were calculated as follows: (width × tion, HCC827 has been reported to have higher MET length × height) × π/6. The normalized tumor volume as expression while PC9 has higher EGFR and FGFR3 follows: (TV /TV ), where TV is the initial expression among NSCLC cell lines harboring mutant j,treated i,control i tumor volume of first administration, and TV is the EGFR status . Resistant cells were established through tumor volume of day j. Animals were sacrificed after the continuous exposure to gefitinib or osimertinib using a final drug administration and tumors were collected for dose-escalation procedure, and finally they exhibited at ex vivo analysis. least 100-fold greater IC than did the parental cells. We found that PC9 and HCC827 cells were initially very Patient-derived xenograft study sensitive to both gefitinib and osimertinib (Fig. 1b). Patient-derived tumor specimens were collected at Interestingly, two gefitinib-resistant cells showed different Yonsei University Severance Hospital. The study protocol sensitivity to osimertinib. Osimertinib effectively inhibited Official journal of the Cell Death Differentiation Association Kim et al. Cell Death and Disease (2019) 10:361 Page 4 of 12 A B C Fig. 1 Overexpression of AXL is observed in EGFR-TKI resistant NSCLC cells. NSCLC cells (PC9, PC9-gef, HCC827, HCC827-gef, and HCC827-osi) were treated with gefitinib or osimertinib for 72 h. Cell viability was measured by MTT assay as described in Methods (a). IC values and subculture conditions were stated (b). The protein expressions of major RTKs, p-AKT, and p-ERK were determined by Western blotting (c). Cells were incubated on the confocal dish for 24 h, fixed, and stained with C-term AXL (red) and DAPI (blue) for visualization of cellular localization (d). Data are presented as the mean fold changes ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test the growth of PC9-gef cells, but not HCC827-gef cells. compensating for EGFR inactivation. In addition, the Moreover, acquired osimertinib resistant cells developed levels of phosphorylated Akt and ERK were elevated from HCC827 also became intrinsically resistant to gefi- compared with those of HCC827 cells (Fig. 1c). The tinib. This suggests acquired resistant cells established expressions of AXL in HCC827, HCC827-gef and from HCC827 cells develop EGFR-independent resistance HCC827-osi cells were also evaluated by immunocy- mechanism. tochemistry staining of C-term AXL (Fig. 1d). AXL was clearly overexpressed in the resistant cells and mainly AXL is a major bypass pathway in osimertinib resistant localized in the cytosol. cells To examine the role of AXL in the resistant cells, the In order to investigate the mechanisms of acquired effects of AXL knockdown on the drug sensitivity of resistance of HCC827-gef and HCC827-osi cells, the EGFR-TKIs in HCC827-gef and HCC827-osi cells were expression levels of major receptor tyrosine kinases evaluated. The efficiency of siRNA was confirmed prior to (RTKs) and major downstream targets were evaluated by the experiments (Fig. 2a). AXL inhibition significantly Western blotting. The activation of phosphorylated Akt restored the sensitivity of gefitinib and osimertinib in and ERK has been reported to drive resistance and both HCC827-gef and HCC827-osi cells (Fig. 2b, c). 27,28 growth in tumor cells . In HCC827-gef and HCC827- Although AXL was overexpressed in PC9-gef cells, osi cells, both AXL and MET were activated in knockdown of AXL was less effective in restoring the Official journal of the Cell Death Differentiation Association Kim et al. Cell Death and Disease (2019) 10:361 Page 5 of 12 HCC827-gef AB 120 120 siNC siNC siAXL siAXL ** 100 100 *** HCC827-osi HCC827-gef *** *** *** *** *** 80 80 HCC827-osi HCC827-gef 1.2 60 60 1.2 ** 1.0 1.0 40 40 0.8 0.8 20 20 0.6 *** 0.6 0 0 0 0.04 0.2 1 5 0 0.04 0.2 1 5 0.4 0.4 Gefitinib (μM) Osimertinib (μM) *** 0.2 0.2 0.0 0.0 HCC827-osi siCTL siAxl siCTL siAxl siNC siAXL siNC siAXL 120 siNC kDa siNC kDa AXL 140 siAXL siAXL AXL 140 100 *** *** *** *** *** *** *** β-actin 42 β-actin 42 ** 0 0.04 0.2 1 5 0 0.04 0.2 1 5 Gefitinib (μM) Osimertinib (μM) HCC827 DE HCC827-gef Time (hr) 01248 16 20 24 Time (hr) 01248 16 20 24 CHX - + ++++++ CHX - + ++++++ kDa kDa AXL AXL 16 140 140 HCC827 HCC827 β-actin 42 β-actin 42 H HCC827-gef CC827-gef HCC827-osi HCC827-osi HCC827-osi Half-life of AXL after Cell lines CHX treatment (hr) * Time (hr) 01248 16 20 24 ** HCC827 1.8 CHX - +++++++ * kDa 2 AXL HCC827-gef > 24 0 AXL AXL GAS6 β-actin HCC827-osi > 24 Fig. 2 The total AXL protein degradation rate is extended in EGFR-TKI