Molecular and Cellular Effects of NEDD8-Activating Enzyme Inhibition in Myeloma

Published OnlineFirst January 12, 2012; DOI: 10.1158/1535-7163.MCT-11-0563

Molecular Cancer Preclinical Development Therapeutics

Douglas W. McMillin1,4, Hannah M. Jacobs1,4, Jake E. Delmore1,4, Leutz Buon1,4, Zachary R. Hunter2, Val Monrose3, Jie Yu5, Peter G. Smith5, Paul G. Richardson1,4, Kenneth C. Anderson1,4, Steven P. Treon2, Andrew L. Kung3, and Constantine S. Mitsiades1,4

Abstract The NEDD8-activating enzyme is upstream of the 20S proteasome in the ubiquitin/proteasome pathway and catalyzes the ﬁrst step in the neddylation pathway. NEDD8 modiﬁcation of cullins is required for ubiquitination of cullin-ring ligases that regulate degradation of a distinct subset of proteins. The more targeted impact of NEDD8-activating enzyme on protein degradation prompted us to study MLN4924, an investiga- tional NEDD8-activating enzyme inhibitor, in preclinical multiple myeloma models. In vitro treatment with ¼ MLN4924 led to dose-dependent decrease of viability (EC50 25–150 nmol/L) in a panel of human multiple myeloma cell lines. MLN4924 was similarly active against a bortezomib-resistant ANBL-6 subline and its

bortezomib-sensitive parental cells. MLN4924 had submicromolar activity (EC50 values <500 nmol/L) against þ primary CD138 multiple myeloma patient cells and exhibited at least additive effect when combined with dexamethasone, doxorubicin, and bortezomib against MM.1S cells. The bortezomib-induced compensatory upregulation of transcripts for ubiquitin/proteasome was not observed with MLN4924 treatment, suggesting distinct functional roles of NEDD8-activating enzyme versus 20S proteasome. MLN4924 was well tolerated at doses up to 60 mg/kg 2 daily and signiﬁcantly reduced tumor burden in both a subcutaneous and an orthotopic mouse model of multiple myeloma. These studies provide the framework for the clinical inves- tigation of MLN4924 in multiple myeloma. Mol Cancer Ther; 11(4); 942–51. 2012 AACR.

Introduction of the entire group of proteins degraded through the Following the successful clinical development of pro- NEDDylation process (1, 2). teasome inhibitors, such as bortezomib for injection, tar- Preclinical and clinical data from our group and others geting protein homeostasis has become an attractive ther- have shown the activity of proteasome inhibitors in plas- apeutic strategy for multiple myeloma and other neo- ma cell dyscrasias such as multiple myeloma and Wal- plasias. Cullin-ring ligases (CRL) have recently become denstroms Macroglobulinemia (WM). This activity sug- targets of interest because they regulate degradation of gests that these cancers may also be sensitive to the proteins more selectively than the 20S proteasome in the inhibition of the ubiquitin/proteasome pathway at other ubiquitin/proteasome protein degradation pathway (1). levels, such as the NEDDylation component of protein Specifically, NEDD8-activating enzyme catalyzes the first degradation. To address this hypothesis, we tested the step in the NEDD8 Neddylation pathway. NEDD8 mod- preclinical activity of the NEDD8-activating enzyme ification of cullins is required for ubiquitination of CRLs inhibitor MLN4924 against multiple myeloma cells. and targeting NEDD8-activating enzyme or Ubc12, the A panel of multiple myeloma cell lines were treated with downstream enzyme in the pathway, allows for inhibition MLN4924 for 72 hours and compared with the colon carcinoma line HCT116. In addition, a longitudinal assessment of viability of MM.1S showed commitment to death within fi 1 Authors' Af liations: Department of Medical Oncology, Jerome Lipper less than 48 hours. We also observed that normal donor Multiple Myeloma Center; 2Bing Center for Waldenstrom's€ Macroglobuli- nemia; 3Department of Pediatric Oncology, Dana-Farber Cancer Institute; peripheral blood mononuclear cells (PBMC) were less 4Department of Medicine, Harvard Medical School, Boston; and 5Millen- sensitive (EC50 > 1000 nmol/L) than the multiple myeloma nium Pharmaceuticals, Inc., Cambridge, Massachusetts cell lines tested, suggesting that this compound exhibits a Note: Supplementary material for this article is available at Molecular rapid, tumor-selective effect at clinically relevant condi- Cancer Therapeutics Online (http://mct.aacrjournals.org/). tions. We also evaluated primary multiple myeloma þ Corresponding Author: Constantine S. Mitsiades, Jerome Lipper Multiple (CD138 ) patient bone marrow cells and observed submi- Myeloma Center, Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School, 450 Brookline Avenue, Boston, MA cromolar activity by MLN4924. In addition, we tested a 02215. Phone: 617-632-1962; Fax: 617-812-7701; E-mail: series of combinations of MLN4924 with dexamethasone, [email protected] doxorubicin, and bortezomib and observed additive activ- doi: 10.1158/1535-7163.MCT-11-0563 ity or greater with MLN4924. Gene expression profiling 2012 American Association for Cancer Research. revealed distinct signatures as well as distinct patterns of

