VII JORNADAS DE MEDICINA GENÓMICA Y ONCOLOGÍA GENYO 2019 Wednesday, 11 th December ORAL COMMUNICATIONS MASTER STUDENT Pedro Luis Justicia Lirio Generation of lentiviral vectors that mimic the expression pattern of TCR in T cells for immunotherapy applications. Alba Ortigosa Palomo Estudio transcriptómico de subpoblaciones celulares en pacientes con cáncer de mama. Juan Sanjuán Hidalgo CRISPR/Cas9-mediated edition of KRAS driver mutations in Non-Small Cell Lung Cancer (NSCLC). PhD STUDENT SESSIONS (<3 YEARS OF RESEARCH EXPERIENCE) Margarita Barriga García-Mauriño Estudio del papel de Cortistatina como regulador endógeno de los procesos de fibrosis asociados a patologías crónicas. Adrián González González Knockdown of TGF-activated NDRG1 inhibits migration and epithelial-mesenchymal transition of metastatic triple negative breast cancer cells. Noelia Maldonado Pérez Assessment of efficacy and safety of universal CD19-CAR T cells. FLASH-TALKS POSTERS
JuanSanjuánHidalgoCRISPR/Cas9-mediatededitionofKRASdrivermutationsinNon-SmallCellLungCancer(NSCLC).PhDSTUDENTSESSIONS(<3YEARSOFRESEARCHEXPERIENCE) MargaritaBarrigaGarcía-MauriñoEstudio del papel de Cortistatina como regulador endógeno de los procesos de fibrosis asociados apatologíascrónicas.AdriánGonzálezGonzálezKnockdown of TGF-activated NDRG1 inhibits migration and epithelial-mesenchymal transition ofmetastatictriplenegativebreastcancercells.
Wednesday,11thDecember ORAL COMMUNICATIONS MASTERSTUDENTORALCOMMUNICATIONS PedroLuisJusticiaLirio,MaríaTristán,NoeliaMaldonado,andFranciscoMartínCentreforGenomicsandOncologicalResearch(GENyO),Granada18007,SpainGenerationoflentiviralvectorsthatmimictheexpressionpatternofTCRinTcellsforimmunotherapyapplications.Genetherapycanbeusedtorepairgeneticalterationsortoprovidenewfunctionstocells.Conventionalcancertreatmentsaregenerallyinefficientduetothedevelopmentofmechanismstoescapefromtheimmunesystem.New strategies based on Immunotherapy (arming the immune system to attack tumor cells) are becomingimportant tools for several typesofcancer, inparticular, theuseof lentiviralvectors (LVs) togeneratechimericantigen receptors (CARs). CAR expression on T cells is very high due to the presence of strong promoters likeelongationfactor1(EF1-).ThereisevidenceindicatingthatuncontrolledexpressionofCARscouldberesponsibleofserioussideeffectsandlackofefficacyinsomepatients.Dr.Sadelain'sgroupdemonstratedthatexpressingtheCARthroughtheTRACpromoterusinggenomeediting (GE) increasedtheantitumorefficacyofCAR-Tcells.Ourpoint was to generate LVs thatmimic the expression pattern of the TCR as an alternative to GE strategy.Wedesigned chimeric promoters in order to achieve TCR-like kinetic expression on T cells. Those promoters werebased on CD4, B2M, LCK andCD247 genes and inserted into LVs to express eGFP. LVswere used to transduceprimaryhumanTcellsandtheeGFPexpressionlevelsanalyzedalongtimeafterCD3/CD28estimulation.Ourdatashowedthatboth,theCD4andB2MchimericpromotersmimickedtheexpressionpatternoftheendogenousTCR,loweringthelevels8hoursafterthestimuliandacontinuousincreasethereafter,reachingnormallevelsafter72hours. Of note, the T cells transduced with B2M-based LVs were almost completely silenced 8h after T cellactivation,andthiscouldbeanimportantaspecttoreduceTcellexhaustionifapplytoCAR-T.WeproposetheuseB2M-regulatedLVsfortheexpressionofCARinTcells,aswaytoreduceTcellexhaustion.
