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. 2015 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)substrates, 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 2015 American Association for Cancer Research. associated with transformation from both the benign state of

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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 multi- p21 knockout fi ple myeloma cells. Our study identi es a novel function for The lentiviral CRISPR/Cas9 GeCKO system was used for spe- MLN4921 in the upregulation of p21 expression, which is cific p21 knockout in CKS1B-overexpressing multiple myeloma independent of the CKS1B-mediated SCFSkp2 complex forma- Skp2 cells. Two pairs of p21 single guide RNAs (sgRNA) were annealed, tion and NEDD8-dependent SCF activation, resulting in phosphorylated, and ligated into the lentiCRISPR lentiviral killing multiple myeloma cells. sgRNA vector: p21 forward primer: 50-CACCG CCGCGACTGT- GATGCGCTAA-30; reverse, 50-AAACTTAGCGCATCACAGTCG- Materials and Methods CGGC-30. The U6 forward primer: 50-GACTATCATATGCT- 0 Gene expression profiling TACCGT-3 was used for sequencing sgRNA. Lentiviruses contain- The data of gene expression profile (GEP) were collected from a ing the sgRNAs were obtained by cotransfection of the lenti- publicly available website that include 351 newly diagnosed CRISPR-p21sgRNA vector with pVSVg and psPAX2 vectors with patients with multiple myeloma who participated in the Total packaging vectors into HEK-293T cells. Viral supernatant was Therapy 2 (TT2) clinical trial [Zhan (18); Shaughnessy (15)] and harvested after 48 hours. ARP-1 and OCI-My5 multiple myeloma 264 relapsed multiple myeloma in the Assessment of Proteasome cells were transduced with lentivirus containing the annealed p21 Inhibition for Extending Remission (APEX) phase III clinical trial sgRNAs. All primers were purchased from Integrated DNA [Mulligan (19)]. Permutation analyses were performed to corre- Technologies. late NAE1, UBA3, UBC12, and NEDD8 expression with patient survival in the APEX trial (n ¼ 264). The significance of gene RT-PCR expression with drug response in the APEX trial was analyzed by a mRNA was extracted using the RNeasy Mini Kit (Bio-Rad) from Student t test. wild-type, empty vector-transfected, CKS1B-overexpressing, and

