Foretinib Overcomes Entrectinib Resistance Associated with The

Published OnlineFirst February 20, 2018; DOI: 10.1158/1078-0432.CCR-17-1623

Cancer Therapy: Preclinical Clinical Cancer Research Foretinib Overcomes Entrectinib Resistance Associated with the NTRK1 G667C Mutation in NTRK1 Fusion–Positive Tumor Cells in a Brain Metastasis Model Akihiro Nishiyama1, Tadaaki Yamada1,2, Kenji Kita1, Rong Wang1, Sachiko Arai1, Koji Fukuda1, Azusa Tanimoto1, Shinji Takeuchi1, Shoichiro Tange3, Atsushi Tajima3, Noritaka Furuya4,5, Takayoshi Kinoshita4, and Seiji Yano1

Abstract

Purpose: Rearrangement of the neurotrophic tropomyosin Results: KM12SM-ER cells, which showed moderate resis- receptor kinase 1 (NTRK1) gene, which encodes tyrosine receptor tance to entrectinib in vitro, had acquired the G667C mutation kinase A (TRK-A), occurs in various cancers, including colon in NTRK1. The kinase inhibitor foretinib inhibited TRK-A cancer. Although entrectinib is effective in the treatment of central phosphorylation and the viability of KM12SM-ER cells bearing nervous system (CNS) metastases that express NTRK1 fusion the NTRK1-G667C mutation in vitro. Moreover, foretinib proteins, acquired resistance inevitably results in recurrence. The markedly inhibited the progression of entrectinib-refractory CNS is a sanctuary for targeted drugs; however, the mechanism by KM12SM-ER–derived liver metastases and brain tumors in which CNS metastases become entrectinib-resistant remains elu- animal models, predominantly through inhibition of TRK-A sive and must be clariﬁed to develop better therapeutics. phosphorylation. Experimental Design: The entrectinib-resistant cell line Conclusions: These results suggest that foretinib may be effec- KM12SM-ER was developed by continuous treatment with entrec- tive in overcoming entrectinib resistance associated with the tinib in the brain metastasis–mimicking model inoculated with NTRK1-G667C mutation in NTRK1 fusion–positive tumors in the entrectinib-sensitive human colon cancer cell line KM12SM, various organs, including the brain, and provide a rationale for which harbors the TPM3-NTRK1 gene fusion. The mechanism of clinical trials of foretinib in cancer patients with entrectinib- entrectinib resistance in KM12SM-ER cells was examined by next- resistant tumors harboring the NTRK1-G667C mutation, includ- generation sequencing. Compounds that overcame entrectinib ing patients with brain metastases. Clin Cancer Res; 24(10); 2357–69. resistance were screened from a library of 122 kinase inhibitors. 2018 AACR.

Introduction partners, such as LMNA, TPM3, SQSTM1,andBCAN, have been reported (3–6). The NTRK1 fusion gene products promote The neurotrophic tropomyosin receptor kinase 1 (NTRK1) signal transduction through the PI3K-AKT, PLCg-PKC, and gene, located at 1q21–q22, encodes tyrosine receptor kinase A SHC-RAS-MAPK signaling pathways and play crucial roles in (TRK-A), which is a member of the TRK family (1). The cell survival (7). Several compounds, including crizotinib and rearrangement of the NTRK1 locusisrecognizedasanonco- entrectinib, have been shown to inhibit the growth of tumor genic driver and occurs across different tumors, including non– cells that express NTRK1 fusion proteins and have demonstrat- small cell lung cancer (NSCLC) and colon cancer, though at a ed remarkable clinical response in patients with NTRK1 fusion– low incidence (1%; ref. 2). At least four NTRK1 fusion positive tumors (8). Entrectinib is an orally available inhibitor of multiple tyro- 1Division of Medical Oncology, Cancer Research Institute, Kanazawa University, sine kinases, including ALK, ROS1, and TRK-A/TRK-B/TRK-C Kanazawa, Japan. 2Department of Pulmonary Medicine, Graduate School of (9). Entrectinib has been shown to be active and well tolerated Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan. in NTRK1 fusion–positive tumor patients, including patients 3Department of Bioinformatics and Genomics, Graduate School of Advanced with central nervous system (CNS) metastases (10), and is Preventive Medical Sciences, Kanazawa University, Kanazawa, Japan. 4Graduate 5 currently being developed in a phase II clinical trial School of Science, Osaka Prefecture University, Osaka, Japan. Kissei Pharma- (NCT02568267). However, the response to entrectinib is lim- ceutical, Nagano, Japan. ited in time due to acquired resistance. Recent reports have Note: Supplementary data for this article are available at Clinical Cancer demonstrated that the NTRK1-G595R and G667C mutations Research Online (http://clincancerres.aacrjournals.org/). drive acquired resistance to entrectinib in colon cancer with Corresponding Author: Seiji Yano, Cancer Research Institute, Kanazawa NTRK1 fusion products (11). No effective drug that overcomes University, 13-1, Takaramachi, Kanazawa, Ishikawa 920-0934, Japan. Phone: the resistance associated with the NTRK1-G595R and G667C 81-76-265-2780; Fax: 81-76-234-4524; E-mail: [email protected] mutations has been developed yet. doi: 10.1158/1078-0432.CCR-17-1623 Targeted drugs, such as EGFR tyrosine kinase inhibitors (EGFR- 2018 American Association for Cancer Research. TKI) and ALK-TKIs, are generally effective in the treatment of

