reovirus as aviable therapeutic optionfor the treatment of...

Cancer Therapy: Preclinical

Reovirus as a Viable Therapeutic Option for the Treatment ofMultiple Myeloma

Chandini M. Thirukkumaran1,2, Zhong Qiao Shi2, Joanne Luider3, Karen Kopciuk1,2, He Gao4, Nizar Bahlis1,2,Paola Neri1,2, Mark Pho4, Douglas Stewart1,2, Adnan Mansoor3, and Don G. Morris1,2

AbstractPurpose: Despite the recent advances made in the treatment of multiple myeloma, the disease still

remains incurable. The oncolytic potential of reovirus has previously been shown and is currently in phase

III clinical trials for solid tumors. We tested the hypothesis that reovirus can successfully target human

multiple myeloma in vitro, ex vivo, and in vivowithout affecting human hematopoietic stem cell (HHSC) re-

population/differentiation in a murine model that partially recapitulates human multiple myeloma.

Experimental Design:Humanmyeloma cell lines and ex vivo tumor specimens were exposed to reovirus

and oncolysis andmechanisms of cell deathwere assessed. RPMI 8226GFPþ cells were injected intravenously

to non-obese diabetic/severe combined immune deficient (NOD/SCID)mice and treated with live reovirus

(LV) or dead virus (DV). Multiple myeloma disease progression was evaluated via whole-body fluorescence

and bone marrow infiltration. HHSCs exposed to LV/DV were injected to NOD/SCID mice and re-

population/differentiation was monitored.

Results: A total of six of seven myeloma cell lines and five of seven patient tumor specimens exposed to

reovirus showed significant in vitro sensitivity. Tumor response ofmultiplemyeloma by LV, but not DV, was

confirmed by comparison of total tumor weights (P ¼ 0.05), and bone marrow infiltration (1/6, LV; 5/6,

DV).Mice injectedwith LV- orDV-exposedHHSCsmaintained in vivo re-population/lineage differentiation

showing a lack of viral effect on the stem cell compartment. Reovirus oncolysis was mediated primarily by

activation of the apoptotic pathways.

Conclusions: The unique ability of reovirus to selectively kill multiple myeloma while sparing HHSCs

places it as a promising systemic multiple myeloma therapeutic for clinical testing. Clin Cancer Res; 18(18);

4962–72. �2012 AACR.

IntroductionMultiple myeloma is a clonal neoplasm of plasma cells

that accounts for approximately 10% of all hematologicalmalignancies (1). The incidence of multiple myeloma hasdoubled in the last 5 decades (2), with a current actuarialaverage of 17 years life lost per diagnosis (3). Despite recentadvances in the understanding of multiple myeloma biol-ogy and the availability of newer therapeutics such asthalidomide, revlimid, and bortezomib, which haveimproved responses and survival (4), the disease remains

incurable and treatment resistance/toxicities associatedwith these agents ultimately result in patient demise (5,6). Thus, it is imperative to findnovel,more effective, better-tolerated therapeutics for multiple myeloma and of equalimportance is the elucidation of these novel therapeuticsmechanisms of action.

Oncolytic viruses are a group of therapeutics that exhibitan extensive spectrum of anticancer activity with minimalhuman toxicity. One such virus, the reovirus, is an ubiqui-tous, non-enveloped double-stranded RNA virus with neg-ligible pathogenicity in humans (7). The reovirus’s exten-sive preclinical efficacy has been documented by our groupand others and it is presently undergoing phase III clinicaltrial testing for head andneck tumors. The viral sensitivity ofa wide array of tumor histologies is possibly mediated viamultiple permissive oncogenic signaling pathways thatallow the reovirus to target malignancies while sparingnormal cells (8–10). Thus, the "oncogenic addiction" ofmultiple myeloma associated with its survival and prolif-eration creates an environment conducive to reovirustherapy.

Recently, it has been shown that mutated ras and con-stitutively active Akt delineate distinctive oncogenic

Authors' Affiliations: 1Department of Oncology, University of Calgary;2Tom Baker Cancer Center; 3Calgary Laboratory Services, Calgary; and4Department of Public Health Sciences, School of Public Health, Universityof Alberta, Alberta, Canada

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

Corresponding Author: Dr. Don G. Morris, Department of Oncology, TomBaker Cancer Centre, 1331, 29 Street N.W. Calgary, Alberta, T2N 4N2,Canada. Phone: 403-521-3347; Fax: 403-283-1651; E-mail:[email protected]

doi: 10.1158/1078-0432.CCR-11-3085

�2012 American Association for Cancer Research.