resistant cells. The efficiency of siAXL was confirmed using real-time PCR and Western blotting (a). The effects of gefitinib or osimertinib on the cell viability were evaluated after cells were transfected with siNC or siAXL compared with control (no transfection) in HCC827-gef cells (b) and HCC827-osi cells (c). Cycloheximide (CHX) was treated to stop the protein production and the protein expression of AXL (140 kda) was detected by Western blotting over the indicated time points. The expressions of AXL and β-actin were quantified by densitometry using ImageJ. AXL expressions were normalized to β-actin and further compared with 0 h expressions (d). mRNA expression of AXL and GAS6 were detected by real-time PCR using β-actin as a reference gene (e). Data are presented as the mean fold changes ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test gefitinib sensitivity (Supplementary Fig. S1a–c). Together, AXL than that of HCC827 parent cells (Fig. 2d). Western these results suggest AXL is the major bypass pathway blotting bands of AXL expression was normalized by and plays a significant role in HCC827-gef and HCC827- β-actin expression to quantify half-life. The half-life of osi cells which are resistant to both gefitinib and AXL protein was calculated to be 1.8 h in HCC827 cells, osimertinib. while it was longer than 24 h in the resistant cells. A ligand of AXL, GAS6 binds and activates AXL receptor Overexpression of AXL is associated with extended protein signaling thereby stimulating the cell proliferation .We degradation rate found that the gene copy number of AXL and its ligand To further assess the status of AXL in the resistant cells genes, GAS6, were increased in the resistant cells (Fig. 2e). compared with the parent cells, we measured the AXL AXL was found to be overexpressed in PC9-gef cells, so protein degradation rate using Western blotting analysis. we also compared the half-life of AXL in PC9 and PC9-gef The protein degradation rate of full-length AXL was cells. The overexpression of AXL was also correlated with measured after blocking new protein synthesis using extended degradation rate in PC9-gef cells (Supplemen- cycloheximide (CHX). Both HCC827-gef and HCC827- tary Fig. S1d). These results indicated that resistant cells osi cells displayed extended protein degradation rates of exhibit higher GAS6 and AXL expression and AXL Official journal of the Cell Death Differentiation Association Normalized mRNA Normalized mRNA expression of Axl expression of AXL Normalized mRNA Normalized mRNA expression of AXL expression of Axl Cell Viability (% of control) Cell Viability (% of control) Normalized mRNA expression Cell Viability (% of control) Cell Viability (% of control) Kim et al. Cell Death and Disease (2019) 10:361 Page 6 of 12 overexpression is closely associated with extended protein pathway were further examined in both sensitive and degradation rates. resistant cells. YD concentration-dependently inhibited full-length AXL and its downstream targets including YD inhibits cell proliferation and by AXL degradation in phosphorylated Akt and ERK without affecting the total resistant cells protein expression (Fig. 3d). Interestingly, YD suppressed YD, derived from the flowers of Daphne genkwa, inhi- the levels of phosphorylated MET and total MET expressions. Collectively, YD efficiently regulates AXL bits AXL expression by targeting protein degradation (Fig. 3a) . To access the effects of AXL degradation on degradation, MET, and their downstream signaling in cell viability, we examined the effects of YD in HCC827, both EGFR-TKI-sensitive and EGFR-TKI-resistant cells. HCC827-gef, and HCC827-osi cells. The treatment of YD for 72 h reduced cell viability of HCC827, HCC827-gef, YD resensitizes HCC827-gef and HCC827-osi cells to EGFR- and HCC827-osi with IC value of 7.06 nM, 2.92 nM, and TKIs 9.2 nM, respectively (Fig. 3b). Moreover, YD treatment AXL kinase inhibitors are reported to resensitize EGFR- accelerated the degradation of AXL protein of resistant TKIs resistant cells to EGFR-TKIs . We further evaluated cells. The calculated half-life of AXL protein after YD whether AXL degrader, YD, could resensitize resistant treatment was 1.2 h in HCC827-gef cells and 2.7 h in cells to gefitinib and osimertinib. Treatment of YD with HCC827-osi cells which became similar to that of parent gefitinib or osimertinib for 72 h inhibited cell growth cells (Fig. 3c). The effects of YD on the AXL signaling more than any single drug treatment (Fig. 4a left and B Fig. 3 YD can regulate the degradation of AXL and effectively inhibit