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p21 knockout cultured cells treated with or without bortezomib, and 10ug of total protein lysate was separated on a Mini-PRO- MLN4924, or vehicle for 24 hours. Reverse-transcription PCR was TEAN TGX precast acrylamide gel (BIO-RAD). Gels were trans- performed with 5 iScript reverse transcription supermix (Bio- ferred to activated nitrocellulose membranes. Membranes were Rad) according to the manufacturer's protocols. Quantitative real- blocked with 5% nonfat dry milk in Tris buffered saline (TBS) time PCR (qRT-PCR) was used to detect p21 and GAPDH expres- containing 0.05% Tween-20 (TBS-T). Membranes were blotted sion in. Primers used for qRT-PCR were as follows: p21 forward, with the indicated primary antibodies. All primary antibodies 50-TGT CCG TCA GAA CCC ATG C-30, p21 reverse, 50-AAA GTC were diluted according to the manufacturer's directions. CKS1B, GAA GTT CCA TCG CTC-30, and GAPDH forward, 50-GAC ACC CUL1, p27 and all secondary antibodies were purchased from CAC TCC TCC ACC T-30, reverse, 50-ATG AGG TCC ACC ACC CTG Santa Cruz Biotechnology, primary antibodies p21, P-RB(S608), T-30. GAPDH transcript levels were used to normalize the amount P-RB(S780), P-RB(S807) and b-Actin were purchased from Cell of p21 cDNA in each sample. Signaling Technology. b-Actin was used to normalize the amount of protein for each sample. Protein bands were visualized using Cell proliferation assay HRP-conjugated secondary antibodies and SuperSignal West Pico A total of 1.5 105 cells/mL were seeded into 6-well plates in (Pierce). Blots were subsequently stripped and reprobed for b 2-mL media in triplicate. Cells were untreated, treated with -actin as loading control. Uncropped blots are presented in 5 nmol/L bortezomib, or 1 mmol/L MLN4924. Media were Supplementary Fig. S1. replenished with fresh RPMI-1640 with or without drugs every 2 days. Cell proliferation and viability were evaluated for 7 days at Statistical analyses the indicated time point using a hemocytometer paired with One-way ANOVA ( three groups) and Student t tests were Trypan blue exclusion. Cell viability is expressed as the ratio of used to determine significance between experimental groups. live cells relative to total cells. Two-way ANOVA paired with the Tukey–Kramer method for multiple comparisons was used to compare multiple groups with Soft-agar clonogenic formation assay multiple treatment levels. Significance was defined as P < 0.05. ARP-1 and OCI-My5 multiple myeloma cells (1 105) were seeded in 12-well plates with or without bortezomib or MLN4924 Results at varying doses in fresh RPMI-1640. Cells were resuspended in Multiple myeloma cells overexpressing CKS1B are resistant to 0.33% agar in MyeloCult H5100 cultures/10% FBS and allowed bortezomib but sensitive to MLN4924 to clonally expand for 3 weeks. Cells were fed twice each week by CKS1B expression is increased in relapsed multiple myeloma placing two drops of medium on the layer with or without drugs. and confers drug resistance, including resistance to bortezomib Images of all plates were taken on a Nikon Eclipse Ti microscope, (5). In an attempt to overcome this drug resistance and toxi- and colony numbers were counted using the ImageJ. The colony cities associated with pan-proteasomal inhibitors, more specif- efficiency was calculated as (colony number in the image) (well ically targeted inhibitors of protein degradation have emerged, area)/(actual area the photo represent)/10,000 100%. including the NAE inhibitor MLN4924. Because of the established drug-resistance against bortezomib of multiple myeloma b Senescence -galactosidase cell staining cells overexpressing CKS1B, we wanted to examine whether b Senescence -galactosidase staining was used to detect cells overexpressing CKS1B were resistant to treatment with b -galactosidase activity, a known characteristic of senescent MLN4924. cells. OCI-My5 and ARP-1 cells with or without the stable We compared the effects of bortezomib and MLN4924 on overexpression of CKS1B or stable knockdown of p21 were the proliferation and survival of ARP-1 and OCI-My5 multiple grownin6-wellplates.Cellsweremocktreatedortreatedfor myeloma cell lines stably transfected with empty vector or 48 hours with bortezomib or MLN4924, were harvested, washed CKS1B expression vector. We found that while cells expressing fi b with PBS, and xed. Cells were resuspended in -Galactosidase basal levels of CKS1B were susceptible to treatment with both Staining Solution (5 mmol/L K3Fe(CN)6, 5 mmol/L K4Fe bortezomib and MLN4924 (Fig. 1A, left), cells overexpressing (CN)6 3H2O, 2 mmol/L MgCl2, PBS up to 500 mL, pH 6.0) CKS1B treated with bortezomib continued to proliferate in a and seeded into 6-well plates. Plates were sealed with parafor- manner comparable with untreated cells (Fig. 1A, right) while b maldehyde and incubated at 37 C. The -galactosidase staining cells overexpressing CKS1B treated with MLN4924 failed to solution was removed and staining was assessed under a micro- proliferate appreciably (Fig. 1A, right, green line). In examining scope. Stained cells were counted and expressed relative to total cell viability, treatment with either bortezomib or MLN4924 in cells. Graphs represent the average and standard deviations from cells expressing basal levels of CKS1B resulted in diminished four separate plates. This method was also used to assess senes- viability (Fig. 1B, left). In cells overexpressing CKS1B, treatment cence in CKS1B-overexpressing multiple myeloma cells treated with bortezomib resulted in viability comparable with mock with empty vector or stable p21 knockdown with or without the treatment (Fig. 1B, right) but treatment with MLN4924 yielded treatment of MLN4924. Representative plates are depicted in rapid collapse leading to total cell death within 6 days (Fig. 1B, Supplementary Fig. S3. right, green line). These results highlight that cells with high CKS1B are resistant to bortezomib treatment but remain sen- Immunoblots sitive to MLN4924. Protein expression levels were determined by Western blot In order to assess the role of p53 on proliferation and cellular analysis. Briefly, cells were lysed using a Mammalian Cell Extrac- viability, we performed experiments using the p53-positive tion Kit with protease inhibitor cocktail (Biovision). Protein KMS28PE multiple myeloma cell line with or without the stable concentrations were determined on a NanoDrop 1000 (Thermo) overexpression of CKS1B. We found that KMS28PE cells exhibited

