Epithelial-To-Mesenchymal Transition Is a Mechanism of ALK Inhibitor

Published OnlineFirst February 8, 2019; DOI: 10.1158/0008-5472.CAN-18-2052

Cancer Translational Science Research

Epithelial-to-Mesenchymal Transition Is a Mechanism of ALK Inhibitor Resistance in Lung Cancer Independent of ALK Mutation Status Koji Fukuda1, Shinji Takeuchi1, Sachiko Arai1, Ryohei Katayama2, Shigeki Nanjo1, Azusa Tanimoto1, Akihiro Nishiyama1, Takayuki Nakagawa1, Hirokazu Taniguchi1, Takeshi Suzuki3,Tadaaki Yamada4, Hiroshi Nishihara5, Hironori Ninomiya6,Yuichi Ishikawa7, Satoko Baba7, Kengo Takeuchi7, Atsushi Horiike8, Noriko Yanagitani8, Makoto Nishio8, and Seiji Yano1

Abstract

Mutations in the ALK gene are detectable in approximately resistant lung cancer cells showed that EMT associated with 40% of ALK-rearranged lung cancers resistant to ALK inhibi- decreased expression of miR-200c and increased expression of tors. Although epithelial-to-mesenchymal transition (EMT) is ZEB1 caused cross-resistance to new-generation ALK inhibi- a mechanism of resistance to various targeted drugs, its tors alectinib, ceritinib, and lorlatinib. Pretreatment with the involvement in ALK inhibitor resistance is largely unknown. histone deacetylase (HDAC) inhibitor quisinostat overcame In this study, we report that both ALK-mutant L1196M and this resistance by reverting EMT in vitro and in vivo. These EMT were concomitantly detected in a single crizotinib- findings indicate that HDAC inhibitor pretreatment followed resistant lesion in a patient with ALK-rearranged lung cancer. by a new ALK inhibitor may be useful to circumvent resistance Digital PCR analyses combined with microdissection after constituted by coexistence of resistance mutations and EMT in IHC staining for EMT markers revealed that ALK L1196M was the heterogeneous tumor. predominantly detected in epithelial-type tumor cells, indicating that mesenchymal phenotype and ALK mutation can Significance: These findings show that dual inhibition of coexist as independent mechanisms underlying ALK inhibi- HDAC and ALK receptor tyrosine kinase activities provides a tor–resistant cancers. Preclinical experiments with crizotinib- means to circumvent crizotinib resistance in lung cancer.

Introduction kinase inhibitor (TKI), shows dramatic clinical efficacy against ALK-rearranged NSCLC. Recent clinical trials demonstrated the ALK rearrangement, most commonly EML4-ALK, is detected in response rate of approximately 60% and progression-free survival approximately 3%–5% of unselected non–small cell lung cancer (PFS) of approximately 12 months (3–5). However, almost all (NSCLC; refs. 1, 2). Crizotinib, a first-generation ALK tyrosine patients who strongly responded to crizotinib acquire resistance over time. The most defined mechanism of crizotinib resistance is secondary or acquired ALK mutations. Unlike the EGFR-TKI 1Division of Medical Oncology, Cancer Research Institute, Kanazawa University, resistance in EGFR-mutated NSCLC, where one secondary resis- 2 Kanazawa, Japan. Cancer Chemotherapy Center, Japanese Foundation for tance mutation (EGFRT790M) is detected in approximately 60% of 3 Cancer Research, Tokyo, Japan. Division of Functional Genomics, Cancer resistant patients (6), diverse ALK resistance mutations (e.g., Research Institute, Kanazawa University, Kanazawa, Japan. 4Department of Pulmonary Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan. L1196M, I1171T/N/S, L1152P/R, F1174C/L/V, C1156Y/T, 5Genomics Unit, Keio Cancer Center, Keio University School of Medicine, Tokyo, I1171T/N/S, S1206C/Y, G1269A/S, V1180L, and G1202R) can Japan. 6Department of Pathology, The Cancer Institute Hospital, Japanese be detected in about 20%–25% of resistant patients. The Foundation for Cancer Research, Tokyo, Japan. 7Pathology Project for Molecular second-generation ALK-TKIs, alectinib (7–10) and ceriti- Targets and Division of Pathology, The Cancer Institute, Japanese Foundation nib (11), which have different spectrum of sensitivity to these 8 for Cancer Research, Tokyo, Japan. Department of Thoracic Medical Oncology, ALK resistance mutations, have been approved to treat crizo- The Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo, ALK Japan. tinib-resistant -rearranged NSCLC. In addition, other mechanisms of ALK-TKI resistancehavebeenreported,such Note: Supplementary data for this article are available at Cancer Research as ALK gene amplification (12, 13), activation of bypass sig- Online (http://cancerres.aacrjournals.org/). naling (e.g., HGF-Met, EGFR, c-KIT, IGF-1R, and HER3; K. Fukuda and S. Takeuchi contributed equally to this article. refs. 14–17), acquisition of other driver oncogenes (including Corresponding Authors: Seiji Yano, Cancer Research Institute, Kanazawa Univer- mutated EGFR and BRAF; ref. 18), and limited drug penetration sity, 13-1 Takaramachi, Kanazawa-shi, Ishikawa-ken 920-0934, Japan. Phone: 817- due to blood–brain barrier and overexpression of P-glycopro- 6265-2780; Fax: 817-6234-4524; E-mail: [email protected]; and Shinji tein (19). In these cases, alternative treatment strategies have Takeuchi, E-mail: [email protected] been proposed (14–17, 19). doi: 10.1158/0008-5472.CAN-18-2052 Epithelial-to-mesenchymal transition (EMT) is an increasingly 2019 American Association for Cancer Research. recognized driver of tumor progression, and a cause of both innate

