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Cancer Therapy: Preclinical

Bortezomib-Induced Unfolded Protein Response IncreasesOncolytic HSV-1 Replication Resulting in SynergisticAntitumor Effects

Ji YoungYoo1, Brian S.Hurwitz1,2, ChelseaBolyard3, Jun-GeYu4, JianyingZhang5, KaruppaiyahSelvendiran6,Kellie S. Rath6, Shun He7, Zachary Bailey10, David Eaves10, Timothy P. Cripe8, Deborah S. Parris9,Michael A. Caligiuri7, Jianhua Yu7, Matthew Old4, and Balveen Kaur1

AbstractBackground:Bortezomib is an FDA-approved proteasome inhibitor, andoncolytic herpes simplex virus-

1 (oHSV) is a promising therapeutic approach for cancer. We tested the impact of combining bortezomib

with oHSV for antitumor efficacy.

Experimental Design: The synergistic interaction between oHSV and bortezomib was calculated

using Chou–Talalay analysis. Viral replication was evaluated using plaque assay and immune fluores-

cence. Western blot assays were used to evaluate induction of estrogen receptor (ER) stress and unfolded

protein response (UPR). Inhibitors targeting Hsp90 were utilized to investigate the mechanism of cell

killing. Antitumor efficacy in vivowas evaluated using subcutaneous and intracranial tumor xenografts of

glioma and head and neck cancer. Survival was analyzed by Kaplan–Meier curves and two-sided log-rank

test.

Results: Combination treatment with bortezomib and oHSV (34.5ENVE), displayed strong synergistic

interaction in ovarian cancer, head and neck cancer, glioma, and malignant peripheral nerve sheath tumor

(MPNST) cells. Bortezomib treatment induced ER stress, evident by strong induction of Grp78, CHOP,

PERK, and IRE1a (Western blot analysis) and the UPR (induction of hsp40, 70, and 90). Bortezomibtreatment of cells at both sublethal and lethal doses increased viral replication (P < 0.001), but inhibition ofHsp90 ablated this response, reducing viral replication and synergistic cell killing. The combination of

bortezomib and 34.5ENVE significantly enhanced antitumor efficacy in multiple different tumor models

in vivo.

Conclusions: The dramatic synergy of bortezomib and 34.5ENVE is mediated by bortezomib-induced

UPR and warrants future clinical testing in patients. Clin Cancer Res; 20(14); 3787–98. �2014 AACR.

IntroductionOncolytic herpes simplex virus-1 (oHSV) therapy utilizes

viruses that are engineered to infect and replicate in cancercells with minimal damage to non-neoplastic tissue. Thistherapy is currently being evaluated for safety and efficacy inmultiple phase I, II, and III clinical trials (1). The resultsfrom a phase III testing of T-Vec (an oHSV developed byAmgen) have shown promising results in tumor shrinkage.Although theoverall survival data have yet tobe established,there is a significant need to optimize this promising ther-apy in vivo. Although second- and third-generation virusesare being created and tested inpreclinical studies, drug-viruscombinations can be rapidly translated to clinical trials tomaximize efficacy and minimize toxicity (2).

The proteasome is a cellular organelle that controls deg-radation and recycling of a wide variety of proteins thatregulate diverse cellular functions, including cell-cycle pro-gression, cell death, gene expression, signal transduction,metabolism,morphogenesis, differentiation, antigenpresen-tation, and neuronal function. Inhibition of the proteasome

Authors' Affiliations: 1Department of Neurological Surgery, DardingerLaboratory for Neuro-oncology and Neurosciences; 2Biomedical Sci-ence Major; 3Integrated Biomedical Sciences Graduate Program;4Department of Otolaryngology, Head & Neck Surgery; 5Center forBiostatistics; 6Division of Gynecologic Oncology, Department ofObstetrics and Gynecology; 7Division of Hematology, Department ofInternal Medicine, The Ohio State University Wexner Medical Center;8Department of Pediatrics, Center for Childhood Cancer and BloodDiseases, The Research Institute at Nationwide Children's Hospital andthe Division of Hematology/Oncology/BMT, Nationwide Children's Hos-pital; 9Department of Molecular Virology Immunology Medical Genetics,The Ohio State University, Columbus; and 10Division of Oncology,Cincinnati Children's Hospital Medical Center, University of Cincinnati,Cincinnati, Ohio

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Authors: Balveen Kaur, Department of Neurological Sur-gery, Dardinger Laboratory for Neuro-oncology and Neurosciences, TheOhio State University, 385-DOSUCCC, 410West 12th Avenue, Columbus,OH 43210. Phone: 614-292-3984; Fax: 614-688-4882; E-mail:[email protected]; and Matthew Old, E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-14-0553

�2014 American Association for Cancer Research.

ClinicalCancer

Research

www.aacrjournals.org 3787

on June 18, 2021. © 2014 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst May 9, 2014; DOI: 10.1158/1078-0432.CCR-14-0553

http://clincancerres.aacrjournals.org/

can result in cellular aggregation of unfolded proteins whichinduce estrogen receptor (ER) stress and apoptosis. Cancercells have increased metabolic demands and are thought tobe constantly at the brink of ER stress. Thus, proteasomeinhibition has been investigated as a potential way to targetmalignant cells.