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gene expression changes that were induced by MLN4924 by Dr. Robert Orlowski (MD Anderson Cancer Center, versus bortezomib. Last, animal studies showed that Houston, TX). These lines were characterized by short MLN4924 was tolerated and active against multiple mye- tandem repeat profiling and compared with the known loma xenografts in both subcutaneous and diffuse lesion ATCC database and German Collection of Microorganisms mouse models of multiple myeloma disease. and Cell Cultures databases (3) and used within 6 months from receipt and/or characterization. Cells were grown in either RPMI-1640 (Mediatech Inc.) or Dulbecco’s Modified Materials and Methods Eagle’s Medium (Mediatech Inc.) media with 100 U/mL Cell lines penicillin, 100 mg/mL streptomycin, and 10% fetal calf We evaluated a panel of human multiple myeloma cell serum (GIBCO/BRL), unless stated otherwise. lines, including Dox40 (kindly proved by Lori Hazlehurst; Compounds Lee H. Moffit Cancer Center, Tampa, FL), H929 (Deutsche MLN4924 and bortezomib (Fig. 1A; refs. 4, 5), were Sammlung von Mikroorganismen und Zellkulturen provided by Millennium Pharmaceuticals, resuspended GmbH; DSMZ), INA-6 (kindly provided by Renata Bur- in dimethyl sulfoxide (DMSO) at 10 mmol/L and used at ger, University of Kiel, Kiel, Germany), JJN3 (DSMZ), the concentrations indicated. Dexamethasone and doxo- KMS-18 [Japanese Collection of Research Bioresources rubicin were purchased from Sigma, resuspended in (JCRB)], KMS-34 (JCRB), MM.1S (Steve Rosen, North- DMSO, and used at the concentrations indicated. western University, Chicago, IL), OPM2 (DSMZ), and the colon carcinoma cell line HCT116 [American Type Cul- Correlation of NEDD8 transcript levels with clinical ture Collection (ATCC)]. The bortezomib-sensitive ANBL- outcome 6 cell line and their subline ANBL-6-V5R, which exhibits We evaluated the expression levels of NEDD8 transcript decreased response to bortezomib, were kindly provided (201840_at) in the log2 transformed and median-centered

ABBortezomib

100 Ph2 Bort - low NEDD8 Ph2 Bort - high NEDD8 75

P MLN4924 50 = 0.0173

Figure 1. Levels of NEDD8 progression 25

transcript in primary multiple % of patients without myeloma tumor cells correlate with 0 0 100 200 300 400 clinical outcome. A, bortezomib and MNL4924 structures are shown for Days these studies. Kaplan–Meier curves CD of PFS for bortezomib-treated 100 Ph3 Bort - low NEDD8 100 Ph3 Bort - low NEDD8 multiple myeloma patients with low Ph3 Bort - high NEDD8 Ph3 Dex - low NEDD8 versus high levels of NEDD8 75 75 transcript in the phase 2 SUMMIT P (B; P ¼ 0.0173, log-rank test) and the P = 0.0482 = 0.0012 phase 3 APEX trials (C; P ¼ 0.0482, 50 50 log-rank test). Kaplan–Meier curves 25 25 progression

of PFS for bortezomib- versus progression dexamethasone-treated multiple % of patients without patients of % myeloma patients with low without patients of % 0 0 0 100 200 300 400 0 100 200 300 400 (D; P ¼ 0.0012, log-rank test) versus Days Days high (E; P ¼ 0.6149, log-rank test) levels of NEDD8 transcript in E the phase 3 APEX trial. Dex, 100 Ph3 Bort - high NEDD8 dexamethasone; Bort, bortezomib. Ph3 Dex - high NEDD8 75