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and acquired resistance to various cytotoxic and targeted drugs, tumorigenic variant of A925L was established (22), and the including EGFR-TKIs (20). Previous studies reported that in resistant cells were derived and characterized as described previ- patients whose EGFR-TKI resistance was associated with EMT, ously (17). Original A925L were established from a surgical the prognosis was much worse than that of patients whose specimen obtained from a Japanese male patient (T2N2M0, stage EGFR-TKI resistance was due to EGFRT790M mutation in EGFR- IIIA). The human lung adenocarcinoma cell lines H2228 and mutated NSCLC (21). A recent study (13) reported that EMT H3122 (23) containing a EML4-ALK fusion protein were used in component and ALK resistance mutation were simultaneously this research. The H2228 was purchased from the ATCC. The detected in a single tumor lesion in patients with ALK-rearranged H3122 was kindly provided by Dr. Jeffrey A (Novartis Institutes lung cancer who were resistant to ALK-TKIs. However, it is still for BioMedical Research, Cambridge, MA). The human lung unknown whether ALK-TKI resistant tumor cells combine mes- embryonic fibroblast lines MRC-5 and IMR-90 were obtained enchymal phenotype with ALK resistance mutation, or each of the from RIKEN Cell Bank. H1975 was kindly provided by Dr. John D. mesenchymal type tumor cells and ALK resistance mutation– Minna (University of Texas Southwestern Medical Center, Dallas, positive tumor cells coexist in a single lesion. In any of these TX). HCC827 cells were obtained from the ATCC. All cells were cases, no therapy for EMT-associated targeted drug resistance has maintained in RPMI-1640 medium supplemented with 10% FBS, yet been established, being considered an urgent unmet need. penicillin (100 U/mL), and streptomycin (10 mg/mL) in a humid- In this study, we intensively examined crizotinib-resistant ified CO2 incubator at 37 C. Cells were passaged for <3 months tumor lesions obtained from a patient with ALK-rearranged lung before renewal from frozen, early-passage stocks. Cells were cancer. We found that mesenchymal-type tumor cells and ALK regularly screened for Mycoplasma by using MycoAlert Mycoplas- resistance mutation can coexist in a single tumor lesion as inde- ma Detection Kits (Lonza). Cell lines authentication was per- pendent mechanisms underlying the ALK inhibitor–resistant formed by a short tandem repeat analysis in a laboratory at the cancers. Utilizing human EML4-ALK lung cancer cell lines, we National Institute of Biomedical Innovation (Osaka, Japan). further explored the mechanism by which EMT was induced Crizotinib, alectinib, ceritinib, lorlatinib, vorinostat, and quisi- during acquisition of crizotinib resistance and show the thera- nostat were obtained from Selleck Chemicals. SB43152 was peutic strategy to overcome EMT-mediated ALK-TKI resistance in purchased from Cayman Chemical. vitro and in vivo. Antibodies and Western blot analysis The primary antibodies, including anti-ALK (C26G7), anti- Materials and Methods phospho-ALK (Tyr1604), anti-phospho-Akt (S473), anti-Akt, Laser capture microdissection and digital PCR analysis anti–E-cadherin, anti–Zo-1, anti-claudin1, antivimentin, anti– Autopsy samples from a patient who acquired resistance to N-cadherin, anti-ZEB1, anti-Snail1, anti-Snai2, anti-Twist, anti- crizotinib were used for these tests. The lung tumor tissues PAI1, anti-MED12, anti–phospho-EGFR, anti–phospho-c-KIT, were first stained with antivimentin for IHC. Epithelial regions anti–c-KIT, anti–phospho-SRC, anti-SRC, anti–phospho-SMAD2, þ þ (ALK , vimentin , and E-cadherin ) and mesenchymal regions anti-SMAD2, and anti–b-actin were obtained from Cell þ þ (ALK , vimentin , and E-cadherin ) in a total of 29 slides (5 mm Signaling Technology. Anti-ERK1/ERK2, p-ERK1/ERK2 (T202/ each) were collected by Micro Laser Systems MBIII (Carl Zeiss Y204), and anti-EGFR were obtained from R&D Systems. Anti- Microscopy). SMAD7 was purchased from Santa Cruz Biotechnology. Western DNA extraction was performed following the manufacturer's blot analysis was as described previously (17). Phospho-RTK array recommendations with slight modifications. Briefly, microdis- Kit (ARY001B) was obtained from R&D Systems. sected formalin-fixed, paraffin-embedded (FFPE) sample were treated with proteinase K (Promega) for 16 hours at 70C. After Cell viability assay adding lysis buffer, DNA isolation was performed using a Max- Cell viability was measured by the MTT dye (24) reduction well16 FFPE Tissue LEV DNA Purification Kit on a Maxwell16 method. Cells, plated at a density of 2 103/100 mL RPMI-1640 Instrument (Promega) according to manufacturer's instructions. plus 10% FBS per well in 96-well plates, were incubated for 24 DNA was eluted in 50 mL of nuclease-free water, and the concen- hours. Crizotinib or alectinib were then added to each well, and tration was measured by a Quantus Fluorometor (Promega). incubation was continued for another 72 hours. Cell growth was measured with MTT solution (2 mg/mL; Sigma), as described Digital PCR reactions previously (17). Briefly, digital PCR reactions were performed using a Bio-Rad QX200 Droplet Digital PCR (ddPCR) platform. Reactions were set In vitro pretreatment with quisinostat up by ddPCR Supermix for Probes (no dUTP; Bio-Rad), LBx Cells were cultured in complete medium without inhibitor for Probe ALK L1196M (A071) for the mutation analysis, and LBx 48 hours, followed by treatment with the histone deacetylase Probe APOB CNV for the CNV analysis (RIKEN GENESIS Co., (HDAC) inhibitor quisinostat in a 10-cm dish for 24 to 72 hours. Ltd.). Thermal cycling was performed on a Veriti thermal cycler Cells were placed in 96-well plates and treated with crizotinib, and (Applied Biosystems) using the following thermal cycling proto- then incubated for another 72 hours for the MTT assay. col: 10 minutes 95C, 40 (30 seconds 94C, 1 minute 58C), 10 minutes 98C with the 50% ramp rate. Results were analyzed and Migration assay exported using the Quantasoft 1.6.6.0320 software. The cell migration assay was performed using Transwell (Sig- ma-Aldrich). A total of 1 105 cells in 200-mL medium were Cell culture and reagents placed in the upper chamber and the lower chamber was filled A human lung adenocarcinoma cell line A925LPE3 with an with 1-mL serum-free media. Cells were incubated for 24 hours, EML4-ALK fusion protein (variant 5a, E2:A20), which is highly nonmigrating cells were removed using cotton swabs, and the

