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Molecular Chaperone Gp96 Is a Novel Therapeutic Target of Multiple Myeloma

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Molecular Chaperone Gp96 Is a Novel Therapeutic Target of Multiple Myeloma Published OnlineFirst September 27, 2013; DOI: 10.1158/1078-0432.CCR-13-2083 Clinical Cancer Cancer Therapy: Preclinical Research Molecular Chaperone gp96 Is a Novel Therapeutic Target of Multiple Myeloma Yunpeng Hua1, Shai White-Gilbertson1, Joshua Kellner1, Saleh Rachidi1, Saad Z. Usmani2, Gabriela Chiosis3, Ronald DePinho4, Zihai Li1, and Bei Liu1 Abstract Purpose: gp96 (grp94) is a key downstream chaperone in the endoplasmic reticulum (ER) to mediate unfolded protein response (UPR) and the pathogenesis of multiple myeloma is closely linked to dysre- gulated UPR. In this study, we aimed to determine the roles of gp96 in the initiation and progression of multiple myeloma in vivo and in vitro. Experimental Design: We generated a mouse model with overexpression of XBP1s and conditional deletion of gp96 in B-cell compartment simultaneously to identify the roles of gp96 in the development of multiple myeloma in vivo. Using a short hairpin RNA (shRNA) system, we silenced gp96 in multiple human multiple myeloma cells and examined the effect of gp96 knockdown on multiple myeloma cells by cell proliferation, cell-cycle analysis, apoptosis assay, immunohistochemistry, and human myeloma xenograft model. The anticancer activity of gp96 selective inhibitor, WS13, was evaluated by apoptosis assay and MTT assay. Results: Genetic deletion of gp96 in XBP1s-Tg mice attenuates multiple myeloma. Silencing of gp96 causes severe compromise in human multiple myeloma cell growth through inhibiting Wnt-LRP-survivin pathway. We also confirmed that knockdown of gp96 decreased human multiple myeloma growth in a murine xenograft model. The targeted gp96 inhibitor induced apoptosis and blocked multiple myeloma cell growth, but did not induce apoptosis in pre-B leukemic cells. We have demonstrated that myeloma growth is dependent on gp96 both genetically and pharmacologically. Conclusions: gp96 is essential for multiple myeloma cell proliferation and survival, suggesting that gp96 is a novel therapeutic target for multiple myeloma. Clin Cancer Res; 1–10. Ó2013 AACR. Introduction pathways including activating transcription factor 6 (ATF6), As an ER chaperone, gp96 (1), also known as grp94 (2), the double-stranded RNA-activated protein kinase-like ER endoplasmin (3), ERp99 (4), and HSP90b1(5), is a para- kinase (PERK), and the spliced form of X box binding logue of HSP90 and its expression can be induced by the protein 1 (XBP1s) to induce the expression of several major accumulation of misfolded proteins (6). It binds to and ER HSPs including gp96, grp78, and calreticulin to enhance hydrolyzes ATP (7–9), and is the most abundant protein in protein folding machinery (11). UPR plays critical roles for the endoplasmic reticulum (ER) lumen. gp96 is a key plasma cell differentiation as demonstrated by lack of downstream chaperone in the ER to mediate unfolded plasma cells in the absence of XBP1 (12–14). Moreover, protein response (UPR; refs. 10). UPR is an evolutionally gp96 is induced more than 10-fold during B-cell activation conserved mechanism to maintain protein quality control (4), and it has been shown to participate in the assembly of in the secretory pathway. Accumulation of misfolded pro- B-cell receptor complexes through its association with teins in the ER triggers the activation of three well-known immunoglobulin-a (Iga) molecules (15). In addition to the critical roles of UPR in plasma cell differentiation, a picture has recently emerged of the roles of UPR, particu- larly XBP1s, in myeloma pathogenesis. XBP1s and down- 1 Authors' Affiliations: Department of Microbiology and Immunology, stream ER chaperones are consistently upregulated in mye- Hollings Cancer Center, Medical University of South Carolina, Charleston, South Carolina; 2Levine Cancer Institute, Charlotte, North Carolina; loma cells (16). Most strikingly, transgenic expression of 3Department of Molecular Pharmacology, Memorial Sloan Kettering Can- XBP1s in the B-cell compartment of mice results in plasma cer Center, New York, New York; and 4The University of Texas MD Anderson Cancer Center, Texas cell dyscrasia with evidence of increased monoclonal anti- bodies ("M-spike"), lytic bone lesions, plasmacytosis, and Corresponding Author: Bei Liu, Department of Microbiology and Immu- nology, Hollings Cancer Center, Medical University of South Carolina, 86 kidney damage (17). However, the roles of individual ER Jonathan Lucas Street, Charleston, SC 29425. Phone: 843-792-8994; HSPs in myeloma have not been reported. Fax: 843-792-9588; E-mail: [email protected] Using genetic strategies, we recently found that gp96 is doi: 10.1158/1078-0432.CCR-13-2083 an obligate master chaperone for multiple Toll-like recep- Ó2013 American Association for Cancer Research. tors (TLR) and integrins (18–21). However, under the www.aacrjournals.org OF1 Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst September 27, 2013; DOI: 10.1158/1078-0432.CCR-13-2083 Hua et al. (IL)-6. U266B1 cells