the growth of both EGFR-TKI sensitive and resistant cells. Chemical structure of yuanhuadine (YD) (a). Indicated cell lines were treated with YD for 72 h and viability was measured. (b). The protein expression of AXL (140 kda) was detected after co-treatment of CHX and YD by Western blotting analysis over the indicated time points to test the effects of YD on the protein degradation (c). The expressions of AXL and β-actin were quantified by densitometry using ImageJ. AXL expressions were normalized to β-actin and further compared with 0 h expression (d). Cells were treated with indicated concentrations of YD for 24 h (e). Data are presented as the mean fold changes ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test Official journal of the Cell Death Differentiation Association Kim et al. Cell Death and Disease (2019) 10:361 Page 7 of 12 YD 0 nM YD 1 nM HCC827-gef YD (nM) YD 5 nM YD 10 nM 510 HCC827-gef HCC827-osi Gefitinib (μM) CI YD -+ -+ YD - + - + 1 0.81 0.87 Gefitinib - - + + kDa Osimertinib - - + + 5 0.67 0.46 p-AXL p-AXL 10 0.41 0.34 (Y702) (Y702) Gefitinib (µM) AXL AXL 140 YD 0 nM p-MET p-MET YD 1 nM (Y1234/1235) HCC827-osi YD (nM) (Y1234/1235) YD 5 nM YD 10 nM MET MET 145 Osimertinib p-Akt p-Akt (μM) CI (S473) (S473) 0.5 0.69 0.33 Akt Akt 1 0.67 0.46 5 0.54 0.50 p-p70S6 p-p70S6 70 Osimertinib (µM) p70S6 p70S6 70 p-ERK1/2 p-ERK1/2 HCC827-gef or HCC827-osi ERK1/2 ERK1/2 21 days (administration 6 days per week) 54 p-JNK p-JNK JNK JNK Vehicle YD 46 Vehicle YD Snail 29 Snail Gefitinib Combination Osimertinib Combination β-actin 42 β-actin 800 p-Akt/Akt p-Akt/Akt HCC827-gef HCC827-osi p-ERK/ERK p-ERK/ERK xenograft 700 xenograft p-p70S6/p70S6 p-p70S6/p70S6 1.4 1.4 p-JNK/JNK p-JNK/JNK 400 600 1.2 1.2 1.0 1.0 400 0.8 0.8 0.6 0.6 200 300 0.4 0.4 0.2 0.2 0.0 0.0 CTL YD Osi Combi CTL YD Gef Combi 0 7 14 21 0 7 14 21 Days of administration Days of administration Fig. 4 YD and EGFR-TKIs synergistically inhibit the growth of gefitinib and osimertinib cross-resistant cells in in vitro and in vivo. Cell viability was measured after combined treatment of YD and gefitinib or osimertinib for 72 h in HCC827-gef (a) and HCC827-osi (b), respectively. Based on the cell viability results, CI values were calculated to demonstrate the combination effects in each cell line. The effects of combination treatment compared with single treatment were accessed using western blot. Cells were treated with YD (10 nM) and/or gefitinib (1 μM) in HCC827-gef cells, and YD (10 nM) and/or osimertinib (500 nM) in HCC827-osi cells (c). HCC827-gef cells were subcutaneously implanted into the flanks of Balb/c-nude mice (n = 5). Mice were orally administered with compounds 6 times per week for 21 days and doses indicated are 1 mg/kg for YD, 10 mg/kg for gefitinib. HCC827-osi cells were subcutaneously implanted into the flanks of Balb/c-nude mice (n = 5). Mice were orally administered with compounds 6 times per week for 21 days and doses indicated are 0.5 mg/kg for YD, 5 mg/kg for osimertinib (d). Data are presented as the mean fold changes ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test left). These synergistic effects were evaluated by combi- strongly inhibited the expressions of p-Akt, p-ERK, p- nation index (CI) analysis using the Chou-Talalay p70S6, and p-JNK which regulate cell proliferation and method, which is based on the median-effect equation survival (Fig. 4c). Moreover, YD and combined treatment 23,32 and also provide algorithms for computer simulation inhibited snail expression, which is one of the major EMT (CI < 1: synergism, CI = 1: additive; CI > 1: antagonism). target genes regulated by AXL signaling . In search of The formula can be applied for any dose-effect analysis potential application of YD in a combination therapy, we and even suitable for small-scale experiments. Various also investigated the combined effects of YD and other doses of compounds were tested to accurately determine kinase inhibitors such as PHA-665752 (c-MET inhibitor), the effects of drug combinations. The calculated CI values SP600125 (JNK inhibitor), and LY294002 (PI3K inhibitor) were smaller than 1, which indicates synergism (Fig. 4a which were showed limited efficacy as a single agent in the right and b right). resistant cells. Combined treatment with YD resulted in Treatment with either gefitinib or osimertinib alone did significant suppression of cell viability and showed not have significant effects on the expression of AXL, synergism with all of three kinase inhibitors (Supple- MET, and downstream signaling in resistant cells. On the mentary Fig. S2). These