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cells that migrated to the lower chamber were stained with 0.1% Sports, Science, and Technology, Japan. The protocol was crystal violet for 5 minutes. Cells were counted under a micro- approved by the Committee of Ethics of Experimental Animals, scope. The data presented are representative of three independent and the Advanced Science Research Center, Kanazawa University experiments. (Kanazawa, Japan).

Transfection of siRNA Luciferase expression analyses with the IVIS imaging system siRNA oligonucleotides specific to ALK and E-cadherin were After inoculation, the quantity of tumors was tracked in live obtained from Santa Cruz Biotechnology. siRNA specific to ZEB1 mice by repeated noninvasive optical imaging of tumor-specific (#1, #2) and siRNA negative control were purchased from Thermo luciferase activity using the IVIS Lumina XR Imaging System Fisher Scientific. Introduction of siRNA was performed using (PerkinElmer) as described previously (17, 22). Lipofectamine2000 (Thermo Fisher Scientific) in accordance with the manufacturer's instructions. Results Reporter assay EMT and ALK resistance mutation coexisted independently in a The miR-200c-141 promoter (1057 bp) was amplified from #4 single lesion in a patient with crizotinib-treated ALK- cells and ligated into the pNL2.2 [NlucP/Hygro] vector (4832 bp) rearranged lung cancer to generate the pNL2.2-miR200c construct. The selection of We first evaluated the presence of mesenchymal-type tumor correct clones was confirmed by restriction map analysis and cells, representing EMT, and ALK resistance mutation in clinical sequencing. Cells were transfected with pNL2.2-miR200c by specimens obtained from a patient before and after crizotinib Lipofectamine 2000. Twenty-four hours after transfection, cells treatment. The female patient was diagnosed by biopsy specimen were treated with drugs and after 24 hours, the NanoLuc luciferase from a subclavian lymph node metastasis as stage IV primary lung activity was measured using Nano-Glo Luciferase Assay System adenocarcinoma with ALK rearrangement. She received crizotinib (Promega). Each transfection was carried out in triplicate in treatment and showed partial response. However, 4 months after 96-well plates. the initiation of crizotinib, she died due to acute disease progression. Specimens from the primary lung tumor, as well as from the qPCR brain and subcutaneous metastases were obtained at autopsy. In For RNA quantification, reverse transcription of the collected all specimens, the presence of ALK-rearranged tumor cells was RNAs was performed using SuperScript VILO cDNA synthesis Kit confirmed by IHC for ALK. In a pre-crizotinib tumor obtained and Master Mix (Invitrogen). TaqMan Gene Expression Assays from the diagnostic subclavian lymph node, we found that (Applied Biosystems) for MED12 (#1726657) were used accord- almost all tumor cells were E-cadherin–positive/vimentin- ing to the manufacturer's protocol. qPCR analysis was performed negative, indicating the epithelial phenotype (Fig. 1A). In this with ViiA7 Real-Time PCR system (Applied Biosystems). The tumor, known ALK mutations associated with ALK-TKI resistance DDC relative miRNA levels were calculated using the formula 2 t. were not detected. On the other hand, in specimens from the The data were normalized with respect to the expression of ACTB crizotinib-treated primary lung tumor, brain and subcutaneous (#4331182). For miRNA quantification, TaqMan MicroRNA metastases, we found both the E-cadherin–positive/vimentin- Assays (Applied Biosystems) for miR-200c (#002300) and negative tumor cells and the E-cadherin–negative/vimentin- miR-141 (#000463) were used according to the manufacturer's positive tumor cells, indicating coexistence of epithelial and protocol. qPCR analysis was performed with a 7900HT Fast Real- mesenchymal phenotype tumor cells in the same lesion Time PCR System (Applied Biosystems). The relative miRNA (Fig. 1A; Supplementary Fig. S1). Unexpectedly, ALKL1196M, the DD levels were calculated using the formula 2 Ct. The data were gatekeeper mutation, which confers resistance to crizotinib, was normalized with respect to the expression of RNU6B (#001093). detected in all crizotinib-treated tumors obtained at autopsy (Fig. 1A). These results indicate that mesenchymal-type tumor Pleural carcinomatosis model in SCID mice cells may exist after crizotinib treatment even in the ALK resistance We used 5-week-old female SCID mice (Clea) for the study. For mutation–positive tumor lesion. the pleural carcinomatosis model (22), the skin and subcutane- To investigate whether the mesenchymal type tumor cells ous tissue on the right side of the chest were cut and the parietal acquire ALKL1196M mutation, we sought to measure the copy pleura were exposed. Tumor cells (1 106/100 mL) were then number of ALKL1196M in epithelial and mesenchymal-type tumor injected into the right thoracic cavity through the parietal pleura lesions, separately. We first stained the primary lung tumor for by using a 27-G needle. Subsequently, the incisions were sutured E-cadherin and vimentin, respectively, by IHC (Fig. 1B), followed to close the wound. One week after inoculation, mice were treated by isolated the E-cadherin–positive/vimentin-negative lesions orally with ALK-TKIs, 50 mg/kg of crizotinib, or 20 mg/kg of and the E-cadherin–negative/vimentin-positive lesions, respec- alectinib. Then, the mice were randomized into three arms for the tively, by laser capture microdissection (LCM; Fig. 1C). Pathol- ALK-TKI continuation, 10 mg/kg quisinostat for 5 days followed ogists carefully confirmed that almost all cells in the lesions by no treatment, or 10 mg/kg quisinostat for 5 days followed by selected for LCM were tumor cells. We then evaluated the copy the ALK-TKI treatment. The luciferase expression of tumors and number of ALKL1196M and wild-type ALK in the epithelial and mouse body weights were measured twice per week. All surgeries mesenchymal type tumor cell lesions, separately. Very interest- were carried out under sodium pentobarbital anesthesia, and ingly, more than 12 copies of ALKL1196M mutation were detected efforts were made to minimize the suffering of the animals. All in 1 mg of total DNA from the epithelial-type tumor cell lesion animal experiments in this study were performed in strict accor- (accounting for 27% of all ALK alleles including the wild-type dance with the recommendations in the Guide for the Care and ALK), by sharp contrast, ALKL1196M mutation was hardly detected Use of Laboratory Animals of the Ministry of Education, Culture, (only less than 2 copies in 1 mg DNA and accounting for 5% of all