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group in 1999. KM12SM cells are a highly metastatic variant of Translational Relevance KM12C cells, which present the TPM3-NTRK1 gene rearrange- The central nervous system (CNS) is a sanctuary site from ment (17). The KM12SM cell line (which generates spontaneous targeted drugs. However, the mechanism of targeted drug metastasis) was established from liver metastases after orthotopic resistance in CNS tumors is largely unknown. Entrectinib, an cecum inoculation of cultured KM12C cells in nude mice (19). inhibitor of multiple kinases such as ALK, ROS1, and TRK, is KM12SM cells were maintained and cultured in RPMI-1640 clinically effective in the treatment of cancer patients with medium (Thermo Fischer Scientific K.K.) with 10% FBS, penicillin rearrangements in ALK, ROS1, and NTRK1. Although entrec- (100 units/mL), and streptomycin (50 mg/mL), in a humidified tinib has initial activity against CNS metastases, resistance CO2 incubator at 37 C. Ba/F3, Ba/F3 transfected with wild- usually develops. Here, we found that the NTRK1-G667C type TPM3-NTRK1 (Ba/F3_WT), Ba/F3 transfected with TPM3- mutation, which causes moderate resistance to entrectinib NTRK1 G595R (Ba/F3_G595R), and Ba/F3 transfected with in vitro, could be detected in a brain metastasis–mimicking TPM3-NTRK1 G667C (Ba/F3_G667C) cells were gifted by model when acquired resistance to entrectinib was induced. Dr. R. Katayama (Division of Experimental Chemotherapy, Can- Furthermore, we demonstrated that foretinib could inhibit the cer Chemotherapy Center, Japanese Foundation for Cancer phosphorylation of tyrosine receptor kinase A with the G667C Research, Tokyo, Japan). Ba/F3 cells were cultured in DMEM mutation and overcome entrectinib resistance in the animal containing 10% FBS with 0.5 ng/mL IL3 (Invitrogen). Ba/F3_WT, models for liver and brain metastases. Our findings provide a Ba/F3_G595R, and Ba/F3_G667C cells were cultured in DMEM rationale for clinical trials with foretinib in cancer patients containing 10% FBS. Possible mycoplasma infection in the cells with entrectinib-resistant tumors harboring the NTRK1- was regularly checked using a MycoAlert Mycoplasma Detection G667C mutation, including patients with brain metastases. kit (Lonza). Cell line authentication was performed by a short tandem repeat analysis in a laboratory at the National Institute of Biomedical Innovation (Osaka, Japan) in May 2015. Human dermal microvascular endothelial cells were purchased from patients with CNS diseases, such as brain metastasis and lepto- KURABO, maintained in HuMedia-MvG with growth supple- in vitro meningeal carcinomatosis, with no history of TKI administration ments (KURABO), and used for assays at passages two to fi (TKI-na€ve). However, patients frequently experience acquired ve. Entrectinib, foretinib, and nintedanib were purchased from resistance to targeted drugs during the progression of CNS metas- Selleck Chemicals. tases (12). The CNS is therefore called "the sanctuary site of targeted drugs" (13). The mechanisms of targeted drug resistance Cell viability assay in brain lesions may be different from those in extracranial Cells were seeded at a density of 2 103/100 mL RPMI-1640 lesions. For instance, the EGFR-T790M mutation is responsible plus 10% FBS per well in 96-well culture plates and incubated for 50% to 60% of the acquired resistance in extracranial lesions overnight. The next day, the indicated compound was added to from EGFR-mutated NSCLC treated with EGFR-TKIs, whereas it is each well at a concentration gradient (0, 0.003, 0.01, 0.1, 0.3, 1, 3, much less frequent in CNS lesions from the same patients (14). 10, and 30 nmol/L), and incubation was continued for Although poor penetration of targeted drugs into the CNS, an additional 72 hours. Cell viability was measured using a because of the blood–brain barrier (BBB), is thought to be CCK-8 kit (Dojindo Laboratories). Each experiment was per- associated with the drug resistance (15), the mechanism of formed at least 3 times, with triplicate samples. Ba/F3, Ba/F3_WT, resistance in CNS lesions, whose understanding is important to Ba/F3_G595R, and Ba/F3_G667C cells were seeded at a density of establish better therapeutics, remains elusive. 3 103/100 mL DMEM plus 10% FBS with or without IL3 per well We previously reported (16) that KM12SM cells, a highly in 96-well culture plates and incubated overnight. The following metastatic variant of the KM12C colon cancer cell line, which procedures were the same. have been shown to express the TPM3-NTRK1 fusion protein (17), have a high potential to produce brain metastases after internal carotid artery inoculation in nude mice. The incidence of Antibodies and Western blotting brain metastases in colon cancer, previously considered rare, has Cells were lysed with Cell Lysis Buffer (Cell Signaling Technol- now increased and is associated with increased survival owing to ogy) containing 1% (v/v) phosphatase inhibitor cocktail 3 (Sig- improved treatments (18). Based on these considerations, we ma) and distilled water. Cell extracts (20 mg per lane) were examined the mechanism of entrectinib resistance in brain separated by SDS-PAGE using Mini-PROTEAN TGX Precast Gels, lesions, utilizing a brain metastasis–mimicking model obtained and the separated proteins were electrophoretically transferred to through the injection of KM12SM cells. We found that the NTRK1- Immun-Blot PVDF membranes (Bio-Rad). The primary antibo- G667C mutation caused entrectinib resistance in brain lesions. dies used were: TRKA (14G6), phospho-TRKA (Tyr490), AKT, Furthermore, we demonstrated, for the first time, that foretinib phospho-AKT (Ser473), MET (25H2), VEGFR2 (55B11), cleaved can overcome entrectinib resistance associated with the NTRK1- PARP (Asp214), b-actin (all from Cell Signaling Technology), G667C mutation in the liver metastasis model and the brain ERK1/ERK, phospho-ERK1/ERK2 (Thr202/Thr204, both from metastasis–mimicking model. R&D System), and TRKA (C-14; Santa Cruz Biotechnology). The membranes treated with the primary antibodies were incubated fi Materials and Methods for 1 hour at temperature range with species-speci c horseradish peroxidase–conjugated secondary antibodies. Immunoreactive Cell lines, cell culture, and compounds bands were visualized with SuperSignal West Dura Extend Dura- The human colon cancer cell line KM12SM was kindly gifted by tion Substrate, an enhanced chemiluminescent substrate (Pierce Dr. I.J. Fidler (MD Anderson Cancer Center, Houston, TX) to our Biotechnology).

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Foretinib Overcomes Entrectinib Resistance in Brain Tumor