results indicate targeting AXL other hand, the combination of YD and EGFR-TKIs degradation by YD can resensitize resistant cells not only Official journal of the Cell Death Differentiation Association Tumor volume (mm3) Cell Viability (% of control) Cell Viability (% of control) Tumor volume (mm ) Fold change compared to control cells Fold change compared to control cells Kim et al. Cell Death and Disease (2019) 10:361 Page 8 of 12 Acquired gefitinib resistant patient derived tumors (YHIM-1009) 31 days (administration 7 days per week) PDX model Vehicle YD Gefitinib Combination 500 24 4 8 12 16 20 24 28 31 4 8 12 16 20 24 28 31 Days of administration Days of administration Fig. 5 YD and EGFR-TKIs synergistically inhibit the growth in acquired gefitinib-resistant PDX models. When tumor volumes reached 200 to 250 mm3, mice engrafted with YHIM-1009 tumors were segregated into appropriate treatment groups (three to five mice per group). Mice were orally administered with compounds every day for 31 days and doses indicated are 1 mg/kg for YD, 10 mg/kg for gefitinib (a). *P < 0.05, **P < 0.01, ***P < 0.005 by t-test to EGFR-TKIs, but also potentially to c-MET inhibitor, activity of single agent administration and combination JNK inhibitor, or PI3K inhibitor. administration in acquired gefitinib-resistant patient derived xenograft (PDX) model. The third mouse gen- Combined administration of YD with EGFR-TKIs eration of YHIM-1009 (EGFR 19del mutation/ PIK3CA synergistically inhibits tumor growth in xenograft model E542K) tumor model, an acquired-gefitinib resistant bearing HCC827-gef and HCC827-osi cells tumor, was used for drug efficacy testing. The previous To further confirm the synergistic activity of YD and study demonstrated that YHIM-1009 did not show EGFR-TKIs using in vivo model, we examined their effi- response to both gefitinib (25 mg/kg) and osimertinib cacy in a xenograft model engrafted with HCC827-gef and (6.25 mg/kg) administration . When tumor volumes HCC827-osi cells. The drugs were orally administered reached 200 to 250 mm , animals were administrated with once a day, 6 days per week, for 3 weeks. Resistant cells vehicle, YD (1 mg/kg), gefitinib (10 mg/kg), or YD and developed in vitro were implanted in the right flank of gefitinib every day for 31 days. Combined administration nude mice. After one week, mice were randomly assigned of YD and gefitinib notably inhibited the tumor growth to each group. Consistent with in vitro data, the combi- derived from acquired gefitinib-resistant patient tumor nation administration effectively inhibited the tumor more efficiently than a single treatment (Fig. 5a). Body- group in both HCC827-gef and HCC827-osi xenograft weight was measured to monitor the toxicity, and there tumor model (Fig. 4d). YD as a single treatment group was no significant change in bodyweight for all groups. resulted in tumor-regression after 14 days in HCC827-gef Unlike the results of HCC827-gef xenograft model, YD tumor model and 17 days in HCC827-osi tumor model. single administration resulted in only slight inhibition of Collectively, the combination of YD and EGFR-TKIs tumor growth. These results indicate that YD potentially shows potential for both inhibitions of in vitro cell pro- more useful for combination therapy than as a single liferation and in vivo tumor growth via AXL degradation. agent. Combined administration of YD with gefitinib Combined administration of YD with gefitinib delays the synergistically inhibits tumor growth of gefitinib-resistant emergence of resistance in long-term xenograft model PDX model bearing HCC827 cells In order to assess the combination effects of YD and Because AXL is regarded as an acquired resistance gefitinib in more clinically relevant setting, we compared mechanism to EGFR-TKIs, we hypothesized that AXL Official journal of the Cell Death Differentiation Association Tumor volume (mm ) Body weight (g) Kim et al. Cell Death and Disease (2019) 10:361 Page 9 of 12 Vehicle YD A B Vehicle YD Gefitinib Combination Gefitinib Combination 2400 600 2000 500 1600 400 1200 300 1000 8 800 200 *** 11 46 60 90 *** 400 100 Days of administration *** Combi- Day Vehicle YD Gefitinib nation 0 10 20 30 40 50 60 0 15 30 45 60 75 90 11 7.3 4.0 1.3 0.9 Days of administration 46 27.6 9.0 2.8 1.0 60 32.6 9.5 5.3 1.6 YD -+ -+ 90 42.4 10.6 12.5 8.1 Gefinib -- + + Vehicle YD AXL Gefitinib Combination Fig. 6 The combined administration of YD and gefitinib can delay the emergence of resistance and inhibit AXL overexpression in a long- term in vivo xenograft model. HCC827-Luc cells were