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Figure 1. Multiple myeloma cells overexpressing CKS1B are resistant to bortezomib but sensitive to MLN4924. A, effects of bortezomib and MLN4924 on the in vitro proliferation of ARP-1 and OCI-My5 multiple myeloma cell lines with and without the overexpression of CKS1B. Cells were treated with either bortezomib (5 nmol/L) or MLN4924 (1 mmol/L) for 7 days. Proliferation was assessed by hemocytometer cell counts. B, effects of bortezomib and MLN4924 on cell viability of ARP-1 and OCI-My5 multiple myeloma cell lines with and without the overexpression of CKS1B. Cells were treated with either bortezomib (5 nmol/L) or MLN4924 (1 mmol/L) for 7 days. Cell viability is expressed as the ratio of viable cells to the total number of cells. C, effects of bortezomib and MLN4924 on the clonogenic capacity of ARP-1 and OCI-My5 multiple myeloma cell lines with and without the overexpression of CKS1B was assessed by counting colony formation in soft-agar. D, the effects of CKS1B expression on senescence in multiple myeloma cell lines was assessed by b-galactosidase staining. E, the effects of bortezomib and MLN4924 on senescence in ARP-1 and OCI-My5 cells overexpressing CKS1B was assessed by b-galactosidase staining. In all experiments, data are mean SD of three separate experiments. very similar proliferative patterns to both OCI-MY5 and ARP1 p27 and increased cellular senescence (20–22). CKS1B is an cells when treated with MLN4924 (Supplementary Fig. S2B). We essential accessory protein for the function of SCFSkp2.With also found that viability was affected very similarly to OCI-MY5 a known role for CKS1B in SCFSkp2 function and because of cells of the same CKS1B background (Supplementary Fig. S2C). the important role for SCFSkp2 in senescence, we examined the Together, these findings suggest that p53 status does not contrib- effects of bortezomib and MLN4924 on senescence in CKS1B- ute greatly to MLN4924 sensitivity. overexpressing multiple myeloma cell lines. We examined We examined the ability of CKS1B-overexpressing cells to the effect of CKS1B knockdown on senescence in ARP-1 and form colonies in soft agar in the presence of bortezomib or OCI-My5 multiple myeloma cell lines using shRNA to deplete MLN4924. ARP-1 and OCI-My5 cell lines with or without CKS1B expression (Fig. 1D). In each cell line, knockdown of CKS1B overexpression were seeded in the presence or absence CKS1B resulted in greatly increased senescence relative to treat- of bortezomib or MLN4924. Treatment of either EV cell line ment with a scrambled control. These results suggest that the with bortezomib or MLN4924 resulted in decreased colony role for CKS1B in SCFSkp2 integrity relates to senescence and formation (Fig. 1C). However, upon CKS1B overexpression, that a loss of CKS1B has comparable effects to the loss SCFSkp2 colony formation significantly increased upon botezomib chal- in the promotion of senescence. lenge. Conversely, colony formation remained very low in Next, we examined the effects of bortezomib and MLN4924 CKS1B-overexpressing cells upon challenge with MLN4924. on senescence in ARP-1 and OCI-My5 cell lines stably over- (Fig. 1C). These results indicate that cells with basal levels of expressing CKS1B. In both cell lines, the overexpression of CKS1B are sensitive to both bortezomib and MLN4924, where- CKS1B slightly increased senescence in untreated cells relative as cells overexpressing CKS1B are resistant to bortezomib but to cells transfected with empty vector (Fig. 1E). Treatment of remain sensitive to MLN4924. parental cell lines with either bortezomib or MLN4924 resulted Skp2 is a critical component of the E3 ligase complex in a marked increase in senescence. However, upon CKS1B SCFSkp2. Targeting Skp2 leads to accumulation of p21 and overexpression, there was a significant decrease in senescence