50 P = 0.6149

progression 25 % of patients without 0 0 100 200 300 400 Days

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gene expression data set of tumor cells from evaluable plated in 384-well plates and allowed to attach overnight. bortezomib-treated multiple myeloma patients enrolled multiple myeloma cells were then plated and treated with in phase II and III trials of relapsed/refractory multiple MLN4924 for 48 hours at the doses indicated. Following myeloma (6, 7). Patients were classified as having high (top incubation, luciferin substrate (Xenogen Corp) was added quartile of expression) versus low (lower 3 quartiles of to the culture and the resulting bioluminescence signal was expression) levels of NEDD8 and Kaplan–Meier survival measured using an Envision luminometer (Perkin Elmer). analyses comparing the 2 groups were carried out by SPSS 17.0 and visualized with GraphPad Prism 5.0. Osteoclast differentiation/coculture PBMCs were collected from collars of normal donors, Cell viability assessment ficoll gradient separated, resuspend in a-MEM media Cell viability was measured by MTT (Chemicon Inter- containing 10% FBS and Pen/Strep. Cells were cultured national) colorimetric survival assay, as previously pub- for 16 hours to allow attachment and nonadherent cells lished (8) or CellTiterGlo (CTG; drug combination studies). were removed. Fresh media supplemented with MCSF In brief, multiple myeloma cell lines were plated in 48-well (25 ng/mL; R&D Systems #216-MC-005) and RANKL plates at a density of 20,000 cells per well. MLN4924 (50 ng/mL; Peprotech #310-01) was added and cells were was added at the concentrations indicated and compared cultured for 3 to 4 weeks to differentiate mature osteo- with vehicle-treated controls. Cultures were then incubat- clasts. Half of media was changed 2 per week, contain- ed for 72 hours in a 37 C incubator with 5% CO2. MTT was ing fresh MCSF and RANKL. At 3 to 4 weeks, cells were added to each well for the final 4 hours of treatment, trypsinized, replated, and stained with TRAP stain to followed by addition of isopropanol containing 0.04 determine the differentiation of osteoclasts. Mature osteo- N HCl to dissolve the crystals (at volume 1.5- to 3-fold of clasts were plated in 96-well plates, allowed to attach the total culture volume). Optical absorbance of the culture overnight, and used in coculture experiments as indicated medium was then measured at 570/630 nm with a spec- in the figure legends. trophotometer (Molecular Devices Corp.). Peripheral blood samples from healthy donors were Animal studies collected with heparinized tubes and processed by Ficoll The in vivo anti-multiple myeloma activity of MLN4924 density gradient centrifugation (Amersham Biosciences) to was evaluated in a subcutaneous xenograft model of isolate PBMCs, which were plated at 10,000 cells per well RPMI8226/S multiple myeloma cell line in CB.17-severe and prestimulated with 5 mg/mL phytohemagglutinin combined immunodeficient (SCID) mice. The antitumor (PHA; Sigma-Aldrich) for 1 hour. MLN4924 was admin- activity and pharmacodynamic study in live mice was istered at the doses indicated in the respective experi- conducted at Oncodesign S.A (Dijon). In brief, 2 107 ments for 72 hours, followed by the addition of CTG and RPMI 8226 cells were injected into the right flank of CB.17 measurement as described above. Following consent, mye- SCID mice and studies were initiated once tumors had loma patient samples were collected, Ficoll gradient sep- formed to approximately 200 mm3. MLN4924 was dosed arated, and selected using CD138 microbeads (Miltenyi subcutaneously at 10, 30, and 60 mg/kg twice a day for 21 Biotec). Cells were cultured in RPMI-1640 (Mediatech Inc.) days and tumor volume monitored. To evaluate the phar- with 100 U/mL penicillin, 100 mg/mL streptomycin, and macodynamic effects of MLN4924, a single subcutaneous 20% fetal calf serum and treated as indicated in the figures. dose of MLN4924 was administered and the effects on the Viability was measured by CTG assay, as described above. NEDD8-activating enzyme pathway were examined as described previously (4, 10). Cell death commitment assay The in vivo anti-multiple myeloma activity of MLN4924 The minimum exposure of multiple myeloma cells to was evaluated in a previously established model of dif- þ MLN4924 that is required to commit them to death was fuse GFP/luc multiple myeloma lesions in SCID/NOD evaluated by incubating cells in 24-well plates with mice. Briefly, male (6- to 8-week old) SCID/NOD mice MLN4924 (500 nmol/L) up to 72 hours. Following incuba- were obtained from Charles River; housed and monitored tion, the cells were washed with drug-free medium to re- in the Animal Research Facility of the Dana-Farber Cancer move any residual drug, and then incubated in drug-free Institute; g irradiated (150 rads) using Cs137 g-irradiator medium for an additional 3 days, resulting in equal length source; and received (24 hours postirradiation) tail i.v. of incubation for all experimental conditions. Multiple mye- injections of 106 MM.1S-GFP/luc cells per mouse. Mice loma cell survival was quantified by CTG and expressed as were monitored regularly for changes in body weight, percentage of the value obtained from respective controls. signs of infection or paralysis, and with weekly bioluminescence imaging (11). Stromal cell coculture The luciferase (luc)-expressing cell lines, MM.1S- Immunoblotting analysis mCherry/luc and OPM2-mCherry/luc, were generated For immunoblotting analyses, MM.1S cells (10 106 by retroviral transduction and used for coculture experi- cells per condition) were plated in RPMI-1640 medium ments with the luc-negative human stromal line HS-5, as with 10% FBS, penicillin, and streptomycin as previously described previously (9). Briefly, HS-5 stromal cells were described. MLN4924 was added at a concentration of