RNA sequencing KM12SM cells using Lipofectamine LTX (Invitrogen) according to Total RNA was extracted from tumor cells using an RNeasy Plus the manufacturer's instructions. The cells were then selected with Mini kit (Qiagen). Quantity and quality of the extracted RNAs puromycin. were evaluated with the Qubit RNA BR Assay Kit (Thermo Fisher Scientific) and the RNA 6000 Nano LabChip on the Agilent 2100 Docking analyses Bioanalyzer (Agilent Technologies). One microgram of total RNA The coordinates of TRK-A for the docking study of foretinib and was used for the construction of a strand-specific RNA sequencing entrectinib were prepared from the X-ray structures with DFG-out library. The library was prepared using the TruSeq Stranded conformation (PDB code: 5JFS) and DFG-in conformation (PDB mRNA LT Sample Prep Kit (Illumina) according to the manu- code: 4AOJ), respectively. The coordinate of the G667C mutant facturer's instructions. The individually indexed libraries were for the docking study of foretinib was prepared using the Dis- pooled and then diluted to 10 pmol/L for cluster formation with covery Studio (version 4.5, BIOVIA). The three-dimensional con- the TruSeq PE Cluster kit v3-cBot-HS (Illumina), using an Illu- formations of compounds were generated by OMEGA (version mina cBot fluidics station. RNA sequencing analysis was per- 3.141592-1.23.2.3, OpenEye Scientific Software) with default formed using a HiSeq2000 sequencer (Illumina) with a 2 settings. Docking was performed using FRED (version 3.0.1, 100 bp paired-end module. The alignment of the paired-end OpenEye Scientific Software) with constraints making a hydrogen FASTQ files to the human reference genome (GRCh37) was bond with Met592. The displayed docking models had the best performed using TopHat2 software (20) and visualized using FRED score. Integrative Genomics Viewer software (21). The FASTQ files were further analyzed, to find known and/or novel fusion transcripts, using FusionCatcher software (22). To validate the presence of the Animal experiments TPM3-NTRK1 fusion transcript and a mutation in the kinase All animal experiments in this study were performed in strict domain, the total RNA extracted from the two cell lines was also accordance with the recommendations in the Guide for the Care subjected to reverse transcription-PCR (RT-PCR) and Sanger and Use of Laboratory Animals of the Ministry of Education, sequencing analysis. In brief, complementary DNAs (cDNA) for Culture, Sports, Science and Technology, Japan. The protocol was PCR templates were synthesized from the total RNA with the approved by the Committee of Ethics of Experimental Animals, SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific). and the Advanced Science Research Center, Kanazawa University, DNA fragments encompassing both the TPM3-NTRK1 fusion Kanazawa, Japan (approval number: AP-081088). Five-week-old break point and the mutation site were generated by PCR ampli- female SCID mice (C.B-17/Icr-scid/scidJc) and male Hairless fication using TaKaRa Ex Taq DNA polymerase (TAKARA BIO). SCID (SHO) mice were purchased from CLEA and Charles River Nucleotide sequences of the fragments were determined with the Laboratories Japan, respectively. BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher For the liver metastasis model, tumor cells were inoculated into Scientific) on an Applied Biosystems 3130 Genetic Analyzer the spleen as reported previously (24). In brief, the SCID mice (Thermo Fisher Scientific). were anesthetized with tribromoethanol and inhalation of 1.5% isoflurane. After an incision was made in the skin, subcutaneous RNA interference tissue, and peritoneal membrane on the left side of the abdomen, Duplexed Silencer Select siRNAs against NTRK1 (s9745, the spleen was exposed. A suspension of tumor cells (1 106/ s9746), Silencer Select siRNAs against VEGFR2 (s7822, s7833), 50 mL) was injected into the spleen using a 27-G needle. The siRNAs targeting MET (HSS106478), and Silencer Select Negative incision was closed by suture. Control #1 siRNA (Ambion) were used for RNAi assays. Briefly, For the brain metastasis–mimicking model, tumor cells were 5 aliquots of 1 10 cells in 2 mL of antibiotic-free medium were inoculated into the brain as reported previously (23). In brief, the plated into each well of a 6-well plate and incubated at 37 C for 24 SHO mice were anesthetized with tribromoethanol. The scalp was hours. The cells were transfected with siRNA (250 pmol) or sterilized with 70% ethanol, and a small hole was created in the scrambled RNA using Lipofectamine RNAiMAX (5 mL; Invitrogen) skull, 0.5 mm anterior and 3.0 mm lateral to the bregma, using a in accordance with the manufacturer's instructions. After 24 dental drill. A suspension of tumor cells (1.5 105/1.5 mL) was hours, the cells were washed twice with PBS and incubated for injected into the right striatum, 3 mm below the surface of the an additional 48 hours in antibiotic-containing medium. The brain, using a 10-mL Hamilton syringe with a 26-G needle. The tumor cells were then used for cell proliferation assays. NTRK1, scalp was closed using an Autoclip Applier (BD). VEGFR2, and MET knockdowns were confirmed by Western blot. In both models, the development of tumors was tracked in live mice by repeated noninvasive optical imaging of tumor-specific Kinase inhibitor library screen luciferase activity using the IVIS Lumina XR Imaging System We used the Kinase Inhibitor Library (L1200-01 and -02) from (PerkinElmer) as described previously (25). The intensity of the Selleck Chemicals for the screening of kinase inhibitors. Briefly, bioluminescence signal was analyzed using Living Image 4.0 cancer cells were seeded (2,000 cells/well) overnight into 96-well software (PerkinElmer) by serially quantifying the peak photon plates and incubated with 0.1 and 1 mmol/L of 122 kinase flux in a selected region of interest (ROI) within a given tumor. The inhibitors for 72 hours. The inhibitory activity of each drug was intensity of the bioluminescence signal was corrected to consider assessed with a CCK-8 kit. the total area of the ROI and the elapsed time for which bioluminescence signals were read with a CCD camera. The corrected EGFP-Eluc gene transfection value was then expressed as photons/s. The expression vector GFP-Eluc [Enhanced green fluorescent protein (EGFP) from the pIRES-EGFP Vector and Emerald Luc Liver. Tumor luminescence and mouse body weight were mea- (Eluc) from Emerald Luc Vector; ref. 23] was transfected into sured twice a week. Thirteen days after inoculation, mice were