subcutaneously implanted into the flanks of Balb/c-nude mice (five mice per group). Drugs were orally administered 6 times per week for 90 days and doses indicated are 0.5 mg/kg for YD, 10 mg/kg for gefitinib (a). The graph represents tumor volumes of indicated days which were normalized to the initial tumor volume for comparison (b). Bioluminescence images of mice at the final day before sacrifice were measured and mice were injected with Firefly D-luciferin before imaging (c). Immunohistochemical analysis of AXL was performed in tumor tissue (d). The schematic diagram illustrating the significance of combined YD with EGFR-TKI treatment in NSCLC (e). *P < 0.05, **P < 0.01, ***P < 0.005 by t-test inhibition with EGFR-TKIs from the beginning of treat- the initial size and started to grow rapidly after 35 days of ment would prevent the emergence of acquired resistance, drug administration (Fig. 6a right). The tumor volumes on enhance the therapeutic efficacy, and extend drug day 11, 46, 60, and 90 were normalized to the initial tumor response time. Therefore, we performed a long-term volume before the administration to quantify the effects (90 days) xenograft experiment using a xenograft model (Fig. 6b). Tumors in the combination group later started implanted with EGFR-TKI-sensitive HCC827 cells (Fig. 6a to grow after day 60, but their final volumes were still left). Not every vehicle group survived throughout the smaller than those of the YD group or gefitinib group long-term experiment due to the tumor burden, so (Fig. 6c). Although tumor size in the vehicle-administered remaining mice were still used to record tumor size and group was much larger, AXL expression was undetected body weights. We observed no change in body weights in in immunohistochemistry staining. These data confirm combination group and gefitinib group, while the body that AXL increases in response to the long-term admin- weight was only slightly reduced in YD group (data not istration of gefitinib regardless of tumor size (Fig. 6d). shown). Combination of YD and gefitinib both almost Combined treatment with YD and gefitinib was not completely prevented the tumor growth for 60 days, while resulted in the AXL expression in the tumors. However, the tumors of the gefitinib administration group exceeded the tumor sizes of the combination treatment group also Official journal of the Cell Death Differentiation Association Tumor Volume ( Tumor Volume ( Normalized tumor volume Kim et al. Cell Death and Disease (2019) 10:361 Page 10 of 12 increased at later points in time, suggesting that tumors without toxicity . Therefore, N-terminal fragment gen- may have acquired resistance by additional mechanisms. erated by AXL may prevent binding of GAS6 and AXL Further studies are needed to investigate the mechanism receptor thereby enhancing the AXL blockade activity. involved with resistance to the combination treatment of YD inhibited AXL, as well as MET protein expression YD and gefitinib. Collectively, these results suggest that which resulted in more sustained inhibition of down- using a combined treatment as a first-line therapy may stream signaling pathway. Previous study also reported delay the emergence of acquired-resistance and as a that tumor suppressor OPCML inactivates AXL- second-line therapy may overcome resistance (Fig. 6e). dependent oncogenic signaling in ovarian cancer and also prevent transactivation of other RTKs such as EGFR Discussion and cMET . Although the mutation and activation of EGFR are It is well reported that AXL inhibition using kinase recognized as important oncogenic drivers in NSCLC inhibitors can sensitize or synergistically work with sev- patients, targeting EGFR using currently existing EGFR- eral drugs such as antimitotic drugs, erlotinib, and ima- 8,17,19 TKIs has shown limited success due to the emergence of tinib . In addition, several studies suggested that acquired resistance . Osimertinib, a third-generation there are crosstalks between AXL and other RTKs; (1) TKIs, has been developed to treat mutant EGFR or/and AXL clusters together with other cell surface RTKs in the T790M-positive NSCLC patients and also has been mesenchymal cells which leads to crosstalk-amplified reported to have superior efficacy as first-line treatment . signaling network and a limited response to single TKIs A recent study showed the activation of AXL as an treatment . (2) When head and neck and esophageal adaptive response to osimertinib, and SPRY4 as a poten- squamous cell carcinomas become resistant to PI3Kα tial negative regulator of AXL signaling in lung