ClinicalCancer

Research

Clin Cancer Res; 18(18) September 15, 20124962

on May 8, 2018. © 2012 American Association for Cancer Research. clincancerres.aacrjournals.org Downloaded from

Published OnlineFirst July 3, 2012; DOI: 10.1158/1078-0432.CCR-11-3085

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pathways in this malignancy, which independently contri-butes to multiple myeloma survival (11). Activating muta-tions in the ras gene family are common in multiple mye-loma, with N- and K-rasmutations being the most frequent(12, 13). In addition, multiple other cancer-promotingsignaling pathways have been shown to be active in mul-tiple myeloma such as the MEK/ERK, phosphoinositide3-kinase (PI3K)/Akt, mTOR/p70S6-kinase, and NF-kBpathways (14–16) that may be permissive to reovirus lyticinfection, thereby increasing the spectrum of potentiallytreatable patients. In susceptible tumor cells, reovirus isthought to exploit these activated neoplastic signaling path-ways in a p53-independent/-dependent manner (17, 18)and involves NF-kB activation/trafficking (17, 19), auto-crine release of the TNF-related apoptosis-inducing ligand(TRAIL; refs. 20, 21), and activation of endoplasmic retic-ular (ER) stress (22).To date, only 2 studies have examined the preclinical

effects of reovirus on multiple myeloma. Our group haspreviously shown the ability of the reovirus to purge mul-tiple myeloma successfully during autologous transplanta-tions (23) and Kelly and colleagues (22) have investigatedthe potential of using reovirus in combination therapy withbortezomib on subcutaneous multiple myeloma tumorestablished in a murine model as well as in a 5TGM1syngeneic murine system. Although there is a lack of con-sensus about the best in vivo preclinical model of multiplemyeloma, that is, immunocompetent syngeneic or humanxenograft murine model or fetal bone marrow (BM)implant-multiplemyeloma xenograft (SCID-hu), a relative-ly straightforward intravenous/intracardiac approach to thedelivery of multiple myeloma should be considered. Sub-cutaneous models fail to consistently home to the bonemarrow (22), and murine multiple myeloma cell linesinvolved in syngeneic models fail to authentically replicatehuman genetic lesions/aberrations found in human mul-tiplemyeloma. The SCID-humodel, although itmimics thehuman BM environment, does not produce disseminateddisease as seen in advanced human multiple myeloma.To represent clinical relevance, it is imperative to study

the effects of reovirus given systemically onhumanmultiplemyeloma in vivo in the BMmicroenvironment. In this study,we showed the oncolytic potential of the reovirus against a

large cohort of humanmultiplemyeloma cell lines (HMCL)and ex vivo patient specimens and provide evidence thatintravenous treatment of reovirus is effective in targetingdisseminated human multiple myeloma in vivo and thatreovirus exposure does not abrogate human hematopoieticstem cell re-population/differentiation in the BM compart-ment. This is the first report to show the efficacy of reovirustargeting of humanmultiplemyeloma in the BMmilieu andhuman stem cell re-population post-reovirus treatment inan in vivo setting, and thus support the rapid translation ofpreclincial data to a future phase I clinical trial. In additionto confirming that reovirus orchestrates cells death viaapoptosis, we showed for the first time that reovirus infec-tion of multiple myeloma leads to the modulation ofautophagy which is of further clinical relevance as under-standing mechanisms of reovirus oncolysis of multiplemyeloma will lead to optimal use of this therapeutic,especially for the treatment of therapy-resistant disease.

Materials and MethodsCell lines

RPMI 8226, U266,MM1S, andH929were obtained fromthe American Type Culture Collection (ATCC, Virginia).OPM2 was obtained from the German collection of micro-organisms and cell cultures (Braunschweig, Germany).KMS-11, INA-6, and RPMI 8226 GFPþ cells were kindlyprovided by Drs. Irene Ghobrial (Dana Farber CancerInstitute, Boston, MA), Renate Burger (University of Erlan-gen-Nuernberg, Erlangen, Germany), and Linda Pilarsky(University of Alberta, Canada), respectively. RPMI 8226,U266, MM1S, H929, KMS11, and OPM2 were maintainedin RPMI1640medium (Gibco BRL) containing 10%FBS. Inaddition, the medium was supplemented with either G148(100 mg/mL), interleukin 6 (IL-6; 2.5 ng/mL), or 0.05mmol/L 2-mercaptoethanol for RPMI 8226 GFPþ, INA-6,and H929 cells, respectively. Multiple myeloma primarytumor cells were obtained from BM aspirate/biopsies. Diag-nosiswasbasedonhistopathology, immunohistochemistry,and immunophenotype. All procedures involving patientswere approved by the Conjoint Health Research EthicsBoard, University of Calgary, Calgary, Alberta, Canada.