Bortezomib is a peptide-based, reversible proteasomeinhibitor, which is currently FDA approved either as asingle agent or in combination with other chemo-/radio-therapeutic agents for multiple myeloma. It is also used asa second-line treatment for ovarian and head and neckcancers and is currently under clinical evaluation for thetreatment of several other cancer types. Recent evidenceindicates that most patients do not respond to this drugwhen it is used as a single agent, and several strategiestesting its efficacy in combination with other drugs arebeing pursued (3, 4).

The combination of bortezomib and oHSV is intriguingbecause HSV-1 exploits the host proteasome during its lifecycle (5, 6), but proteasome-mediated degradation of viralcapsids in infected macrophages is also thought to beimportant for stimulating antiviral IFN responses in thesecells (7). In addition, bortezomib treatment has also beenshown to induce Epstein–Barr virus and Kaposi sarcomavirus lytic gene expression, suggesting that bortezomibtreatment could also improve virus replication in vivo (8).These results suggest that bortezomib may have opposingeffects on oHSV efficacy.

In this study, we demonstrate for the first time that theinduction of the unfolded protein response (UPR) afterbortezomib treatment improved oHSV replication and syn-ergistically improved cancer cell killing in vitro and in vivo.These findings demonstrate that the synergistic interactionbetween oHSV and bortezomib improves overall therapeu-

tic efficacy via augmenting the cancer cell killing, providingadvanced rationale for combining these agents in a clinicaltrial. This is the first study to demonstrate the utility ofcombining a clinically relevant proteasome inhibitor toaugment replication of oHSV and to achieve synergistic cellkilling.

Materials and MethodsCell lines, viruses, antibodies, and molecular biologyreagents

All cancer cell lines with the exception of ovarian cancercells were cultured in DMEM (Gibco BRL) supplementedwith 10% FBS (Gibco BRL). Ovarian cancer cell lines weremaintained in RPMI medium (Gibco BRL) supplementedwith 10% FBS. Human glioma cell lines (U251T3 andLN229), head and neck cancer cell lines (CAL27,UMSCC11A, and UMSCC74A), and ovarian cancer celllines (PA1, A2780-CS, A2780-CR, and SKOV3) have beencultured in our laboratory and U251T3 cells were obtainedas a tumorigenic clone of U251 cells by serial passage ofthese cells three times in mice. Normal human astrocyte(NHA), human umbilical vein endothelial cell (HUVEC),and hepatocyte cells were purchased from SciencellResearch Laboratories and cultured according to manufac-turer’s instruction. U87DEGFR cell line expresses a truncat-ed, constitutively active,mutant formof EGFRvIII. GBM169are primary patient-derived neurosphere cultures main-tained in neurobasal medium. Human ovarian cancerpatient ascites cells were harvested from patient ascitesunder an Institutional Review Board-approved protocol.All cell culture media were supplemented with 2 mmol/LL-glutamine, 100 IU/mL penicillin, and 100 mg/mL strep-tomycin. All cell lines were maintained at 37�C in a humid-ified atmosphere at 5% CO2. The construction and gener-ation of 34.5ENVE (viral ICP34.5 Expressed by Nestinpromotor and Vstat120 Expressing) and HSV1716(a mutant HSV-1 deleted in both copies of RL1 gene whichencodes the protein ICP34.5) has beenpreviously described(9). Virus was propagated in Vero African green monkeykidney cells (ATCC) as previously described (10).

Cytotoxicity assayThe cytotoxicity of bortezomib and34.5ENVE in each cell

line was determined by a standard MTT assay (Roche) asmanufacturer’s instructions. Tomeasure 50% effective dose(ED50) of each bortezomib or 34.5ENVE alone, cells wereplated onto 96-well plates of approximately 50% conflu-ence on day zero and then treated with bortezomib or PBSfor 16 hours before drug washout and infection with34.5ENVE or Hank’s Balanced Salt Solution (HBSS). Cellviability was measured 72 hours postinfection. To measuresynergistic cell killing, cells were treated with bortezomiband 34.5ENVE (as detailed above) at serially diluted con-centrations of 0.0625, 0.125, 0.25, 0.5, 1, 2, and 4 times oftheir ED50 in a constant ratio. Cell viabilitywasmeasured72hours postinfection. All assays were performed in triplicate.For rescue assay with geldanamycin and 17-AAG, cells were

Translational RelevanceThis study describes synergy between proteasome

inhibition and oncolytic herpes simplex virus-1 (oHSV).Induction of the cellular unfolded protein response(UPR) is usually associated with resistance to protea-some inhibition and other chemotherapies. Here, wefound that proteasome inhibition-induced UPR sensi-tizes cells to oncolysis. Interestingly HSV-1 is known toexploit the proteasome during its replication cycle andproteasome inhibitors have been suggested as antiviralagents. In contrast, this study shows that bortezomibtreatment increases viral replication, a finding addition-ally supported by the clinical observation of increasedincidence of latent virus reactivation in patients treatedwith bortezomib. Because bortezomib is FDA approved,its combination with oHSV can be rapidly translated inpatients withmultiple solid tumors. This study paves theway for a combination treatment strategy that can utilizesuboptimal doses of bortezomib in conjunction withoHSV to maximize efficacy with minimal toxicity.

Yoo et al.

Clin Cancer Res; 20(14) July 15, 2014 Clinical Cancer Research3788




pretreated with either agent for 1 hour before bortezomibtreatment.

