The p97 Inhibitor CB-5083 Is a Unique Disrupter of Protein … · Small Molecule Therapeutics The p97 Inhibitor CB-5083 Is a Unique Disrupter of Protein Homeostasis in Models of Multiple

Small Molecule Therapeutics

The p97 Inhibitor CB-5083 Is a Unique Disrupterof Protein Homeostasis in Models of MultipleMyelomaRonan Le Moigne1, Blake T. Aftab2, Stevan Djakovic1, Eugen Dhimolea3, Eduardo Valle1,Megan Murnane2, Emily M. King3, Ferdie Soriano1, Mary-Kamala Menon1, Zhi Yong Wu1,Stephen T.Wong1, Grace J. Lee1, Bing Yao1, Arun P.Wiita4, Christine Lam4, Julie Rice1,Jinhai Wang1, Marta Chesi5, P. Leif Bergsagel5, Marianne Kraus6, Christoph Driessen6,SzerenkeKiss vonSoly1, F.Michael Yakes1, DavidWustrow1, LauraShawver1, Han-JieZhou1,Thomas G. Martin III2, Jeffrey L.Wolf2, Constantine S. Mitsiades3, Daniel J. Anderson1, andMark Rolfe1

Abstract

Inhibition of the AAA ATPase, p97, was recently shown tobe a novel method for targeting the ubiquitin proteasomesystem, and CB-5083, a first-in-class inhibitor of p97, hasdemonstrated broad antitumor activity in a range of bothhematologic and solid tumor models. Here, we show that CB-5083 has robust activity against multiple myeloma cell linesand a number of in vivo multiple myeloma models. Treatmentwith CB-5083 is associated with accumulation of ubiquiti-nated proteins, induction of the unfolded protein response,and apoptosis. CB-5083 decreases viability in multiple mye-loma cell lines and patient-derived multiple myeloma cells,including those with background proteasome inhibitor (PI)

resistance. CB-5083 has a unique mechanism of action thatcombines well with PIs, which is likely owing to the p97-dependent retro-translocation of the transcription factor,Nrf1, which transcribes proteasome subunit genes followingexposure to a PI. In vivo studies using clinically relevantmultiple myeloma models demonstrate that single-agentCB-5083 inhibits tumor growth and combines well withmultiple myeloma standard-of-care agents. Our preclinicaldata demonstrate the efficacy of CB-5083 in several multiplemyeloma disease models and provide the rationale for clinicalevaluation as monotherapy and in combination in multiplemyeloma. Mol Cancer Ther; 16(11); 2375–86. �2017 AACR.

IntroductionInhibition of the b5 peptidase of the 20S proteasome with

bortezomib provided clinical validation for targeting proteinhomeostasis and transformed the standard of care in both mul-tiple myeloma and mantle cell lymphoma (1, 2). The recentclinical successes of the second-generation proteasome inhibitors(PI), carfilzomib and ixazomib, have built upon this therapeuticstrategy and confirmed the clinical susceptibility of multiple

myeloma to inhibitors of the ubiquitin proteasome system (UPS;ref. 3). The dependency of multiple myeloma on protein homeo-stasis pathways is owing to the fact that they retain the charac-teristic biology of normal plasma cells, namely synthesis andsecretion of large amounts of immunoglobulins (4). However,multiple myeloma remains an incurable disease, even with theintroduction of new therapeutic modalities over the past twoyears, andhence, there is a need to developnewagents interveningin the myeloma cell's protein homeostasis pathways at differentpoints compared with PIs.

Recently, inhibition of p97 has emerged as a new approach fortargeting protein homeostasis in tumor cells (5, 6). Also known asvalosin-containing protein, p97 is an essential and conservedmember of the AAA family of adenosine triphosphatases (AAAATPases) and is a key regulator of protein homeostasis. p97generates mechanical force via ATP hydrolysis, and this force isused to extract proteins from macromolecular complexes andorganelles. p97 is involved in multiple biological processes,including protein homeostasis, endoplasmic reticulum–associat-ed degradation (ERAD), autophagy, chromatin remodeling, andGolgi reassembly through its interaction with cofactors (7).Reducing p97 levels with siRNA causes ER stress and activatesapoptosis through the unfolded protein response (UPR), a path-way that acts both to resolve unfolded protein stress and to triggercell death when the buildup of such unfolded proteins becomesirresolvable (5, 8, 9).

1Cleave Biosciences, Inc., Burlingame, California. 2Department of Medicine,Division of Hematology & Oncology, University of California San Francisco, SanFrancisco, California. 3Department of Medical Oncology, Dana-Farber CancerInstitute, Harvard Medical School, Boston, Massachusetts. 4Department ofLaboratory Medicine, University of California, San Francisco, San Francisco,California. 5Comprehensive Cancer Center, Mayo Clinic, Scottsdale, Arizona.6Experimental Oncology and Hematology, Department of Oncology and Hema-tology, St. Gallen, Switzerland.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

R. Le Moigne and B.T. Aftab contributed equally to this article.

Corresponding Author: Ronan Le Moigne, Cleave Biosciences, 866 MalcolmRoad, #100, Burlingame, CA 94010. Phone: 650-443-3014; Fax: 855-347-2137;E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0233

�2017 American Association for Cancer Research.

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CB-5083 is a potent and highly selective inhibitor of the D2ATPase domain of p97 (10, 11). Inhibition of p97 by CB-5083activates the UPR and subsequently induces apoptosis in a widevariety of hematologic and solid tumor cell lines. CB-5083 wasshown to be the first inhibitor of p97 to demonstrate significanttumor growth inhibition in vivo andwas also active in solid tumormodels where PIs were inactive (10). As CB-5083 disrupts proteinhomeostasis upstream of the proteasome, we wanted to assesswhether targeting p97 would have different effects from PIs invarious in vitro and in vivo models of multiple myeloma. Ourstudies described here suggest there is a therapeutic rationale fortargeting p97 in multiple myeloma and that inhibition of p97 isbiologically distinct from targeting the proteasome.