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orally administered vehicle, entrectinib (15 mg/kg), or foretinib cultured it in vitro. The expanded tumor cells were named (30 mg/kg) daily, for 10 consecutive days. KM12SM-ER (Fig. 1A). We found that the KM12SM-ER cells were resistant to treatment with 30 mg/kg entrectinib in our brain Brain. After 7 days, the mice were randomized and treated orally metastasis–mimicking model (Supplementary Fig. S2). with saline, entrectinib (15 mg/kg), or foretinib (30 mg/kg), daily. Next, we examined the sensitivity of KM12SM-ER cells in vitro. The mouse body weights were measured twice per week. As expected, KM12SM-ER cells were discernibly resistant to entrectinib compared with KM12SM cells (Fig. 1B; IC50 KM12SM-ER, Immunohistochemistry 56.1 nmol/L; IC50 KM12SM, 1.70 nmol/L). To assess the resis- Formalin-fixed, paraffin-embedded tissue sections (4-mm- tance mechanism, we examined the effect of entrectinib on the thick) were deparaffinized. Proliferating cells were detected by phosphorylation of TRK-A and its downstream molecules by incubating tissue sections with a Ki-67 antibody (Clone MIB-1; Western blot. Entrectinib inhibited phosphorylation of TRK-A DAKO Corp.). Apoptotic cells were detected by terminal deox- and its downstream molecules AKT and ERK, and thereby induced ynucleotidyl transferase–mediated dUTP nick end labeling apoptosis, indicated by the expression of cleaved PARP (c-PARP) (TUNEL) staining (In Situ Cell Death Detection Kit; Roche) of in KM12SM cells, but not in KM12SM-ER cells (Fig. 1C). These the tissue sections. Antigens were retrieved by microwaving the results suggest that entrectinib failed to inhibit TRK phosphory- tissue sections in 10 mmol/L citrate buffer (pH 6.0) and Tris-EDTA lation in KM12SM-ER cells, which were therefore resistant to the buffer (pH 9.0). After incubation with a secondary antibody, drug. We next determined the effect of TRK-A knockdown in peroxidase activity was visualized via a DAB reaction using a KM12SM-ER cells utilizing siRNAs specific for NTRK1. Three Histofine Simple Stain MAX-PO(R) kit (Nichirei). The sections different siRNAs for NTRK1 successfully knocked down TRK-A were counterstained with hematoxylin. All sections were also expression and reduced the viability of both KM12SM and stained with hematoxylin and eosin. KM12SM-ER cells (Fig. 1D and E). These results strongly suggest that KM12SM-ER cells acquired entrectinib resistance through a – Quantification of immunohistochemistry results TRK-A dependent mechanism, possibly through the acquisition NTRK1 The five areas containing the highest numbers of stained cells of resistance-inducing mutations in . within each section were selected for histologic quantification, via KM12SM-ER cells had the NTRK1-G667C mutation light or fluorescence microscopy at 400 magnification. All We next performed next-generation sequencing using the RNA results were independently evaluated by two authors (T. Yamada extracted from the KM12SM/Eluc and KM12SM-ER cells. The and A. Nishiyama). sequencing analysis yielded 29,876,111 and 28,133,224 paired-end reads for the KM12SM/Eluc and KM12SM-ER cells, Statistical analysis respectively. Both the KM12SM/Eluc and KM12SM-ER cells pos- Data from the viability assays and the tumor progression in the sessed the TPM3-NTRK1 fusion gene (Fig. 2A), whose presence xenograft model are expressed as mean SD. The statistical was further confirmed by direct sequencing of cDNA (Fig. 2C) and significance of differences was analyzed by one-way ANOVA and RT-PCR (Fig. 2B). Importantly, we found the mutation G667C in Spearman rank correlations performed using GraphPad Prism exon 10 of the NTRK1 gene from the KM12SM-ER cells; this Ver. 6.0 (GraphPad Software, Inc.). For all analyses, a two-sided P mutation was not present in the KM12SM/Eluc cells. A recent value less than 0.05 was considered statistically significant. study demonstrated two resistance-inducing mutations, G595R and G667C, in the NTRK1 gene, through the analysis of entrecti- Results nib-resistant KM12 cells and clinical specimens from liver metas- Establishment of KM12SM-ER cells that acquired in vivo tases and plasma DNA of patients with colon cancer who had resistance to entrectinib acquired entrectinib resistance (11). The G595R and G667C We previously confirmed that both the KM12C and KM12SM mutations were reported to induce high and moderate resistance cells had the TPM3-NTRK1 gene rearrangement and were equally to entrectinib, respectively (11). Therefore, we concluded that sensitive to TRK inhibitors, such as crizotinib and entrectinib KM12SM-ER cells acquired entrectinib resistance through the (unpublished data). In this study, we first established KM12SM/ NTRK1-G667C mutation. Eluc cells by transfecting a plasmid expressing the EGFP/Eluc gene into KM12SM cells. KM12SM and KM12SM/Eluc cells were Foretinib and nintedanib can inhibit TRK-A bearing a G667C similarly sensitive to entrectinib in vitro (Supplementary Fig. S1), mutation indicating that the transfection of EGFP-Eluc had no effect on the Because no effective drug has been identified, so far, as being sensitivity to entrectinib. To establish resistant tumor cells that capable of inhibiting TRK-A with a G667C mutation, we next had acquired entrectinib resistance in the microenvironment of sought to screen compounds which might have such activity using the brain, KM12SM/Eluc cells were inoculated into the brain of a Kinase Inhibitor Library that includes 122 inhibitors (Fig. 3A). SCID mice. The treatment with entrectinib (15 mg/kg), com- Cell viability assays on KM12SM-ER cells revealed that some menced 7 days after tumor cell inoculation, discernibly delayed inhibitors could inhibit the viability of KM12SM-ER cells. Spe- the progression of tumors in the brain and prolonged the survival cifically, 13 compounds inhibited the viability by 40% compared of the mice, indicating the antitumor effect of entrectinib on with the control. Of the 13 compounds, 5, including foretinib and KM12SM/Eluc cells in the brain. However, the tumor lumines- nintedanib, are known to inhibit VEGFR-2 (Fig. 3B). Because cence gradually increased, despite the continued entrectinib treat- foretinib and nintedanib are currently used in clinical trials or ment, suggesting that the tumor cells in the brain had acquired clinical practice, we further assessed the effect of these two drugs. resistance to entrectinib. We harvested the brain tumor from mice As expected, foretinib (IC50 ¼ 1.95 nmol/L) and nintedanib on day 45 (entrectinib treatment was given for 37 days) and (IC50 ¼ 27.5 nmol/L), but not entrectinib (IC50 ¼ 56.1 nmol/L),