In vitro viral replication assayCells were treated � bortezomib (at indicated doses)

for 16 hours, and following drug washout cells wereinfected with 34.5ENVE (at indicated doses) for 2 hours.Seventy-two hours postinfection, cells and supernatantwere collected, and the number of infectious particlespresent in the resulting supernatant was determined aspreviously described (9).

Western blot analysis and antibodiesCell lysates were fractionated by SDS-PAGE and trans-

ferred to polyvinylidenedifluoride membranes. In UL30detection experiments, cell and nuclear fractions were sep-arated by NE-PER Nuclear and Cytoplasmic ExtractionReagents (Thermo Scientific, Cat# 78835). Blocked mem-branes were then incubated with antibodies against Anti-LC3B, calnexin, PERK, IRE1a, PDI, Bip/GRP78, CHOP,Ero1-La, HSP40, HSP70, HSP90, and GAPDH (Abcam;each diluted 1:1,000); horseradish peroxidase (HRP)-con-jugated secondary anti-mouse antibody (each diluted1:1,000; GE Healthcare); HRP-conjugated secondary goatanti-rabbit antibody (each diluted 1:1,000; Dako), and theimmunoreactive bands were visualized using an enhancedchemiluminescence (GE Healthcare). UL30 antibody(diluted 1:100) used was purified rabbit IgG raised againstrecombinant Escherichia coli-expressed bacteriophage T7gene 10 protein fused with the C-terminal portion of theHSV-1 pol gene (11).

Animal surgeryAll mice experiments were housed and handled in accor-

dance with the Subcommittee on Research Animal Care ofthe Ohio State University (Columbus, OH) guidelines andadhered to the NIH Guide for the Care and Use of Labo-ratory Animals. Female athymic nu/nu mice (CharlesRiver Laboratories) were used for all in vivo experiments.Nude mice with subcutaneous tumors (100 mm3) wererandomized to be treated with either intraperitoneal PBS orbortezomib (0.8 mg/kg) twice a week. For intracranialtumor studies, anesthetized nudemicewere implantedwithtumor cells as described (9). Three days following cellimplantation, mice were randomized to receive PBS orbortezomib (0.8 mg/kg) via intraperitoneal injection twicea week. Seven days later, mice with intracranial or subcu-taneous tumors were inoculated with 34.5ENVE or HBSS[intracranial: 5 � 104 plaque-forming unit (pfu) or forsubcutaneous tumors: 1 � 105 pfu]. Intraperitoneal borte-zomib or PBS injections continued for the duration of theexperiment, and animals were observed daily and eutha-nized at the indicated time points or when they showedsigns of morbidity (hunched posture and weight loss). Forsubcutaneous studies, tumor volume was calculated on thebasis of tumor length and width using the following for-mula: volume ¼ 0.5 LW2 as described (9). For histologicanalysis, subcutaneous CAL27 tumors in nude mice were

treated with/without bortezomib (0.8mg/kg) twice a week.Seven days later, virus was intratumorally injected, and theninjected again 2 days later. Three days after the second virusinjection, tumors were harvested from each treatmentgroup. Representative sections were stained with hematox-ylin and eosin (H&E) and anti-HSV-1 antibody (Dako,1:200 dilution) and then examined by microscopy.

Statistical analysisTo compare two independent treatments for continuous

endpoints, the Student t test was used. When multiplepairwise comparisons aremade, one-wayANOVAwas used.Log-rank test was used to compare survival curves forsurvival data. When median survival was not observed fortwo or more groups in an experiment, survival probabilityon the last day of observationwas calculated for each group.P values were adjusted for multiple comparisons by Holms’procedure. A P value 0.05 or less is considered significant.For all synergy analysis, cancer cells were seeded into a 96-well plate, and were treated with vehicle or bortezomib for18 hours followed by drug washout, and then virus infec-tion. Three days after infection, cell viability was measuredusing a standard MTT assays (Roche Applied Sciences). Forsynergy analysis, dose–response curves and ED50 valueswere determined for each individual treatment (drug orvirus). Fixed ratios of ED50 of drug and virus were then usedto treat the cells, either with dual treatment or with indi-vidual treatments (as controls). CompuSyn software pro-gram algorithm assessed the combination index (CI). Com-bined dose–response curves were fitted to Chou–Talalaylines, which are derived from the law of mass action. CI < 1indicates synergistic interaction, whereas CI > 1 is antago-nistic and CI ¼ 1 is considered additive (12).

ResultsInteraction of bortezomib treatment with oHSV

The sensitivity of head and neck squamous cell carcino-mas, ovarian cancer, malignant peripheral nerve sheathtumors (MPNST), and glioma to bortezomib or 34.5ENVEalone in vitro was evaluated. Cells were treated with indi-cated doses of bortezomib and/or oHSV and cell death wasmeasured by a standard MTT assay. Figure 1A shows thatcombination treatment increased cell killing comparedwith the predicted additive effects (dotted line), suggestingsynergistic cell killing.