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500 nmol/L for 0 to 72 hours. Cell pellets were collected Results and treated with Triton X-100 lysis buffer containing NEDD8 transcript levels correlates with clinical 1 PBS, Triton X-100 (1% v/v), sodium deoxycholate outcome in multiple myeloma patients (0.5% w/v), SDS (0.1% w/v), EDTA (1 mmol/L), 1 mmol/L We evaluated the expression levels of NEDD8 tran- phenylmethylsulfonyl fluoride, 1 mmol/L sodium fluo- script (201840_at) in gene expression data set of tumor ride, 1 mmol/L sodium orthovanadate, 1 mg/mL aprotinin, cells from evaluable bortezomib-treated multiple myelo- 5 mg/mL leupeptin, and 5 mg/mL pepstatin A. The samples ma patients enrolled in phase II and III trials of relapsed/ were cleared by centrifugation (14,000 rpm, 30 minutes, refractory multiple myeloma (6, 7). We observed that 4 C) and assessed for protein concentration by Bradford patients with high expression of NEDD8 transcript had assay (Sigma). SDS-PAGE (12%) was carried out (30–50 mg significantly shorter progression-free survival (PFS) com- of protein per lane), and proteins were electroblotted pared with patients with low NEDD8 transcript levels. onto polyvinylidene difluoride membranes. After 1 hour Among patients treated in the phase III trial of bortezomib incubation in blocking solution (5% milk in TBS-T buffer), versus dexamethasone, those with low levels of NEDD8 membranes were exposed to primary antibody over- transcript had significantly longer PFS with bortezomib night at 4 C. Following washing in TBS-T, the respective than with dexamethasone however, in patients with high secondary horseradish peroxidase–labeled antibody was levels of NEDD8 transcript, there was no significant dif- added at 1:20,000 dilution for 1 hour at room temperature. ference in patients treated with bortezomib versus dexa- The membrane was then washed with TBS-T for 45 to methasone (Fig. 1). 60 minutes with multiple changes of the wash buffer, and the protein expression was visualized with the ECL tech- In vitro activity of MLN4924 against multiple nique. The primary antibodies used for immunoblotting myeloma cell lines and primary patient samples were purchased from Santa Cruz Biotechnology, Upstate We treated a panel of multiple myeloma cell lines with Biotechnologies, or Cell Signaling. Secondary antibodies MLN4924 (0–1000 nmol/L) for 72 hours and evaluated were purchased from Jackson ImmunoResearch. viability with MTT colorimetric survival assays. The major- ity of cell lines had EC50 values less than 500 nmol/L (Fig. Gene expression profiling of cells treated with 2A). The most sensitive cells lines, such as OPM2, INA-6, MLN4924 and KMS34, exhibited EC50 values less than 100 nmol/L. Total RNA extraction and purification, cDNA synthesis In addition, we evaluated the impact of MLN4924 on the and cRNA labeling, Affymetrix chip (human HG-U133 plus colorectal carcinoma cell line HCT116, previously reported 2.0 array) hybridization and data analysis were carried out to be MLN4924 responsive (4) for comparison. þ as previously described (12–14). Briefly, MM.1S cells were In addition to cell lines, CD138 primary multiple mye- treated with 250 nmol/L MLN4924, 10 nmol/L bortezomib, loma patient samples were evaluated for sensitivity to or with DMSO as a control for the indicated time points; MLN4924. Multiple myeloma patient bone marrow was þ total RNA was then extracted and purified, cDNA synthe- collected, ficolled, and then selected for CD138 cells. Cell sized and cRNA labeled before hybridization to the HG- were then plated and treated with increasing doses of U133 plus 2.0 arrays. Gene expression data have been MLN4924 for 72 hours. Of the 6 samples treated, we observ- deposited in Gene Expression Omnibus (GEO, GSE33577). ed activity of the drug against 4 samples with EC50 values Transcripts suppressed by MLN4924 were analyzed in less than 600 nmol/L. Two samples, however, remained publically available databases for relevance to clinical out- insensitive to the drug up to 1000 nmol/L of drug (Fig 2B). come and/or differential expression in various stages of For further understanding of the activity of MLN4924 plasma cell dyscrasias. We specifically analyzed the log2 against normal tissues, PBMCs from healthy donors were transformed median centered values (derived from the isolated, PHA stimulated to induce cycling and prolifer- Oncomine database) of different MLN4924-suppressed ation, then treated with MLN4924 for 72 hours (Fig. 2C). genes in the GEO (15) data sets GSE5900 [comparison of The percentage of viable PBMCs was 90% or greater gene expression of bone marrow plasma cells from healthy following MLN4924 treatment in the absence of PHA donors, monoclonal gammopathy of undetermined signi- stimulation. Sensitivity of PBMCs to MLN4924 was ficance (MGUS), and smoldering myeloma (SMM; ref. 16)]; increased as a result of PHA stimulation, but remained GSE2658 (17); GSE2113 (comparison of gene expression less than the most sensitive multiple myeloma cell lines. profiles of purified plasma cells obtained from MGUS, We also tested in vitro the activity of MLN4924 against newly diagnosed multiple myeloma, and from plasma cell the bortezomib-sensitive multiple myeloma cell line leukemia patients; ref. 18); GSE4452 (gene expression pro- ANBL6 and its subline ANBL6-V5R, which exhibits files of multiple myeloma patients before treatment (19). decrease response to bortezomib. We did not observe Differential expression between normal plasma cells and increased resistance of ANBL6-V5R cells to MLN4924 MGUS and SMM; between MGUS and newly diagnosed compared with the parental cell line (Fig. 2D). multiple myeloma and plasma cell leukemia patients; or Cell death commitment assays were conducted on between patients with differential patient outcome were Dox40, MM.1S, OPM2, and KMS34 cells by exposing them evaluated using 1-way ANOVA or unpaired T test, as stated to MLN4924 (500 nmol/L) for up to 72 hours, followed by in the figure legends using Prism software (GraphPad). drug washout and incubation in drug-free medium for an

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AB 0 15.625 31.25 62.5 125 250 500 1,000 nmol/L EC50 (nmol/L) OPM2 33.7 INA-6 25.3 150 BCWM1 53.3 KMS-34 70.2 HCT116 95.8 100 H929 157.9 JJN3 195.6 MM1S 439.8 WSU-WM 615.6 % Survival 50 KMS-18 948.8 RPMI8226 1287.3 Dox40 2004.0 0

100 % Survival

70 40 90 80 10 50 60 20

3 0 200 400 600 800 1,000

0 0 MLN4924 (nmol/L)