Materials and MethodsCell culture and reagents

Bortezomib- and carfilzomib-resistant AMO-1 cell lines weregenerated as described previously (12, 13). Cancer cell lines wereobtained from ATCC or Deutsche Sammlung von Mikroorganis-men und Zellkulturen GmbH between 2011 and 2015 and werecultured according to the manufacturer's instructions. Fresh bonemarrow samples fromadult patientswithmultiplemyelomawereextracted at the respective hospital centers during the patient'sregular treatment schemes, following clinical practice at thecenter. These samples were treated with CB-5083, ex vivo, andviability of the CD38/CB138-negative and positive populationswere analyzed with the ExviTech platform (14). Lenalidomide,bortezomib, carfilzomib, and ixazomib were obtained from Sell-eck Chemicals. Thapsigargin and dexamethasone were obtainedfrom Sigma.

ImmunofluorescenceCells were cultured in clear bottomed, tissue culture–treated

384-well plates and treated with compound in well duplicates.For immunofluorescence staining, paraformaldehyde (4% final)was added to plates after 8 hours of treatment. Cells were blockedin PBS with 1% BSA, 0.3% Triton X-100 and Hoechst (1:10,000)for 1 hour and then incubated in primary antibodies at 4�C for 16hours. Cells were washed three times in PBS, and secondaryantibodies were added for 2 hours at 25�C. Cells were washedfour times in PBS and imaged with an automated wide fieldfluorescence microscope (Cell Insight, Thermo Fisher Scientific).Automated image analysis was written in MATLAB (MathWorks)to count nuclei, mask cellular compartments, and measure fluo-rescence intensities within cellular compartments.

Western blottingCells or ex vivo lysates were lysed with RIPA buffer supplemen-

ted with protease inhibitors (Roche Applied Science) and phos-phatase inhibitors (Sigma). Lysates were cleared and protein wasquantified by Pierce BCA Protein Assay Kit. Western blotting wasperformed using the Novex NuPAGE SDS-PAGE Gel system.Briefly, 10 mg of protein was resolved on 4% to 12% Bis-Trisgradient gels and then transferred onto nitrocellulose mem-branes. Membranes were blocked for 1 hour at room temperaturein TBS þ 0.1% Tween (TBST) with 5% nonfat dry milk. Mem-branes were probed overnight at 4�C with primary antibodies(Supplementary Table S1). Membranes were washed three timesin TBST and then incubated with goat anti-rabbit or goat anti-mouse secondary antibodies (Supplementary Table S1) for 1hour

at room temperature. Membranes were then washed three timeswith TBST and developed with SuperSignal West Dura Chemilu-minescent Substrate. Images were taken using a Bio-Rad Gel DocImager system, exported as TIF files and cropped using NIHImageJ.

Cell viability assaysViability assays conducted in myeloma cell lines, stably trans-

duced with firefly luciferase, were carried out as described previ-ously (15, 16). For coculture assays, short-term ex vivo cultures ofCD138� fractions from bone marrow aspirates were cultured forthree passages. HS5, HS27A, or patient-derived cultures wereseeded into assay plates 16 hours prior to addition of multiplemyeloma cells. Cocultureswere grown for 24hours, and then afterdrug addition, multiple myeloma cell viability was monitoredusing luciferase as described above. GI50 values were derived fromdose–response curves plotted and fitted using Prism (GraphPad).CellTiter-Glo assays were conducted as described previously (10).

Gene expression analysisMean gene expression values were calculated for all solid

tumor–derived cell lines versusmultiple myeloma cell lines usingthe CCLE (https://portals.broadinstitute.org/ccle/home) geneexpression data. Difference in gene expression between the twocell groups was calculated using a Student t test. Genes over-expressed in multiple myeloma with a P >0.001 were analyzedusing ENRICHR (http://amp.pharm.mssm.edu/Enrichr/).

Transcriptome analysisMultiple myeloma cells were treated with CB-5083, bortezo-

mib, or with DMSO as a control. RNA samples were convertedinto cDNA libraries using the Illumina TruSeq Stranded mRNASample Preparation Kit (Illumina # RS-122-2103). The pipelinemRNAv8-RSEM (EA-Quintiles) was used to analyze RNAsequencing (RNA-seq) data. Total RNA extraction, purification,and data analysis were performed as described previously (10).TheRNA-seq data havebeendeposited inNCBI'sGene ExpressionOmnibus (17) and are accessible through GEO Series accessionnumber GSE101923 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc¼GSE101923). For gene ontology analysis, sampleswere processed as described previously (10).

In vivo modelsAll experiments involving animals were approved by an insti-

tutional Animal Care and Use Committee. Female athymic nudemice and SCID beige mice, 5 to 8 weeks of age and weighingapproximately 20 to 25 gwere purchased fromEnvigo. Cancer cellline xenografts were established by implanting 1 � 106 to 20 �106 cells subcutaneously or intravenously. For the ortho-meta-static models, biweekly luciferase imaging was conducted usingthe Xenogen IVIS Spectrum Imaging System and Living Imagesoftware. Overall tumor burden was both quantified in photons/second and visualized by heatmap representations of radiance(photons/sec/cm2/sr). CB-5083, lenalidomide, ixazomib, and theCB-5083 vehicle (0.5% methyl cellulose in water) were admin-istered by oral gavage, bortezomib and carfilzomib were dosed bytail vein injection, and dexamethasone was administered intra-peritoneally. Tumor volume and body weights were measuredtwice weekly and daily, respectively, throughout the study.Vk�MYCmicework andSPEP analysiswasperformed asdescribedpreviously (18).