To further evaluate synergistic interaction between bor-tezomib and oHSV, wemeasured sensitivity of each cell lineto bortezomib and oHSV. Briefly, cells were treated withbortezomib at concentrations ranging from 0.1 to 500nmol/L for 16 hours, followed by drug washout. To testsensitivity to 34.5ENVE, cells were infected with differentdoses of oHSV ranging from 0.001 to 0.5 multiplicity ofinfection (MOI) defined as virus pfu/cell. Seventy-twohours following bortezomib washout or 34.5ENVE infec-tion, cell viability was measured by a standard MTT assay.The ED50 of bortezomib and 34.5ENVEwas each defined asthe dosage yielding 50% cell viability at 72 hours following

Oncolytic HSV-1 Virotherapy Synergizes with Bortezomib

www.aacrjournals.org Clin Cancer Res; 20(14) July 15, 2014 3789




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Figure 1. Effect of bortezomib and oHSV on cancer cell killing. A, treatment of ovarian cancer, head and neck cancer, and glioma cells with bortezomiband oHSV showed more than additive cell killing. Briefly, the indicated cells were treated with sublethal doses of bortezomib or PBS for 16 hours,followed by washout drug and treatment with sublethal dosed of 34.5ENVE or PBS. Seventy-two hours later from 34.5ENVE infection, cell viability wasmeasured via MTT assay. (Continued on the following page.)

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treatment as compared with untreated controls. Supple-mentary Table S1 shows the sensitivity of each cell line tobortezomib and 34.5ENVE. To study the interactionbetween bortezomib and 34.5ENVE, cells were treated withbortezomib for 16 hours, followed by drug washout andtreatment with 34.5ENVE at concentrations of 0.0625,0.125, 0.25, 0.5, 1, 2, and 4 times of their ED50 in a constantratio. Cell viability was measured 72 hours after virusinfection. The results were analyzed using themedian-effectmethod of Chou–Talalay (COMPUSYN; ref. 12). Figure 1Bshows combination index plots of Chou–Talalay. The plotsshow synergistic cell killing between bortezomib and34.5ENVE (combination) in PA1, A2780-CS (cisplatin sen-sitive), SKOV3, and A2780-CR (cisplatin resistant) ovariancancer cells CAL27, UMSCC11A, and UMSCC74A, headand neck squamous cell carcinomas and in U251T3,LN229, and U87DEGFR glioma cells (Fig. 1B). In a separateexperiment, similar synergistic cell killing was observedwhen clinical grade oHSV1716 was used in combinationwith bortezomib to treat MPNST cells (Supplementary Fig.S1; CI values less than 1 at all fractions affected). To evaluatethe effect of bortezomib on oHSV safety, we compared thecytotoxic ability of oHSV toward NHA, HUVEC, and hepa-tocyte cells treated with/without ED50 concentrations ofbortezomib (Fig. 1C). In all three normal cells tested, noadditive (dotted line) or synergy was observed betweenbortezomib and 34.5ENVE. These data support the conclu-sion that the combination of bortezomib and oHSV signif-icantly increased sensitivity to cell death for awide variety ofsolid tumor cells but not for normal cells.

Impact of bortezomib on oHSV replicationTo investigate the impact of bortezomib on 34.5ENVE

replication, U251T3 glioma cells were treated with suble-thal and ED50 doses of bortezomib (2 and 12 nmol/L)followed by 34.5ENVE infection (Fig. 2A and B). Fluores-cent microscopy was used to image GFP-positive (inf-ected) cells, 48 hours postinfection. Figure 2A showsincreased GFP-positive infected cells in both concentra-tions of bortezomib, indicating that bortezomib treatmentincreased viral replication in vitro. In addition, quantifi-cation of viral titers showed a significant increase in viraltiter in the both sublethal and ED50 doses of bortezomib(Fig. 2B–C). Quantification of viral titers in cells treatedwith or without bortezomib revealed a significant increasein viral replication in the multiple cell lines treated withsublethal doses of bortezomib in all cells tested: ovarian:SKOV3 cells: 20.3-fold, head and neck cancer cells: CAL27:1.8-fold, UMSCC74A: 1.8-fold, and glioma: U87DEGFR:8.4-fold (Fig. 2C and supplementary Table S2). Collectiv-

ely, these data demonstrate that bortezomib increasedviral replication.

Efficacy of combination treatment in patient-derivedprimary tumor cells ex vivo

Next, we tested synergistic interaction of bortezomib and34.5ENVE in ovarian cancer patient ascites derived tumorcells and glioblastoma (GBM) patient-derived primaryGBM neurospheres (GBM169) ex vivo (Fig. 2D). Briefly,cellswere treatedwithbortezomib for 16hours, followedbydrug washout and treatment with 34.5ENVE at concentra-tions of 0.0625, 0.125, 0.25, 0.5, 1, 2, and 4 times of theirED50 in a constant ratio. Cell viability was measured 72hours after virus infection. The results were analyzed usingthemedian-effect method of Chou–Talalay (COMPUSYN).Synergistic cell killing was obvious in both patient-derivedascites and primary GBM neurosphere cells (Fig. 2D).Patient-derived primary GBM neurosphere cells (GBM169)pretreated with or without bortezomib were labeled withred cell tracker and treated with 34.5ENVE. Fluorescentmicroscopy images showed increased GFP-positive infectedcells in bortezomib-treated cells (Fig. 2E). Quantification ofviral titers in cells treated with or without bortezomibrevealed a significant increase in viral replication in bothpatient ascites and primary GBM neurospheres: Ovarianpatient ascites: 4.3-fold and GBM169: 1.9-folds increaseover oHSV alone (Fig. 2F).