were cultured in RPMI medium sup- Translational Relevance plemented with 15% heat-inactivated FCS, 1 mmol/L sodi- Multiple myeloma is an incurable plasma cell neo- um pyruvate (Gibco), 10 mmol/L HEPES (Gibco) and plasm whose pathogenesis is closely linked to dysregu- penicillin–streptomycin (Gibco). All cells were cultured in lated unfolded protein response (UPR) in the endoplas- 5% CO2 incubator. Gp96-mutant and WT pre-B-cell lines mic reticulum (ER). In this study, we demonstrated that were provided by Brian Seed (Harvard University, Cam- the persistence of plasma cells as well as the development bridge, MA). of myeloma is critically dependent on gp96, a major molecular chaperone in the ER in vivo. Furthermore, our Reagents results demonstrated that gp96 knockdown causes Antibodies used for flow cytometry were obtained from severe compromise in human multiple myeloma cell BD Biosciences and eBioscience. Antibodies against LRP6, growth through attenuating Wnt-LRP-survivin pathway. b-catenin, survivin, c-myc, and caspase-9 were purchased Targeting gp96 in multiple myeloma cells by a gp96- from Cell Signaling Technology, cyclin D1 was obtained specific inhibitor also blocks proliferation and induces from Abcam, and gp96 antibody was bought from Enzo Life apoptosis. This is the first study to uncover the critical Sciences, Inc. Ig levels were determined by a sandwich ELISA roles of gp96 in the initiation and progression of mul- kit from Southern Biotechnology Associates. All other che- tiple myeloma, suggesting that blockade of gp96 is a micals were obtained from Sigma-Aldrich and Fisher Sci- novel therapeutic strategy against this disease. entific. IL-4 and IL-21 were purchased from PeproTech. WS13, a gp96-specific Hsp90 inhibitor of the purine-scaf- fold class (22), was synthesized as previously described (23, 24) and the structure was recently reported (25). steady-state conditions, gp96 is not required for B-cell activation, germinal center formation, plasma cell differ- Flow cytometry and in vitro plasma cell differentiation entiation, and class-switching or affinity maturation (19). Surface staining of cells and analysis on FACSCalibur and It is unclear whether gp96 is required for plasma cell FACSVerse (Becton-Dickinson) were done as described biology and for the development of myeloma during (18, 26). B cells were purified from spleens using CD19 chronic ER stress conditions. In this study, we addressed magnetic beads according to the manufacturer’s protocol the roles of gp96 in myeloma using XBP1s-transgenic (Miltenyi Biotec). Purified murine splenic B cells were mice as well as multiple human multiple myeloma (MM) labeled with carboxyfluorescein succinimidyl ester (CFSE), cell lines. We demonstrated that gp96 is required for and then stimulated with anti-m antibody, agonistic CD40 myeloma progression both in vitro and in vivo.Mecha- antibody, and IL-21 for 3 days. This was followed by flow nistically, we found that gp96 critically controls the Wnt- cytometric analysis of expression levels of cell surface LRP6-survivin pathway. Our results indicated that gp96 is CD138 by dividing cells. a novel therapeutic target for multiple myeloma. Viral vectors and transduction Human gp96 and surivin short hairpin RNA (shRNA) Materials and Methods lentiviral vectors as well as control vector were purchased Mice and cell lines from Open Biosystems. Survivin retroviral vector and empty B-cell–specific gp96-deficient mice and wild-type control vector control were obtained from Origene Technologies. littermates have been described (19). B-cell–specific XBP1s- Cells were seeded in a 12-well plate and spin-infected with transgenic and gp96-deficient mice were generated by cross- recombinant virus (3,000 rpm, 32C, 90 minutes) in a ing our B-cell–specific gp96-deficient mice with XBP1s desktop centrifuge as reported (26). transgenic mice. SCID mice were kindly provided by Jenni- fer Wu (Medical University of South Carolina, Charleston, Protein extraction and Western blot analysis SC). Mice were bred and maintained according to the Protein extraction and immunoblot were performed as established guidelines and an approved protocol by Med- described previously (20). Briefly, cells were washed three ical University of South Carolina Institutional Animal Care times with ice-cold PBS and lysed in radioimmunoprecipi- and Use Committee. Human multiple myeloma cell line tation assay lysis buffer (0.01 mol/L sodium phosphate, pH RPMI-8226, U266B1, MM.1S, and MM.1R were purchased 7.2, 150 mmol/L NaCl, 2 mmol/L EDTA, 1% NP-40, 1% from American Type Culture Collection. OPM1, JK-6L, and sodium deoxycholate, 0.1% SDS, 2 mmol/L AEBSF, 130 INA-6 were kindly provided by Yubin Kang (Medical Uni- mmol/L bestatin, 14 mmol/L E-64, 0.3 mmol/L aprotinin, versity of South Carolina). Multiple myeloma cells were and 1 mmol/L leupeptin). Total cell lysates was resolved on cultured in RPMI medium (Sigma-Aldrich) supplemented denaturing and reducing 10% to 12% SDS-PAGE, and the with 10% heat-inactivated fetal calf serum (FCS; Atlas proteins were transferred from the gel onto Immobilon-P Biologicals), 55 mmol/L 2- mercaptoethanol (2-ME, Gibco), membranes.
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