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In vivo protein analysisTumors were excised, flash frozen, and stored at �60�C to

�90�C. Following thawing, tissues were processed as describedpreviously (10). Determination of cleaved PARP and CHOPaccumulation in tumor lysates was performed using commercial-ly available MesoScale Kits (MSD). Determination of K48-poly-ubiquitin accumulation in tumor lysates was performed by devel-oping a MesoScale assay with a K48-Ubiquitin antibody (Milli-pore). Total protein concentrations were determined with a BCAassay (BCA Protein Assay #23225, Thermo Fisher Scientific) andthen normalized to 2 mg/mL. Aliquots were added to the ELISA/MSD plate, and the remaining assay procedures were carried outaccording to the manufacturer's protocol. Determination of cir-culating lambda light chain in mouse plasma was performedusing a commercially available kit (Biovendor).

Statistical analysisFor in vivo studies, statistical analysis was performed using two-

way ANOVA on delta tumor values in Prism (GraphPad).

ResultsCB-5083 treatment leads to potent and rapid death of multiplemyeloma cell lines compared with solid tumor cell lines

To characterize the potency of cell death following exposureto CB-5083, 18 human multiple myeloma cell lines weretreated with CB-5083. Following 72-hour exposure, CB-5083showed GI50s ranging from 96 to 1,152 nmol/L, with a medianGI50 of 326 nmol/L (Fig. 1A). CB-5083 was less potent inimmortalized or primary patient-derived bone marrow stromalcultures compared with multiple myeloma cell lines (Figs. 1A

Figure 1.

Effect of CB-5083 on cell growth and survival inmultiplemyelomaand solid tumor cell lines.A,Multiplemyelomacell lines expressingFF-Lucwere treatedwith adosetitration of CB-5083 for 72 hours, followed bymeasurement of viability using BLI. B,A panel of solid tumor cell lines (n¼ 153) andmultiple myeloma cell lines (n¼ 7)were treated with CB-5083 for 24, 40, and 72 hours, and cell viability (box plot � min and max) was determined using the Cell Titer Glow assay. C, Genesoverexpressed inmultiplemyelomawith aP value<0.001were analyzedusingENRICHR.Meangeneexpression valueswere calculated for all solid tumor–derived celllines versus multiple myeloma cell lines using the CCLE gene expression data. Difference in gene expression between the two cell groups was calculatedusing a Student t test. D, Time course evaluation of viability of cell lines treated with 2.5 mmol/L CB-5083 for various amounts of time with cell death read-out at 72hours using the Cell Titer Glow assay. E, Time course evaluation of caspase-3/7 induction in cell lines treated with 2.5 mmol/L CB-5083 by caspase 3/7-Gloassay. All data are representative of � two experiments.

CB-5083 Preclinical Activity in Multiple Myeloma

www.aacrjournals.org Mol Cancer Ther; 16(11) November 2017 2377




and 6D). CB-5083 has potent antitumor activity in several solidtumor cell lines (10); however, multiple myeloma cell linesshowed significantly greater overall sensitivity to CB-5083 thansolid tumor cell lines (Fig. 1B). In an attempt to understand thedifference in sensitivity of solid tumor versus multiple myelo-ma cell lines, we analyzed publicly available genomic data(CCLE) via ENRICHR (http://amp.pharm.mssm.edu/Enrichr/).GO Biological Process showed the myeloma cells to havesignificant enrichment of ER/UPR–related pathways comparedwith the solid tumor cells (Fig. 1C). Frequently, the levels of adrug target can influence sensitivity to its inhibitor. Therefore,we analyzed p97 protein levels in a subset of three multiplemyeloma and three solid tumor cell lines (Supplementary Fig.S1A) and found no correlation between p97 levels and CB-5083 sensitivity. We next analyzed the kinetics of cell death inthis subset of cell lines. The multiple myeloma cell linesMM.1S, AMO-1, and RPMI8226 died far more rapidly thanthe colon tumor cell lines HCT116 and DLD-1 and the pan-creatic cancer cell line, CFPAC-1 (Fig. 1D). These data wereconsistent with the induction of apoptosis that peaked between8 and 16 hours in the multiple myeloma cell lines versus 36 to48 hours in the colon and pancreatic tumor cell lines (Fig. 1E).This rapid kinetic of cell death was confirmed using an inde-pendent viability assay (time-lapse bioluminescence; Supple-mentary Fig. S1B and S1C). In conclusion, we found thatmultiple myeloma cells grown in the presence of CB-5083 arehighly sensitive, die rapidly, and have a baseline gene expres-sion profile that is distinct from less sensitive solid tumor celllines.

CB-5083 treatment activates the UPR, induces irresolvable ERstress, and activates apoptosis in vitro and in vivo

p97 knockdown or inhibition with small molecules hasbeen shown to induce endoplasmic reticulum (ER) stress andactivate all three arms of the UPR (5, 19). CB-5083 caused astrong induction of the UPR as measured by the phosphor-ylation of PERK and accumulation of sXBP1 and BiP (Fig.2A; Supplementary Fig. S2). CB-5083 treatment led to a

robust induction of the transcription factor CCAAT/enhanc-er-binding protein homologous protein (CHOP), which washigher than the maximum response observed with bortezo-mib and similar to what was seen with the ER stress agentthapsigargin.

K48-linked polyubiquitin accumulation, a hallmark of inhibi-tion of protein degradation (20, 21), was followed as a pathwaymarker for p97 inhibition (Fig. 2A and B). CB-5083, like borte-zomib, induced a rapid accumulation of K48-linked polyubiqui-tinated proteins in cultured cells and xenografted multiple mye-loma tumors. In RPMI8226 xenografts, a single dose of CB-5083led to plasma levels of CB-5083 that ranged from 2 to 20 mmol/Lover a period of 24 hours, sufficient to maintain significant K48polyubiquitin accumulation (Fig. 2B). This dose was sufficient toactivate the UPR, and CHOP protein expression increased up to2.7-fold compared with baseline levels. Consequently, apoptosiswas activated, as demonstrated by an increase in cleaved PARP(cPARP) levels (Fig. 2C).