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Figure 1. ALK mutation was predominantly detected in the epithelial type of the tumor cells from a patient who acquired resistance to crizotinib. A, Biopsy samples from patients with EML4-ALK lung cancer before crizotinib treatment and autopsy samples from lung and subcutaneous metastases of the patient who acquired resistance to crizotinib were stained with anti-ALK, E-cadherin, and vimentin for IHC analysis. Summary of EMT status and ALK-L1196M mutation is shown. Scale bar, 100 mm. B, Epithelial (E-cadherinþ, vimentin) and mesenchymal (E-cadherin, vimentinþ) regions in the lung tumor are marked, respectively. Scale bar, 2mm. C, The laser-dissected regions of the tumor tissue that was picked up and separated for analysis by digital PCR. Scale bar, 2 mm. ALK-L1196M gene copy number in the lung tumor (D)andALK-L1196M mutation ratio (E) of dissected epithelial and mesenchymal regions in the lung tumor and the subcutaneous metastasis as estimated by digital PCR (n ¼ 5). WT, wild type. Error bars, SEM. , P < 0.001.

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ALK alleles including the wild-type ALK) in the mesenchymal type and increased, respectively, in the most resistant #4 cells, com- tumor cell lesion (Fig. 1D and E). EML4-ALK fusion gene has been pared with the parental A925LPE3 cells (Fig. 3C). In addition, the known to be heterozygous on the chromosome, and we found resistant cells increased cell motility than the parental A925LPE3 that ALK gene copy number was not increased in the crizotinib- cells (Fig. 3D and E). These findings indicate that the three clones, resistant specimens based on the digital PCR analysis. If hypoth- #3, #4, and #8, acquired crizotinib resistance predominantly esizing that the lesion consisted of only tumor cells, only 10% of by EMT. the mesenchymal-type tumor cells had the ALKL1196M mutation, indicating that about 90% of tumor cells acquired crizotinib Downregulation of miR-200 family members resulted in resistance in the absence of ALK mutation in the mesenchymal- induction of EMT in ALK-rearranged NSCLC cells type lesion. Furthermore, in the subcutaneous metastasis, we We next sought to clarify the mechanism by which these three isolated the E-cadherin–positive/vimentin-negative lesions and clones developed a mesenchymal phenotype. A recent study the E-cadherin–negative/vimentin-positive lesions, respectively, reported that the classical EMT inducer, TGF-b,causedALK by LCM. We found that ALKL1196M mutation was detected only in inhibitor resistance in an ALK-rearranged NSCLC cell line, the epithelial tumor cell lesions, not in the mesenchymal type H2228 (25). We observed that TGF-b1treatmentreduced tumor lesions (Fig. 1E). These results strongly show that EMT is an E-cadherin expression and slightly induced crizotinib resistance independent resistant mechanism against ALK-targeted therapy. in H2228 cells, but not in other ALK-rearranged NSCLC cell lines, such as A925LPE3 or H3122 (Supplementary Fig. S3A and Establishment of crizotinib-resistant EML4-ALK lung cancer S3B). In addition, treatment of TGF-b1 inhibitor SB431542 did cells associated with EMT not revert EMT and restore the sensitivity against crizotinib in To investigate the mechanisms by which EMT cause ALK-TKI these clone cells (Supplementary Fig. S3C and S3D). As shown resistance, we sought to establish human ALK-rearranged lung in Fig. 3A and Supplementary Fig. S3E, the expression cancer cell lines, which acquire EMT-dependent crizotinib resis- of MED12, whose suppression was reported to induce mesen- tance. We first induced crizotinib resistance in a pleural effusion chymal phenotype through activation of TGF-b signaling in mouse model inoculated with an EML4-ALK lung cancer cell line, ALK-rearranged lung cancer cells (26), was not suppressed in the A925LPE3 (22). After continuous treatment of the recipient mice resistant clones, compared with the parental A925LPE3 cells. with crizotinib, they experienced the relapse of pleural carcino- Furthermore, these three clones did not produce detectable matosis, which acquired resistance to crizotinib. We then gener- levels of TGF-b, and the expression of multiple TGF-b target ated a cell line, named APE-CR, from the pleural effusion, and also genes including PAI-1, Smad7 in these clones were not activated established 15 clones by limiting dilution. Of these, we focused (Fig. 3A). These data suggest that EMT in these three clones was on clones #3, #4, and #8 because they showed a spindle-like induced by mechanisms other than activation by TGF-b. mesenchymal morphology, whereas the parental A925LPE3 cells ZEB1 is known to be an E-cadherin transcriptional repressor had the cobblestone-like epithelial morphology (Fig. 2A). Next, and plays a crucial role in EMT (27), and we found that the we examined the sensitivity of selective ALK inhibitors to the expression of ZEB1 was increased in the three clones, whereas generated cells. As shown in Fig. 2B, the three clones were over six Snail1, Snail2, and Twist, which have been reported as transpri- times more resistant to crizotinib compared with parental tional factors associated with EMT were not increased in these A925LPE3 cells, and cross-resistant to the next-generation ALK- clones (Fig. 3A). Thus, we first transfected cells from clone #4 with TKIs alectinib, ceritinib, and lorlatinib. Furthermore, crizotinib ZEB1-specific siRNA and analyzed the expression of E-cadherin. effectively inhibited ALK phosphorylation in both the parental As we expected, ZEB1-specific siRNA reduced the expression of