ReovirusReovirus serotype 3 (strain Dearing) was propagated in

L929 cells and purified as previously described (10).Human multiple myeloma cells lines (HMCL) grown tosubconfluence were infected with either no virus (NV), livereovirus (LV), or UV-inactivated reovirus (dead reovirus,DV) at a multiplicity of infection (MOI) of 40 plaque-forming units (PFU)/cell for 24, 48, and 72 hours. Reoviruscytotoxicity was monitored using the WST-1 (4-[3-(4-Iodo-phenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate) assay (24). Similarly, patient BM samples thatwere found to contain more than 3% multiple myelomacells were washed in PBS and cultured in RPMI1640 medi-um containing 20% FBS and antibiotics and exposed to 40or 100 MOI of LV or DV for 24 hours. The higher reovirusMOI was used as themarrow samples were not enriched for

Translational RelevanceThis study examines the potential use of the oncolytic

virus, the reovirus, as a therapeutic agent for multiplemyeloma, a disease for which there is no cure at presentdespite several new therapeutics being brought to theclinic. This work utilizes human multiple myeloma celllines, ex vivopatient tumors, and in vivo animalmodels toensure that the observations are as clinically relevant to atranslational clinical setting. It lays the foundation tosupport an early-phase clinical trial for multiple myelo-ma with reovirus.

Reovirus as a Therapeutic for Multiple Myeloma

www.aacrjournals.org Clin Cancer Res; 18(18) September 15, 2012 4963




the multiple myeloma cells, thus diluting the effectiveconcentration of reovirus.

Flow cytotmetric analysis of apoptosis and autophagyHMCLs were infected with either 40 MOI of DV, LV, or

NV for 24, 48, and 72 hours and harvested. Followingcentrifugation, the cell pellets were washed in PBS twicebefore subsequent staining with propidium iodide (PI)/RNase A or Annexin-V-Alexa Fluor 488 (A13201; Invitro-gen)/7AAD (A07704; Beckman Coulter) as previouslydescribed (10). Flow cytometric analysis was carried out toassess DNA fragmentation and phosphatidyl serine expres-sion using a Navios flow cytometer (Beckman Coulter) andFCS Express software (De Novo Software).

For caspase inhibition, cells were pretreated with Z-VAD-FMK-001 (R&D systems), 50 mmol/L for 1 hourbefore infection with reovirus. Harvested cells werestained for rabbit anti-active caspase 3 V450 (BectonDickinson Biosciences) using Beckman Coulter’s Intra-

Prep intracellular staining kit according to the manufac-turer’s instructions. Autophagy was detected using theCyto-ID Autophagy detection kit (ENZ-51031-k200;Enzo Life Sciences) in NV-, DV-, or LV-treated RPMI8226 cells at 0, 24, and 48 hours. An autophagy inhibitor,3-MA (SC-205596; Santa Cruz), was added to cells at10 mmol/L for 1 hour before infection with reovirus. Toidentify vesicles colocalizing with LC3-II, a marker ofautophagosomes, 1 � 106 cells were centrifuged andresuspended in 1 mL of Assay Buffer. Then, 0.5 mL ofa 1:2,000 dilution of the Cyto-ID Green DetectionReagent was added to the cell suspension, and the mixturewas incubated at 37�C for 30 minutes in the dark andanalyzed immediately in the LFL1 detector.

Apheresis product and reovirus effects on humanCD34þ stem cells

All apheresis products (AP) used in the present studywereobtained from patients registered at the Tom Baker Cancer

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Figure1. A,HMCLcellswere cultured in thepresenceof live (LV) or dead (DV) reovirus (40MOI) andvialbility assesedvia theWSTassayat 48and72hours. Datarepresent themean� 95%CI of 3 independent experiments. B, effect of reovirus onmultiple myeloma (MM) patient specimens. Patient MM tumor from bonemarrow aspirates was cultured in the presence of LV or DV. After 24 hours of virus exposure, cell viability (CD 138þ56þ) was assessed using flow cytometry.C, representative flow cytometric scatterplots of a reovirus sensitive, moderately sensitive, and resistant patients.

Thirukkumaran et al.