The target engagement and pathway protein modulationsseen in multiple myeloma cell lines in vitro and in vivo inresponse to CB-5083 treatment were consistent with the cellu-lar responses to p97 inhibition, with differences noted whencomparing with bortezomib.

Sensitivity and transcriptional response to CB-5083 is distinctfrom PIs in multiple myeloma

To evaluate the similarities or differences in protein homeo-stasis disruption via PIs versus p97 inhibition, we compared therelative sensitivity of 18 multiple myeloma cell lines to borte-zomib, carfilzomib, and CB-5083 by correlating GI50 valuesacross the cell line panel for all pairwise comparisons ofinhibitors. The correlation for relative sensitivities of the mul-tiple myeloma cell lines to bortezomib and carfilzomib wassignificant (P ¼ 0.0001), reflecting the similar mechanism ofaction of both drugs (Fig. 3C). However, the relative responseand sensitivity across the multiple myeloma lines were notsignificantly correlated between CB-5083 and either PI (P ¼0.613 and 0.118, respectively; Fig. 3A and B).

Figure 2.

CB-5083 activates UPR and apoptosisof multiple myeloma cell lines in vitroand in vivo. A, RPMI8226 cells weretreated with DMSO, CB-5083 (0.3125–20 mmol/L), bortezomib (6.25–400nmol/L), or thapsigargin (12.5–800nmol/L) during 8 hours. Protein lysateswere subjected to immunoblotting withanti-BiP, XBP1s, PERK, CHOP, K48ubiquitin, or GAPDH antibodies. B, SCIDbeige mice bearing RPMI8226 tumors(n ¼ 3–4/group) received a single oraldose of CB-5083 at 60 mg/kg. Tumorswere collected at 1, 6, and 24 hourspostdose, and tumor lysates weresubjected to MSD analysis for K48ubiquitin. Plasma from the same micewas collected, and levels of CB-5083were determined by LC-MS and plottedwith K48-Ub modulation. C, The sametumor lysates were subjected tomeasurement of CHOP and cPARPprotein levels by MSD.

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Next, we analyzed gene expression in response to either borte-zomiborCB-5083 inRPMI8226, AMO-1, andMM.1S cell lines. Inthese experiments, cross-comparisons of ranked gene lists asso-ciated with each agent were clearly distinct (Supplementary Fig.S3A–S3B). Although transcripts that were most strongly inducedin response to CB-5083 were also induced by bortezomib (Sup-plementary Fig. S3A), transcripts that were most strongly inducedin response to bortezomib showed a mixed profile in response toCB-5083 (Supplementary Fig. S3B). More significant differenceswere noted upon analysis of ranked gene identities for bortezo-mib-induced transcripts, which indicated that many of the heatshock protein family members and chaperones from the Bagfamily typically induced by bortezomib (22) were decreased inresponse to CB-5083 (Fig. 3D). Using RNA-seq data, gene ontol-ogy analysis was performed to identify the pathways that werealtered after bortezomib or CB-5083 treatment. In bothRPMI8226 (Fig. 3E) and AMO-1 (Fig. 3F) cell lines, the geneontology analysis showed both compounds induced similar

pathways, but CB-5083 responses were predominantly focusedon ER stress and UPR, whereas bortezomib responses werefocused more on general protein stress. Altogether, these cellviability and transcriptomic results suggest that the mechanismof action of CB-5083 and the transcriptional response it producedhas some unique features compared with PIs.

Combination with PIs improves potency of CB-5083 inmultiple myeloma cells

Wenext investigated whether the blockade of both p97 and theproteasome could lead to enhanced cytotoxicity. RPMI8226 cellswere treated with a range of CB-5083 concentrations in thepresence of various dose levels of bortezomib (Fig. 4A) or carfil-zomib (Fig. 4B). When PIs were combined with CB-5083, a lowerGI50 of CB-5083 was observed, suggesting enhanced cell killingactivity. These data were confirmed in three additional multiplemyeloma cell lines (Supplementary Fig. S4A–S4C). The combi-nation of CB-5083 with PIs was then studied in vivo. After a single

Figure 3.

CB-5083 activity is different than PIs. A–C, The viability GI50 for the 18 multiple myeloma cell line panel was generated for carfilzomib (A), bortezomib (B), andCB-5083 (C), then plotted and compared for each pair of agents. D, Transcriptomic data were generated in RPMI8226, MM.1S, and AMO-1 cells treated for6 hours with a high (1 mmol/L for RPMI8226 and MM.1S; 500 nmol/L for AMO-1) or a low (500 nmol/L for RPMI8226 and MM.1S; 100 nmol/L for AMO-1) dose ofCB-5083, or bortezomib (10 nmol/L). Gene expression heatmaps were generated using GENE-E software. E and F, Gene ontology analysis was performed ongene lists obtained after RNA-seq analysis, sorted bymagnitude of differential expression after bortezomib or CB-5083 treatment in RPMI8226 (E) or AMO-1 (F) cells,by GO biological processes enrichment analysis using Enrichr.






dose of CB-5083 combined with a single dose of bortezomib, thecombination arm showed a much more dramatic effect on theinduction of K48-ubiquitin and cPARP in comparison with eachsingle agent alone (Fig. 4C and D). The sustained K48-ubiquitinand cPARP accumulation translated into significantly improvedantitumor activity (Fig. 4E and F). The combination of CB-5083and bortezomib in the AMO-1 model was highly active (TGI108%– Supplementary Fig. S4D), and when represented in awaterfall plot, the individual data showed an increased responserate with the combination when compared with single-agent

bortezomib or CB-5083 (Fig. 4E). In RPMI8226 tumors, thecombination of suboptimal doses of CB-5083 and carfilzomibshowed superiority to both single agents (P¼ 0.0004; Fig. 4F). Insummary, when CB-5083 was combined with PIs, enhancedantitumor effects were observed.