125 CDANBL6-wt 100 ABNBL6-5VR Donor 1 75

500 Donor 1 + PHA 50

Donor 2 % Survival 25 400 Donor 2 + PHA 0 0 20 40 60 80 Donor 3 300 Bort (nmol/L) Donor 3 + PHA 125 Donor 4 ANBL6-wt 200 100

% Survival ABNBL6-5VR Donor 4 + PHA 75 100 50

0 % Survival 25 0 200 400 600 800 1,000 0 0 250 500 750 1000 MLN4924 (nmol/L) MLN4924 (nmol/L)

Figure 2. Activity of MLN4924 on multiple myeloma cells. Multiple myeloma (OPM2, INA-6, KMS-34, H929, JJN3, MM.1S, KMS-18, and Dox40), WM (BCWM1 and WSU-WM), and colon carcinoma (HCT116) cell lines were treated with MLN4924 (0–1000 nmol/L for 72 hours) and viability was assessed by MTT assay. A, the most sensitive cell lines (OPM2 and INA-6) have EC50 values less than 40 nmol/L, whereas the most resistant multiple myeloma cell line (Dox40) has an EC50 value more than 2 mmol/L. Bone marrow aspirates from multiple myeloma patients were processed using Miltenyi anti-CD138 microbeads for puriﬁcation of multiple myeloma cells, which were then cultured in RPMI1640 media containing 20% serum. B, CD138þ selected cells were treated with increasing doses of MLN4924 for 72 hours, and viability was assessed by CTG. To access the sensitivity of nonmalignant cells, PBMCs were isolated from normal donors and screened in the presence and absence of PHA stimulation (5 ng/mL) for 72 hours. C, the number of viable cells in each condition is expressed as percentageofthe non–PHA-stimulated drug-free control. The multiple myeloma cell line ANBL6 and its subline ANBL6-V5R were tested for sensitivity to bortezomib and MLN4924. D, ANBL6-V5R was more resistant to bortezomib, but not MLN4924, compared with the parental cell line. Bort, bortezomib.

additional 72 hours. MLN4924 treatment for 48 hours Activity of MLN4924 in animal studies resulted in commitment to death in OPM2, KMS34, and MLN4924 showed dose-dependent anti-multiple MM.1S (Supplementary Fig. S1), suggesting that molec- myeloma activity in the RPMI8226/S xenograft model ular events initiating cell death can occur in less than 48 in CB.17 SCID mice (Fig 4A). Complete inhibition of hours of MLN4924 treatment. tumor growth was achieved at 60 mg/kg twice a day dose level, which is below the MTD on this schedule. In MLN4924 overcomes stroma and osteoclast addition, MLN4924 showed dose-dependent inhibition protection of multiple myeloma cells of Nedd8-cullin levels with a concomitant increase Lucþ MM.1S and OPM2 cells were cultured in the in levels of 2 CRL substrates, Nrf-2 and pIKBa (Supple- presence and absence of HS-5 stromal cells or differenti- mentary Fig. S2). We also evaluated the in vivo anti- ated osteoclasts for 72 hours. Cell viability was normal- multiple myeloma activity of MLN4924 in sublethally ized to each respective drug-free control. Results show irradiated SCID/NOD mice injected i.v. with MM.1S- that MLN49424 was similarly active both in the presence GFP/luc cells for establishment of diffuse multiple and absence of HS-5 stromal cells (Fig 3A and B) and myeloma bone lesions. MLN4924 treatment was asso- presence and absence of osteoclasts (Fig. 3C and D), ciated with decrease in tumor burden (Fig. 4B) without a indicating that MLN4924 can overcome the protective signiﬁcant decrease in average body weight in the mice effects of bone microenvironmental factors. (data not shown).

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AB125 MM.1S 125 OPM2 No stroma No stroma 100 Plus stroma 100 Plus stroma

75 75 Figure 3. MLN4924 overcomes the protective effect of nonmalignant 50 50 % Survival accessory cells of the bone % Survival microenvironment. MM.1S and 25 25 OPM2 multiple myeloma cells were cultured in the presence and 0 0 0 200 400 600 800 1,000 0 200 400 600 800 1,000 absence of HS-5 stromal cells (A and B) or differentiated osteoclasts (C MLN4924 (nmol/L) MLN4924 (nmol/L) and D) and treated with MLN4924 for 72 hours. The resulting cell viability CD was normalized to each respective 125 MM.1S 125 OPM2 no drug control (n ¼ 4). Comparable No osteoclasts No osteoclasts activity was observed against 100 Plus osteoclasts 100 Plus osteoclasts MM.1S and OPM2 cells both in the presence or absence of these bone 75 75 microenvironment cells with 50 50 treatment with MLN4924. % Survival % Survival 25 25

0 0 0 200 400 600 800 1,000 0 200 400 600 800 1,000 MLN4924 (nmol/L) MLN4924 (nmol/L)

