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NEDD8 Inhibition Overcomes CKS1B-Induced Drug Resistance by Upregulation of P21 in Multiple Myeloma Junwei Huang1,2, Yi Zhou1, Gregory S

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NEDD8 Inhibition Overcomes CKS1B-Induced Drug Resistance by Upregulation of P21 in Multiple Myeloma Junwei Huang1,2, Yi Zhou1, Gregory S Published OnlineFirst July 8, 2015; DOI: 10.1158/1078-0432.CCR-15-0254 Cancer Therapy: Preclinical Clinical Cancer Research NEDD8 Inhibition Overcomes CKS1B-Induced Drug Resistance by Upregulation of p21 in Multiple Myeloma Junwei Huang1,2, Yi Zhou1, Gregory S. Thomas1, Zhimin Gu1, Ye Yang1, Hongwei Xu1, Guido Tricot1, and Fenghuang Zhan1 Abstract Purpose: CKS1B is significantly upregulated in multiple mye- Results: Cells overexpressing CKS1B were resistant to borte- loma and associated with poor prognosis. The identification of zomib but sensitive to MLN4924. Treatment of CKS1B-over- novel therapies is essential for effective treatment of patients expressing cells with MLN4924 decreased proliferation, resistant to chemotherapy. The NEDD8 inhibitor MLN4924 clonogenicity, and induced senescence. MLN4924, but not selectively targets SCFSkp2 activation and offers a more specific bortezomib, induced stabilization of p21 and knockdown of approach to protein degradation inhibition than total proteaso- p21 resulted in loss of MLN4924 sensitivity. Patients with mal inhibition. The goal of this study was to evaluate whether MGUS and multiple myeloma exhibited increased expression MLN4924 is effective in high CKS1B conditions and identify of NEDD8 pathway genes relative to normal plasma cells. mechanisms regulating drug potency. Multiple myeloma patients with high NEDD8 expression were Experimental Design: Bortezomib and MLN4924 sensitivity linked to bortezomib resistance in clinical trials, and had was assessed through proliferation, viability, clonogenic poten- inferior outcomes. tial, and senescence induction in cells overexpressing CKS1B. The Conclusions: Our data demonstrate that cells with elevated mechanism for MLN4924 sensitivity was elucidated by immu- CKS1B expression are resistant to bortezomib but sensitive noblot analysis of SCFskp substrates and confirmed by shRNA to MLN4924 and offer a mechanism through the stabilization knockdown. The clinical relevance of the NEDD8 pathway was of p21. These findings provide rationale for targeting the examined in gene expression profiles (GEP) derived from healthy NEDD8 pathway in multiple myeloma patients exhibiting people, patients with monoclonal gammopathy of undetermined elevated expression of CKS1B. Clin Cancer Res; 21(24); 5532–42. significance (MGUS), and multiple myeloma. Ó2015 AACR. Introduction associated with global proteasomal inhibition and resistance to bortezomib in multiple myeloma are major concerns, prompt- Multiple myeloma is a malignancy of plasma cells that accu- ing the further development of novel therapies. These improved mulate in the bone marrow, secrete antibody, and interfere with inhibitors are designed to more specifically target critical factors the production of normal blood cells. Multiple myeloma is now in protein turnover and are now part of ongoing clinical trials. the second most common hematologic malignancy in the United One such molecule, MLN4924, is an inhibitor of NEDD8- States, with a 5-year survival rate less than 50% (1). In the past activating enzyme (NAE), preventing the conjugation of the decade, proteasome inhibition and immunomodulators have small ubiquitin-like protein NEDD8 (neural precursor cell revolutionized multiple myeloma therapies. In particular, borte- expressed developmentally downregulated protein 8) to zomib, which targets the 26S proteasome subunit b5, has induced cullin-RING ubiquitin E3 ligases (CRL). This inhibitor has a high level of positive response rates (2, 3). However, toxicities shown promise against multiple myeloma in vitro by inhibiting the degradation of skp1/cullin-1/F-box SKP2 (SCFSkp2)sub- strates, including p27 and p21 (4). 1Department of Internal Medicine, Carver College of Medicine, Univer- CDC28 kinase subunit 1 (CKS1B) is a necessary cofactor of the sity of Iowa, Iowa City, Iowa. 2Institute of Cancer Research, School of CRL complex, SCFSkp2, which regulates cellular entry into S-phase Basic Medical Sciences, Southern Medical University, Guangzhou, and possesses antiapoptotic activity through p27-dependent and China. -independent pathways, and is a critical factor in multiple mye- Note: Supplementary data for this article are available at Clinical Cancer loma drug resistance (5). CKS1B is an essential protein for normal Research Online (http://clincancerres.aacrjournals.org/). cell division and growth (6), and is expressed at a high level in Junwei Huang, Yi Zhou, and Gregory S. Thomas contributed equally to this various cancer tissues, including hepatocellular carcinoma (7), article. colon (8), lung (9), oral squamous cell carcinoma (10), breast Corresponding Author: Fenghuang Zhan, Department of Internal Medicine, cancer (11), and others. In multiple myeloma, amplification of University of Iowa, 3269A CBRB 285 Newton Road, Iowa, Iowa City, IA 52242. region 1q21, which includes the CKS1B gene, identifies a sub- Phone: 319-384-0066; Fax: 319-353-8377; E-mail: [email protected] population with poor prognosis, an aggressive clinical course, and doi: 10.1158/1078-0432.CCR-15-0254 few therapeutic options (12, 13). CKS1B amplification is also Ó2015 American Association for Cancer Research. associated with transformation from both the benign state of 5532 Clin Cancer Res; 21(24) December 15, 2015 Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst July 8, 2015; DOI: 10.1158/1078-0432.CCR-15-0254 Overcoming CKS1B-Induced Drug Resistance with MLN4924 Compounds Translational Relevance MLN4924 and bortezomib were provided by Millenium Pahr- A subpopulation of patients with overexpression of CKS1B maceuticals. Each was resuspended in dimethyl sulfoxide is resistant to available therapeutics, and correlated with poor (DMSO) to a concentration of 10mmol/L and used at the con- prognosis, highlighting the immediate need for novel, effec- centrations indicated. tive therapeutics. Current therapeutic options, including the pan-proteasomal inhibitor bortezomib, are associated with Cell lines and cell culture fi toxicitiy arising from nonspeci c proteasomal inhibition. Human multiple myeloma cell lines ARP-1, OCI-My5, and MLN4924 inhibits the neddylation and activation of KMS28PE with or without CKS1B overexpression (expression Skp2 SCF offering a more targeted method of inhibiting protein levels previously detailed in ref. 5) were cultured in RPMI-1640 degradation than bortezomib. Here, we report that cells with (Life Technologies). HEK-293T cells were cultured in DMEM (Life elevated expression of CKS1B are resistant to bortezomib but Technologies). All media were supplemented with 10% heat- remain sensitive to MLN4924 treatment, highlighting the inactivated fetal bovine serum (FBS) and 1% penicillin/strepto- fi ef cacy of neddylation inhibition in high-risk multiple mye- mycin. All cells were cultured in humidified incubators at 37 C loma patients. We further define the mechanistic role for p21 with 5% CO2. in MLN4924 sensitivity in elevated CKS1B environments. Examination of gene expression profiles (GEP) from patients Establishment of stable multiple myeloma cell lines with multiple myeloma and monoclonal gammopathy of undetermined significance (MGUS) reveal elevation of Overexpressing CKS1B. The construction of multiple myeloma cell NEDD8 machinery expression and further reinforce the lines stably overexpressing CKS1B has been described previously fl importance of targeting SCFSkp2 activation. Our data support (5). Brie y, the cDNA sequence for human and mouse CKS1B is fi the further examination of MLN4924 in preclinical combina- equivalent. CKS1B was ampli ed from the PCDH-mouse-CKS1B- torial studies with currently available treatments for multiple EF1-RFP vector using the following primers: CKS1B Xba1 forward: Bam myeloma. GTGtctagaATGTCCCACAAACAAATCTACTATTCG,CKS1B H1 reverse: GTGggatccTCATTTCTTTGGCTTCTTGGGC. CKS1B cDNA was cloned into a modified pCDH vector (Systems Biology). Lentiviruses were obtained by cotransfection of the pCDH- CKS1B vector with packaging vectors into HEK-293T cells monoclonal gammopathy of undetermined significance (MGUS) following the standard protocol of ProFection Mammalian to multiple myeloma and further progression to plasma cell Transfection System (Promega). Viral supernatant was har- leukemia (14). Previously, we identified CKS1B as one of 70 vested after 36 hours and concentrated by ultracentrifugation. high-risk genes inversely associated with survival in newly diag- ARP-1andOCI-My5multiplemyelomacellsweretransfected nosed multiple myeloma (15) and high nuclear expression of with lentivirus containing CKS1B cDNA to yield CKS1B-over- CKS1B is an adverse prognostic factor for relapsed/refractory expressing multiple myeloma cells. The efficiency of lentiviral multiple myeloma patients (16). These findings provide compel- transfection was determined by measuring the expression of ling evidence that CKS1B represents a strong candidate target gene green fluorescent protein (GFP) using flow cytometry. Approx- for therapy. imately 95% transduction efficiency of multiple myeloma cells Skp2 Neddylated SCF represents the activated complex essen- was consistently achieved. CKS1B-OE multiple myeloma cells tial for the ubiquitination of CRL substrates (17). In this were screened by treatment with puromycin to select for study, we test the efficacy of MLN4924 in multiple myeloma CKS1B-overexpressing cells. cells overexpressing CKS1B. We explore how MLN4924 affects cell viability and senescence in CKS1B-overexpressing
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