Clin Cancer Res; 18(18) September 15, 2012 Clinical Cancer Research4964




Centre, Calgary, Canada, after informed consent. AP cellswere washed in PBS and treated with ammonium chlo-ride for 10 minutes to induce lysis of red blood cells.Mononuclear cells resuspended in Robosep buffer (cat #20104; Stem Cell Technologies) were depleted of line-age-committed cells using the Robosep automatedimmunomagnetic cell-separation system (cat # 20000;Stem Cell Technologies). The RoboSep progenitorenrichment antibody cocktail (Catalog # 18056; StemCell Technologies) was used to enrich for CD34þ cells.The isolated cells were seeded at a density of 2 to 3 � 103

/mL in StemSpan SFEM (Stem Cell Technologies) con-taining cytokines and growth factors (23) and then

incubated in the presence of LV or DV (40 MOI) for72 hours at 37�C in a humidified incubator with 5% CO2

. Harvested cells were assayed for viability and purity(CD34þ and CD45þ cells) using flow cytometry aspreviously described (23). We have previously shownno significant difference between DV and NV treatments(23, 25) in different tumors as well as CD34þ stem cellsand AP. Therefore, DV was selected as the most stringentcontrol for all reovirus experiments.

PCR for detection of human "Alu" sequencesTo confirm "human cell" re-population in vivo, DNA was

isolated from mouse blood and BM using QIAamp

Figure 2. HMCLs were exposedto 40 MOI of LV or DV for 24, 48, and72hours andapoptosiswas assayed.A, DNA fragmentation assessed by PIinorporation using flow cytometry. B,AnnexinV and 7AAD expression inDV- and LV-treated HMCLs. C,Annexin V expression by flowcytometry for the multiple myeloma(MM) cell lline U266.

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DNA mini kits (Qiagen) and PCR was carried out againsthuman-specific "Alu" primers (AAGTCGCGGCCGC-TTGCAGTGAGCCGAGAT; ref. 26). The PCR product wasseparated in 2% agarose gels containing ethidium bromideand "human Alu" bands were visualized.

AnimalsSix- to 8-week-old female severe combined immune

deficient/non-obese diabetic (SCID/NOD) mice were pur-chased from Jackson Laboratories, Bar Harbor, Maine. Theanimals were housed in a biocontainment animal facilityand provided food andwater ad libitum. All procedures werereviewed and approved by the University of Calgary AnimalCare Committee.

Evaluation of reovirus efficacy in human multiplemyeloma under in vivo settings

Twelve SCID/NOD mice were sublethally irradiated(1.25 Gy total body g-irradiation from a 137Cs source)and administered an intraperitoneal injection of 50 mL ofanti-asialo GM1 antibody for elimination of naturalkiller (NK) cells. Mice were injected with 2.5 � 106

RPMI 8226-GFPþ cells via tail vein for tumor establish-ment. On day 7, mice in 2 groups were given a singleinjection of either LV or DV (1 � 107 PFU) intravenous-ly. Animals were assessed for weight loss, groomingperformance, and signs of visible tumor 3 times perweek. On day 35, when animals exhibited greater than20% weight loss or required euthanasia, all animals were

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Figure 3. A and B, caspase inhibition leads to inhibition of DNA fragmentation caused by reovirus. RPMI 8226 cells were pretreated with Z-VAD-FMK-001,50 mmol/L for 1hour before infectionwith 40MOI of DV, LV, or No virus (NV) for 0 and 24 hours. Harvested cells were assayed for DNA fragmentation using flowcytometry. Flow cytometric scatterplots (A), % DNA fragmentation, mean (n ¼ 3) � SE (B).






sacrificed and assessed for multiple myeloma diseaseprevalence.Sacrificed animals were subjected to whole-body fluores-

cence imaging for detection of GFPþ multiple myelomalesions in the skeleton and subcutaneous tissues. Liver,spleen, lung, brain, heart, small, and large intestines wereanalyzed for metastatic deposits by GFP fluorescence.Femurs from animals were flushed in PBS þ 10% FBS andthe collected BM cells were analyzed for GFP þ/CD38þ/138þ staining.

Human stem cell re-populating ability in vivoSixteen NOD/SCID mice were sublethally irradiated and

treated with anti-asialo GM1 antibody as described above.Animalswere divided into 3 groups, and thefirst 2 groups (n¼ 6)were administered 200 mL of LV- orDV-treatedCD34þstem cells (1–5� 106, isolated from patient AP)mixed with2� 105 of AP cells that have been impaired for proliferationthrough irradiation (2 Gy; carrier cells) via the tail vein. The3rd group (n¼ 4) was given only carrier cells (2� 105). Toprevent reovirus-related morbidity and inflammatory vas-culitis in the extremities seen in immunocompromisedanimals beyond day 30 (never seen in nude mice or immu-nocompetent mice), all animals were treated with purifiedpolyclonal rabbit antireovirus serum (100 mg, tail vein) ondays 14, 21, and 28. Bodyweights and grooming abilities ofanimals were recorded. On days 25 and 40, 3 animals ineach group were subjected to cardiac puncture, and periph-

eral blood was collected in tubes containing heparin (Sig-ma-Aldrich). Femurs and tibia were removed from thesacrificed mice and BM cells were flushed out into salineand combined for each mouse. Using a CD45/LSS gatingstrategy and a CD19 gate, harvested BM and blood wereimmunophenotyped by flow cytometry with the following2- and 5-color antibody combinations: CD45/CD34, CD8/CD4/CD45/CD16þ56/CD3, Kappa/Lambda/CD19/CD5/CD20, and CD64/CD13/CD45/CD33/CD16 (BeckmanCoulter).