Molecular sequelae of MLN4924 treatment MGUS patients (Supplementary Fig. S3F). These results Immunoblotting of MLN4924-treated MM.1S showed support the notion that the molecular sequelae of MLN4924 upregulation (as early as 8 hours) of p27, CTD1, and NRF2, treatment include genes with putative roles in the progres- which are known targets of the NEDD8 pathway, followed sion and clinical outcome of multiple myeloma patients. by markers of cell death, such as cleavage of PARP and caspase-3 at later time points (as early as 32 hours after MLN4924 combinations with other anti-multiple initiation of treatment; Fig 5A). We also evaluated the myeloma agents gene expression profiling changes triggered in MM.1S We next evaluated combinations of MLN4924 with cells treated with MLN4924 versus bortezomib. We agents used for clinical management of multiple myeloma observed that the profile of MLN4924-triggered transcrip- (dexamethasone, bortezomib, doxorubicin). Although no tional changes was distinct from the one induced by bor- synergy was observed between MLN4924 and bortezo- tezomib exposure (Fig. 5B). Indeed, known transcriptional mib (Fig. 6A), doxorubicin (Fig. 6B) or dexamethasone responses to 20S proteasome inhibition, such as upregula- (Fig. 6C), there also was no antagonism observed, sug- tion of proteasome subunits (Fig. 5C) or heat shock proteins gesting that MLN4924 does not adversely impact the anti- (data not shown), were minimal or absent in the multiple myeloma activity of these approved agents. MLN4924-treated cells (Supplementary Table). In addition, we observed that several MLN4924-suppressed genes Discussion were associated with higher expression in multiple mye- The preclinical and clinical development of the protea- loma patients with adverse clinical outcome or were ex- some inhibitor bortezomib for the treatment of multiple pressed at higher levels in patients with plasma cell leu- myeloma led to significant improvements in the clinical kemia or multiple myeloma compared with MGUS or outcome of patients with this presently incurable plasma normal plasma cells. For example, KLF2 (Supplementary cell dyscrasia. The clinical success of bortezomib validat- Fig. S3A), PHACTR1 (Supplementary Fig. S3B), and ed the notion that therapeutic targeting of ubiquitination FAM1985 (Supplementary Fig. S3C) were expressed at and proteasome function is feasible without catastrophic higher levels in SMM and MGUS compared with normal toxicities. It also led to further efforts to identify other plasma cells; FMA1985 was expressed higher in multiple druggable targets in this pathway, particularly at molec- myeloma patients who were deceased versus alive at ular levels situated upstream of the proteasome. These 5 years (Supplementary Fig. S3D); PSMC3IP deceased ex- targets have included the CRLs, which regulate degrada- pressed at higher levels in patients who were deceased tion of proteins more selectively than the chymotryptic- versus alive at 1 year after diagnosis (Supplementary Fig. like site of the 20S proteasome core. NEDD8-activating S3E); and PSMC3IP was expressed at higher levels in enzyme has been the focus of particular attention because plasma cell leukemia versus multiple myeloma versus it catalyzes the first step in the NEDD8 neddylation

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A A 1,000 Vehicle MM.1S 10 mg/kg BID 0 8 16 2432 48 72 h MLN4924 (500 nmol/L) 30 mg/kg BID 800 Parp )

3 60 mg/kg BID 600 Casp 3 cleaved

400 CTD1 volume (mm Average tumor 200 NRF2

p27

0 3 6 9 14 17 20 GAPDH Days of treatment

B 5,000 Vehicle * BC 60 mg/kg BID 4,000

3,000

2,000

Tumor burden 1,000 (bioluminescence) 0 0 10 20 30 40 Days of treatment

Figure 4. In vivo anti-multiple myeloma activity of MLN4924. RPMI8226/S cells were injected subcutaneously in CB.17-SCID mice and treatment was initiated once tumors had reached approximately 200 mm3. MLN4924 was dosed subcutaneously at 10, 30, and 60 mg/kg twice a day (BID) for 21 days and tumor volume monitored (N ¼ 12 per group). There was a statistically signiﬁcant difference in tumor burden only when comparing the control with 60 mg/kg group (A; P < 0.05; 1-way ANOVA). In addition, an orthotopic model of multiple myeloma was evaluated. Sublethally irradiated NOD/SCID mice injected i.v. with MM.1S multiple myeloma cells were randomly assigned to receive MLN4924 treatment (60 mg/kg BID i.p.) or vehicle control. B, tumor burden was evaluated by whole-body bioluminescence imaging weekly and tumor burden calculated using Living Image software. Treated mice had lower tumor burden compared with vehicle treated controls (P ¼ 0.1186 day 33; P ¼ 0.0269 day 38; N ¼ 6 per group).

pathway. NEDD8 modification of cullins is required for ubiquitination of CRLs and targeting NEDD8-activating enzyme or Ubc12, the downstream enzyme in the pathway, allows for inhibition of the entire group of proteins degraded through the neddylation process. Our interest on the therapeutic targeting of NEDD8- Figure 5. Immunoblotting analysis and transcriptional profiles of activating enzyme was further enhanced by our observa- MLN4924-treated multiple myeloma cells. A, MM.1S cells treated with tion that multiple myeloma patients with high levels MLN4924 show upregulation (as early as 8 hours) of p27, CTD1, and of NEDD8 transcript had shorter PFS upon treatment NRF2, which are known targets of the NEDD8 pathway, followed by markers of cell death, such as cleavage of PARP and caspase-3 at later with bortezomib. We also observed that, in the random- time points (as early as 32 hours after initiation of treatment). The gene ized phase III APEX trial which compared bortezomib expression profiling changes triggered in MM.1S cells by MLN4924 with dexamethasone treatment in relapsed or refractory versus bortezomib were evaluated using the HG-U133plus2.0 multiple myeloma, bortezomib-treated patients with high oligonucleotide microarray. B, the profile of MLN4924-triggered NEDD8 transcriptional changes was distinct from the one induced by levels of transcript did not have a significant bortezomib exposure. C, known transcriptional responses to 20S advantage over dexamethasone-treated patients in terms proteasome inhibition, such as upregulation of proteasome subunits, of PFS. This suggests that perturbation of the NEDD8 were minimal or absent in the MLN4924-treated cells. Bort, bortezomib.