A925LPE3 cells and these three clones, whereas downstream ZEB1 and increased the expression of E-cadherin (Fig. 4A). Inter- phospho-AKT and phospho-ERK were inhibited in parental estingly, we found that the knockdown of ZEB1 restored the A925LPE3 cells, but not in the three clones (Fig. 2C). Consistently, sensitivity to crizotinib (Fig. 4B; Supplementary Fig. S4A and knockdown of ALK by specific siRNA inhibited the viability of S4B) in these three clones. Because ZEB1 expression can be parental A925LPE3 cells, but not of the three clones (Fig. 2D). In regulated by miRNAs (28, 29), we then conducted a miRNA addition, known resistance mutations in ALK were not detected in profile analysis of the parental and #4 cells. As shown the parental A925LPE3 cells or these three clones. These findings in Fig. 4C, the expression of miR-200 family members including indicate that these three clones acquire resistance by an ALK- miR-141 and miR-200c was remarkably decreased in #4 cells independent resistance mechanism. compared with the parental cells. miR-200 family is known to EGFR activation is a known mechanism of crizotinib resis- play an important role in regulation of EMT (28). In addition, tance (12); however, we did not observe EGFR activation in these overexpression of miR-200c was reported to cause mesenchymal- three clones (Supplementary Fig. S2A). Hence, combined use of to-epithelial transition (MET; ref. 30). Consistently, we observed EGFR inhibitors did not restore crizotinib sensitivity of these that overexpression of miR-141 or miR-200c by transfection clones (Supplementary Fig. S2B). Furthermore, other receptor decreased the ZEB1 expression and increased the E-cadherin tyrosine kinases reported as ALK-TKI resistance mechanisms expression in #4 cells (Supplementary Fig. S5A). These findings including FGFR, HER3, MET, IGF-1R, c-KIT, and SRC were not indicate that these clones acquired crizotinib resistance via EMT activated in these clones (Supplementary Fig. S2A and S2C). On mediated by decreased expression of miR-200 family. the other hand, these clones showed typical mesenchymal phenotype, such as decreased epithelial marker (E-cadherin, Zo-1, Restored miR-200c expression reversed EMT and resensitized and claudin1) expression, increased mesenchymal marker tumor cells to crizotinib (vimentin, N-cadherin, and ZEB1) expression (Fig. 3A and B). We thus hypothesized that chemical restoration of miR-200c/ Microarray analysis confirmed that the expression of several 141 may reverse EMT and resensitize the tumor cells to ALK-TKIs. epithelial and mesenchymal phenotype–related genes decreased The miR-200c/141 is transcribed as one RNA molecule and then

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Figure 2. Crizotinib-resistant A925LPE3 clones were generated in vivo. A, A925LPE3 parental and the resistant clones (#3, #4, and #8) were evaluated to determine any morphologic changes consistent with EMT using a light microscope. Scale bar, 100 mm. B, Growth inhibition assessed by MTT assay of A925LPE3, APE-CR, and the clones treated with crizotinib, alectinib, ceritinib, or lorlatinib for 72 hours, respectively (n ¼ 3). Results are

expressed as mean SEM. IC50 values were calculated and demonstrated. C, A925LPE3 parental and the resistant clones were treated with 1 mmol/L crizotinib for 2 hours. The changes of phosphorylation of indicated proteins were analyzed by Western blotting. D, Control and ALK siRNA were introduced into parental and resistant cells, and ALK knockdown was conﬁrmed by Western blot analysis. Cell viability was analyzed by MTT assay 72 hours later (n ¼ 3). , P < 0.001.

cleaved as miR-200c and miR-141, respectively (29). Therefore, generation" HDAC inhibitor, has a high potency toward class I to identify compounds that stimulate the miR-200c/141 promo- HDACs with a more prolonged pharmacodynamics response tor activity, we first transfected #4 cells with a nano-luc (Nluc) in vivo than "first-generation" HDAC inhibitors (35–37). We expression vector (Supplementary Fig. S5B and S5C) and per- indeed confirmed that the treatment of quisinostat remarkably formed a reporter assay on a 200 kinase inhibitor library (Sup- increased the expression of miR-200c and miR-141 (Fig. 4F) and plementary Table S1). As shown in Fig. 4D, CUDC-101, an decreased that of ZEB1 (Fig. 4G). Furthermore, transfection of inhibitor of EGFR, HER2, and class I/class II HDACs (31, 32), antagomiR-200c, but not antagomiR-141, canceled the down- had the highest potential to increase miR-200c/141 promotor regulation of ZEB1 by quisinostat in the #4 cells (Fig. 4G). These activity. HDAC2, a member of class I HDACs, has been reported to findings suggest that quisinostat decreases ZEB1 expression pre- play a critical role in suppression of the miR-200 family in breast dominantly by upregulating miR-200c. cancer cells (33). In addition, mocetinostat, an inhibitor of class I HDACs, was reported to cancel ZEB1-associated drug resistance in Quisinostat reversed EMT and restored the sensitivity to several types of tumor cell lines (34). Thus, we further investigated ALK-TKIs the effect of various class I HDAC inhibitors on the miR-200c/141 We next tested the hypothesis that quisinostat may restore promotor activity. As shown in Fig. 4E, quisinostat most potently ALK-TKI sensitivity by reverting EMT in the #4 cells. To determine increased the miR-200c/141 promotor activity among the tested the optimum concentration for quisinostat, we first used the HDAC inhibitors, including CUDC-101. Quisinostat, a "second- normal lung fibroblast lines, MRC-5 and IMR-90. Because