Statistical AnalysisAll analyses were carried out using the statistical software

program R (Boston, MA; ref. 27). For the WST assay experi-ments, 3 independent experiments were carried out witheach cell line in triplicate. The resulting colorimetric read-ings were averaged and background readings were sub-tracted to provide the corresponding number of viable cells.The data is presented as themeanwith their 95%confidenceintervals (CI).

To assess reovirus treatment on multiple myelomatumor, harvested tumor from DV- and LV-treated animalswas subjected to an exact Wilcoxon Mann–Whitney RankSum test, a nonparametric, robust alternative to the 2sample t-test, appropriate when data may be non-nor-mally distributed. To explore the significance of reovirus-induced DNA fragmentation and annexin V expression,we calculated 2-factor Analysis of Variance (ANOVA)

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Figure 3. (Continued ) C, reovirus infection modulates autophagy in human meyloma. RPMI 8226 cells were infected with 40 MOI of DV or LV for 0, 24, and 48hours. Harvested cells were stained with Cyto-ID and assayed for LC3-II colocalization to the autophagosomes by using flow cytometry. D, 3-MAtreatment suppresses reovirus-induced autophagy in human meyloma. RPMI 8226 cells were pretreated with 10 mmol/L of autophagy inhibitor 3-MA for 1hour before infection with 40 MOI of DV, LV, or No virus (NV) for 0, 24, and 48 hours. Harvested cells were stained with Cyto-ID and assayed for LC3-IIexpression using flow cytometry. Bars represent percentage changes in median channel fluorescence relative to the NV controls, mean (n ¼ 3) � SE.






models within each of the 3 cell lines. Levene’s test forvariance homogeneity based on the median values wasused to check this assumption.

To confirm caspase and autophagy activation duringreovirus infection ofmultiplemyeloma,DV- and LV-treatedmultiple myeloma cells with and without caspase andautophagy inhibitors were also compared using their meanand 95% CIs, with planned t-test comparisons for specificquestions of interest.

ResultsReovirus sensitivity of HMCLs and ex vivo specimens

All HMCLs displayed significant sensitivity toward reo-virus, showing less than 12% viability at 72 hours, exceptfor OPM2 (Fig. 1A). Reovirus sensitivity could not becorrelated with immunoglobulin H (IgH) translocationsor ras mutational status of HMCLs (Supplementary TableS1). Interestingly, theonly reovirus-resistantHMCL,OPM2,harbored a PTEN mutation.

When reovirus sensitivity on ex vivo patient-derivedtumor was investigated, 5 out of 7 patient multiple myelo-ma cells exhibited reovirus sensitivity, highlighting itspotential to be used as a systemic therapeutic (Fig. 1B). Itwas difficult to grow the ex vivo tumor in RPMI mediumbeyond 48 hours and, therefore, only data for 24 hours arepresented. Supplementary Table S2 presents data indicatingthat reovirus sensitivity does not correlate with the prog-nostic characteristics of the 7 patient tumors [individual t(4:14), 13q(-) and TP53]. Flow cytometric scatterplotsrepresentative of sensitive, moderately sensitive, and resis-tant patients are shown in Fig. 1C.

Reovirus oncolysis is mediated through apoptosisTo investigate the mechanism of reovirus-mediated

cell death, RPMI8226, NCI-H929, U266, and OPM2 cellswere treated with DV or LV and assessed for apoptosis.All 3 reovirus-sensitive HMCLs exposed to LV showedsignificantly elevated features of apoptosis comparedwith DV-treated cells (i.e., membrane phosphatidyl ser-ine flipping and DNA fragmentation). Significant tem-poral changes on DNA fragmentation with LV treatmentwere seen in all 3 HMCLs (F-tests for interaction;P-values < 0.01; Fig. 2A) and in annexin V binding inU266 cells (P < 0.006; Fig. 2B). Significant differenceswere seen between LV and DV at all time points inRPMI8226 and NCI-H929 cells (F-tests for treated cells;P < 0.0001); these collective results confirm the presenceof reovirus oncolysis.