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Figure 1. Establishment of KM12SM-ER cells with acquired resistance to entrectinib in vivo. A, We transfected the EGFP/Eluc gene into KM12SM cells to establish KM12SM/Eluc cells. KM12SM/Eluc cells were inoculated into the brain of SCID mice. The mice were treated daily with or without entrectinib (15 mg/kg) for 37 days until the bioluminescence increased. Mean SE of total flux are shown in the bottom plot. Then, the entrectinib-treated brain tumor was harvested at the point indicated by the orange triangle and cultured in vitro. The expanded tumor cells were named KM12SM-ER. B, The sensitivity of KM12SM-ER and KM12SM cells to entrectinib was determined through cell viability assays, using a CCK-8 kit. The data (mean SD of triplicate cultures) shown are representative of three independent experiments with similar results. C, Tumor cells were treated with entrectinib (10 nmol/L) for 4 hours or c-PARP for 48 hours, and harvested lysates were assessed by Western blotting. Data shown are representative of three independent experiments with similar results. D, KM12SM and KM12SM-ER cells were treated with siRNA specificforNTRK1 (siNTRK) or a scrambled control (siSCR). Then, cell viability was determined with a CCK-8 kit. Data (mean SD) shown are representative of three independent experiments, with similar results. , P < 0.05, as determined by the Student t test, compared with siSCR. E, KM12SM and KM12SM-ER cells were transfected with NTRK1-specific siRNA (siNTRK) or a siSCR. After 48 hours, the cell lysates were harvested and evaluated for protein expression by Western blotting. Three independent experiments were performed. inhibited the viability of KM12SM-ER cells at a concentration phosphorylation of TRK-A and its downstream molecules, AKT lower than 1 nmol/L (Fig. 3C). In addition to entrectinib, foretinib and ERK, in KM12SM cells, and entrectinib was the most potent and nintedanib also inhibited the viability of KM12SM cells. inhibitor. Foretinib and nintedanib inhibited the phosphoryla- Interestingly, KM12SM-ER cells were more sensitive to foretinib tion of TRK-A with the G667C mutation in KM12SM-ER cells and nintedanib, compared with KM12SM cells. The kinase inhi- more efficiently than that of the wild-type TRK-A in KM12SM cells bition profile assay also showed that both foretinib and ninte- (Supplementary Fig. S4). Interestingly, foretinib and nintedanib danib were able to inhibit TRK-A, although their activity was remarkably inhibited the phosphorylation of TRK-A, AKT, and weaker than that of entrectinib. The activity of foretinib and ERK in KM12SM-ER cells (Fig. 3D), whereas entrectinib mildly nintedanib against TRK-B and TRK-C was much weaker than that suppressed the phosphorylation of TRK-A with the G667C muta- against TRK-A (Supplementary Fig. S3). Western blot analyses tion and transiently inhibited the phosphorylation of ERK (Sup- revealed that entrectinib, foretinib, and nintedanib inhibited the plementary Fig. S5). With regard to cell viability and TRK-A

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Figure 2. KM12SM-ER cells harbored the NTRK1-G667C mutation. A, Schematic representation of RNA-sequencing reads indicating the presence of TPM3-NTRK1 fusion transcripts. FusionCatcher software was used to count the number of paired-end reads relative to fusion transcripts ("Spanning pairs") or mapping on the fusion junction ("Spanning unique reads"). The red-colored transverse line indicates the RT-PCR target region, encompassing the TPM3-NTRK1 fusion junction and the site of the acquired p.G667C mutation (denoted by the asterisk). B, RT-PCR followed by agarose gel electrophoresis conﬁrmed the presence of TPM3-NTRK1 fusion transcripts in KM12SM/Eluc and KM12SM-ER cells. C, Sanger sequencing of the RT-PCR products showed the identity of the fusion junctions in the TPM3-NTRK1 fusion transcripts from the two cell lines. D, Integrative Genomics Viewer images illustrating mapped RNA sequencing reads, which indicate the presence of the NTRK1 sequence alteration p.G667C (NM_002529.3: c.1999G>T) in KM12SM-ER cells (bottom), but not KM12SM/Eluc cells (top). E, Sanger sequencing electropherograms of RT-PCR products validating the acquired mutation p.G667C in the TPM3-NTRK1 fusion transcripts only in KM12SM-ER cells.

phosphorylation, entrectinib induced apoptosis (assessed by of Ba/F3_G595R cells. Importantly, foretinib, but not entrectinib, expression of c-PARP) in KM12SM cells, and foretinib and ninte- inhibited Ba/F3_G667C cell viability (Fig. 4A). Western blotting danib caused apoptosis more discernibly than entrectinib in revealed that foretinib and entrectinib inhibited the phosphory- KM12SM-ER cells. These results indicate that foretinib and nin- lation of TRK-A, AKT, and ERK in Ba/F3_WT cells, whereas tedanib can inhibit the phosphorylation of TRK-A even in the entrectinib was more potent than foretinib. However, foretinib, presence of the G667C mutation and suppress the viability of but not entrectinib, inhibited the phosphorylation of TRK-A and KM12SM-ER cells in vitro. AKT in Ba/F3_G667C cells (Fig. 4B). These results clearly indicated that foretinib inhibited TRK-A with G667C mutation, Foretinib overcomes resistance associated with NTRK1-G667C whereas it appeared to be less potent than entrectinib against mutation, not NTRK1-G595R mutation wild-type TRK-A. To further assess whether foretinib directly inhibited TRK-A with or without G667C mutation, we examined the effects Docking analysis of TRK-A with or without G667C mutation of foretinib compared with those of entrectinib using Ba/F3, and foretinib/entrectinib Ba/F3_WT, Ba/F3_G595R, and Ba/F3_G667C cells. Both foretinib We examined whether foretinib could bind to TRK-A with and entrectinib inhibited the viability of Ba/F3_WT cells, but not G667C mutation by docking simulation analysis. Inhibitor-

