Ciarcia R

Costa A

Forte IM

Giordano A

Iannuzzi CA

Indovina P

Malfitano AM

Montagnaro S

Pentimalli F

Portella G

English

Archivio della ricerca - Università degli studi di Napoli Federico II

Oncolytic Viruses as a Possible Therapeutic Strategy against Malignant Pleural MesotheliomaIM Forte 1, F Pentimalli1, P Indovina2,3, S Montagnaro4, A Costa5, CA Iannuzzi1, AM Malfitano 6, R Ciarcia4, AGiordano3,5, G Portella6.1 Cell BiologyandBiotherapyUnit, IstitutoNazionaleTumori,IRCCS,FondazioneG. Pascale,I-80131Naples,Italy; 2 Institutefor High PerformanceComputingand Networking,National ResearchCouncil of Italy (ICAR-CNR),I-80131Naples,Italy;3 SbarroInstitutefor CancerResearchandMolecularMedicine,Centerfor Biotechnology,Collegeof ScienceandTechnology,TempleUniversity,Philadelphia,PA 19122, USA; 4 Departmentof VeterinaryMedicineandAnimalProductions,Universityof Naples ³ ) H G H U L F R , , ´ Napoli, Italia;5 Departmentof MedicalBiotechnologies,Universityof Siena,I-53100Siena,Italy; 6 DipartimentoScienzeMedicheTraslazionali,Universitàdi Napoli  ³ ) H G H U L F R , , ´ I-80131Naples,Italy.Introduction and AimMalignant pleural mesothelioma(MPM) is a very aggressivecancercorrelatedtoasbestosexposure. MPM is resistantto thecurrenttherapeuticstrategiesanddespitemulti modality treatmentsits prognosisremainsdismal,with a mediansurvivalof 9-17 monthsfrom diagnosis[1]. As the incidenceof MPM is predictedto increaseinthe next decade[2], the identificationof new effectivetherapeuticapproachesis anurgentneed.Cancer therapy through oncolytic viruses (OVs) has always been consideredapromisingtherapeuticapproachthat haverecentlyfound a successfulapplicationinthe clinical setting [3]. MPM representsan ideal candidatefor virotherapy fornumerousreasonsincluding the frequently localized pattern of growth and thepleurallocation,which allowsdirectaccessfor theintra-tumoralinjectionof theOvs[4].1. TsaoAS, WistubaI, et al. Malignantpleural mesothelioma. J Clin Oncol20092. Alpert N et al. Epidemiologyof mesotheliomain the 21 st centuryin Europeandthe United States,40 yearsafterrestricted/bannedasbestosuse. TranslLung CancerRes20203. RussellSJ,PengKW, andBell JC. Oncolyticvirotherapy. Nat Biotechnol20124. PeaseDF, KratzkeRA. OncolyticViral Therapyfor Mesothelioma. FrontOncol20175. HeiseC, HermistonT, et al. An adenovirusE1A mutantthat demonstratespotentand selectivesystemicanti-tumoralefficacy. Nat Med 20006. S. Tusell Wennier et al. Bugs and Drugs: Oncolytic Virotherapy in Combinationwith ChemotherapyCurrPharmBiotechnol. 20127. Indovina P, Marcelli E, Di Marzo D, Casini N, Forte IM, Giorgi F, Alfano L, Pentimalli F, GiordanoA.Abrogating G2/M checkpointthrough WEE1 inhibition in combinationwith chemotherapyas a promisingtherapeuticapproachfor mesothelioma. CancerBiol Ther. 20148. Rodrigues R et al. Bovine herpesvirus type 1 asa novel oncolytic virus . Cancer GeneTher 2010Our preliminary data on the potential use of virotherapybasedon thedl922-947oncolyticvirus andon CpHV-1 virus suggesthatOV couldbean effectivestrategyfor the treatmentof MPM. We plan to further carryon thepreclinicalcharacterizationof OVs usebothassingleagentandincombinationstrategiesand we will analyzethe molecularmechanismsunderlyingapoptosisinductionandwhetherviral entry into the cells andviral replicationareaffectedby combinatorialapproaches. We will thenassesswhetherthisstrategyis feasiblealsoonorthotopicMM models.dl922-947inducesapoptosisand hyperdiploidy in MPM cell linesAnalysis of dl922-947effectson MPM cell linesWe focusedon adenoviruseswith a 24 bp deletion in theE1A-conservedregion 2, which binds and inactivatestheretinoblastomaprotein, resulting in a virus (dl922-947) [5]thatcannottrigger S phaseentry in normalcells,but canstillreplicatein cells with an aberrantG1-S checkpoint,a defectobservedin over 90% of human cancers,including MPM(Figure 1). First, we assessedthe effects of the virus on apanelof MPM representativeof themaindifferenthistotypes:the epithelioid NCI-H28 and NCI-H2452, the biphasicMSTO-211H and the sarcomatoid NCI-H2052. BysulforhodamineB (SRB) assay we determinedthe IC50values at 5 days following