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upon bortezomib treatment. This decrease was not exhibited p21, p27, and senescence, we hypothesized that the drug resis- upon MLN4924 treatment. These results indicate that MLN4924, tance of CKS1B-overexpressing cells against bortezomib, but not but not bortezomib, is able to induce senescence in CKS1B- the NAE inhibitor MLN4924, stems from an increase in the overexpressing cells. ubiquitin-mediated degradation of downstream targets of the Taken together, these data illustrate that multiple myeloma CRL–SCF complex. Therefore, we sought to examine the effect cells over expressing CKS1B are resistant to treatment with the of drug treatment on SCFSkp2-regulated proteins in cells over- proteasomal inhibitor bortezomib but sensitive to the NAE expressing CKS1B. inhibitor MLN4924. MLN4924 overcomes CKS1B-induced drug resistance by CKS1B regulates neddylation-related signaling inhibiting p21 ubiquitin-mediated degradation In order to examine the relationship between CKS1B and In order to examine the effect of drug treatment on SCFSkp2 neddylation, we examined the neddylation of Cullin-1 in ARP- -regulated proteins in cells overexpressing CKS1B, we performed 1 and OCI-My5 multiple myeloma cell lines upon the modula- immunoblot analysis of known downstream targets of CRL/SCF tion of CKS1B expression. In both cell lines, the overexpression of (Fig. 3A). CKS1B led to increased neddylation of Cullin-1 as evidenced Consistent with Fig. 2, the use of a Cullin-1–speciﬁc antibody using an antibody targeted against NEDD8 (Fig. 2A). Conversely, showed that the overexpression of CKS1B led to an increase in the knockdown of CKS1B expression with shRNA led to a decrease in neddylated form of Cullin-1 and treatment with MLN4924 neddylated Cullin-1 in each cell line (Fig. 2B). Taken together, completely blocked the neddylation of Cullin-1 (Fig. 3A, row these data highlight the intimate relationship between CKS1B and 2). In both cell lines, inhibition of SCFSkp2 activation by blocking the neddylation of Cullin-1. We next explored the effects of Cullin-1 neddylation with MLN4924, independent of CKS1B MLN4924 on the neddylation of Cullin-1 in ARP-1 and OCI-My5 expression, led to increased expression of p27 and p21proteins cells overexpressing CKS1B. Consistent with the role of MLN4924 relative to expression seen in cells without drug treatment (Fig. 3A, as an NAE inhibitor, treatment with MLN4924 in each cell line led rows 7 and 8). Treatment of cells with basal level CKS1B expres- to a substantial decrease in the neddylation of Cullin-1 (Fig. 2C). sion with bortezomib resulted in increased levels of p27 and p21 ﬁ Paired with our earlier ndings, these results demonstrate that as we expected. Although we expected the overexpression of drug-resistant cells with increased CKS1B also have increased CKS1B to lead to the downregulation of p27 and p21 in borte- Cullin-1 neddylation. Because increased neddylation of Cullin- zomib-treated cells, we observed downregulation of p21 but Skp2 1 leads to increased activation of SCF , a negative regulator of continued overexpression of p27 (Fig. 3A, row 7 vs. row 8). Together, these results imply that bortezomib-induced increases in p27 are insensitive to CKS1B overexpression and suggest a mechanism of p27 regulation distinct from SCFSkp2 regulation of p21 and p27. We further examined the effects of drug treatment in cells with basal and elevated levels of CKS1B expression on the mRNA expression of the p21 gene CDKN1A. Consistent with the immunoblots, MLN4924 increased the low basal level expression of p21 dramatically in both parental and CKS1B- overexpressing cells, whereas treatment with bortezomib had negligible effects on the mRNA expression of p21 (Fig. 3B). CKS1B-overexpressing cells are sensitive to MLN4924, but not bortezomib, and p21 is differentially regulated from p27 in CKS1B-overexpressing cells upon bortezomib. These results suggest a critical role for p21 in the regulation of CKS1B drug resistance. Accordingly, we investigated the role of p21 in drug sensitivity in multiple myeloma cell lines.

p21 is necessary for MLN4924 sensitivity in CKS1B- overexpressing cells In order to investigate the role of p21 in drug sensitivity, we modulated p21 expression using cells stably expressing shRNA against p21. Because low basal expression of p21 is well established (20, 21), we conﬁrmed the induction of p21 expression Figure 2. CKS1B regulates neddylation-related signaling. A, protein expression was upon treatment of cells with MLN4924, an established DNA- measured in ARP-1 and OCI-My5 cells stably transfected with empty damaging agent and activator of p21 (22, 23). CKS1B-overexpres- vector (EV) or CKS1B (OE). A 30 mg/lane of whole-cell lysates were resolved sing OCI-My5 multiple myeloma cells expressing shRNA against on an SDS-PAGE gel and analyzed by immunoblotting with the indicated p21 substantially knocked down p21 protein expression upon antibody. B, immunoblot of protein lysates derived from ARP-1 and OCI-My5 MLN4924 treatment relative to cells transfected with empty vector cells stably transduced with either scrambled control or CKS1B-targeted (Fig. 4A). shRNA. A 30-mg whole-cell lysate was resolved using SDS-PAGE and lysates were blotted with the indicated antibodies. C, ARP-1 or OCI-My5 We examined cell viability in OCI-My5 cells overexpressing cells with or without the overexpression of CKS1B were immunoblotted for CKS1B with and without knockdown of p21 protein expression. Cullin-1 with or without MLN4924 (1 mmol/L) treatment (1 hour). The knockdown of p21 decreased cell sensitivity to MLN4924 in

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Figure 3. MLN4924 overcomes CKS1B-induced drug resistance by inhibiting p21 ubiquitin-mediated degradation. A, immunoblot analysis of protein expression following 48- hour treatment with bortezomib (5 nmol/L) or MLN4924 (1 mmol/L) in ARP-1 and OCI-My5 multiple myeloma cell lines stably transfected with empty vector (EV) or CKS1B (OE). Whole-cell lysates were resolved on an SDS-PAGE gel and analyzed by immunoblot using the indicated antibodies. B, p21 mRNA expression was determined in ARP-1 and OCI-My5 multiple myeloma cell lines stably transfected with empty vector (EV) or CKS1B (OE). qRT-PCR was performed to assess p21 mRNA expression following 48-hour treatment with bortezomib (5nmol/L) or MLN4924 (1 mmol/L) and results were normalized to the expression of GAPDH. Values are expressed relative to untreated cells transfected with empty vector. Data are mean SD of four separate experiments.