ER stress and unfolded protein response in cellstreated with bortezomib and oHSV

Bortezomib is reversible proteasome inhibitor thatblocks the chymotrypsin-like activity of the proteasomeresulting in the accumulation of unfolded proteins. Accu-mulation of unfolded proteins induces ER stress leading tocaspase-dependent apoptosis (13–15). Treatment of bothglioma and head and neck cancer cells with bortezomibshowed induction of proteins indicative of ER stress in adose-dependent manner (Fig. 3A and Supplementary Fig.S2A). In contrast, oHSV is known to disarm ER stress duringinfection (16). To test the impact of combination treatmentof oHSVandbortezomibonER stress, wemeasured changesin levels of cellular proteins that are induced during ERstress. Increased expression of PERK, Calnexin, IRE1a,CHOP, Ero1-La, and GRP78 proteins was observed inoHSV-infected and uninfected glioma and head and neckcancer cells after bortezomib treatment (Fig. 3B and Sup-plementary Fig. S2B).

Along with ER stress, unfolded proteins also lead to theinduction of an UPR, which constitutes increased expres-sion of HSP and cellular chaperone proteins. Both glioma

(Continued.) Data shown are % cell death � SD of cells treated with bortezomib/PBS for 16 hours before media washout alone or with oHSV infection.B, Chou–Talalay analysis of combining bortezomib with 34.5ENVE in ovarian cancer, head & neck cancer, and glioma cells. Briefly, cells were treatedwith bortezomib for 16 hours, followed by drug washout and treatment with 34.5ENVE at concentrations of 0.0625, 0.125, 0.25, 0.5, 1, 2, and 4 times oftheir ED50 in a constant ratio. Data shown as fraction affected (fa) versus CI plots. CI < 1 indicate synergy, CI ¼ 1 indicate additive, and CI > 1 indicate anantagonistic interaction. C, normal human astrocytes (NHA), HUVEC, and hepatocyte cells were plated and treated with/without ED50 concentration ofbortezomib for 16 hours, followed by drug washout and treatment with 34.5ENVE. 72 hours following 34.5ENVE infection, cell viability wasmeasured byMTTassay. Data points represent the mean % cell viability relative to uninfected cells and error bars indicate � SD for each group.






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Figure2. Effectofbortezomib treatmentonviral replication.U251T3cellswere treatedwith2or12nmol/Lbortezomib for16hoursbeforedrugwashoutand34.5ENVEinfection (MOI ¼ 0.002 with 2 nmol/L bortezomib and 0.01 for 12 nmol/L bortezomib) and evaluated for virus replication. A, bright field (Top) and fluorescencemicroscopic images (bottom) of GFP-positive infected cells 48 hours postinfection demonstrate increased virus replication following bortezomib treatment at bothsublethal (2 nmol/L) and ED50 (12 nmol/L) concentrations relative to no bortezomib-treated cells (magnification, �100). (Continued on the following page.)

Yoo et al.





and head and neck cancer cells treated with bortezomibshowed a dose-dependent induction of HSP proteins (Fig.3C and Supplementary Fig. S2C). Consistent with this, weobserved induction ofHSPs (HSP40, 70, and90a) in oHSV-infected and uninfected glioma and head and neck cancercells pretreated with bortezomib (Fig. 3D and Supplemen-tary Fig. S2D). Hsp90a induction was observed at both lowandhigh concentrations of bortezomib treatment of gliomaand head and neck cancer cells. HSP90 has been previouslyshown to be important for localization of HSV polymeraseto the nucleus (17), thus we hypothesized that bortezomib-inducedHSP90 induction could be critical for the increasedviral replication and cell killing following combinationtreatment. To test this hypothesis, we examined the impactof geldanamycin, an HSP90 inhibitor, on combinationtreatment-induced tumor cell cytotoxicity. Figure 4A showscombination index plots of Chou–Talalay of glioma cellstreated with bortezomib and 34.5ENVE in the presence orabsence of geldanamycin. Treatment of glioma cells withgeldanamycin reduced synergistic cell killing interactionsbetween 34.5ENVE and bortezomib (Fig. 4A). The syner-gistic interaction of the combination treatment was alsoreduced in cells treated with 17-AAG, a less toxic alternativeHSP90 inhibitor (Fig. 4B), supporting the significance ofHSP90 in oHSV and bortezomib synergistic cell killing.Consistent with the importance of HSP90, both 17-AAGand geldanamycin treatment resulted in a loss of theincreased viral replication achieved by bortezomib treat-ment (Fig. 4C). HSP90 is thought to increase HSV poly-merase localization to the nucleus. To directly test whetherbortezomib treatment affected nuclear localization of HSVpolymerase (UL30), we compared the cytosolic and nuclearfractions (C.F and N.F, respectively) of oHSV-infected cellstreated without/with bortezomib at the indicated concen-trations (Fig. 4D). Increased nuclear localization of UL30was observed in bortezomib-treated cells. Collectively,these results demonstrate that the induction of HSP90 bybortezomib is essential for the synergy with 34.5ENVEtreatment.

Combination treatment increased therapeutic efficacyin vivoWe next examined the therapeutic efficacy of bortezomib

in subcutaneous U251T3 glioma model (Fig. 5A). Mice

bearing the U251T3 glioma tumor xenograft displayedtumor growth inhibition of 91.7% in response to combi-natorial treatment. Moreover, at day 23 after treatment, sixout of eight tumors treatedwith bortezomib plus 34.5ENVEhad completely regressed.