AlbaOrtigosaPalomoEstudiotranscriptómicodesubpoblacionescelularesenpacientesconcáncerdemama.Elcáncerdemamaeselsegundotipodecáncermáscomúnenelmundoyelmásfrecuenteenmujeres.Elinterésen el control de la diseminación del cáncer en general, y del cáncer de mama en particular, ha impulsado eldesarrollodenuevasestrategiasdedetecciónypronósticode laenfermedadyderespuestaa los tratamientos.Entreestasestrategiasdestacaprincipalmentelabiopsialíquida(BL),unatécnicanoinvasivaquepermiteobtenerinformación tumoral en base a la determinación de biomarcadores en un fluido corporal. Entre esosbiomarcadoresdeBLseencuentran,entreotros,lascélulastumoralescirculantes(CTCs),lasplaquetaseducadasportumor(TEPs,delinglésTumorEducatedPlatelet)ylosleucocitos.Estetrabajotienecomoprincipalobjetivoelestudiotranscriptómicodelassubpoblacionesleucocitariasyplaquetariastantoenpacientesdecáncerdemamacomo en donantes sanos para determinar biomarcadores diagnóstico en este tipo de cáncer. Además, sedeterminó la presencia de CTCs en estas poblaciones y se correlacionaron con variables clinicopatológicas. LosresultadospreliminariesdeterminanquelosnivelesdemiRNA5690yFHL3enleucocitosyGLYATL2,HLA-B,CTSW,CDK16ySH3PXD2Aenplaquetassonpotencialesbiomarcadoresdiagnósticodelaenfermedad.
Juan SanjuánHidalgo1, Álvarez Pérez, Juan Carlos1,2 Rodríguez Lara,María Isabel1,3 ArenasMolina,AlbertoManuel1,2BaliñasGavira,Carlos1,2PeinadoFernández,Paola1,2AndradesDelgado,Álvaro1,2CuadrosCelorrio,Marta1,3MedinaVico,Pedro1,21:GENYO.CentreforGenomicsandOncologicalResearch.AndalusianRegionalGoverment/UniversityofGranada/Pfizer2:DepartmentofBiochemistryandMolecularBiology(I).FacultyofSciences,UniversityofGranada.3:DepartmentofBiochemistryandMolecularBiology(III).FacultyofMedicine,UniversityofGranada.CRISPR/Cas9-mediatededitionofKRASdrivermutationsinNon-SmallCellLungCancer(NSCLC).INTRODUCTIONLung cancer is the leading cause of cancer worldwide, being Non-Small Cell Lung Cancer (NSCLC) the mostprevalent subtype. It is estimated that 30% of these tumors harbor mutations in Kirsten Rat Sarcoma viraloncogene (KRAS),whicharemainly located in the12thcodon.Thesemutationsareknownasdrivermutations,becausetheyconstituteanoncogenicformoftheKRASproteinwhichcontinuouslypromotescellproliferationandeventuallyleadstotumordevelopment.GiventhehighfrequencyoftheseKRASmutationsinNSCLCandthelackofsuccessfulcompoundsforitstreatmenttothedate,wehavedevelopedaClusteredRegularlyInterspacedShortPalindromicRepeats(CRISPR)/CasgenetargetingstrategyagainstG12CandG12Ddrivermutations,whichaimtobeanalternativeapproachforfuturelungcancertreatment.METHODOLOGYDesignedgRNAs targetingG12CandG12Dmutationswere tested inKRASmutant andKRASwildtype cell lines.CRISPRtoolsweredeliveredtocellsasaribonucleoprotein(RNP)complex.SpecificityofgRNASandKRASlosswereassessedthroughT7endonucleaseassayandWesternBlot,respectively.RESULTSWe have demonstrated that the CRISPR/Cas system can be implemented to selectively target KRASmutationswithout affecting its wildtype sequence. Consequently, the edition ofmutant KRAS in NSCLC cell lines (H1792,SKLU-1 and A427) displays a significant reduction in tumor phenotype, in terms of cell viability and clonogenicability.Thus,ourresultsprovideanewinsightandaverypromisingtooltotreatlungcancer,byselectivelyeditingthemostfrequentmutationsofKRASwithoutaffectingthewildtypeversionofthisoncogene.