CB-5083 prevents Nrf1 regulation of proteasome transcriptionUpon exposure to PIs, mammalian cells increase expression of

multiple proteasome subunits, which elevate proteasome contentand promote survival (22, 23). This process is driven by the

Figure 4.

Combinationof CB-5083with bortezomib showedenhancedcytotoxic effect inmultiplemyeloma.A andB,Viability of RPMI8226 cells over a range ofCB-5083dosesas a single agent (black) and in the presence of varying doses of bortezomib (A) or carfilzomib (B; colored). Percentage viability was determined by normalizing toviability obtained in the DMSO control group. Cells were treated continuously with CB-5083 and PIs for 72 hours and then assessed for viability by usingCell Titer GlowAssay.C andD, Pharmacodynamic evaluation in SCID beigemice bearing RPMI8226 tumors (n¼ 3–4/group). Micewere received a single oral dose ofCB-5083 at 60 mg/kg, bortezomib at 0.8 mg/kg, or the combination of both agents. Tumors were collected at 1, 6, and 24 hours postdose, and tumorlysateswere subjected toMSDanalysis for K48-Ub (C) or cPARP (D).E,Waterfall plot representation of tumor response at individualmouse level inAMO-1 xenograftstreated with CB-5083, bortezomib, or both agents in combination (n ¼ 8–9/group). For each animal, the best average response was calculated as theminimal value of tumor volume change at time t to its baseline, with t >10 days. CB-5083 was dosed 4 days on/3 days off, orally (PO) at 50 mg/kg, and bortezomibtwice per week, i.v. at 0.8 mg/kg. Traditional growth curve of the same experiment is represented in Supplementary Fig. S4. F, RPMI8226 cells (1 � 107) weresubcutaneously inoculated into SCID beige mouse hind flank region. When the tumors reached 200 to 300 mm3, mice were randomized in treatment groups(9mice in each group) and treatedwith CB-5083 and carfilzomib vehicles, CB-5083 (30mg/kg –4days on/3 days off), carfilzomib (3mg/kg, 2 days on/5 days off), orcombination of both agents for 18 days. All data are representative of �2 experiments.

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transcription factor Nrf1/NFE2L1. It was recently demonstratedthat p97 is required for this Nrf1-mediated proteasome bounce-back response owing to its activity in retro-translocating Nrf1from the ER membrane to the cytosol (24). As this proteasomebounce-back has been described to limit the ability of PIs to killmyeloma cells (25), blocking such a compensatory responsethrough p97 inhibition may provide a mechanism to enhancethe efficacy of PIs by coadministration with a p97 inhibitor, suchas CB-5083. Therefore, we treated RPMI8226 and AMO-1 cellswith either CB-5083, bortezomib, or the combination of bothagents (CB-5083 treatment 1 hour prior to bortezomib; Fig. 5A).Treatment with bortezomib for 1 hour was not long enough tostabilize Nrf1 (Fig. 5A). At later time points, a low molecularweight band of Nrf1 accumulated in cells treated with bortezo-mib, whereas after CB-5083 treatment, a higher molecular weightband of Nrf1 accumulated. In the combination experiment ofboth agents, most of the Nrf1 was found as the higher molecularweight band. The lowmolecular weight band represents a cleavedand active formofNrf1 that is translocated to the nucleus, where itactivatesmany proteasome subunit genes (PSM; see later Fig. 5C).CB-5083 treatment leads to the accumulation of an uncleavedhighermolecular weight form of Nrf1, which corresponds toNrf1that is retained in the ERmembrane and cannot translocate to the

nucleus (Fig. 5C and D). The CB-5083–mediated retention ofNrf1 in the ER was observed in additional multiple myeloma celllines (Supplementary Fig. S5A) and the dominant CB-5083phenotype in combination with bortezomib was recapitulatedin vivo in RPMI8226 xenografts (Fig. 5B). In A549 cells, CB-5083prevented the bortezomib-induced translocation of Nrf1 to thenucleus in a dose-dependent manner (Fig. 5C and D).

Consistent with a block in Nrf1 activation, bortezomib-induced expression of proteasome genes PSMA6, PSMC6, andPSMD11 is prevented by cotreatment with CB-5083 (Supplemen-tary Fig. S5B–S5D). In conclusion, combining CB-5083 withbortezomib prevented the activation of Nrf1 and the downstreamactivation of proteasome subunit expression.

CB-5083 remains equipotent in models of acquired resistanceto PIs

CB-5083 is a protein homeostasis disruptor with a differenti-ated mechanism of action compared with the PIs and has thepotential to be active in multiple myeloma cells resistant to PIs.Therefore, CB-5083was tested against AMO-1 cell line clones thatare resistant to bortezomib or carfilzomib (12). AMO1-BtzR has abortezomibGI50>100-fold higher thanAMO1-WT.A similar shiftin sensitivity was seen in the AMO1-CfzR cell line with respect to

Figure 5.

The Nrf1 upregulation induced by bortezomib is inhibited by CB-5083. A, AMO-1, RPMI8226, and MM.1S cells were treated with either bortezomib (10 nmol/L), CB-5083 (1 mmol/L), or in combination, with CB-5083 administered 1 hour prior to bortezomib. Protein lysates were immunoblotted at different time points withanti- Nrf1 and GAPDH antibodies.B, SCID beigemice bearing RPMI8226 tumors (n¼ 3–4/group) received a single oral dose of CB-5083 at 60mg/kg, and 1 hour later,a single dose of bortezomib at 0.8 mg/kg or the combination of both agents. Tumor were collected at 2, 6, and 24 hours after CB-5083 dose, and tumorlysates were subjected to immunoblot of Nrf1. C, Immunofluorescence of Nrf1 and nucleus in A549 adherent cells. Cells were treated with 1 mmol/L of bortezomib,10 mmol/L of CB-5083, or with the combination of both agents for 8 hours. D, The levels of Nrf1 in the nucleus were measured.






carfilzomib (Fig. 6B and C). Interestingly, each clone possessedsignificant cross-resistance to the alternative PI. However, CB-5083 potency was similar in the wild-type AMO-1 cells and theAMO1-BtzR and AMO1-CfzR clones (Fig. 6A).