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Figure 3. Screening of compounds that inhibit the viability of KM12SM-ER cells. A, The effect of 122 compounds from a Kinase Inhibitor Library on the viability of KM12SM-ER cells was assessed using a CCK-8 kit. The 122 kinase inhibitors (100 nmol/L) are rank-ordered from the highest to the lowest according to their inhibitory effect on the viability of KM12SM-ER cells. The effects of the top 13 compounds indicated by a red line with an arrowhead are further shown in B. B, Red bars indicate the compounds that are known to have anti–VEGFR-2 activity. C, The effect of entrectinib, nintedanib, and foretinib on the viability of KM12SM and KM12SM-ER cells was determined with a CCK-8 kit. Data (mean SD of triplicate cultures) shown are representative of three independent experiments with similar results. D, KM12SM and KM12SM-ER cells were incubated with the indicated drugs (10 nmol/L) for 4 or 48 hours (for c-PARP), and the cell lysates were harvested. The expression of indicated molecules was determined by Western blot analysis. Data shown are representative of three independent experiments with similar results. binding structures of protein kinase, including TRK-A, are gener- interact with the Ca atom of Gly595 in wild-type TRK-A. There- ally divided into DFG-in and DFG-out conformations, which are fore, the G595R mutation would likely provoke a severe steric determined by a flip-flop motion of the highly conserved DFG hindrance in the binding of both inhibitors to TRK-A (Supple- motif (Asp668-Phe669-Gly670 in TRK-A; ref. 26). The DFG-in mentary Fig. S6). This is consistent with the fact that entrectinib conformer is rigid as it is ready for substrate ATP binding, whereas and foretinib did not show any inhibitory activity against the the DFG-out conformers as autoinhibition states are divergent, G595R mutant. The calculated configuration in the DFG region of particularly those of TRK-A markedly increase the flexibility of the TRK-A possesses certain unreliability due to the remarkably higher DFG motif through Gly667. Docking-simulated models based on flexibility. the crystal structures of the foretinib/EphA2 and entrectinib/Alk complexes (27, 28) support the inhibitory effects of foretinib and The viability of KM12SM-ER cells was not affected by entrectinib on the wild-type and G667C mutant of TRK-A. Entrec- knockdown of VEGFR-2 or MET tinib binds to the DFG-in conformation of the wild-type TRK-A, Foretinib and nintedanib are known kinase inhibitors for and the difluorobenzene group of the inhibitor closely matches VEGFR-2/MET and VEGFR-2, respectively (29, 30). To rule out and forms a van der Waals contact with the Ca atom of Gly667 the possibility that the effect of foretinib and nintedanib was (Fig. 4C, left). However, the side chain of the replacing cysteine mediated by the inhibition of VEGFR-2 or MET, we assessed the may be a serious impediment in entrectinib binding to the TRK-A effect of knockdown of VEGFR-2 or MET in KM12SM-ER cells. G667C mutant (Fig. 4C, right). It is highly unlikely that the rigid VEGFR-2 protein was undetectable in KM12SM-ER cells (Supple- DFG-in conformation is adapted for entrectinib binding to the mentary Fig. S7B). Treatment with siRNA specific for NTRK1 and G667C mutant. Foretinib binds to the DFG-out conformation of MET successfully knocked down TRK-A and MET, respectively the wild type and G667C of TRK-A accompanying the structural (Supplementary Fig. S7D). Under the same experimental condi- adjustment but fits better to G667C compared with the wild type. tions, treatment with specific siRNA for VEGFR-2 or MET did not The fluorobenzene group of foretinib forms the weakly repulsive inhibit the viability of KM12SM-ER cells, whereas the treatment interactions with Phe521 of the wild-type but not with that of the with siRNA for NTRK1 did (Supplementary Fig. S7A and S7C). G667C mutant (Fig. 4D). Entrectinib and foretinib strongly These results strongly suggest that the inhibitory effect of foretinib

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Figure 4. Sensitivity of Ba/F3, Ba/F3_WT, Ba/F3_G595R, and Ba/F3_G667C cells to entrectinib and foretinib, and structural analysis of TRK-A with or without G667C mutation and foretinib/entrectinib. A, The sensitivity of Ba/F3, Ba/F3_WT, Ba/F3_G595R, and Ba/F3_G667C cells to foretinib or entrectinib was determined through cell viability assays using a CCK-8 kit. The data (mean SD of triplicate cultures) shown are representative of three independent experiments with similar results. B, Ba/F3_WT and Ba/F3_G667C cells were incubated with the indicated drugs (3 nmol/L) for 4 hours, and the cell lysates were harvested. The expression of indicated molecules was determined by Western blot analysis. Data shown are representative of three independent experiments with similar results. C, Binding structures of entrectinib with the wild-type (left) and TRK-A with G667C mutation (right). D, Binding structures of foretinib with the wild-type (left) and TRK-A with G667C mutation (right).

and nintedanib in KM12SM-ER cells was predominantly due to not entrectinib, inhibited the phosphorylation of TRK-A and AKT inhibition of TRK-A, not VEGFR-2 or MET. in liver tumors (Fig. 5C), consistent with the data from bioluminescence analysis. Foretinib caused the regression of liver metastases produced by We next evaluated tumor cell proliferation and apoptosis in KM12SM-ER cells liver metastasis using immunohistochemistry (IHC). The treat- We next sought to evaluate the effect of such drugs in vivo. ment with foretinib remarkably reduced the number of prolifer- Specifically, we decided to focus on foretinib, because, compared ating tumor cells and increased the number of TUNEL-positive with nintedanib, it reduced the viability of KM12SM-ER cells tumor cells in liver tumors, compared with the control and more potently at concentrations close to Cmax [foretinib 90.5 ng/ entrectinib-treated groups, although the treatment with entrecti- mL (140 nmol/L) and nintedanib 34.8 ng/mL (53.6 nmol/L)], as nib slightly decreased the number of proliferating tumor cells and shown in clinical trials (31, 32). The liver is the organ that is increased the number of TUNEL-positive tumor cells compared affected the most by colon cancer metastases. Therefore, for our with the control (Fig. 5D and E). These results indicate that in vivo experiments, we first investigated the effect of foretinib in foretinib inhibited liver metastases by suppressing tumor cell a liver metastasis model obtained through injection of KM12SM- proliferation and inducing apoptosis of entrectinib-refractory ER cells. After the intrasplenic inoculation of KM12SM-ER cells, KM12SM-ER cells, and inhibited TRK-A phosphorylation. bioluminescence in the liver became detectable by day 7. Then, we commenced treatment with entrectinib (15 mg/kg) or foretinib Foretinib inhibited the progression of brain tumor metastases (30 mg/kg), daily, from day 13 to day 22. Entrectinib treatment caused by KM12SM-ER cells slightly delayed the progression of liver metastasis but did not We next evaluated the efficacy of foretinib on brain tumors decrease the bioluminescence, indicating resistance to entrectinib in a brain metastasis–mimicking model obtained through injec- due to the KM12SM-ER cells in the liver metastasis model (Fig. 5A tion of KM12SM-ER cells. After the intracerebral inoculation of and B). Under the same experimental conditions, foretinib treat- KM12SM-ER cells, bioluminescence in the brain became detect- ment decreased bioluminescence, indicating the capability of able by day 7. When the total flux in the brain lesions reached 1 foretinib to reduce liver metastases produced by entrectinib- 105 photons/sec, we randomized the mice into three groups and resistant KM12SM-ER cells (Fig. 5A and B). None of the mice commenced daily treatment with entrectinib (15 mg/kg) or showed significant weight loss (Supplementary Fig. S8A). Western foretinib (30 mg/kg) in two of the groups (treated groups). In blot analyses of liver metastatic lesions revealed that foretinib, but both the control and entrectinib groups, bioluminescence