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Figure 3. EMT is induced in the crizotinib- resistant A925LPE3 clones. A, The expression of EMT markers was analyzed by Western blotting with the indicated antibodies. B, Immunoﬂuorescence assay of EMT markers. Scale bar, 100 mm. C, Heatmap representation of microarray data showing the expression levels of several key EMT-related genes in parental A925LPE3 and #4 cells. D, Wound closure was evaluated for 48 and 72 hours after scratch in A925LPE3 or the resistant clones. Scale bar, 400 mm. E, The migration assays were performed using Transwell. Cells were incubated for 24 hours, and the cells that penetrated through the ﬁlter were stained and counted using a light microscope. n ¼ 3. Error bars, SEM. , P < 0.001.

0.03 mmol/L quisinostat had almost no effect on the viability of in the resistant cells. However, when we pretreated #4 cells with the fibroblast lines, we utilized this concentration of quisinostat quisinostat, the phosphorylation of AKT was strongly inhibited by in the in vitro experiments (Supplementary Fig. S6A). As shown crizotinib (Supplementary Fig. S8A), suggesting that AKT signal- in Fig. 5A and B, 0.03 mmol/L quisinostat upregulated the ing plays an important role in the acquisition of crizotinib expression of E-cadherin and downregulated the expression of resistance in the cells with a mesenchymal phenotype. N-cadherin, vimentin, and ZEB1 in a time-dependent manner. To further investigate whether resensitization to ALK-TKIs by Accordingly, treatment of #4 cells with quisinostat for 3 days quisinostat depends on the expression of EMT marker proteins, #4 reduced their motility and induced epithelial morphology (Sup- cells were transfected with E-cadherin-specific siRNA and then plementary Fig. S6B and S6C). To investigate whether quisinostat treated with quisinostat for 72 hours. We found that siRNA could reverse the sensitivity to crizotinib, we pretreated #4 cells successfully blocked the quisinostat-induced E-cadherin expres- with quisinostat for 3 days, and then treated them with crizotinib sion (Fig. 5D), and almost completely canceled the effect of for another 3 days (Fig. 5C). Importantly, pretreatment of quisi- quisinostat on restoring the sensitivity to crizotinib (Fig. 5E). nostat restored sensitivity to crizotinib in these three clones to the These results indicate that quisinostat can resensitize ALK- level observed in the parental A925LPE3 cells. Furthermore, rearranged lung cancer cells with mesenchymal phonotype to pretreatment of quisinostat also restored sensitivity to the sec- ALK-TKIs by reverting the EMT. ond-generation ALK-TKIs, alectinib, ceritinib, and lorlatinib The same phenomenon was observed in the EGFR-mutated (Fig. 5C; Supplementary Fig. S7). As mentioned in Fig. 2C, cri- NSCLC cell lines, HCC827KGR and H1975OR, which acquired zotinib alone did not inhibit the phosphorylation of AKT or ERK resistance to gefitinib and osimertinib (AZD9291), respectively,

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Figure 4. Quisinostat reverted EMT by upregulating miR-200c expression through inhibition of ZEB1. A, Control and ZEB1 siRNA were introduced into #4 cells, and cell lysates were analyzed by Western blot analysis with the indicated antibodies 48 hours later. B, After the transfection of ZEB1 siRNA for 48 hours, cells were

treated with crizotinib, and the cell viability was measured 72 hours later by MTT assay (n ¼ 4). Each IC50 of crizotinib is shown. C, Heatmap representation of miRNA array data showing the expression levels of miRNAs in decreasing order of fold change more than 10 between parental A925LPE3 and #4 cells. The levels of the miR-200a, 200b, and 429 included in miR-200 family members are shown in the lower columns of the heatmap (n ¼ 2). D, Screening of drugs that induce expression of miR-200c-141 in #4 cells is shown on the heatmap. The #4 cells were transfected with pNL2.2-miR200c and treated with a library including 200 kinase inhibitors. The luciferase activity was measured after 24 hours. E, Change of miR200c-141 promoter activity in #4 cells treated with 6 HDAC inhibitors (n ¼ 3). Error bars, SEM. , P < 0.01. F, The #4 cells were treated with 0.03 mmol/L quisinostat and examined for miR-200c and miR-141 expression by qRT-PCR (n ¼ 3). Error bars, SEM. , P < 0.01. G, The #4 cells were treated with antago–miR-200c or antago–miR-141, followed by treatment of 0.03 mmol/L quisinostat for 72 hours. Cell lysates were analyzed by Western blotting with the indicated antibodies.