One potential mechanism of resistance to PIs is throughinteraction of multiple myeloma cells with the supportivebone marrow stromal compartment (BMSC; refs. 26, 27). Toassess the influence of BMSCs on CB-5083's anti-myelomaactivity, compartment-specific bioluminescence (CS-BLI;ref. 15) assays were performed with MM.1S cells coculturedin the presence of the BMSC cell lines HS5 and HS27A orprimary multiple myeloma patient-derived BMSC culture, BM-61. In these assays, the MM.1S GI50 was 0.53 mmol/L and wasunaffected by the presence or absence of BMSCs (Fig. 6D).Furthermore, the GI50 against the MM.1S cell compartmentwas 3- and 6-fold lower than the immortalized and primaryBMSC compartment GI50 alone (1.58 mmol/L for HS5 and 3.18mmol/L for BM-61). In contrast, carfilzomib demonstrated amarked reduction in potency and decreased anti-myeloma

activity in the context of all BMSCs, with its GI50 shifting1.7- to 2.4-fold, from 21 nmol/L to 36–51 nmol/L in coculturewith BMSCs (Fig. 6E).

Extending the above observations from CS-BLI into patient-derived samples, CB-5083 activity was assessed in bone marrowaspirates from multiple myeloma patients. Twenty-three sam-ples were assessed by flow to measure apoptosis in CD138þ

and CD138� cells, 14 from newly diagnosed patients and 9from patients that had been previously treated with bortezo-mib. In the newly diagnosed samples, CB-5083 activity variedwidely with GI50s ranging from 0.47 to 36.3 mmol/L. However,CB-5083 had GI50s below 1.6 mmol/L in all but one previouslytreated patient samples (Fig. 6F). In the same bone marrowsample, analysis of CB-5083 activity in CD138� cells showedlower sensitivity compared with the CD138þ cell population in16 of 18 samples, where both types were analyzed (Fig. 6G).These findings suggest that CB-5083 has more cytotoxic activityagainst multiple myeloma cells compared with the normal cellpopulation of bone marrow.

Figure 6.

CB-5083 activity in models of proteasome adaptation. A–C, CB-5083 (A), bortezomib (B), or carfilzomib (C) were tested in AMO-1 WT, bortezomib-resistant, andcarfilzomib-resistant cell lines, treated 72 hours with each agent. D and E, MM.1S cells were cultured alone or with BMSCs in the presence of either CB-5083(D) or carfilzomib (E), and viability was measured by compartment-specific bioluminescence (CS-BLI) assays. F, Fourteen chemotherapy-na€�ve and nine refractoryor resistant primary multiple myeloma specimens were treated with a dose titration of CB-5083 for 24 hours, and their viability was measured by flowcytometry. A–F, Data representative of multiple experiments. G, Primary multiple myeloma samples (CD38þ/138þ) and their matching normal population(CD38�/138�) were treated with CB-5083 for 24 hours. The EC50 of each patient sample was then matched between the CD38þ/138þ and CD38�/138� population.

Le Moigne et al.





In conclusion, CB-5083 was shown to be active against PIadapted cell line models, retained potent anti-myeloma activityin BMSC coculture models, and had potent activity in ex vivopreviously treated multiple myeloma patient samples.

CB-5083 is highly active in mouse models relevant to multiplemyeloma

Previously, CB-5083was shown to be active in a variety of solidtumormodels (10).We used anortho-metastaticmousemodel ofmultiple myeloma to assess efficacy of CB-5083. Mice wereinjected intravenously with MM.1S (Fig. 7A) or RPMI8226 cells(Supplementary Fig. S6A) that were modified to express lucifer-ase. Oral treatment with CB-5083 at a dose of 60 mg/kg on a 4days on/3 days off schedule inhibited tumor progression to an

extent that compared favorably (P < 0.0001) to intravenousbortezomib over a 3-week dosing cycle (Fig. 7B). CB-5083 dem-onstrated a similar level of efficacy in the disseminated RPMI8226tumor model, which again, compared favorably with an effectiveregimen of intraperitoneal carfilzomib (Supplementary Fig. S6B).

In a subcutaneous RPMI8226 model, CB-5083 was comparedwith bortezomib, carfilzomib, and ixazomib. In this model, allagents inhibited RPMI8226 growth with CB-5083 showing thehighest TGI (Fig. 7C). After three cycles of dosing with CB-5083,tumors were allowed to regrow and were rechallenged withCB-5083 once they reached 700 mm3 (Supplementary Fig. S6C).CB-5083 was again able to induce 50% regression of these tumors.

Next, CB-5083 activity was investigated in combination withdexamethasone and lenalidomide, two active anti–multiple

Figure 7.

