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4040.Full.Pdf Published OnlineFirst February 25, 2020; DOI: 10.1158/1078-0432.CCR-19-2460 CLINICAL CANCER RESEARCH | PRECISION MEDICINE AND IMAGING Discovery of Berberine that Targetedly Induces Autophagic Degradation of both BCR-ABL and BCR-ABL T315I through Recruiting LRSAM1 for Overcoming Imatinib Resistance Zhao Yin1,2,3,4, Guiping Huang1,3,4, Chunming Gu1,2,3,4, Yanjun Liu1,3,4, Juhua Yang1,3,4, and Jia Fei1,2,3,4 ABSTRACT ◥ Purpose: Imatinib, the breakpoint cluster region protein (BCR)/ Results: We discovered that BBR bound to the protein tyrosine Abelson murine leukemia viral oncogene homolog (ABL) inhibitor, is kinase domain of BCR-ABL. BBR inhibited the activity of widely used to treat chronic myeloid leukemia (CML). However, ima- BCR-ABLandBCR-ABLwiththeT315Imutation,anditalso tinib resistance develops in many patients. Therefore, new drugs with degraded these proteins via the autophagic lysosome pathway by improved therapeutic effects are urgently needed. Berberine (BBR) is a recruiting E3 ubiquitin-protein ligase LRSAM1. BBR inhibited potent BCR-ABL inhibitor for imatinib-sensitive and -resistant CML. the cell viability and colony formation of CML cells and pro- Experimental Design: Protein structure analysis and virtual screen- longed survival in CML mouse models with imatinib sensitivity ing were used to identify BBR targets in CML. Molecular docking and resistance. analysis, surface plasmon resonance imaging, nuclear magnetic res- Conclusions: The results show that BBR directly binds to and onance assays, and thermoshift assays were performed to confirm the degrades BCR-ABL and BCR-ABL T315I via the autophagic BBR target. The change in BCR-ABL protein expression after BBR lysosome pathway by recruiting LRSAM1. The use of BBR is a treatment was assessed by Western blotting. The effects of BBR were new strategy to improve the treatment of patients with CML with assessed in vitro in cell lines, in vivo in mice, and in human CML bone imatinib sensitivity or resistance. marrow cells as a potential strategy to overcome imatinib resistance. See related commentary by Elf, p. 3899 Introduction both in vitro and in vivo. BBR is a clinically important natural isoquinoline alkaloid derived from the plant Berberis vulgaris that is Chronic myeloid leukemia (CML) results from the transformation known to have multiple pharmacologic activities, including anticancer of primitive hematopoietic cells by the breakpoint cluster region effects (5–7). However, the mechanisms underlying the effects of BBR protein (BCR)/Abelson murine leukemia viral oncogene homolog on cancer cells have not been fully elucidated. In this study, BBR (ABL) oncogene. Imatinib, a tyrosine kinase inhibitor that binds to significantly inhibited CML cell viability and colony formation and the ATP-binding site of ABL, is remarkably effective at treating CML. prolonged survival in both imatinib-sensitive and -resistant CML However, resistance, which develops in many patients, is the main mouse models. Hence, BBR is a promising new inhibitor for use in barrier to prolonged survival, and the use of a single tyrosine kinase CML treatment, particularly for imatinib-resistant CML. inhibitor cannot cure CML. Thus, the development of novel targeted therapeutic agents, or the use of imatinib in combination with other drugs, is required to improve response rates and overcome imatinib Materials and Methods – resistance (1 4). Human samples and cell lines Here, we describe the identification and characterization of the Healthy peripheral blood mononuclear cells (PBMC) and CML mechanism of action of berberine (BBR) as an ABL-binding agent that bone marrow (BM) mononuclear cells were obtained from adult can capable of degrading BCR-ABL and overcome imatinib resistance donors in Guangdong Provincial Emergency Hospital/the Guangdong Second Provincial General Hospital after written informed consent was obtained according to the institutional guidelines and the Dec- 1Department of Biochemistry and Molecular Biology, Medical College of Jinan University, Guangzhou, China. 2Institute of Chinese Integrative Medicine, Med- laration of Helsinki. These cells were cultured in medium containing ical College of Jinan University, Guangzhou, China. 3Engineering Technology 100 ng/mL stem cell factor (SCF), 100 ng/mL granulocyte-colony Research Center of Drug Development for Small Nucleic Acids, Guangdong, stimulating factor (G-CSF), 20 ng/mL FMS-like tyrosine kinase 3 þ China. 