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Figure 5. Pretreatment of quisinostat reverted EMT and restored sensitivity to crizotinib. A and B, The change of EMT markers in #4 cells treated with 0.03 mmol/L quisinostat was analyzed by Western blotting (A) and by immunoﬂuorescence (B). Scale bar, 100 mm. C, The #4 cells were pretreated with 0.03 mmol/L quisinostat for 3 days, followed by crizotinib or alectinib for 72 hours. Cell viability was measured by MTT assay (n ¼ 3). Error bars, SEM. , P < 0.01. D, Control and E-cadherin siRNA were introduced to #4 cells, followed by pretreatment with 0.03 mmol/L quisinostat for 72 hours. The expression of E-cadherin was conﬁrmed by Western blotting. E, After pretreatment with quisinostat, cells were treated with 1 mmol/L crizotinib, and cell viability was measured 72 hours later using the MTT assay (n ¼ 3). Error bars, SEM. , P < 0.01.

associated with EMT. We found that pretreatment of Sequential therapy with quisinostat resensitized the tumors to HCC827KGR and H1975OR cells with quisinostat reversed the crizotinib or alectinib in the in vivo pleural carcinomatosis EMT and sensitized the cells to geﬁtinib and osimertinib, model respectively (Supplementary Fig. S8B and S8C). These results To examine the effect of quisinostat in vivo in the pleural suggest that quisinostat may overcome EMT-associated TKI carcinomatosis model, we inoculated #4 cells into the thoracic resistance in several types of lung cancer cells with different cavities, where this clone acquired crizotinib resistance, in the driver oncogenes. SCID mice. The mice were continuously treated with crizotinib or

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Figure 6. Sequential therapy with quisinostat and ALK-TKI in vivo. A and B, The #4 cells (1 106/100 mL) were injected into the right thoracic cavity of SCID mice, and after a week of the inoculation, the mice were treated with 50 mg/kg of crizotinib (A) or 20 mg/kg of alectinib (B). The mice were randomized into three arms for the ALK-TKI continuation, 10 mg/kg quisinostat for 5 days followed by no treatment, or 10 mg/kg quisinostat for 5 days followed by the ALK-TKI treatment. The luciferase expression of tumors and mouse body weights were measured twice per week (n ¼ 4). Error bars, SEM. , P < 0.05. #, mice treated with quisinostat for 5 days without crizotinib retreatment became moribund by day 32. C, The expression of EMT marker proteins of the pleural tumor from a mouse treated with crizotinib/quisinostat and from a mouse treated with alectinib/quisinostat was collected on day 26 and day 59, respectively. D, The expression of E-cadherin of the pleural tumor from a mouse treated with crizotinib/quisinostat was analyzed by IHC. E, Summary scheme outlining the heterogeneity of crizotinib resistance mechanisms in ALK-rearranged lung cancer and therapeutic strategy for overcoming the resistance.

alectinib, and we confirmed the progression of pleural carcino- (Fig. 6A and B). During the treatment with quisinostat, no matosis, indicating ALK-TKI resistance. On the basis of the results macroscopic changes or loss in body weight were observed in of the in vitro experiments, we then pretreated the mice with or the mice compared with control. Furthermore, we confirmed that without quisinostat (10 mg/kg) for 5 days, and thereafter, treated quisinostat treatment for 5 days increased the expression of them with crizotinib or alectinib. As shown in Fig. 6A, the mice E-cadherin and decreased that of vimentin in tumor cells obtained treated with quisinostat for 5 days without crizotinib retreatment from the thoracic cavity (Fig. 6C; Supplementary Fig. S9A). became moribund by day 32, indicating that quisinostat treat- Similar results were also observed by IHC: E-cadherin was restored ment alone was not enough for controlling crizotinib-resistant to the levels observed in parental A925LPE3 tumors after the pleural carcinomatosis. Interestingly, pretreatment with quisino- quisinostat treatment (Fig. 6D). In addition, repeated CT scans stat followed by crizotinib or alectinib successfully regressed confirmed disappearance of pleural effusion in mice pretreated pleural carcinomatosis, whereas exclusive continuous treatment with quisinostat followed by crizotinib (Supplementary Fig. S9B). with crizotinib or alectinib failed to control disease progression These findings strongly indicate that quisinostat is capable of