cancer . inhibition, AXL dimerizes with EGFR and activates However, little has been studied about the roles of over- EGFR/PKC/mTOR pathway . (3) AXL not only forms a expressed AXL in acquired resistance mechanisms against complex with EGFR but also may promote nuclear 47,48 osimertinib. Our study report that extended degradation translocation of the EGFR . (4) MGCD265, which is a rate of AXL is pivotal in the development of resistance to multi-targeted TKIs targeting MET, AXL, and PDGFR, gefitinib, osimertinib; thus, we investigated therapeutic completely inhibited the growth in only tumors with high approaches using drug combination to overcome resis- MET gene copy gains, and combination of MGCD265 and tance by targeting AXL degradation. erlotinib effectively inhibited the tumor growth in most of Targeting protein degradation may have an advantage triple breast cancer cells and tumors . (5) AXL kinase over kinase inhibition in the regulation of cancer cell inhibitor (R428) suppressed EMT-induced metastasis and growth; thus the efficacy of drugs with proteolysis- combined treatment enhanced the effects of lapatinib or targeting chimera (PROTAC) technology is under inves- trastuzumab in HER2-overexpressed breast cancer PDX 37,38 50 tigation . Several studies with EGFR, HER2, and c- model . These studies support combination treatment MET inhibitors have confirmed that targeted protein ought to be superior than single drug treatment. degradation may have an advantage because it yields more In this study, we confirmed that AXL protein degrada- sustained loss of functions and prevents kinome re- tion with EGFR-TKIs synergistically suppresses the wiring . Along these lines, we utilized the natural anti- growth of EGFR mutant cells and tumors. The effects of tumor compound YD to target AXL degradation which combined administration were very significant in PDX exhibits anti-proliferative activities against various human model. The tumor model used in PDX harbors EGFR lung cancer cells by regulating AXL, SerpinB2, NNMT, mutation, as well as PI3KA mutation, which may explain 22,39–42 and BMP4 . YD exhibits nanomolar IC in NSCLC results of inefficient YD single administration group. cell lines which is much lower than currently existing Nonetheless, the combined administration showed very AXL kinase inhibitors based on their reported IC potent tumor growth inhibition. As previously reported, value . tumor tissues obtained from gefitinib-administrated We previously found that YD cleaves AXL independent group in a long-term xenograft study with HCC827 cells of kinase, metalloprotease, proteasome and lysosome showed significantly higher AXL expression from the 30 17 activities . Treatment of YD can release N-terminal into immunohistochemistry results . Although tumor size in the culture medium and sequentially generate intracel- the vehicle-administered group was much bigger in long- lular domain thereby degrading the full-length AXL. term xenograft, the AXL expression was negligible. These When N-terminal of AXL is cleaved, it may work as a findings suggest that AXL is upregulated in response to decoy receptor to inhibit the binding of GAS6 and AXL the long-term administration of gefitinib, regardless of receptor. For example, an engineered AXL decoy recep- tumor size, and affect sensitivity of tumors to EGFR-TKIs. tor, which antagonizes the GAS6/AXL system by cap- When administrated in combination of YD and gefitinib, turing GAS6, can inhibit cancer cells and tumor growth AXL was not detected in the tumors from the Official journal of the Cell Death Differentiation Association Kim et al. Cell Death and Disease (2019) 10:361 Page 11 of 12 immunohistochemistry results. These results indicate that 9. Axelrod, H. & Pienta, K. J. Axl as a mediator of cellular growth and survival. Oncotarget 5,1–35 (2014). YD completely suppressed the AXL activation and was 10. Lemke, G. Biology of the TAM receptors. CSH Perspect. Biol. 5, a009076 (2013). able to delay the tumor regrowth. 11. Ammoun, S. et al. Axl/Gas6/NFkappaB signalling in schwannoma pathological In conclusion, we have elucidated AXL as a driver of proliferation, adhesion and survival. Oncogene 33,336–346 (2014). 12. Antony, J., Huang,R.Y.J.&AXL-Driven,E.M.T.State as a targetableconduit in cross-resistance to gefitinib and osimertinib and demon- cancer. Cancer Res. 77, 3725–3732 (2017). strated the efficacy of combining YD with gefitinib and/or 13. Antony, J. et al. The GAS6-AXL signaling network is a mesenchymal (Mes) molecular subtype-specific therapeutic target for ovarian cancer. Sci. Signal. 