CKS1B-overexpressing cells and increased cell viability while We also explored the importance of p21 in the induction of prolonging survival relative to cells expressing p21 (Fig. 4B). senescence in cells overexpressing CKS1B. Although treatment of Importantly, we have performed comparative proliferation assays CKS1B-overexpressing cells with MLN4924 resulted in increased between cells with and without knockdown of p21. We find no senescence relative to untreated cells, the amount of cells in significant differences in proliferation, suggesting that changes in senescence was dramatically reduced upon the knockdown of viability are not an artifact of altered proliferation rates (data not p21 (Fig. 4D). shown). These results demonstrate that MLN4924 sensitivity on cell We further examined the clonogenic ability of p21 knockout proliferation and survival, clonogenic capacity, and senescence cells to form colonies in soft agar in the presence of MLN4924 in CKS1B-overexpressing cells is dependent upon p21 and a relative to cells expressing basal p21 levels. Consistent with a loss of p21 shifts CKS1B-overexpressing cells toward insensi- role for p21 in sensitivity to MLN4924, colony formation tivity to MLN4924. Taken together, these findings reveal, for the significantly increased in cells with p21 knockdown compared first time, a crucial role for p21 in regulating cell sensitivity to with cells expressing p21 upon MLN4924 treatment (Fig. 4C). MLN4924. This increase was not seen in untreated cells. These results Neddylation machinery is dysregulated and associated with indicate that cells with diminished levels of p21 are less drug resistance and poor clinical outcomes in multiple myeloma. sensitive to MLN4924 and have a higher clonogenic capacity Having demonstrated the importance of the NEDD8 pathway in the presence of MLN4924. in regulating SCFSkp2 activity in multiple myeloma cell lines, we

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Figure 4. p21 is necessary for MLN4924 sensitivity in CKS1B-overexpressing cells. A, immunoblot analysis of p21 expression in OCI-My5-CKS1B OE multiple myeloma cells stably transfected with empty vector (EV) or p21 shRNA-expressing vector (p21KO) following 24-hour treatment with MLN4924. B, the effects of bortezomib and MLN4924 on cell viability were assessed in OCI-My5-CKS1B–overexpressing cells transfected with either empty vector (EV) or shRNA targeting p21 expression (p21KO). Cells were seeded in the presence of MLN4924 (0.5 mmol/L, 1 mmol/L) and viability was monitored for 6 days. Cell viability was assessed by hemocytometer cell counts using trypan blue exclusion staining and is expressed as the ratio of viable cells to the total number of cells. Data are mean SD of three separate experiments. C, the effect of MLN4924 (10 nmol/L, 100 nmol/L, 500 nmol/L) on the clonogenic capacity of OCI-My5-CKS1B–overexpressing multiple myeloma cells transfected with empty vector or shRNA targeting p21 (p21 KO) was assessed by counting colony formation in soft-agar. Data are mean SD of three separate experiments. D, the effects of MLN4924 (100 nmol/L) on senescence in OCI-My5-CKS1B–overexpressing cells with the expression of empty vector or shRNA targeting p21 (p21 KO) was assessed by b-galactosidase staining. Data are mean SD of three separate experiments.

investigated the clinical importance of the NEDD8 pathway in and thalidomide (data not shown; ref. 24). However, patients multiple myeloma. with elevated expression of proteins involved in the neddyla- Although there is a broad distribution of gene expression in tion and activation SCFSkp2 had decreased overall survival each group from TT2, we found that the mRNA expression of relative to patients with low expression of neddylation pathway NAE1, UBA3, and UBC12 was, on average, significantly elevated in proteins. The elevated expression of UBA3 (P ¼ 0.003), UBC12 plasma cells from patients with MGUS and patients newly diag- (P ¼ 0.001), or CKS1B (P ¼ 0.015) each correlated with signif- nosed with multiple myeloma (24) relative to normal plasma icantly poorer prognoses than patients with low expression of the cells (NPC) from healthy donors (Fig. 5A). same gene in the APEX trial, a phase III study that compared We also examined NEDD8 mRNA expression in patients from bortezomib with dexamethasone in post-relapse multiple mye- APEX trials that were responsive or unresponsive to standard loma patients (Fig. 6A). Populations were further classified on two multiple myeloma therapies (i.e., bortezomib and dexametha- variables of CKS1B and UBA3 expression (Fig. 6B, left) and CKS1B sone; refs. 19, 25). Although great variance was seen in popula- and UBC12 expression (Fig. 6B, right). In both scenarios, patients tions, the average expression of NEDD8 was significantly elevated with (high/high) expression of either CKS1B-UBA3 (P ¼ 0.029) or in patients exhibiting decreased response to clinical treatment CKS1B-UBC12 (P ¼ 0.006) demonstrated significantly decreased (P ¼ 0.015; Fig. 5B). This result is further segregated into response survival from patients with heterogenous (high/low) or (low/ to either bortezomib or dexamethasone. As shown in Fig. 5C, high) expression. In both cases, greatest survival was seen in (low/ average NEDD8 expression was significantly elevated in patients low) CKS1B-UBA3 or CKS1B-UBC12 populations, suggesting exhibiting decreased clinical response to bortezomib (P ¼ synergistic effects of CKS1B with either UBC12 or UBA3 in patient 0.0005), but was not significantly altered in patients resistant to outcome. dexamethasone (P ¼ 0.61). These results identify a subset of patients with increased NEDD8 expression who are, on average as a population, resistant to bortezomib but not to dexamethasone Discussion in the clinic. A marked increase in the frequency of high-risk designation in The effect of NAE1, UBA3, UBC12, and NEDD8 expression multiple myeloma patients from 13% at diagnosis to 76% at on patient outcome was further analyzed using the Kaplan– relapse provides strong evidence for disease evolution in multiple Meier survival and log-rank tests. We did not find any signif- myeloma (15), suggesting the outgrowth of a drug-resistant icance in the TT2 trial that includes treatment with melphalan subpopulation of multiple myeloma cells. Previously, we have