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Antimyeloma Activity of NEDD8-Activating Enzyme Inhibition

A 0 3.906 7.813 15.63 31.25 62.5 125 250 MLN4924 (nmol/L; 48 h) 0 100.0 95.7 82.4 74.8 68.1 60.3 50.2 35.0 1 98.3 94.1 83.7 76.1 66.1 58.1 49.8 34.4 2 90.2 79.6 74.4 62.3 53.9 41.8 35.5 19.4 3 45.3 47.1 40.4 28.2 19.1 11.0 7.7 1.9 4 24.2 24.9 20.6 13.9 7.2 3.6 1.8 0.5 6 6.0 7.7 5.5 3.1 1.4 0.7 0.3 0.1 8 2.0 2.3 2.0 1.1 0.5 0.3 0.1 0.1 10 0.5 0.5 0.4 0.3 0.2 0.1 0.1 0.1

Bort (nmol/L; 24 h) 100 % Survival 90 80 70 60 50 40 30 20 10 Figure 6. Combinations of 0 MLN4924 with established anti-multiple myeloma agents. MLN4924 treatment was combined 0 3.906 7.813 15.63 31.25 62.5 125 250 MLN4924 (nmol/L; 48 h) with agents used for clinical B 0 100.0 102.0 88.7 83.9 71.1 69.9 55.8 38.6 management of multiple myeloma, 2.5 104.3 100.5 98.6 90.0 75.1 67.6 59.0 38.2 namely bortezomib (A), doxorubicin 5 112.8 108.7 99.0 87.7 80.5 72.9 58.1 39.6 (B), and dexamethasone (C). Activity 10 84.3 86.1 88.3 78.7 73.2 61.6 52.4 33.1 was observed with these 12 84.6 85.9 82.0 83.4 73.8 72.3 59.0 40.4 combinations at drug doses relevant 15 55.1 60.9 57.7 62.9 63.5 64.3 56.4 36.8 to those achieved clinically in the 18 23.6 35.4 32.0 40.6 39.0 46.9 45.2 32.6 treatment of patients. Bort, 20 9.5 18.1 17.5 22.4 25.8 30.7 35.5 24.0 bortezomib; Doxo, doxorubicin; Dex, Doxo (ng/mL; 48 h) dexamethasone. C 0 3.906 7.813 15.63 31.25 62.5 125 250 MLN4924 (nmol/L; 48 h) 0 100.0 92.6 80.0 67.2 66.9 60.2 49.7 35.2 7.8125 98.8 94.2 84.0 69.5 66.7 51.3 46.3 36.7 15.625 93.6 83.2 76.3 67.5 62.0 56.9 46.0 36.4 31.25 93.3 84.8 74.1 67.3 66.4 56.6 47.1 36.0 62.5 96.4 82.4 81.0 72.3 66.4 62.3 52.6 39.2 125 92.2 83.4 77.7 66.6 68.7 63.1 53.9 38.6 250 84.2 78.4 72.4 67.9 69.3 64.9 53.4 36.5 500 76.4 71.3 66.2 57.6 66.6 60.1 49.0 30.2 Dex (nmol/L; 72 h)

cascade may characterize a subset of multiple myeloma significantly reduced the tumor burden of both subcuta- patients who do not receive maximum benefit from borte- neous and orthotopic multiple myeloma lesions in immu- zomib-based therapies. For those subsets of patients, ther- nocompromised mice. sapeutic targeting of the NEDD8 pathway could be useful. Mechanistically, NEDD8-activating enzyme inhibition We evaluated the antimyeloma activity of the NEDD8- with MLN4924 treatment was associated with early activating enzyme inhibitor MLN4924 in our in vitro increase in protein levels of p27, CTD1, and NRF2. This preclinical models and observed a dose- and time-depen- observation is compatible with the known role of NEDD8- dent reduction in viability of multiple myeloma cell lines activating enzyme in regulating intracellular levels of these þ as well as primary CD138 multiple myeloma tumor cells proteins. A more open-ended analysis of the transcription- isolated from multiple myeloma patients. The EC50 values al profile of MLN4924-treated multiple myeloma cells of MLN4924 against multiple myeloma cell lines and showed changes distinct from those triggered by bortezo- primary tumor cells was comparable with or lower than mib. For instance, although bortezomib triggers major the values observed in preclinical studies of other neo- increases in transcript levels for diverse heat shock proteins plasias considered to be highly responsive to NEDD8- family members and proteasome subunits, MLN4924 trig- activating enzyme inhibition (4, 10, 20). Importantly, this gered minimal, if any, such changes. This difference likely activity observed in vitro against multiple myeloma cells reflects the distinct functional consequences of inhibiting could be achieved with exposure to MLN4924 for 48 hours the b5 subunit of the 20S proteasome core versus NEDD8- or less. Furthermore, we observed that the response of activating enzyme. The b5 subunit of the proteasome core multiple myeloma cells to MLN4924 was similar when regulates the intracellular levels of a larger pool of ubiqui- these cells were cultured alone versus cocultured with tinated proteins. In contrast, NEDD8-activating enzyme bone marrow stromal cells or osteoclasts. This indicates regulates the neddylation of a more restricted set of pro- that NEDD8-activating enzyme inhibition can circumvent teins. Because the targets of NEDD8-activating enzyme the proliferative/antiapoptotic stimulation provided to regulation are enriched for proteins with critical roles multiple myeloma cells by the interactions with non- in proliferation, survival, or drug resistance, MLN4924 malignant accessory cells of the bone marrow micro- achieves antitumor responses, even though it does not environment. Consistent with this observation, MLN4924 induce the pronounced intracellular proteotoxic stress