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Figure 5. Therapeutic effect of foretinib and entrectinib in a liver metastasis model obtained using KM12SM-ER cells. A, KM12SM-ER cells were inoculated into the spleen of SCID mice, and the mice were randomized on day 13. Mice were treated orally with entrectinib (15 mg/kg) or foretinib (30 mg/kg) daily, from days 13 to 21. Bioluminescence was evaluated twice a week, from days 7 to 22. Data shown are mean SE for 5 mice in each group. B, Representative bioluminescent and photographic merged images of mice. C, Mice were sacrificed on day 22, and liver metastatic lesions were harvested. The protein lysates extracted from the liver lesions were used for Western blot assays to detect the expression of the indicated molecules. D, Proliferating tumor cells (Ki-67) and apoptotic cells (TUNEL) in the liver metastases were identified by IHC. Bar, 100 mm. E, Quantification of proliferating and apoptotic cells in liver metastases, determined by IHC in D. The data shown are the mean SD of five areas. , P < 0.05 as determined with the Mann–Whitney U test. increased rapidly, and there was no statistically significant differ- proliferating tumor cells and increased the number of TUNEL- ence between these two groups. Moreover, all mice lost body positive tumor cells in brain tumors as compared with the weight drastically and became moribund by day 14 (7 days after untreated group and the entrectinib-treated group. The effect of the beginning of the treatment). Under the same experimental the entrectinib treatment was only marginal compared with the conditions, treatment with foretinib remarkably delayed the control (Fig. 6D and E). These results indicate that foretinib elevation of bioluminescence, compared with the other two inhibited tumor progression by suppressing tumor cell prolif- groups (Fig. 6A and B). The body weight loss in the foretinib- eration and inducing apoptosis of entrectinib-refractory treated group was only marginal until day 17 (Supplementary Fig. KM12SM-ER cells, and inhibiting TRK-A phosphorylation, in S8B). These results clearly indicate that foretinib is also effective in the brain metastasis–mimicking model as well as in the liver the brain metastasis–mimicking model obtained using entrecti- metastasis model. nib-resistant KM12SM-ER cells. Western blot analyses further revealed that foretinib, but not entrectinib, inhibited the phos- Discussion phorylation of TRK-A in the brain tumors, consistent with its antibrain tumor effect observed in this model (Fig. 6C). Recent reports have described the development of a new We next performed IHC analyses on the brain tumors. The compound, LOXO195, which could overcome larotrectinib treatment with foretinib remarkably reduced the number of (a TRK inhibitor) resistance associated with TRK-A-G595R and

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Figure 6. Therapeutic effect of foretinib and entrectinib in a brain metastasis–mimicking model obtained using KM12SM-ER cells. A, KM12SM-ER cells were inoculated into the brain of SHO mice, and the mice were randomized on day 7. Mice were treated orally with entrectinib (15 mg/kg) or foretinib (30 mg/kg) daily, from days 7 to 24. Bioluminescence was evaluated twice a week, from days 7 to 24. Data shown are mean SE for 5 mice in each group. B, Representative bioluminescent and photographic merged images of mice. C, When mice became moribund, they were sacrificed and brain tumor lesions were harvested. The protein lysates extracted from the brain lesions were used for Western blots to detect expression of the indicated molecules. D, Proliferating tumor cells (Ki-67) and apoptotic cells (TUNEL) in the brain metastases were identified by IHC. Bar, 100 mm. E, Quantification of proliferating and apoptotic cells in brain metastases, determined by IHC in D. The data shown are the mean SD of five areas. , P < 0.05 as determined with the Mann–Whitney U test.

TRK-C-G623R in 2 patients (33). However, the effect of LOXO195 A secondary mutation in a target gene is the most common against TRK-A-G667C is weaker than that against TRK-A-G595R. mechanism of targeted drug resistance (34). Interestingly, in In addition, the effect of LOXO195 on CNS lesions is still NTRK1, different secondary mutations, such as the G667C and unknown. The present study demonstrated three major ﬁndings: G595R mutations, could be acquired by continuous exposure (1) KM12SM-ER cells acquire entrectinib resistance through to entrectinib at different concentrations (11). Speciﬁcally, the the NTRK1-G667C mutation in a brain metastasis–mimicking G667C and G595R mutations were acquired by exposure to model; (2) the multiple kinase inhibitor foretinib, which is low (30–100 nmol/L) and high (1–2 mmol/L) concentrations under evaluation in some clinical trials, inhibits the viability of of entrectinib in a KM12 cell model, respectively (11). We KM12SM-ER cells bearing the NTRK1-G667C mutation and is speculate that, despite entrectinib is initially effective on more effective in these cells than in KM12SM cells; (3) foretinib patients with CNS lesions, the KM12SM-ER cells with the overcomes entrectinib resistance in both liver metastasis and NTRK1-G667C mutation acquired entrectinib resistance in the brain metastasis–mimicking models obtained using KM12SM- brain presumably through the exposure to low concentration of ER cells. These results suggest that, in tumors expressing NTRK1 entrectinib because of its relatively lower penetration in the fusion products, foretinib may be effective in overcoming entrec- braincomparedwiththatinextracranialtumors.Thishypoth- tinib resistance associated with the G667C mutation in tumors of esis is supported by evidence, from the present study, that various organs, including the brain. entrectinib had a marginal effect in a liver metastasis model