5538 Clin Cancer Res; 21(24) December 15, 2015 Clinical Cancer Research

Overcoming CKS1B-Induced Drug Resistance with MLN4924

Figure 5. The neddylation pathway is dysregulated and associated with drug resistance in multiple myeloma. A, examination of the mRNA expression proﬁles of neddylation pathway genes. Signal levels from NAE1, UBA3, UBC12,andNEDD8 are shown on the y-axis. GEP signals were derived from participants in TT2 clinical trials of different clinical backgrounds. Patients were designated as healthy donors with NPCs, MGUS patients (MGUS), or multiple myeloma patients (MM) and are sorted on the x-axis. B, an examination of NEDD8 expression in patients categorized as unresponsive (No Response) or responsive (Response) to treatment with dexamethasone and/or bortezomib. Mean signal levels of patients and P values of differences in expression are given. C, GEPs from 5B were further segregated by treatment with bortezomib or dexamethasone. NEDD8 expression signal levels are plotted on the y-axis. The mean signal level and P value of differences are given between unresponsive and responsive patients. demonstrated CKS1B expression results in increased multidrug disease. The existence of a subset of patients who do not receive resistance, while, clinically, CKS1B expression is increased in maximum beneﬁt from currently available treatments in multiple relapsed multiple myeloma patients (5), providing molecular myeloma highlight the critical need for improved therapeutic evidence for CKS1B in a more drug-resistant multiple myeloma options.

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Figure 6. Dysregulation of the neddylation pathway is associated with poor prognosis in multiple myeloma. A, the Kaplan–Meier estimates of overall survival in patients with varied expression of neddylation pathway genes. Patients with elevated expression of UBA3 (left) showed poorer overall survival than those with low expression (29% vs. 81%; P ¼ 0.003). Patients with elevated expression of UBC12 (middle) showed poorer overall survival than those with low expression (28% vs. 48%; P ¼ 0.001). Patients with elevated expression of CKS1B (right) showed poorer overall survival than those with low expression (35% vs. 47%; P ¼ 0.015). B, the Kaplan–Meier estimates of overall survival in patients with sorted CKS1B and neddylation enzyme GEPs. Left, patient survival sorted by high/low CKS1B expression and high/low UBA3 expression is depicted. Patients high in expression of CKS1B and UBA3 exhibited signiﬁcantly (P ¼ 0.029) poorer overall survival (26%) than those with either high CKS1B/low UBA3 expression (40%) or low CKS1B/highUBA3 expression (31%). Patients with low CKS1B/low UBA3 demonstrated the greatest survival rate (29%). Right, patient survival sorted by high/low CKS1B expression and high/low UBC12 expression is depicted. Patients high in expression of CKS1B and UBC12 (left) exhibited signiﬁcantly (P ¼ 0.006) poorer overall survival (20%) than those with either high CKS1B/low UBC12 expression (44%) or low CKS1B/high UBC12 expression (36%). Patients with low CKS1B/low UBA3 demonstrated the greatest survival rate (54%).