Figure 2C represents flow cytometric analysis of U266apoptosis with nearly 90% of the LV-treated cells showingannexin V and 7-AAD positivity by 72 hours. In significantcontrast to these HMCLs, no evidence of any apoptoticactivity was detected in reovirus-resistant OPM2 whentreated with either LV or DV (data not shown). To confirmthat reovirus-induced apoptosis is mediated via terminalcaspase 3,multiplemyeloma cells were treatedwith caspaseinhibitor Z-VAD-FMK-001 and DNA fragmentation wasassessed. As depicted in Figs. 3A and B, treatment with Z-

VAD-FMK-001 led to a significant downregulation of frag-mentedDNA in the sub-G0/G1 peak in LV-treated cells at 24hours (t-test P ¼ 0.0012).

Reovirus modulates autophagy induction in multiplemyeloma

Although apoptosis induction was prominent in reovirus-infected HMCLs, complete reovirus oncolysis could not beattributable to this process. We, therefore, examined autop-hagy involvement post-reovirus infection of myeloma.Autophagy activity was similar in DV- and LV-treated RPMI8226 cells at 0 hours (Fig. 3C). Autophagy induction wasevident at 24 hours in LV-treated cells and, at 48 hours, thiswas more pronounced with a distinctive shift in medianchannel fluorescence of stained cells with Cyto-ID. To con-firm the autophagy process, we treated RPMI 8226 cells withautophagy inhibitor 3-MA and verified autophagy activitypost virus infection.Distinct temporal increases inautophagyinduction were evident in LV-treated cells in comparison toDV-treatedcells (Fig. 3D).Treatmentwith3-MAconsiderablydownregulated this increase in autophagy caused by LV,resulting in a 4-fold reduction by 48 hours, thus indicatingthat autophagy might also contribute to cell death.

Intravenous reovirus treatment successfully targetsdisseminated human multiple myeloma

Toexplorewhether intravenous reovirus treatmentwouldtarget disseminated human multiple myeloma in a mousemodel, animals in 2 groups with established disease weretreatedwith a single intravenous injectionofDVor LV. All ofthe 6 mice injected with DV showed symptoms of malig-nancy (weight loss, neurological deficits, etc.) beyond days20 and, therefore, all animals were sacrificed at day 35.MultipleGFPþmultiplemyeloma lesionswere seen in5outof 6 DV-treated mice (Fig. 4A), with the majority of lesionsseen in the axial skeleton, skull, and plasmacytoma-likelesions. In contrast, 4 of the 6mice treatedwith LV appearedto be disease free with no visible GFPþ lesions detected.Comparison of total tumorweights between the 2 groups ofmice showed significant differences (P¼ 0.05, exact Mann–Whitney Rank Sum test; Fig. 4B). Mouse BM analyzed forGFPþ/CD38þ/CD138þ multiple myeloma cells showedtumor in 5 of 6 DV-treated mice and only in 1 LV-treatedmouse (Table 1). Figure 4C presents the flow cytometricscatterplots of 2 mice from each group that showed thehighest amount of tumor burden in BM. The tumor burdenseen inDV-treatedmousewas >10-fold than that of the onlyLV-treated mouse detected with human multiple myelomainBM.Thesemicewere treatedonlywitha single injectionofreovirus and, if multiple LV injections were given, completeeradication of disease would be anticipated.

Reovirus exposure of human CD34þ hematopoieticstem cells does not abrogate re-population/differentiation in mice

Mice of both groups (DV- or LV-treated) showedhuman stem cell re-population at 25 to 30 days postinfusion (Fig. 5A). In addition, DV- or LV-treated stem cells






showed lineage differentiation in mouse BM. As lymphoiddifferentiation preceedsmyeloid differentiation, themajor-ity of differentiated cells were noted to be of the myeloidlineage as expected. In significant contrast to LV- or DV-treated stem cells-infused mice, no human stem cells wereseen in mouse BM in recipients that received carrier cellsalone (Figs. 5A and 5B). To further confirm the presence ofhuman cells, DNA was extracted from mouse BM andblood. PCR conducted against human "Alu" sequencesconfirmed human cell re-population only in animals thatreceived DV- or LV-treated stem cells. (Fig. 5C).