9, osimertinib in overcoming EGFR-TKI resistance in cells, ra97 (2016). resistant tumor xenograft and PDX model, and in delaying 14. Aguilera, T. A. et al. Reprogramming the immunological microenvironment the emergence of acquired resistance. These results sug- through radiation and targeting Axl. Nat. Commun. 7, 13898 (2016). gest combination use of YD and EGFR-TKIs may repre- 15. Brand, T. M. et al. AXL mediates resistance to cetuximab therapy. Cancer Res. 74,5152–5164 (2014). sent a potential therapeutic strategy for NSCLC patients. 16. Muller, J. et al. Low MITF/AXL ratio predicts early resistance to multiple tar- geted drugs in melanoma. Nat. Commun. 5, 5712 (2014). Acknowledgements 17. Zhang,Z.F.etal. Activation ofthe AXL kinase causes resistance to EGFR- The authors thank Byoung Chul Cho (JE-UK Institute for Cancer Research, JEUK targeted therapy in lung cancer. Nat. Genet. 44, 852 (2012). Co. Ltd., Gumi-City, Kyungbuk, Republic of Korea /Division of Medical 18. Wilson, C. et al. AXL inhibition sensitizes mesenchymal cancer cells to anti- Oncology, Department of Internal Medicine, Yonsei Cancer Center, Yonsei mitotic drugs. Cancer Res. 74,5878–5890 (2014). University College of Medicine, Seoul, Republic of Korea) for carrying out the 19. Ben-Batalla, I. et al. Axl blockade by BGB324 inhibits BCR-ABL tyrosine kinase PDX study. This research was supported by a National Research Foundation of inhibitor-sensitive and -resistant chronic myeloid leukemia. Clinical Cancer Res. Korea (NRF) grant funded by the Korean Government (MSIP) 23,2289–2300 (2017). (2016R1C1B1008540 and 2016M3A9B6903499). 20. Boshuizen, J. et al. Cooperative targeting of melanoma heterogeneity with an AXL antibody-drug conjugate and BRAF/MEK inhibitors. Nat. Med. 24,203 Data availability and materials (2018). Please contact authors for data request. 21. Kariolis, M. S. et al. An engineered Axl ‘decoy receptor’ effectively silences the Gas6-Axl signaling axis. Nat. Chem. Biol. 10,977–983 (2014). Conflict of interest 22. Hong, J. Y., Nam,J.W., Seo, E. K. &Lee,S.K.Daphnanediterpene esters with The authors declare that they have no competing interest. anti-proliferative activities against human lung cancer cells from Daphne genkwa. Chem. Pharm. Bull. 58,234–237 (2010). Ethics approval and consent to participate 23. Chou, T. C. Theoretical basis, experimental design, and computerized simu- In vivo tumor xenograft experiments followed the guidelines of and approved lation of synergism and antagonism in drug combination studies. Pharmacol. by Institute of Laboratory Animal Resources of Seoul National University (SNU- Rev. 58,621–681 (2006). 161108-2). 24. Kang, H. N. et al. Establishment of a platform of non-small-cell lung cancer patient-derived xenografts with clinical and genomic annotation. Lung Cancer 124,168–178 (2018). 25. Kim, W. K. et al. Esculetin suppresses tumor growth and metastasis by tar- Publisher’s note geting Axin2/E-cadherin axis in colorectal cancer. Biochem. Pharmacol. 152, Springer Nature remains neutral with regard to jurisdictional claims in 71–83 (2018). published maps and institutional affiliations. 26. Karachaliou, N. et al. Common co-activation of AXL and CDCP1 in EGFR- mutation-positive non-smallcell lung cancer associated with poor prognosis. Supplementary Information accompanies this paper at (https://doi.org/ Ebiomedicine 29,112–127 (2018). 10.1038/s41419-019-1601-6). 27. Jacobsen, K. et al. Convergent Akt activation drives acquired EGFR inhibitor resistance in lung cancer. Nat. Commun. 8, 410 (2017). Received: 13 February 2019 Revised: 25 March 2019 Accepted: 17 April 28. Normanno, N. et al. The MEK/MAPK pathway is involved in the resistance of breast cancer cells to the EGFR tyrosine kinase inhibitor gefitinib. J. Cell Physiol. 207,420–427 (2006). 29. Varnum, B. C. et al. Axl receptor tyrosine kinase stimulated by the vitamin K- dependent protein encoded by growth-arrest-specificgene6. Nature 373, 623–626 (1995). References 30. Bae,S.Y., Hong,J.Y., Lee, H. J.,Park, H. J. &Lee,S.K.Targeting thedegradation 1. Kim, T. Y.,Han,S.W.&Bang,Y.J.Chasing targetsfor EGFR tyrosine kinase of AXL receptor tyrosine kinase to overcome resistance in gefitinib-resistant inhibitors in non-small-cell lung cancer: Asian perspectives. Exp. Rev. Mol. non-small cell lung cancer. Oncotarget 6,10146–10160 (2015). Diagnost. 7,821–836 (2007). 31. Giles, K. M. et al. Axl mediates acquired resistance of head and neck cancer 2. Liam, C. K., Pang, Y. K. & Poh, M. E. EGFR mutations in Asian Patients with cells to the epidermal growth factor receptor inhibitor erlotinib. Mol. Cancer advanced lung adenocarcinoma. J. Thorac. Oncol. 