Combination treatment also improved survival of micebearing intracranial GBM169 tumors (Fig. 5B). All micetreated with PBS, bortezomib alone, or 34.5ENVE alonedied with a median survival of 31 days (bortezomibalone: HR of survival ¼ 1.18, P ¼ 0.927; 34.5ENVE alone:HR of survival ¼ 0.89, P ¼ 0.797). Whereas, by day 100following treatment, 70% (median survival > 100 days;HR of survival ¼ 0.11, P < 0.001) of the animals in thebortezomib combined with 34.5ENVE group were stillviable (Fig. 5B).

Similar tumor growth suppression was observed in acombination with 34.5ENVE in the CAL27 head and neckxenograft model established in nude mice (Fig. 5C and D).Untreated tumors grew rapidly, leading to a tumor volumeof 1,407.36 mm3 by day 33 (95% confidence interval ¼1,142.36 to 1,672.36 mm3), whereas mean tumor volumesof bortezomib, 34.5ENVE, and combination treated micewere 920.43 mm3 (difference with PBS ¼ �486.93 mm3, P¼ 0.004), 217.23 (difference with PBS¼�1190.13mm3, P< 0.001), and 116.46 mm3 (difference with PBS ¼�1290.90 mm3, P < 0.001), respectively (Fig. 5C). Thesedata correlate to 34.6, 84.6, and 91.7% tumor growthinhibition, respectively, compared with control PBS-treatedmice. In addition, combination treatment resulted inincreased survival of mice (Fig. 5D). Together these resultshighlight the translational significance of combining bor-tezomib treatment with oHSV therapy for both glioma andhead and neck cancers.

Combination treatment increased viral replication andnecrotic cell death in vivo

The apparent enhanced antitumor efficacy and survivalbenefit resulting from combinatorial treatment was furtherinvestigated by immunohistological examination of CAL27subcutaneous tumor xenografts 3 days after virus treatment.As shown in Fig. 6, there was significantly increased HSVstaining in tumors treated with combination treatment ascompared with tumors treated with oHSV alone. A largeportion of tumor sections derived from tumors treated with

(Continued.) B, quantification of virus yield in the both sublethal and ED50 concentration of bortezomib-treated cells. Both cells and media wereharvested 48 hours (12 nmol/L) Asterisks indicate and 72 hours (2 nmol/L) after infection, and viral titers were determined by standard plaque assay. C, theindicated cell lines were treated with the indicated doses of bortezomib 16 hours before 34.5ENVE infection. Both cells and media were harvested 72 hoursafter viral infection and viral titers were determined by standard plaque assay. Data points represent the mean, and error bars indicate � SD for eachgroup. statistically significant differences between indicated pairs. D, Chou–Talalay analysis of combining bortezomib with 34.5ENVE in primary ovariancancer ascites derived tumor cells and in primary GBMpatient derived tumor cells maintained as undifferentiated neurospheres in vitro. Briefly, primary tumorcells were treated with bortezomib for 16 hours, followed by drug wash out and treatment with 34.5ENVE at serially diluted concentrations of 0.0625, 0.125,0.25, 0.5, 1, 2, and 4 times of their ED50 in a constant ratio. E, patient-derived neurospheres (GBM169) treatedwith/without bortezomib (2 nmol/L) were stainedwith CellTracker CMTPX and incubated with single cell suspension of GBM169 cells infected with 34.5ENVE (MOI ¼ 0.1) for one hour. Twenty-four hourspostinfection, bortezomib-treated neurospheres showed increased GFP-positive, infected cells relative to untreated cells (magnification, �100). F, theindicated primary patient-derived ovarian cancer ascites and GBM169 cells were treated with/without bortezomib (4 nmol/L for ascites and 2 nmol/L forGBM169) 16 hours before 34.5ENVE infection. Both cells andmedia were harvested 72 hours after viral infection and viral titers were determined by standardplaque assay. Data points represent the mean, and error bars indicate � SD for each group. Asterisks indicate statistically significant differencesbetween indicated pairs.






bortezomib and oHSV were necrotic, as evident with H&Estaining (Fig. 6).

DiscussionoHSV therapy has shown promise in preclinical models

and several clinical studies testing its efficacy in patients areongoing and interim analysis of a large-phase three trial for aGMCSF expressing oncolytic HSV has revealed significantimprovement in durable response rates (18). Proteasomeinhibition has recently emerged as a promising target incancer therapy, but its efficacy in conjunctionwith oHSVhasnot been previously investigated. Here, we report the firststudy to show that proteasome inhibition with bortezomibcan be combined with oHSV to effectively target varioussolid tumors. Such combinatorial strategiesholdgreat prom-ise as cancer treatments because they can enhance efficacywhile minimizing toxicity (19). We utilized Chou–Talalayanalysis and found that bortezomib interacted synergistical-ly with oHSV in vitro in killing various solid cancer cells,including ovarian, head and neck, MPNST, and glioma.Importantly, combination treatment resulted in a greaterthan 2- to 8-fold decrease in the dosage of either bortezomibor oHSV necessary for similar cell killing.