The aim of this study was to evaluate if the NAE inhibitor sing cells identified the differential regulation of the CRL/SCF MLN4924 is effective in treating multiple myeloma in cells with downstream target p21, showing the degradation of p21 upon increased CKS1B expression. Here, we demonstrate that multiple bortezomib treatment but not MLN4924 treatment. The use of myeloma cells overexpressing CKS1B are resistant to bortezomib targeted shRNA against p21 resulted in a loss of sensitivity to but remain sensitive to MLN4924. Our results demonstrate that MLN4924 in cellular proliferation, clonogenic expansion, and the treatment of cells with elevated CKS1B expression with senescence induction. These results confirm a mechanism MLN4924, but not bortezomib, results in decreased cellular through p21 by which CKS1B-overexpressing cells exhibit drug proliferation and survival, reduced clonogenic expansion, and resistance to bortezomib but sensitivity to MLN4924. the induction of cellular senescence, strongly suggesting a role for The clinical potential of MLN4924 inhibition of Cullin-1 NEDD8 regulation of CRL/SCF activation under conditions of activation through the neddylation pathway was highlighted by CKS1B-induced drug resistance. SCFSkp2 is responsible for regu- examining neddylation machinery expression patterns in multi- lating the ubiquitin-mediated degradation of the cell-cycle check- ple myeloma patients. Consistent with GEPs derived from patient point proteins p21 and p27. We found the overexpression of populations, a great deal of variance was exhibited in each CKS1B led to increased neddylation of Cullin-1, whereas targeted examined population. On average, we found that the expression knockdown of CKS1B reduced Cullin-1 neddylation. These find- of the E1 Nedd8-activating enzyme NAE1, the E1 Nedd8-activat- ings demonstrate a novel role for CKS1B in the regulation of ing enzyme UBA3, the E2 Nedd8-conjugating enzyme UBC12,to SCFSkp2 ubiquitin-mediating complex and suggest a mechanism be significantly upregulated in multiple myeloma patients relative for how cells with elevated CKS1B expression may evade growth to patients with MGUS or NPCs derived from healthy donors. In regulation. Finally, extensive immunoblot analysis of SCFSkp2 the APEX trial, we also saw a significant increase in the expression degradation targets upon drug treatment in CKS1B-overexpres- of Nedd8 in patients unresponsive to the combinatorial treatment

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Overcoming CKS1B-Induced Drug Resistance with MLN4924