DiscussionThe present study evaluated reovirus as a viable option

for systemic therapy of human multiple myeloma. Thebroad-spectrum activity of reovirus on HMCLs was con-firmed under in vitro conditions where 6 of the 7 HMCLsshowed sensitivity to reovirus. Four of these harbored rasmutations, but this alone was not predictive of virusoncolysis, as U266 and KMS11 were ras wild type. Inter-estingly, only OPM2 harbored a PTEN mutation and wasreovirus resistant. Testing of 3 reovirus-sensitive HMCLsconfirmed that cell death is manifested through apoptosis

Gate 1

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Figure 4. Systemic reovirus treatment targets disseminated humanmyeloma in vivo.RPMI 8226-GFP cells were injected into sublethally irradiated NOD/SCIDmice and treatedwith a single intravenous injection of DV of LV (1� 107 PFU). A, sacrificedmiceweremonitored for whole-body fluorescene of GFPþmultiplemyeloma (MM) lesions. B, tumor collected from animals were analyzed for statistical differences, mean (n¼ 6)�SE. C, bonemarrow collected frommice wasanlyzed for GFPþ MM cells by flow cytometry.






(annexin V expression and DNA fragmentation) andcorroborate our previous findings with breast and pros-tate cancer (10, 17). Inhibition of caspase 3 with Z-VAD-FMK-001 significantly reduced DNA fragmentationinduced by reovirus confirming the active role of caspase3 in reovirus-induced apoptosis.

The novel finding that reovirus infection of multiplemyeloma leads to significant induction of autophagy, a

catabolic process involved in routine turnover of proteinsand intracellular organelles is of interest (28). Reovirusinfection of RPMI8226 cells lead to a time-dependentincrease in autophagy and this was suppressed by 3-MA,a specific inhibitor of autophagy. Although autophagycould serve a dual purpose, either to protect against canceror contribute to cancer by promoting the survival of nutri-ent-starved cells (29),many studies have indicated its role as

Table 1. Reovirus treatment abrogates human multiple myeloma dissemination in mouse bone marrow orin vivo

Presence of RPMI 8226 myelomacells

Presence of RPMI 8226 myelomacells

LV-treated mice bone marrow other lesions DV-treated mice bone marrow other lesions

M1 � � M7 þ �M2 � � M8 þ þM3 � þ M9 þ þM4 þ � M10 � þM5 � � M11 þ þM6 � þ M12 þ þ

A

Mouse A

(LV- treated )

Mouse B

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Figure 5. Reovirus treatment does not abrogate hematopoitic engraftment in mice. Human CD34þ stem cells were isolated from AP and treated with LV or DVup to 72 hours. 1 to 5 � 106 CD34þcells were mixed with carrier cells and injected into sublethally irradiated SCID/NOD mice. A, mouse bone marrow washarvested and analyzed for human hematopoitic stem cells enumeration using flow cytometry. B, evidence of differentiation by flow cytometricimmunophenotyping carried out on leukocyte populations from representative animals treated with carrier cells, DV or LV treatment. C, DNA was extractedfrombloodandBMof animals that receivedhumanCD34þstemcells treatedwithDVor LVor frommice that receivedonly carrier cells. PCRwascarriedout forhuman "Alu" sequences.






a pro-death pathway post treatment (30–35). Several linesof evidence indicate that ER stress leads to autophagy (36–38), and recent studies have highlighted a prominent rolebetween autophagic cell death induced by Akt–mTOR sig-naling triggered by ER stress (37, 38). Recently, Kelly andcolleagues (22) showed that reovirus infection of multiplemyeloma leads to ER–stress-induced apoptosis. Therefore,it is plausible to hypothesize that viral replication withinmultiple myeloma, in addition to induction of apoptosis,also promotes auotphagic cell death initiated via a signalingmechanism of the ER stress pathway.Analysis of reovirus-treated multiple myeloma ex vivo

tumors revealed that reovirus sensitivity is not restricted tomultiple myeloma cancer cell lines in vitro. Further, 5 of the7 samples showed sensitivity to reovirus, highlighting theapplicability of this virus as a possible systemic therapeuticstrategy. A more robust assessment of reovirus efficacy is toexamine its ability to target disseminatedmultiplemyelomaunder in vivo conditions. All DV-treated mice showed signsof disease and required euthanasia due to tumor burden;however, 5 of the 6 mice treated with LVmaintained healthfor the duration of the experiment (35 days). Mice treatedwith DV showed evidence of GFPþ lesions in bone includ-ing the appendicular and axial skeletons, mimicking theosteotropic nature of multiple myeloma in patients. Miceshowing hind-leg paralysis was likely due to vertebral bonelesions and spinal cord involvement,whichwas the primaryreason for euthanization. In addition to the multiple mye-loma tumor detected in the bodies of animals, multiplemyeloma tumor homing to the bone marrow milieu wasconfirmedwith humanmultiple myeloma detection in 5 ofthe 6 mice subjected to DV treatment. In comparison, asingle injection of 1� 107 PFU of reovirus was able to targetbonemarrow infiltration of multiple myeloma in 5 of the 6mice, highlighting its potential as a systemic agent. Wehypothesize that multiple injections of reovirus wouldcompletely irradicate disease, but our NOD/SCID/irradiat-ed/NK cell-depleted model does not support multiple reo-virus injections due to development of vasculitis beyondday 25.Another important objective of this study was to prove