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reverting EMT and restoring the sensitivity of mesenchymal-type in EGFR-mutated NSCLC. Quisinostat is a second-generation tumor cells to ALK-TKIs even in vivo. HDAC inhibitor, with potent activity to HDAC 1 and 2. However, the precise mechanism by which quisinostat increases miR-200c promotor activity is still unknown. Identification of such mech- Discussion anism may allow the development of compounds that overcome In this study, we demonstrated that although mesenchymal- EMT-associated targeted drug resistance much more efficiently type tumor cells and the ALKL1196M mutation coexisted in a single and selectively. crizotinib-resistant lesion, the ALKL1196M mutation was predom- Recent clinical trials, J-ALEX (9) and ALEX (10), demonstrated inantly detected in epithelial-type tumor cells. These new findings that alectinib given as a first-line treatment remarkably prolonged indicate that the mesenchymal phenotype caused by EMT is an the PFS of advanced NSCLC with ALK rearrangement, as com- independent mechanism that can induce crizotinib resistance in pared with crizotinib. Nevertheless, although alectinib has activity patients with ALK-rearranged lung cancer. We further found over a spectrum of crizotinib-resistant mutations, crizotinib does that the tumor cells with mesenchymal phenotype were cross- not inhibit the majority of alectinib-resistant mutations (13). resistant to the new-generation ALK-TKIs, such as alectinib, cer- Thus, the best sequence of ALK-TKIs for ALK-rearranged NSCLC itinib, and lorlatinib. Very importantly, pretreatment with has not yet been determined (39, 40). If the coexistence of the HDAC inhibitor quisinostat, which upregulated the miR- EMT components and ALK mutations induces resistance to 200c/141 promotor activity, restored miR-200c expression, and first-line crizotinib, the same would be expected in relation to reverted EMT, followed by treatment with an ALK inhibitor resistance to second-generation ALK-TKIs with activity against could circumvent the resistance due to EMT in ALK-rearranged crizotinib-resistant ALK mutations. In this study, we demonstrat- lung cancer cells with mesenchymal phenotype. ed that pretreatment with quisinostat could overcome alectinib A recent study by Gainor and colleagues reported that EMT resistance caused by EMT-associated crizotinib resistance in could be observed in the tumors of patients with ALK- rear- tumor cells from a pleural carcinomatosis model. Therefore, the ranged NSCLC treated with ALK-TKIs, including ceritinib. They therapeutic strategy of pretreatment with quisinostat followed by detected mesenchymal tumor cells, defined by loss of E-cad- second-generation ALK-TKIs may be effective for overcoming herin and expression of vimentin, in five of 12 clinical speci- targeted drug resistance where ALK resistance mutations and EMT mens (13). Of the five specimens, ALK-resistant mutations were components coexist (Fig. 6E). Further studies are warranted to detected in three. Thus, they suggested that EMT may be not the clarify the involvement of EMT in resistance to second-generation sole driver of resistance. However, our digital PCR analyses ALK-TKIs given as the first-line treatment in NSCLC with ALK- combined with LCM after IHC staining for E-cadherin and rearrangement. vimentin demonstrated that the ALKL1196M mutation was not In conclusion, we demonstrated that the mesenchymal phe- detected in about 90% of tumor cells with mesenchymal notype caused by EMT can coexist with ALK mutation as an phenotype in a crizotinib-treated tumor, indicating that EMT independent resistance mechanism in ALK inhibitor–resistant could be an independent crizotinib-resistant mechanism in tumors. Moreover, pretreatment with the HDAC inhibitor quisi- ALK-TKI–resistant tumors. Gainor and colleagues also sug- nostat, which upregulates the miR-200c/141 promotor activity, gested that EMT, which was not observed before ALK-TKI restores miR-200c expression, and reverts EMT, followed by an treatment, might have been the predominant mechanism of ALK inhibitor, can circumvent the resistance due to EMT. These resistance in a tumor also exhibiting a ALKL1196M mutation, findings illustrate the importance of developing compounds that which was heavily treated with crizotinib, alectinib, and cer- selectively and efficiently increase miR-200c expression, and itinib (13). In our preclinical finding, ALK-rearranged lung provide a rationale for their sequential use with new-generation cancer cells with mesenchymal phenotype without ALK-resis- ALK inhibitors to treat TKI-resistant ALK-rearranged NSCLC with tant mutations induced cross-resistance to crizotinib and these ALK-resistant mutation and EMT. second-generation ALK-TKIs, suggesting that only EMT could play a role in conferring the resistance, thus supporting our Disclosure of Potential Conflicts of Interest hypothesis. S. Nanjo reports receiving a commercial research grant and has received Circumvention of EMT-associated resistance, based on under- speakers bureau honoraria from AstraZeneca. T. Nakagawa is an associate standing the underlying mechanisms, is an unmet need of tar- director at Eisai Co., Ltd. Y. Ishikawa is a consultant/advisory board member geted drug therapy. We found that EMT could be induced by for Fujirebio Inc. K. Takeuchi has ownership interest (including patents) as reducing the expression levels of miR-200 family members, royalty from Nichirei and is a consultant/advisory board member for Nichirei. S. including miR-200c and miR-141, which resulted in increasing Yano has received a commercial research grant from Chugai and has received fi fl ZEB1 and decreasing E-cadherin expression in ALK-TKI–resistant speakers bureau honoraria from P zer and Chugai. No potential con icts of interest were disclosed by the other authors. ALK-rearranged NSCLC cells. These observations are in the line with the evidence reported in pancreatic, colorectal, and breast cancers (38). Moreover, we provided the first demonstration that Authors' Contributions the second-generation HDAC inhibitor quisinostat could over- Conception and design: K. Fukuda, S. Takeuchi, S. Yano come EMT-associated ALK-TKI resistance by reverting EMT, which Development of methodology: K. Fukuda, S. Takeuchi, R. Katayama was mediated by enhanced expression of miR-200c but not miR- Acquisition of data (provided animals, acquired and managed patients, 141, via upregulated miR200c/141 promotor activity. We further provided facilities, etc.): K. Fukuda, S. Takeuchi, S. Arai, R. Katayama, showed that pretreatment with quisinostat could overcome resis- S. Nanjo, T. Nakagawa, H. Taniguchi, T. Suzuki, T. Yamada, H. Nishihara, H. Ninomiya, Y. Ishikawa, S. Baba, A. Horiike, N. Yanagitani, M. Nishio tance to both crizotinib and alectinib in the pleural carcinoma- Analysis and interpretation of data (e.g., statistical analysis, biostatistics, tosis model. Very importantly, our findings also suggest that computational analysis): K. Fukuda, S. Takeuchi, R. Katayama, S. Nanjo, pretreatment with quisinostat may overcome EGFR-TKI resistance A. Tanimoto, K. Takeuchi, M. Nishio

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Intratumor Heterogeneity in ALK Inhibitor Resistance

Writing, review, and/or revision of the manuscript: K. Fukuda, S. Takeuchi, S. Yano), and the Project for Cancer Research and Therapeutic S. Nanjo, M. Nishio, S. Yano Evolution (P-CREATE) grant number 16cm0106513h0001 to S. Yano. Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): S. Takeuchi, R. Katayama, A. Tanimoto, A. Nishiyama, K. Takeuchi, M. Nishio, S. Yano The costs of publication of this article were defrayed in part by the Study supervision: S. Takeuchi, S. Yano payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Acknowledgments this fact. This work was supported by grants from the Japanese Society for the Promotion of Science (JSPS) KAKENHI (grant numbers 16K19447 Received July 13, 2018; revised December 14, 2018; accepted January 31, and 18K07261 to K. Fukuda; 17K09649 to S. Takeuchi; 16H05308 to 2019; published ﬁrst February 8, 2019.

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Epithelial-to-Mesenchymal Transition Is a Mechanism of ALK Inhibitor Resistance in Lung Cancer Independent of ALK Mutation Status

Koji Fukuda, Shinji Takeuchi, Sachiko Arai, et al.

Cancer Res 2019;79:1658-1670. Published OnlineFirst February 8, 2019.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-18-2052

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