CB-5083 demonstrates a broad activity in in vivo relevant multiple myelomamodels.A andB,MM.1S–Luc cells (1� 106) were injected by the tail vein in NSGmice. Onday 12, mice were randomized (n ¼ 5/group) in a vehicle group, CB-5083 (60 mg/kg, 4 days on/3 days off) and bortezomib (1 mg/kg, biw). Bioluminescencesignal was captured on days 12 and 25 of treatment, and the flux signal was quantified in graph (B). C, SCID beige mice bearing established RPMI8226 tumors(n ¼ 10/group) were treated with vehicle (0.5% methyl cellulose), CB-5083 [100 mg/kg, orally (PO), 4 days on/3 days off], bortezomib (1 mg/kg, i.v.,twice per week), carfilzomib (5 mg/kg, i.v., 2 days on/5 days off) or ixazomib (11 mg/kg, orally, twice per week) during 18 days. D, SCID beige mice bearingadvanced (>300 mm3) RPMI8226 tumors (n ¼ 10/group) were treated with vehicle, CB-5083 (40 mg/kg, orally, 4 days on/3 days off), a combination ofdexamethasone (0.1 mg/kg, i.p., daily) þ lenalidomide (20 mg/kg, orally, daily), or the triple combination CB-5083/dexamethasone/lenalidomide, during 25 days.E, Three Vk�MYC mice were treated with CB-5083 (30 mg/kg, orally 4 days on/3 days off) during14 days. M-protein was measured by SPEP predose days 0 and 14 posttreatment.






myeloma agents (Fig. 7D). Suboptimal doses of CB-5083 com-bined with suboptimal doses of dexamethasone and lenalido-mide led to regression of tumors, demonstrating the superiority ofthe triple combination arm (P < 0.0001). These findings wereconsistent with the level of lambda light chains quantified in theplasma of the tumor-bearing mice (Supplementary Fig. S6D).

Finally, CB-5083 antitumor activity was evaluated in theVk�MYC model, a transgenic mouse model that reproduces thepathogenesis, biology, and clinical features of multiple myelomaand is predictive of clinical activity (18, 28). Prior to dosing,baseline serum M-spike was determined by serum protein elec-trophoresis (SPEP) in individualmice. CB-5083was administeredorally for 2 weekly cycles in C57/BL6 Vk�MYC mice. On day 14,SPEP analysis of serum showed a 55% decrease in M-spike whencompared with predose levels (Fig. 7E). Taken together, CB-5083showed potent antitumor activity in a wide variety of clinicallyrelevant in vivo multiple myeloma models.

DiscussionIt has been well documented thatmyeloma cells are exquisitely

sensitive to perturbation of protein homeostasis, owing to theirhigh rates of immunoglobulin synthesis and secretion (4). Phar-macologic inhibition of the chymotryptic site of the proteasomeexhibits potent clinical activity in myeloma, Waldenstr€om mac-roglobulinemia, and amyloidosis (1, 29, 30). These observationssuggest that the activity of proteasome inhibition in multiplemyeloma may represent an effect against the intrinsic biology ofplasma cells rather than the specific oncogenic events harbored bythe multiple myeloma cells.

The current study examines the phenotypic consequences ofpharmacologic inhibition of p97/VCP using CB-5083 in preclin-ical models of multiple myeloma. p97 plays important roles inprotein homeostasis upstream of the proteasome, and CB-5083has recently been described as a novel anticancer agent targetingp97. CB-5083 appears to be quite selective for the ERAD-relatedfunctions of p97, with only minor (if any) effects on its othercellular functions. This relative selectivity of CB-5083 for theERAD-related functions of p97 renders it an attractive option forthe treatment of multiple myeloma, which exhibits pronouncedresponsiveness upon pharmacologic perturbation of ERAD. Asp97 was demonstrated to be involved in the regulation of otheroncogenic pathways [including NFkB (31) or HIF1a (32)], fur-ther study of the non-ER functions of p97 that may be affected byCB-5083 andmayhavebeenmissedby the gene ontology analysisis warranted.

On the basis of these considerations, we set out to characterizethe response to CB-5083 in models of multiple myeloma. Weobserved that submicromolar concentrations of CB-5083 werecapable of inducing a significant decrease in viability for themajority of multiple myeloma cell lines tested. The observedGI50 values for multiple myeloma cell lines were significantlylower than those observed for non–multiple myeloma cell linestested in this work or previously (10). The exquisite sensitivity ofmultiplemyeloma cells toCB-5083 could be explained by amuchhigher level of ER stress and UPR at baseline, in comparison withsolid tumor cell lines. In viability assays, there was little correla-tion between the sensitivity of multiple myeloma cell lines to CB-5083 and their sensitivity to PIs. The anti–multiple myelomaactivity of CB-5083 was not significantly attenuated by in vitrointeractions of multiple myeloma cells with BMSCs. This can be

viewed as a favorable phenotypic result, given BMSCs conferresistance to anti–multiple myeloma agents via cell adhesion–mediated drug resistance (15, 26). The preclinical anti–multiplemyeloma activity of CB-5083 was associated with potent induc-tion of canonical markers of the UPR, including phosphorylationof PERK, accumulation of sXBP1 and BiP, and induction ofCHOP, as well as accumulation of K48-linked polyubiquitin, ahallmark of inhibition of protein degradation. Despite some ofthe similarities in gene expression changes seen in response to CB-5083 and PIs, CB-5083 did exhibit some distinct features. Thetranscriptional profile of CB-5083–treated multiple myelomacells lacked the pronounced upregulation of heat shock genes orproteasome subunits, which has been reported to reflect com-pensatory responses mounted by multiple myeloma cells in thecontext of 20S proteasome inhibition (22, 33). These distinctevents for CB-5083 versus PIs may be related to the differentialimpact of these classes of drugs on posttranslational processing,intracellular localization, and transcriptional activity of Nrf1.Unlike bortezomib or carfilzomib, CB-5083 treatment is associ-ated with inhibition of Nrf1 cleavage and its retention in the ERmembrane, thereby preventing its nuclear translocation andtranscription of proteasome subunit genes. Notably, Nrf1-medi-ated induction of proteasome gene expression is thought to limitthe cytotoxic effects of theUPR inmyeloma cells in response to PIs(24, 25, 34, 35).

CB-5083 enhanced the anti–multiple myeloma activity ofbortezomib both in vitro and in vivo and was active in bortezomibresistance models as well as primary multiple myeloma cellsderived from patients who had been treated with bortezomib-based regimens. The observation that CB-5083 decreases themyeloma tumor burden in the Vk�MYC genetically engineeredmousemodel of myeloma is also notable, because it suggests thatp97 inhibition may have utility at different stages of the disease.This model is considered to capture the behavior of multiplemyeloma in its earlier stages, whereas xenografts of humanmyeloma cell lines are considered to be reflective of themolecularfeatures of advanced multiple myeloma (18, 28).