PhDSTUDENTSESSIONS(<3YEARSOFRESEARCHEXPERIENCE) García-MauriñoMargaritaBarrigaGM(1),RaquelBenítez(1), JennyCampos-Salinas(1),MartaCaro(1),GemaRobledo(1),FranciscoO’Valle(2),MarioDelgado(1).(1)InstitutodeParasitologíayBiomedicinaLópez-NeyraCSIC,Granada(2)DepartamentodeAnatomíaPatológicaeHistoriadelaCiencia,FacultaddeMedicina,UniversidaddeGranadaEstudiodel papel deCortistatina como regulador endógenode losprocesosde fibrosis asociados apatologíascrónicas.El45%delasmuertesqueseproducenanivelmundialestánasociadasaprocesosfibróticoscrónicosexacerbados.Existe gran variedad de patologías relacionadas con estos, incluyendo Esclerodermia y Fibrosis pulmonaridiopática,resultandoprioritarialaidentificacióndegenesyfactoresdesusceptibilidadyseveridaddelasmismas.Existenevidenciasqueindicanqueelneuropéptidoanti-inflamatoriocortistatinapodríajugarunpapelclavecomoreguladorendógenodefibrosiscrónicapatológica.Inicialmenteencontramosquecortistatinaysusreceptoresseexpresabanencélulasefectorasfibróticas,comofibroblastosymiofibroblastos,yquesupresenciaestabaalteradaentejidosfibróticos.Generamosratonestotalmente(CST-KO)oparcialmente(CST-HET)deficientesencortistatinay losensayamosenunmodeloexperimentalpreclínicodeEsclerodermia,yobservamosquemostraban lesionesfibróticas cutáneas significativamente más exacerbadas y con marcadores fibróticos más elevados que lasmostradas por ratones CST-WT. Además, la deficiencia en cortistatina iba asociada a un desarrollo de fibrosispulmonar severa en los animales con esclerodermia, un proceso secundario que ocurre en un porcentajesignificativodepacientesconestaenfermedadyquecomprometesucalidaddevida.Igualmente,enunmodelopreclínicode fibrosispulmonar idiopática, constatamosque ladeficienciaencortistatina resultóenunaelevadamortalidad causada por infiltración leucocitaria y fibrosis parenquimatosa pulmonar exacerbada y subsiguientefalloorgánico.Eltratamientoconcortistatinarevirtióestefenotipopatológicoylamortalidadobservadaenambosmodelosexperimentales,normalizandolosprocesosfibróticos.CultivosdefibroblastosprimariosaisladosdepielypulmonesderatonesCST-WT,CST-HETyCST-KOdemostraronque ladeficienciaencortistatina llevabaasociadaunamayor activaciónde las rutasde señalización intracelularesprofibróticas, yunaumentode la expresióndemarcadores fibróticos.Nuestrosdatos indicanquecortistatinapodríaconsiderarseenunfuturounbiomarcadordesusceptibilidadasufrirprocesosfibróticosasociadosapatologíascrónicasyunaalternativaterapéuticaparasutratamiento.AdriánGonzálezGonzález1,2,MaríaValenzuela-Torres1,FranciscaE.Cara1,2,AraceliLópez-Tejada1,JoséL.Blaya-Cánovas1,SergioGranados-Principal1,21GENYO,CentreforGenomicsandOncologicalResearch,Granada,Spain.2UGCdeOncologíaMédica,HospitalUniversitariodeJaén,Jaén,Spain.Knockdown of TGF-activated NDRG1 inhibits migration and epithelial-mesenchymal transition ofmetastatictriplenegativebreastcancercellsBackground:TripleNegativeBreastCancer (TNBC)displays theworstpatientsurvival ratesamongbreastcancersubtypes, mainly due to chemoresistance, cellular heterogeneity and metastases. TNBC exhibits overactiveaggressiveness-relatedsignalingpathwayslikeTGF,whichinducesEMT(Epithelial-MesenchymalTransition)duringmetastasis.NDRG1 (N-mycdownstreamregulatedgene1) isdescribedasametastasis suppressor ina rangeoftumor types, including breast cancer. We have previously observed that TGF induces the phosphorylation ofNDRG1indifferentTNBCcell lines.WeaimedatdeterminingwhetherNDRG1isinvolvedintumorcellmigrationandEMTuponTGFactivation.Methods: TNBC cell lines from pleural effusion (MDA-MB-231, MDA-MB-436) and primary tumor
(SUM159,BT549)wereused.Changesinphospho(p)-NDRG1(Thr346)wereassessedbyWesternblotinSUM159 and MDA-MB-231 cells treated with TGF1 (10ng/ml) for 8, 12, 24, 48 and 72h. NDRG1knockdown(50nMsiRNA)wasperformedinMDA-MB-231,MDA-MB-436,SUM159andBT549cellsfor48h,with/withoutTGF1.CellmigrationandexpressionofEMTmarkers(SNAIL,SLUG,TWIST,Vimentin)weredeterminedbythewoundhealingassayandWesternblot,respectively.Result:Our results showeda sustainedphosphorylationofNDRG1duringTGF1 treatment.Overall,uponTGFactivation,NDRG1knockdownwascorrelatedwithlessexpressionofTWIST,SNAILandSLUGinallcelllines. Interestingly, Vimentinwas reduced only in cells frompleural effusion.Opposite to those fromprimarytumor,migrationwassignificantlydecreasedinpleuraleffusionderivedcells.Conclusions:We are the first to demonstrate that TGF motivates and maintains NDRG1 activation in TNBC.Noteworthy, NDRG1maymediate TGF-inducedmigration only in those cells that have escaped fromtheir primary site and reached distant organs, what could be attributed to the modulation ofEMT.Moreover, we show that EMT process is dispensable for the role of NDRG1 on the TGFpromotedmetastaticevents.