4Antisense Biopharmaceutical Technology Co., Ltd., Guangzhou, China. (FLT3), 20 ng/mL IL3, and 20 ng/mL IL6. Preparation of CD34 from Note: Supplementary data for this article are available at Clinical Cancer umbilical cord blood was performed with the EasySep human cord þ Research Online (http://clincancerres.aacrjournals.org/). blood CD34 selection kit (StemCell Technologies) according to fi Z. Yin, G. Huang, and C. Gu contributed equally to this article. the manufacturer's instructions and incubated in Iscove modi ed Dulbecco medium supplemented with 10% FBS (8, 9). The studies Corresponding Author: Jia Fei, Medical College of Jinan University, 601 Western Huangpu Avenue, Guangzhou 510632, China. Phone: 8620-8522-0256; were approved by Institutional Review Board, Jinan University Fax: 8620-8522-01314; E-mail: [email protected] (Guangzhou, China). The imatinib-sensitive CML cell line K562 was purchased from Clin Cancer Res 2020;26:4040–53 Shanghai Cell Bank (Chinese Academy of Sciences, Shanghai). doi: 10.1158/1078-0432.CCR-19-2460 The imatinib-sensitive CML cell line KCL-22 was kindly provided Ó2020 American Association for Cancer Research. by Dr. Muschen (The Children's Hospital, Los Angeles, CA). The AACRJournals.org | 4040 Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst February 25, 2020; DOI: 10.1158/1078-0432.CCR-19-2460 Berberine Induces Autophagic BCR-ABL T315I Degradation The timing of the procedures was as follows: at 0–260 seconds, the Translational Relevance system was prewashed to infiltrate the surface of the chip with running Acquired imatinib resistance is frequently characterized by buffer. At this point, the resonance intensity was about 0 resonance breakpoint cluster region protein/Abelson murine leukemia units (RU). At 260–520 seconds, binding began, with BBR on the chip viral oncogene homolog (BCR-ABL) mutations that affect imatinib surface starting to capture the protein targets in the cell lysate. At 520– binding and kinase inhibition in patients with chronic myeloid 820 seconds, the chip was washed to gradually remove nonbound and leukemia (CML). In this study, we found that Berberine (BBR), nonspecific molecules, while the target protein that specifically bound a Chinese traditional medicine, significantly inhibited the cell to the BBR remained on the chip surface. Resonance intensity viability and colony formation of CML cells and prolonged survival decreased until it reached a plateau at approximately 542.65 RU. As in CML mouse models with imatinib sensitivity and resistance. nonspecific binding to the nonspotted areas gradually decreased, the Further studies demonstrated that BBR not only inhibit BCR-ABL background resonance intensity gradually decreased to baseline tyrosine kinase activity but also directly bind to ABL1, which (37.72 RU), that is, the chip background noise returned to the induce autophagic degradation of both BCR-ABL and BCR-ABL normal level. T315I through recruiting LRSAM1 for overcoming imatinib resis- tance. Our finding would be of remarkable value for further therapy Molecular docking of CML with BCR-ABL mutation. The molecular docking assays between BBR and ABL1 were gen- erated as described previously (11). Using the Protein Preparation Wizard module of Schrodinger€ Maestro 9.3 software (Schrodinger),€ a model of BBR with ABL1 was processed using default settings. A imatinib-resistant CML cell line SFO2 (which does not express BCR- molecular docking analysis was implemented using the Ligand Dock- ABL) was obtained from Dr. Muschen (The Children's Hospital, Los ing tab of the Glide module. Regarding the docking parameters, Angeles, CA). BaF3-P210 and BaF3-P210-T315I cells were kindly precision was set as extra precision (XP), ligand sampling was set presented by Professor Wen li Feng (Chong qin Medical University, as flexible, the number of poses per ligand was set at 5, and other China). These cells were grown in RPMI1640 medium containing 10% parameters were set at the default values. The residue compounds were FBS and 1% penicillin/streptomycin (all purchased from Gibco). Plat- further subjected to Prime molecular mechanics (MM)-generalized E cells were generously provided by Dr. Jing Xuan Pan (Pharmacy Born surface area (GBSA) calculations and vision analysis. College Jinan University, Guangzhou, China; ref. 10). Imatinib and BBR were purchased from Sigma. Protein expression and purification Protein expression and purification assays were performed accord- Identification and quantification of BBR target proteins using ing to our previous report (12). Escherichia coli BL21 (DE3) was surface plasmon resonance and high-performance liquid transformed with a hexa-histidine–tagged recombinant human pro- chromatography mass spectrometry tein tyrosine kinase (PTK) domain of ABL1 (isoform 2). After bacterial To explore the direct cell target of BBR, we designed an surface growth in Terrific Broth containing 30 mg/L kanamycin at 37Ctoan plasmon resonance-high-performance liquid chromatography-mass optical
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