of bortezomib and dexamethasone after relapse relative to those MLN4924 is a more selective inhibitor of protein degradation with response and these patients had inferior outcomes. than the 20S proteasome inhibitor bortezomib. Targets of SCFSkp2 Although our study examines MLN4924 in a drug-resistant, are enriched for proliferation and survival and inhibition by elevated CKS1B environment that recapitulates a portion of a MLN4924 achieves a disproportionately antitumoral response known high-risk phenotype in clinical multiple myeloma without the cellular toxicity associated with the pan-proteasomal patients, there have been previous studies in multiple myeloma inhibition of bortezomib. Therefore, inhibitors of the neddyla- systems that share consistent findings with us. In examining the in tion pathway, and MLN4924 specifically, offer a promising alter- vitro treatment of multiple myeloma cells with MLN4924, McMil- native to bortezomib in the clinical treatment of multiple mye- lin and colleagues (26) have found MLN4924 to be effective in loma and other cancers. limiting viability a number of multiple myeloma cell lines, It has been demonstrated that the treatment of multiple mye- including in the bortezomib-sensitive ANBL6wt multiple myelo- loma cells with MLN4924 results in a distinct GEP from the ma cell line and the bortezomib-resistant ANBL6-5VR subline. treatment of cells with bortezomib, and that treatment with More recently, Gu and colleagues (27) have demonstrated the MLN4924 does not elicit the compensatory upregulation of utility of MLN4924 in the inhibition of both AKT and mTOR transcripts specific for ubiquitin/proteasome (26). MLN4924 and signaling in multiple myeloma cell lines and primary multiple bortezomib both inhibit proteasomal degradation, yet through myeloma cells. By working in p53 wild-type multiple myeloma distinct pathways. This excitingly suggests the possibility of a cell lines (MM.1S and MM.1R) and multiple myeloma cell lines combinatorial approach between these drugs though, to date, with mutant/null p53 status (LP1, OCI-MY5, RPMI8266, OPM2, preliminary findings remain disparate. Gu and colleagues (27) and U266), their work also showed that sensitivity to MLN4924 is have found synergy between bortezomib and MLN4924 in the not related to p53 status (27), while our work was performed induction of apoptosis. Conversely, in exhaustive titration experi- primarily in OCI-My5 multiple myeloma cells (p53-heterozygous ments, McMillin and colleagues (26) found no synergy but also wt/mutant; refs. 28, 29) and ARP-1 multiple myeloma cells (p53- no antagonism between bortezomib and MLN4924 in affecting null, homozygous deletions; ref. 30) cell lines, our efforts in the the viability of multiple myeloma cell lines. p53-positive KMS28PE multiple myeloma cell line resulted in In summary, our study has demonstrated that MLN4924 is nearly identical effects to cellular proliferation and viability upon effective at limiting cell growth and proliferation and inducing MLN4924 treatment as seen in the OCI-MY5 cell line, consistent senescence in drug-resistant, high CKS1B-expressing cells. We with the previously published findings. demonstrate that p21 is critically important for cell sensitivity to MLN4924 induction of senescence occurs in part from the MLN4924 and loss of p21 expression results in MLN4924 resis- accumulation of factors leading to DNA damage (e.g., CDT1) tance. Our findings highlight the clinical potential of targeting the and the accumulation of cell-cycle regulatory proteins (e.g., p21), NEDD8 pathway and are supported by clinical data demonstrat- the combination of which results in the robust activation of the ing multiple myeloma patients with elevated expression of DNA-damage response, leading to senescence (31). We demon- NEDD8 machinery have inferior clinical outcomes relative to strate the treatment of bortezomib-resistant CKS1B-overexpres- patients with lower expression. Although we have not found a sing cells with MLN4924 results in an increase in senescence in a survival advantage correlated with p21 expression in multiple p21-dependent manner. This important role of p21 in MLN4924- myeloma patients (data not shown), clinical trials paired with induced senescence is well supported by the findings of others MLN4924 have yet to be performed and analyzed. Our findings (32). Although the consensus is that p21 is important for suggest that the expression of p21 may predict MLN4924 efficacy MLN4924-induced senescence, there still remain discrepancies and screening patients for p21 expression may have great merit. between the findings of other groups. One group has found that Further examination of MLN4924 in preclinical and, possibly, in the genetic inactivation of Skp2 was insufficient to induce senes- clinical combinatorial studies with currently available treatments cence alone but critically dependent upon p21, p27, and ATF4 (e.g., bortezomib, dexamethasone, doxorubicin) for multiple (33). Another group found that MLN4924-induced senescence is myeloma is warranted. critically dependent upon p21 but independent of p53, p16, and pRb (34). A third group found that both p21 and p53 are Disclosure of Potential Conflicts of Interest important for initiating senescence but either factor is dispensable No potential conflicts of interest were disclosed. as SA-b-gal staining was diminished, but not absent, in p21 / and p53 / cells (32). Our results are consistent with most Authors' Contributions consistent with the second group (34). The striking differences Conception and design: Y. Zhou, G.S. Thomas, Z. Gu, G. Tricot, F. Zhan between these studies may be accounted for in part by the Development of methodology: J. Huang, Y. Zhou, Z. Gu, Y. Yang, H. Xu Acquisition of data (provided animals, acquired and managed patients, different cell culture models used by the each group. provided facilities, etc.): J. Huang, F. Zhan The overexpression of CKS1B in relapsed multiple myeloma Analysis and interpretation of data (e.g., statistical analysis, biostatistics, patients contributes to bortezomib resistance (5) and relates to computational analysis): J. Huang, G.S. Thomas, Y. Yang, H. Xu, F. Zhan decreased survival, constituting a risk factor and highlighting the Writing, review, and/or revision of the manuscript: J. Huang, G.S. Thomas, need for new therapies. We have shown here that genes in the G. Tricot, F. Zhan neddylation pathway (NAE1, UBA3, UBC12, and NEDD8) are Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): G.S. Thomas upregulated leading to activation of SCFSkp2 and correlated with Study supervision: G. Tricot, F. Zhan inferior survival in multiple myeloma patients. These results are fi consistent with and expand upon the ndings of McMillin and Grant Support colleagues (26) who demonstrated decreased progression-free This work was supported by: R01CA152105 (to F. Zhan) from the NCI; The survival (PFS) in patients with elevated expression of NEDD8. Leukemia & Lymphoma Society Translational Research Program (to F. Zhan; By targeting NAE, the first step in the activation of CRL complexes, 6094-12); institutional start-up funds from the Department of Internal

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Huang et al.

Medicine, Carver College of Medicine, University of Iowa (to F. Zhan and G. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate Tricot); The University of Iowa Holden Comprehensive Cancer Center Support this fact. Grant P30 CA086862. The costs of publication of this article were defrayed in part by the Received January 30, 2015; revised May 6, 2015; accepted June 15, 2015; payment of page charges. This article must therefore be hereby marked published OnlineFirst July 8, 2015.

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5542 Clin Cancer Res; 21(24) December 15, 2015 Clinical Cancer Research

NEDD8 Inhibition Overcomes CKS1B-Induced Drug Resistance by Upregulation of p21 in Multiple Myeloma

Junwei Huang, Yi Zhou, Gregory S. Thomas, et al.

Clin Cancer Res 2015;21:5532-5542. Published OnlineFirst July 8, 2015.

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