HSV-1 encoded ICP0 is an E3ubiquitin ligase that utilizescellular machinery to selectively degrade host proteins thatenhance the innate antiviral immune response (20, 21). Theproteasome has three distinct proteolytic activities: chymo-trypsin-like, trypsin-like, and caspase-like activities. Specif-ically, the chymotrypsin-like activity of the proteasome hasalso been shown to be important for virus entry (22). Inaddition, dysregulation of cellular ubiquitin-proteasomesystem has been shown to augment antiviral activity againstHSV-1 and HSV-2 viruses (23). These results suggest that itmay be counterintuitive to utilize bortezomib to increasethe efficacy of oHSV. However, although ICP0-mediatedmodification leads to degradation of cellular antiviral pro-teins (21), its own proteasomal degradation by SIAH-1, acellular ubiquitin ligase, has alsobeen shown to inhibit viralinfection (24). Clinically, increased incidence of herpeszoster, Varicella zoster virus, and hepatitis B virus reactiva-tionhas alsobeennoted inpatients treatedwithbortezomib(25–28). Interestingly an oncolytic adenovirus was shownto synergize with bortezomib by increasing cellular apo-ptosis in vitro and by its immunomodulatory effects in vivo(29). Bortezomib treatment has also been found to increaseapoptosis of EBV-positive transformed B cells suggestingthat it may be a novel strategy for the treatment of EBV-

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Figure 3. Effect of bortezomib on ER stress and UPR in infected and uninfected cancer cells. A, Dose-dependent induction of ER stress in U251T3 cells.Shown are immunoblot analyses of cell lysates from U251T3 glioma cells treated with bortezomib for 16 hours probed for expression of the indicatedproteins. B, ER stress is not increased in cells treated with both bortezomib and oHSV compared with bortezomib only treated cells. Shown are immuneblot analyses of the U251T3 cells treated with/without bortezomib (12 nmol/L) for 16 hours before 34.5ENVE infection at an MOI of 0.01 or 1. Cells wereharvested 2 and 6 hours postinfection, and cell lysates were probed with antibodies against ER stress-related pathway (calnexin, PERK, IRE1a, Bip/GRP78,CHOP, and Ero1-La) GAPDH was used as a loading control. C, dose-dependent induction of HSP40, 70, and 90a in U251T3 cancer cells. Shown areimmunoblot analyses of cell lysates from the indicated cells treated with bortezomib for 16 hours probed for expression of the indicated proteins. D,bortezomib pretreatment induced HSPs expression in uninfected and oHSV-infected U251T3 cells. U251T3 cells treated with/without bortezomib wereinfectedwith 34.5ENVE (MOI¼ 1) and cells were harvested 2 and 6 hours postinfection. Cell lysates were probedwith antibodies against HSP40, HSP70, andHSP90a. GAPDH was used as a loading control.

Yoo et al.





associated lymphomas (30). Although our data show noevidence of increased apoptosis, we have not examined thecontribution of immunomodulatory effects on virus prop-agation and antitumor immune responses generation invivo. Bortezomib-induced activation of UPR has also beenshown to induce cellular transcription factors that canactivate EBV viral promoters and promote a lytic switch.Direct activation of EBV lytic cycle by bortezomib has alsobeen reported in a variety of tumor cells lines. This effect isthought to be due to the induction of C/EBPb, a cellulartranscription factor that can then initiate the activation of

EBV lytic gene expression (31). Although bortezomib hasbeen shown to increase EBV replication, other studies havealso found that bortezomib inhibits VSV via activation ofNF-kB, resulting in an antiviral state that ultimately inhibitsVSV propagation (32). Consistent with these reports, treat-ment of myeloma cells with bortezomib was found toinhibit VSV replication and show less than additive cellkilling in vitro (33). Interestingly, the authors found thatdespite the antagonistic results in vitro bortezomib and VSVtreatment improved antitumor efficacy in vivo. These seem-ingly contradictory studies make the combination of

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bortezomib and oHSV both promising and intriguing.Here, our results demonstrate increased virus replicationand cancer cell killing when cells are pretreated with borte-zomib before oHSV infection. In infectedmacrophages anddendritic cells in vivo, proteasome-mediated degradation ofviral capsid proteins has also been shown to release viralgenomic DNA into the cytoplasm activating antiviral IFNresponses in macrophages post HSV-1 and CMV infectionsand can aid in viral clearance (7). Immune suppressionwithbortezomibmay account for the increased virus reactivationobserved in patients and in vivo tumor-bearing mice in ourexperiments. In the context of our study, we have notinvestigated the effect of bortezomib on antiviral and anti-tumor immune responses that are generated with oHSV

treatment. Future studies to investigate this interactionwould be of interest.

Resistance tobortezomib remains a clinical challenge andthe induction of the UPR has been correlated with thedevelopment of resistance to apoptosis initiated by protea-some inhibition (34). The UPR leads to the induction ofHSPs: a group of ubiquitously expressed chaperon proteinsthat ensure correct folding and prevent aggregation ofspecific target proteins. These chaperone proteins coordi-nate and rescue ER stress and are thought to contributetoward bortezomib resistance (34). Interestingly, HSP90 isalso utilized by HSV-1–encoded DNA polymerase for itsproper localization to the nucleus (35, 36). Our resultsshow that the induction of HSP90 as part of the UPR