that reovirus treatment does not abrogate human stem cellre-population and differentiation in vivo. Previously, wehave shownnegligible adverse effects of reovirus onpurifiedCD34þ human stem cells or AP under in vitro conditions(23, 25). Human stem cells isolated from AP, treated withLV for 3 days, and transplanted into NOD/SCID miceshowed similar re-population and lineage differentiationin comparison to the DV controls. In contrast, control micethat received only carrier cells didnot showhuman stemcellengraftment. Flow cytometric analysis of mouse bone mar-row showed distinct CD45 þ leukocyte and CD45þ34þhuman stem cell clusters in all DV- or LV-treated mice. TheCD45þ34þ stem cells were viable as shown by the lack ofstaining with the viability dye, 7-AAD. CD45-positive cellsin theCD45dim (Blast),monocyte (Mono), and granulocyte(Gran) regions showed mainly myeloid lineage commit-ment on the basis of their co-expressionofCD13 andCD33.

Additional myelomonocytic lineage-associated markers,including CD64 and CD4dim, were expressed on CD13/33þ cells in themonocyte and granulocyte regions. Cells inthe lymphocyte gate (Lymp) did not show expression of anyantigens other than CD45, confirming that myeloid differ-entiation precedes lymphoid differentiation.

To further confirm that cells of humanoriginwerepresentin mice, DNA extracted from blood and BM of LV- and DV-treated mice was subjected to PCR to detect human specific"Alu" sequences. Indications of "Alu" sequences were iden-tified only in mice that received human CD 34þ cells(irrespective of their treatment) and not in control mice orcommercially availablemouseDNA, further confirming thecapability of the reovirus as a successful systemic agent totreat human multiple myeloma.

The reovirus fulfils many of the attributes of a viabletreatment option for multiple myeloma: selective targetingof disease without affecting the stem cell compartment,enhanced efficacy, and relative ease of manufacturing andstorage. Reovirus is not associated with toxicities in immu-nocompetent animals nor in the over 560 patients treatedwith reovirus to date.

In conclusion, this study represents the first utilization ofreovirus as a clinically relevant systemic agent for thetreatment of human multiple myeloma that could lead torapid bench-to-bedside translation. In an era where a per-sonalized approach to medicine is sought, multiple mye-loma may represent a malignancy where bone marrowaspirates could easily bewithdrawn frompatients and testedfor reovirus sensitivity before initiating a treatment regimen.In addition, this study constitutes a framework for thepossible extension of these findings to other hematologicalmalignancies as well.

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

AcknowledgmentsThe authors acknowledge the Southern Alberta Bone Marrow Transplant

Unit and the Apheresis Unit/Blood Bank of the Foothills Medical Centre fortheir support of this study.

Authors' ContributionsConception and design: C. M. Thirukkumaran, D. MorrisDevelopment of methodology: C. M. Thirukkumaran, Z. Q. Shi, J. Luider,D. MorrisAcquisition of data: C. M. Thirukkumaran, Z. Q. Shi, J. Luider, N. J. Bahlis,P. Neri, D. Stewart, A. Mansoor, D. MorrisAnalysis and interpretation of data: C. M. Thirukkumaran, Z. Q. Shi, K.Kopciuk, H. Gao, N. J. Bahlis, P. Neri, D. MorrisWriting, review, and/or revision of the manuscript: C. M. Thirukku-maran, Z. Q. Shi, J. Luider, K. Kopciuk, D. Stewart, A. Mansoor, D. MorrisAdministrative, technical, or material support: C. M. Thirukkumaran, Z.Q. Shi, M. Pho, A. MansoorStudy supervision: C. M. Thirukkumaran, D. Morris

Grant SupportThis study was financially supported by grants to DM and CMT from the

Alberta Cancer Research Institute (grant number 51797906058).The costs of publicationof this articlewere defrayed inpart by thepayment

of page charges. This article must therefore be hereby marked advertisementin accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 2, 2011; revised June 8, 2012; accepted June 13, 2012;published OnlineFirst July 3, 2012.






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2012;18:4962-4972. Published OnlineFirst July 3, 2012.Clin Cancer Res Chandini M. Thirukkumaran, Zhong Qiao Shi, Joanne Luider, et al. Multiple MyelomaReovirus as a Viable Therapeutic Option for the Treatment of

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