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obtained through injection of KM12SM-ER cells, whereas the sensitivity and resistance mechanisms of target drugs in tumors same treatment had no effect in a brain metastasis–mimicking expressing NTRK1 fusion products. model obtained using the same cell line (Fig. 5B). As indicated in the Introduction, at least four NTRK1 fusion Several compounds from a kinase inhibitor screening library, partners, LMNA, TPM3, SQSTM1, and BCAN, have been reported. including foretinib and nintedanib, which have anti-VEGFR-2 However, the distinct sensitivity of each NTRK1 fusion product to activity, suppressed the viability of KM12SM-ER cells in vitro. TRK-A inhibitors is unknown. In addition, it is unknown whether Because the KM12SM-ER cells did not express detectable tumors with different NTRK1 fusion partners acquire resistance to VEGFR-2 protein and treatment with siRNA specific for VEGFR- TRK inhibitors through different mechanisms. Russo and collea- 2 did not affect the viability of KM12SM-ER cells, the observed gues recently revealed that both LMNA-NTRK1--positive colorec- in vitro effect in KM12SM-ER cells is likely due to the inhibition of tal tumors from patients and the TPM3-NTRK1--positive colon a target other than VEGFR-2. Even in the liver metastasis and brain cancer cell line KM12 (same as the KM12C cell line) acquired metastasis–mimicking models, the antiangiogenic effect of fore- entrectinib resistance through secondary NTRK1 mutations tinib was marginal (Supplementary Fig. S9). On the other hand, (G667C and G595R, respectively; ref. 11). In the present study, foretinib clearly inhibited the phosphorylation of TRK-A in we also found that TPM3-NTRK1--positive KM12SM cells devel- KM12SM-ER cells in vitro and in vivo. Therefore, the antitumor oped entrectinib resistance that was associated with the G667C effect of foretinib in our in vivo models obtained through the mutation in brain tumors. Collectively, these findings suggest that injection of KM12SM-ER cells may be predominantly due to the tumor cells with different NTRK1 fusion partners acquire TRK inhibition of TRK-A in KM12SM-ER cells. The G667 residue is in inhibitor resistance, at least in part, by common mutations that the ATP pocket in TRK-A, and the G667C mutation creates steric induce drug resistance. Previous studies have showed that entrec- hindrance that reduces the binding affinity of entrectinib to the tinib is clinically effective for tumors expressing NTRK1 fusion TRK-A catalytic pocket (11). It is of interest that foretinib inhibited products, including NSCLC expressing SQSTN-NTRK1, glioneur- the viability of KM12SM-ER cells more than KM12SM cells, which onal tumors expressing BCAN-NTRK1, and colon cancer expres- strongly suggest that foretinib has higher affinity for the G667C sing LMNA-NTRK1 (5, 6, 39). However, almost all responders mutant of TRK-A than for the wild-type protein. The results of suffered from recurrent diseases due to drug resistance (5, 6, 39). docking simulation supported this hypothesis. Detailed molec- Because glioneuronal tumors produce cranial lesions and NSCLC ular dynamics treatment and/or crystal structure analysis of the frequently produces brain metastases, the control of CNS lesions foretinib/TRK-A complex would promote further exploration of produced by tumors expressing NTRK1 fusion products may selective inhibitors for the G667C mutant. become more important in the future. Foretinib is under evaluation in clinical trials against various In summary, we demonstrated that entrectinib resistance in types of solid tumors. In a preclinical study, foretinib was reported brain tumors expressing an NTRK1 fusion product could occur to be able to penetrate the BBB (the penetration of the drug in the through the NTRK1-G667C mutation. Foretinib can overcome brain was 14%) and reduce the growth of medulloblastoma cells entrectinib resistance associated with the NTRK1-G667C muta- in an orthotopic CNS dissemination model (35). In line with tion in brain tumors. Our results provide a rationale for clinical these results, we demonstrated that foretinib inhibits the tumor trials with foretinib in cancer patients with brain metastases that progression due to KM12SM-ER cells in a brain metastasis– are entrectinib-resistant and express an NTRK1 fusion protein. mimicking model. These findings suggest that foretinib may be effective for the treatment of patients with brain metastases that Disclosure of Potential Conflicts of Interest are entrectinib-resistant and carry the NTRK1-G667C mutation. T. Yamada reports receiving commercial research grants from Boehringer Recent studies have investigated the sensitivity of different Ingelheim Japan, Inc. No potential conflicts of interest were disclosed by the fusion partners to target drugs, using BaF3 or NIH-3T3 cells with other authors. target gene transfection. In the case of the RET fusion product, cells expressing KIF5B-RET were shown to have similar sensitivity to Authors' Contributions the RET-TKIs vandetanib and lenvatinib, when compared with Conception and design: A. Nishiyama, T. Yamada, S. Takeuchi, S. Yano those expressing CCDC6-RET or NCOA4-RET (36, 37). However, Development of methodology: A. Nishiyama, T. Yamada, K. Fukuda, A. Tanimoto a phase II clinical trial investigating the effect of vandetanib in Acquisition of data (provided animals, acquired and managed patients, NSCLC with RET fusion genes demonstrated that the effect of provided facilities, etc.): A. Nishiyama, K. Kita, R. Wang vandetanib was different in patients expressing KIF5B-RET or Analysis and interpretation of data (e.g., statistical analysis, biostatistics, CCDC6-RET, with response rates of 20% and 83%, respectively computational analysis): A. Nishiyama, A. Tanimoto, S. Tange, A. Tajima, (38), suggesting that the effect of targeted drugs in patients may N. Furuya differ depending on the specific fusion partner in the oncogenic Writing, review, and/or revision of the manuscript: A. Nishiyama, T. Yamada, in vitro T. Kinoshita, S. Yano gene rearrangement. The discrepancy of results between the Administrative, technical, or material support (i.e., reporting or organizing study and the clinical trial may indicate a limitation of the in vitro data, constructing databases): T. Yamada, K. Kita, S. Arai, K. Fukuda, model using cells transfected with target genes. In the present A. Tanimoto, S. Takeuchi, N. Furuya, T. Kinoshita study, we performed experiments using two colon cancer cell lines Study supervision: S. Takeuchi, S. Yano derived from KM12C cells, KM12SM and KM12SM-ER, that express the TMP3-NTRK1 fusion protein, because no other tumor Acknowledgments cell line expressing an NTRK1 fusion product was available, The authors thank Dr. I.J. Fidler (MD Anderson Cancer Center, Houston, TX) presumably owing to the low clinical incidence of NTRK1 fusion for providing the KM12-SM cells. They also thank Dr. R. Katayama (Division of Experimental Chemotherapy, Cancer Chemotherapy Center, Japanese Foun- product-positive tumors in cancer patients. The establishment of dation for Cancer Research, Tokyo, Japan) for providing Ba/F3, Ba/F3_WT, other tumor cell lines expressing NTRK1 fusion products, possibly Ba/F3_G595R, and Ba/F3_G667C. The authors thank Yumi Akiyama and Shoko with fusion partners other than TMP3, will further clarify the Ueda (Department of Bioinformatics and Genomics, Graduate School of

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Advanced Preventive Medical Sciences, Kanazawa University) for technical The costs of publication of this article were defrayed in part by the assistance. They also thank the Advanced Preventive Medical Sciences payment of page charges. This article must therefore be hereby marked Research Center, Kanazawa University, for the use of facilities. This study is advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate supported by grants from the Japan Agency for Medical Research and Devel- this fact. opment, AMED (grant numbers 16Ack0106147h0002, 17cm0106513h0002, and 17ck0106147h0003, to S. Yano), and JSPS KAKENHI (grant number Received June 10, 2017; revised September 28, 2017; accepted February 15, JP16H05308, to S. Yano). 2018; published ﬁrst February 20, 2018.

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Foretinib Overcomes Entrectinib Resistance Associated with the NTRK1 G667C Mutation in NTRK1 Fusion−Positive Tumor Cells in a Brain Metastasis Model

Akihiro Nishiyama, Tadaaki Yamada, Kenji Kita, et al.

Clin Cancer Res 2018;24:2357-2369. Published OnlineFirst February 20, 2018.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-17-1623

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Cited articles This article cites 38 articles, 12 of which you can access for free at: http://clincancerres.aacrjournals.org/content/24/10/2357.full#ref-list-1

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