9,E70–E71 (2014). Ther. 12, 2541–2558 (2013). 3. Min, H. Y. & Lee, H. Y. Oncogene-driven metabolic alterations in cancer. Biomol. 32. Chou, T. C. Drug combination studies and their synergy quantification using Ther. 26,45–56 (2018). the Chou-Talalay method. Cancer Res. 70,440–446 (2010). 4. Soh, J. et al.EGFRmutationstatusin pleural fluid predicts tumor respon- 33. Asiedu, M. K. et al. AXL induces epithelial-to-mesenchymal transition and siveness and resistance to gefitinib. Lung Cancer 56,445–448 (2007). regulates the function of breast cancer stem cells. Oncogene 33,1316–1324 5. Uramoto, H. et al. Resistance to gefitinib. Int. J. Clin. Oncol. 11,487–491 (2006). (2014). 6. Yang, Z. et al. Investigating novel resistance mechanisms to third-generation 34. Wu, S. G. & Shih, J. Y. Management of acquired resistance to EGFR TKI-targeted EGFR tyrosine kinase inhibitor osimertinib in non-small cell lung cancer therapy in advanced non-small cell lung cancer. Mol. Cancer 17,38 (2018). patients. Clin. Cancer Res. 24,3097–3107 (2018). 35. Passiglia, F., Raez,L.E.& Rolfo, C. Moving osimertinibto first-line: the right 7. Zhang, H. et al. Discovery of 2,4,6-trisubstitued pyrido[3,4-d]pyrimidine deri- “strategy” in the chessboard of epidermal growth factor receptor-mutated vativesasnew EGFR-TKIs. Eur. J. Med. Chem. 148,221–237 (2018). non-small cell lung cancer? J. Thorac Dis. 10, S1076–S1080 (2018). 8. Gunther, M. et al. Trisubstituted pyridinylimidazoles as potent inhibitors of the 36. Taniguchi, H. et al. AXL confers intrinsic resistance to osimertinib and advances clinically resistant L858R/T790M/C797S EGFR mutant: targeting of both the emergence of tolerant cells. Nat. Commun. 10, 259 (2019). hydrophobic regions and the phosphate binding site. J. Med. Chem. 60, 37. Burslem, G. M. et al. The advantages of targeted protein degradation over 5613–5637 (2017). inhibition: an RTK case study. Cell Chem. Biol. 25,67–77 e63 (2018). Official journal of the Cell Death Differentiation Association Kim et al. Cell Death and Disease (2019) 10:361 Page 12 of 12 38. Raina,K.etal. PROTAC-induced BET protein degradation as a therapy for 44. Antony, J. et al. The tumour suppressor OPCML promotes AXL inactivation by castration-resistant prostate cancer. Proc. Natl Acad. Sci. USA 113, 7124–7129 the phosphatase PTPRG in ovarian cancer. EMBO Rep. 19, e45670 (2018). (2016). 45. Halmos, B. & Haura, E. B. New twists in the AXL(e) of tumor progression. Sci. 39. Bach, D. H. et al. Targeting nicotinamide N-Methyltransferase and miR-449a in Signal. 9,fs14 (2016). EGFR-TKI-resistant non-small-cell lung cancer cells. Mol. Ther. Nucleic Acids 11, 46. Elkabets, M. et al. AXL mediates resistance to PI3Kalpha inhibition by activating 455–467 (2018). the EGFR/PKC/mTOR axis in head and neck and esophageal squamous cell 40. Bae, S.Y., Park,H.J., Hong,J.Y., Lee, H. J. &Lee,S.K. Down-regulation of carcinomas. Cancer Cell 27,533–546 (2015). SerpinB2 is associated with gefitinib resistance in non-small cell lung cancer 47. Meyer, A. S., Miller, M. A., Gertler, F. B. & Lauffenburger, D. A. The receptor AXL and enhances invadopodia-like structure protrusions. Sci. Rep-Uk 6, 32258 diversifies EGFR signaling and limits the response to EGFR-targeted inhibitors (2016). in triple-negative breast cancer cells. Sci. Signal. 6, ra66 (2013). 41. Bach, D. H. et al. BMP4 upregulation is associated with acquired drug resis- 48. Brand, T. M. et al. The receptor tyrosine kinase AXL mediates nuclear trans- tance and fatty acid metabolism in EGFR-mutant non-small-cell lung cancer location of the epidermal growth factor receptor. Sci. Signal. 10,eaag1064 cells. Mol. Ther. Nucleic Acids 12,817–828 (2018). (2017). 42. Jo, S. K.,Hong, J. Y., Park, H. J. &Lee,S.K.Anticanceractivityofnovel daphnane 49. Linklater, E. S. et al. Targeting MET and EGFR crosstalk signaling in triple- diterpenoids from Daphne genkwa through cell-cycle arrest and suppression negative breast cancers. Oncotarget 7, 69903–69915 (2016). of Akt/STAT/Src signalings in human lung cancer cells. Biomol. Ther. 20, 50. Goyette,M.A.etal. Thereceptortyrosine kinase AXL is required at multiple 513–519 (2012). steps of the metastatic cascade during HER2-positive breast cancer progres- 43. Kariolis, M. S. et al. Inhibition of the GAS6/AXL pathway augments the efficacy sion. Cell Rep. 23,1476–1490 (2018). of chemotherapies. J. Clin. Invest. 127,183–198 (2017). Official journal of the Cell Death Differentiation Association

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