treatmentwith dl922-947 andfound that all MPM cell lines were susceptibleto viraltreatment(Table 1), exceptNCI-H2052 cells, in which viralentry was not efficient, as shown through infection with areporteradenovirustransducingGFP(not shown).We testedthe potentialantitumoreffectsof two theoncolyticstrategies: 1) the adenovirusdl922-947inboth MPM cell lines and mousexenograftsand2)the effectsof a non-humanCaprineHerpesvirus-1(CpHV-1) on a panelof MPM cell lines both aloneandin combinationwith cisplatin. Our datasuggestthat theseOvs,aloneor in combination,could be afeasiblestrategyagainstMPM.ConclusionWe thenanalyzedby cytofluorimetricanalysistheeffectof dl922-947on cell cyclefeaturesof responsivecells andwe found that dl922-947 treatmentat the IC50 andIC50/2 inducesan increaseof the subG1 cell fraction,suggestiveof cell death,andof the hyperdiploid(4N) population,suggestiveof mitotic defects. The dl922-947treatmentalsoinducedtheexpectedS phaseincreasein NCI-H2452andNCI-H28 butnot asignificantincreaseinMSTO-211H at thedosesandtimesanalyzed.Inductionof cell deathwasfurtherconfirmedin NCI-H28 andMSTO-211H throughannexinV assay(Table 2).Figure 1Testing dl922-947 in combination strategiesOncolytic adenovirusescan interact synergisticallywith different drugs [6]. Inparticular,we testeddl922-947 efficacy in combinationwith cisplatin that is usedas first-line treatmentfor MPM and in combinationMK-1775, a first-in-classinhibitor of the WEE1 kinase,which we previouslyfound able to sensitizeMPMcells to cisplatin [7] and which is currently being tested in clinical trials fordifferent tumortypes. By analyzingdifferentschedulesof treatment,we foundthatboth cisplatin treatmentMK-1775 are able to increasethe cytotoxic effect of theoncolyticvirus treatmentupon24h from dl922-947infection(Figure 2).dl922-947 inhibits tumor growthin vivo in a xenograft model ofMPMMPM xenograftswherebyathymicmicewereinoculatedsubcutaneouslywith MSTO-211H cellsandwhenthetumorsbecamepalpable,themiceweredividedinto two groups (of 9 animals each) and treated bi-weekly with dl922-947.Virotherapyprovedextremelyeffective in counteractingtumor growth as early asafter3 weeksof treatment: theanimaltreatedshowedtotal tumorregressionalreadyafter the first week,and other two mice had similar results during the courseoftreatment. Animals thathadshowntotal tumor regressionwerenot sacrificedat theend of the experimentand were observedfor 3 additional months. During thisperiod,no tumorre-growthwasobserved(Figure 3).Figure 3Figure 2Oncolytic Caprine Herpesvirus 1 (Cphv -1) induces apoptosisand synergizes with cisplatin in MPM cell linesAmongthedifferent typesof OVs now available,somenon-humanwild-typeOVs showadvantagesoverhumanOVs, includingtheinability to replicateinnormal humancells while having a natural tropism for humancancercellsand the absenceof pre-existing immunity [8], so we also assessedCpHV-1effectson MPM (NCI-H28, MSTO, NCI-H2052) andnon-tumor mesothelial(MET-5A) cells. We foundthatCpHV-1 reducedcell viability andclonogenicpotentialin all MPM cell lines without affectingnon-tumor cells, in which,indeed,wedid not detectintracellularviral DNA aftertreatment. In particular,CpHV-1 induced MPM cell apoptosis(Figure 4B) and accumulationinG0/G1 or S cell cycle phases. Moreover,CpHV-1 strongly synergizedwithcisplatin and this agentcombinationdid not affect normal mesothelialcells(Figure 4A).Figure 4BFigure 4AReferencesResults non-tumor cells, in which,indeed, we did not detect intracellular viral DNA after treatment. In particular,CpHV-1 induced MPM cell apoptosis (Figure 4B) and accumulation inG0/G1 or S cell cycle phases. Moreover, CpHV-1 strongly synergized withcisplatin and this agent combination did not affect normal mesothelial cells(Figure 4A).Figure 4BFigure 4AReferencesResults

ONCOLYTIC VIRUSES AS A POSSIBLETHERAPEUTIC STRATEGY AGANIST MALIGNANT PLEURAL MESOTHELIOMA

https://www.iris.unina.it/bitstream/11588/874568/1/POSTER%20ASGCT%202021_OVs%20%20in%20MPM.pdf

ONCOLYTIC VIRUSES AS A POSSIBLETHERAPEUTIC STRATEGY AGANIST MALIGNANT PLEURAL MESOTHELIOMA

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