www.aacrjournals.org Mol Cancer Ther; 11(4) April 2012 949

McMillin et al.

triggered by bortezomib. Interestingly, we observed that a have nothing to disclose. J. Yu and P.G. Smith are employees of Millen- nium Pharmaceuticals (The Takeda Oncology Company); P.G. Richardson myeloma cell line model with constitutive resistance to has participated in advisory boards of Millennium, Celgene, Novartis, bortezomib and its isogenic bortezomib-sensitive parental Johnson & Johnson, and Bristol-Myers Squibb. K.C. Anderson has received research grants and honoraria from Millennium and Celgene, has been a line are both responsive to MLN4924. This suggests that consultant for Millennium, Celgene, Novartis, Bristol-Myers Squibb, resistance to inhibition of the b5 subunit of the 20S core by Merck, and Onyx, and is a founder of Acetylon Pharmaceuticals. S.P. bortezomib does not necessarily confer cross-resistance to Treon has received honoraria from and is consultant/member of advisory board for Millennium. C.S. Mitsiades has received in the past consultant inhibition of upstream molecular targets, such as NEDD8- honoraria from Millennium Pharmaceuticals, Novartis Pharmaceuticals, activating enzyme. Bristol-Myers Squibb, Merck & Co., Kosan Pharmaceuticals, Pharmion, On the basis of these observations, we further explored Johnson & Johnson, and Amnis Therapeutics, licensing royalties from PharmaMar, and research funding from Amgen Pharmaceuticals, AVEO combination studies of MLN4924 with established anti- Pharma, EMD Serono, Sunesis, Gloucester Pharmaceuticals, and Johnson myeloma agents, such as bortezomib, doxorubicin, and & Johnson. dexamethasone. These in vitro combinations showed no antagonism between the combined agents, suggesting Acknowledgments that currently available regimens for multiple myeloma The authors thank Dr. Robert Orlowski (MD Anderson Cancer Center, treatment may be combined with MLN4924 in further Houston, TX) for providing the ANBL-6 and ANBL-6-V5R cells. preclinical and potentially clinical studies. Our results indicate that NEDD8-activating enzyme Grant Support inhibition with MLN4924 has in vitro and in vivo activity This work was supported in part by the Chambers Medical Foun- in preclinical models of multiple myeloma. The molecular dation (C.S. Mitsiades and P.G. Richardson), the Steven Cobb Foundation (D.W. McMillin and C.S. Mitsiades), the R.J. Corman impact of NEDD8-activating enzyme inhibition is dis- Fund for Myeloma Research (P.G. Richardson and C.S. Mitsiades), tinct from the sequelae triggered by bortezomib, as the Stepanian Fund for Myeloma Research (P.G. Richardson and evidenced by the differential molecular proﬁles induced C.S. Mitsiades), the Honickman Fund (P.G. Richardson and C.S. Mitsiades), NIH grants RO1-50947 (C.S.MitsiadesandK.C.Ander- by MLN4924 versus bortezomib, as well as the activity son), and PO-1-78378 (K.C. Anderson). C.S. Mitsiades is supported of MLN4924 against bortezomib-resistant cells. These ob- by the Dunkin Donuts Rising Stars Program at the Dana-Farber Cancer Institute. servations provide a platform for further evaluation of The costs of publication of this article were defrayed in part by the NEDD8-activating enzyme inhibitors to inform the design payment of page charges. This article must therefore be hereby marked of clinical trials in multiple myeloma. advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Disclosure of Potential Conﬂicts of Interest D.W. McMillin is a founder and equity holder in Axios Biosciences. H. Received July 25, 2011; revised December 16, 2011; accepted December M. Jacobs, J.E. Delmore, L. Buon, Z.R. Hunter, V. Monrose, and A.L. Kung 20, 2011; published OnlineFirst January 12, 2012.

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www.aacrjournals.org Mol Cancer Ther; 11(4) April 2012 951

Molecular and Cellular Effects of NEDD8-Activating Enzyme Inhibition in Myeloma

Douglas W. McMillin, Hannah M. Jacobs, Jake E. Delmore, et al.

Mol Cancer Ther 2012;11:942-951. Published OnlineFirst January 12, 2012.

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