An important area of investigation relates to the characteriza-tion of molecular markers that may identify myeloma cells withpronounced responses to CB-5083 monotherapy. Indeed, someof the myeloma cell lines tested in our studies exhibited morepotent and/or rapid responses to CB-5083 compared with othercell lines in the panel. Our current study was not designed toidentify such candidate markers, but it is reasonable to hypoth-esize that these markers may prove to be quite distinct from thoseassociated with potent responses to PIs given that the quantitativepatterns of response of multiple myeloma cells to these agentsdiffer substantially from the observed pattern for CB-5083. In aseparate recent study fromour groupon the impact ofCB-5083ona large panel of solid tumor cell lines, mutational activation ofRas/Raf/MAPK signaling exhibited a trend for a higher degree ofresponsiveness to CB-5083 (10). Multiple myeloma also exhibitsfrequent activation of Ras/Raf/MAPK signaling through muta-tions in diverse components of this pathway (36) and/or cellnonautonomous activation in the context of tumor microenvi-ronment interactions (26, 37). We were able to demonstrate on asmaller panel of multiple myeloma cells listed in Anderson andcolleagues' article (AMO-1, RPMI8226, OPM2, MM.1S, andMM.1R) that there was a strong correlation between multiplemyeloma tumor responses and phospho/total ERK ratio[unpublished analysis (10)]. We envision that validation of this

Le Moigne et al.





hypothesis in studies of larger cell line panels or in samples frompatients in clinical trials of CB-5083 may represent a path towardconfirming or refuting whether CB-5083might play an importantrole in the treatment of multiple myeloma patients with muta-tions in the Ras/Raf/MAPK pathway.

In summary, CB-5083 has demonstrated robust preclinicalactivity via a novel and distinctive mechanism of action. Current-ly, clinical trials are ongoing in patients with multiple myelomawho have exhausted other available therapies.

Disclosure of Potential Conflicts of InterestB.T. Aftab is the director at Atara Biotherapeutics, Inc. and reports receiving

commercial research grants from Amgen, Cleave Biosciences, CytomX Thera-peutics, Omniox, Inc., and SanofiOncology. S. Djakovic has ownership interest(including patents) in Cleave Biosciences. Z.Y. Wu is a senior research associateat Cleave Biosciences. A.P. Wiita reports receiving a commercial research grantfrom Cleave Biosciences. L. Shawver is an employee at and has ownershipinterest (including patents) in Cleave Biosciences. C.S. Mitsiades is the directorat Takeda and reports receiving commercial research grants from Abbvie,Janssen/Johnson & Johnson, Novartis, Ono Pharmaceutical, and TEVA. Nopotential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: R. Le Moigne, B.T. Aftab, S. Djakovic, E. Valle,F.M. Yakes, H.-J. Zhou, C.S. Mitsiades, D.J. Anderson, M. RolfeDevelopment of methodology: R. Le Moigne, B.T. Aftab, S. Djakovic,E. Dhimolea, E. Valle, Z.Y. Wu, C. Driessen, D. Wustrow, D.J. AndersonAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): R. Le Moigne, B.T. Aftab, S. Djakovic, E. Dhimolea,E. Valle, M. Murnane, E.M. King, F. Soriano, M.-K. Menon, Z.Y. Wu, S.T. Wong,

G.J. Lee, C. Lam, J. Wang, M. Chesi, P.L. Bergsagel, M. Kraus, C. Driessen,S. Kiss von Soly, L. Shawver, T.G. Martin IIIAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): R. Le Moigne, B.T. Aftab, S. Djakovic, E. Valle,M. Murnane, M.-K. Menon, Z.Y. Wu, S.T. Wong, G.J. Lee, B. Yao, A.P. Wiita,J. Rice, F.M. Yakes, T.G. Martin III, C.S. Mitsiades, D.J. Anderson, M. RolfeWriting, review, and/or revision of the manuscript: R. Le Moigne, B.T. Aftab,Z.Y. Wu, G.J. Lee, C. Driessen, D. Wustrow, L. Shawver, H.-J. Zhou, T.G. MartinIII, C.S. Mitsiades, D.J. Anderson, M. RolfeAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): M. Murnane, S.T. Wong, J. Wang, C. Driessen,D. Wustrow, J.L. WolfStudy supervision: R. Le Moigne, B.T. Aftab, S.T. Wong, F.M. Yakes, H.-J. Zhou,T.G. Martin III, C.S. Mitsiades, D.J. Anderson, M. Rolfe

AcknowledgmentsWe thankDrs. Suzanne Trudel, Joan Levy, Jesse Vargas, andDaniel Auclair for

scientific discussion and contributions to work presented in this manuscript.

Grant SupportThis work was funded in part by theMultipleMyeloma Research Foundation

(to B.T. Aftab), The Myeloma Fund of the Silicon Valley Community Founda-tion (to B.T. Aftab), and The Stephen and Nancy Grand Multiple MyelomaTranslational Initiative (to B.T. Aftab). C. Driessen was supported by a grantfrom Oncosuisse.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 14, 2017; revised June 10, 2017; accepted August 9, 2017;published OnlineFirst September 6, 2017.

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2017;16:2375-2386. Published OnlineFirst September 6, 2017.Mol Cancer Ther Ronan Le Moigne, Blake T. Aftab, Stevan Djakovic, et al. Homeostasis in Models of Multiple MyelomaThe p97 Inhibitor CB-5083 Is a Unique Disrupter of Protein

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The p97 Inhibitor CB-5083 Is a Unique Disrupter of Protein … · Small Molecule Therapeutics The p97 Inhibitor CB-5083 Is a Unique Disrupter of Protein Homeostasis in Models of Multiple