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Figure 5. oHSV and bortezomib treatment enhance antitumor efficacy and survival in vivo. A, athymic nude mice were subcutaneously implanted withU251T3 glioma cells. When tumor size reached around 100mm3, PBS or bortezomib (0.8 mg/kg) were administered via intraperitoneal injection twice a weekfor the duration of the experiment. Seven days after initiation of drug treatment mice were injected intratumorally with 5 � 104 pfu of 34.5ENVE or PBS.Data points represent the mean and 95% confidence intervals of the tumor size in each group at the indicated time points (N ¼ 8/group). B, athymicnudemice with intracranial GBM169 cells were treated with/without bortezomib (0.8 mg/kg administered intraperitoneally twice a week for the duration of theexperiment) and were treated with intratumoral injection of HBSS or 1 � 105 pfu of 34.5ENVE on day 14. Data shown are Kaplan–Meier survival curves ofanimals in each group. (N ¼ 9/group for mice treated with PBS or bortezomib alone, and n ¼ 10/group for mice treated with bortezomib and oHSV). C,athymic nude mice were subcutaneously implanted with CAL27 head and neck cancer cells. When tumor size reached around 100 mm3, PBS or bortezomib(0.8 mg/kg) were administered via intraperitoneal injection twice a week for the duration of the experiment. Following one week of bortezomib treatment,animals were injected intratumorally with HBSS or 1 � 105 pfu of oHSV. Tumor volume was measured regularly after treatment. Data points representthemeanof the tumor size and95%confidence intervals for eachgroupat the indicated timepoints (n¼10/group). D,Kaplan–Meier survival curvesof the datain C. The percentage of surviving mice was determined by monitoring the death of mice over a period of 80 days after treatment.


Yoo et al.




activated by bortezomib is responsible for increased virusreplication, and HSP90 inhibitors ablated the synergisticcell killing and increased viral replication induced by bor-tezomib. Together these results indicate that the combina-tion of oHSV therapy with bortezomib is an attractivestrategy to enhance therapy or deal with the developmentof resistance to bortezomib treatment. Here, we show thatthe UPR induced by bortezomib increases viral replicationin vitro. It is interesting to speculate whether differentproteasome inhibitors with different affinities for distinctproteolytic activities of the proteasome and/or their differ-ent half life of each inhibitor may account for the increasedoHSV replication observed in bortezomib-treated cells butinhibition with wild-type virus observed with other protea-some inhibitors. In this study, the lack of bortezomib aloneto show therapeutic efficacy in mice bearing intracranialglioma is consistent with the lack of response observed inpatients with glioma after bortezomib treatment (37).However, the enhanced survival with combination treat-ment is consistent with our results which show that evensublethal doses of bortezomib can also increase viral rep-lication and improve cell killing in vitro. Although it isinteresting to speculate that tumors which can induce UPRin response to bortezomib would be most sensitive tocombination of bortezomib and oHSV, future studies willidentify biomarkers to predict tumor types that will bemostsensitive to this combination treatment.Recently, bortezomib was found to synergize with onco-

lytic reovirus therapy in the treatment of multiple myelomaby induction of ER stress and NOXA-dependent cellularapoptosis (15). Although reovirus induces ER stress ininfected cells, HSV-1 is known to hijack cellular pathwaysto override ER stress signaling andmaintain ERhomeostasis(38). Here, we show that combination of bortezomib andoHSV did not exacerbate ER stress in cells compared with

cells treated with only bortezomib. To our knowledge, thisis the first report showing synergy between oHSV andbortezomib. This study offers a novel therapeutic treatmentstrategy for cancer therapy that can be rapidly translated inpatients with multiple solid tumors.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

DisclaimerAny opinions, findings, and conclusions expressed in this material are

those of the authors and do not necessarily reflect those of the PelotoniaFellowship Program.

Authors' ContributionsConception and design: J.Y. Yoo, B.S. Hurwitz, C. Bolyard, T.P. Cripe,D.S. Parris, M. Old, B. KaurDevelopment ofmethodology: J.Y. Yoo, B.S. Hurwitz, C. Bolyard, J.-G. Yu,S. He, J. Yu, M. Old, B. KaurAcquisitionofdata (provided animals, acquired andmanagedpatients,provided facilities, etc.): J.Y. Yoo, B.S. Hurwitz, C. Bolyard, J.-G. Yu,K. Selvendiran, K.S. Rath, S. He, Z. Bailey, D. Eaves, T.P. Cripe, M. OldAnalysis and interpretation of data (e.g., statistical analysis, biosta-tistics, computational analysis): J.Y. Yoo, B.S. Hurwitz, C. Bolyard, J.-G.Yu, J. Zhang, Z. Bailey, D. Eaves, T.P. Cripe, D.S. Parris, M.A. Caligiuri,M. Old, B. KaurWriting, review, and/or revision of the manuscript: J.Y. Yoo, B.S. Hur-witz, T.P. Cripe, M.A. Caligiuri, M. Old, B. KaurAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): B.S. Hurwitz, D. Eaves, D.S. ParrisStudy supervision: M.A. Caligiuri, M. Old, B. Kaur

Grant SupportThis work was supported by the NIH grant (R01NS064607,

R01CA150153, P01 CA163205, and P30NS045758) to B. Kaur and(R01CA155521) to J.Y. Yoo, the Comprehensive Cancer Center Viral Oncol-ogy Program Award to Matthew Old and by the Pelotonia FellowshipProgram to B.S. Hurwitz.

The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 5, 2014; revised April 16, 2014; accepted May 3, 2014;published OnlineFirst May 9, 2014.

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Yoo et al.




2014;20:3787-3798. Published OnlineFirst May 9, 2014.Clin Cancer Res Ji Young Yoo, Brian S. Hurwitz, Chelsea Bolyard, et al. EffectsOncolytic HSV-1 Replication Resulting in Synergistic Antitumor Bortezomib-Induced Unfolded Protein Response Increases

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