NoeliaMaldonadoPérez1,MaríaTristán-Manzano1,RocíoMartín-Guerra1,SabinaSánchez-Hernández1,MarinaCortijo1,IrisRamos-Hernández1,ConcepciónMarañon1,MaríaDoloresCarmona2,ManelJuan3,ConcepciónHerrera2,KarimBenabdellah1,FranciscoMartín1.1CentroPfizer-UniversidaddeGranada-JuntadeAndalucíadeGenómicaeInvestigaciónOncológicaGENYO-,GenomicMedicine,Granada18007,Spain.2UnidaddeTerapiaCelularIMIBIC-HURS,HospitalReinaSofía,Córdoba14004,Spain.3DepartamentodeHamatología,HospitalClínicdeBarcelona08036,Spain.AssessmentofefficacyandsafetyofuniversalCD19-CARTcellsCurrentadoptiveimmunotherapystrategiesuseautologousTcellsexpressingaChimericAntigenReceptor(CAR)againstCD19+Bcellsmalignancies.Beyondtheimpressivebenefit,timeandqualityofmanufacturingTcellsfromthe refractory patients constitutes a critical limitation for a successful therapy. On the other hand, severe sideeffects, includingpatientdeathsbycytokinerelease-syndrome(CRS)andappearanceofrelapsedleukemia,havebeenreportedasaconsequenceofastrongandbadregulatedCARexpression.Ourhypothesis is thatallogenicuniversalCAR-Tcellscanovercomethehistocompatibilityproblemandcanbegeneratedfromhealthydonorstotreatseveralpatients,expandingaccessto immunotherapy. Inaddition,expressingtheCARsbyusingpromotersthatmimicthephysiologicalpatternoftheTCRcanreducetoxicityandimprovetherapeuticactivity.InfirstplacewegenerateduniversalprimaryTcellsdisruptingtheTCRmoleculeusingCas9/sgRNAribonucleoprotein(70-90%KOefficiency)withoutaffectingnegatively thephenotypeand theproliferationcapacity.BothWTandTCRKOTcellsexpressing theCD19-CAR ina constitutiveway showedgoodefficacyand selectivity in vitro (>90%specificlysis). In parallel, we have tested a panel of different LV-backbones to investigatewhich onemimic better theTCR/CD3expressionprofileafterTCRstimulation,obtainingthebestresultswiththepromoterfromtheWiskott-Aldrich syndrome (WAS) locus. TCRKO T cells expressing the CAR in a TCR-like fashion displayed specific lyticactivity and a less exhausted phenotype. Regarding edition safety, CRISPR/Cas9 technology could encounterundesired side effects on T cells due to off-targets and on-target repair mechanisms. We didn´t find anysignificativecleavageinsixpredictedoff-targetsinsilico.However,wefoundlargedeletionsonthetargetsiteofupto4kb.Atthispoint,wearegeneratingmousemodelstostudytheefficacyandsafetyofthesenewTCRKO-CD19CARTcellsinvivo.
Wednesday,11thDecember FLASH-TALKS RitaCaracuelPernos1,RedondoSilvia1andRodríguez-ManzanequeJuanCarlos11 GENYO. Centre for Genomics and Oncological Research, Pfizer/Universidad de Granada/Junta deAndalucía,Granada18016,Spain.StudyoftheroleofADAMTS1tomodulatemacrophagepolarizationtowardaprotumorigenicADAMTS1 (a disintegrin andmetalloproteasewith thrombospondinmotifs 1) is an extracellular proteasewhichcleaves proteoglycans modifying the tumor microenvironment. The role of ADAMTS1 in tumorigenesis isambiguous,andbothanti-andpro-tumorigenicactivitieshavebeenreported.Recently,ourgrouphasdescribedthatADAMTS1modulatethetumorimmuneinfiltrateandtheimmunebalanceinspleenandbonemarrow,withconsequences promoting tumor progression. Considering that macrophages are relevant players inducing animmunosuppressivemicroenvironment, in thisworkwehave studied the possible role of ADAMTS1 to educatemacrophagestowardapro-tumorigenicstate.Weworkedwithtwodifferenttumormodels,melanomaB16F1andLungLewisCancer (LLC) cell lines,whose tumorcapacitiesaredifferentlyaffectedby theabsenceofADAMTS1.Bone marrow derived macrophages (BMDM) isolated from C57Bl/6 mice were treated with B161F1 and LLCconditioned medium, containing or not ADAMTS1. In both models, ADAMTS1 promotes a pro-tumorigenicpolarization of macrophages, but the effect was more pronounced with LLC conditionedmedium. In addition,endogenousADAMTS1also showed contribute topolarizeBMDM.To reduce variability and complexity derivedfrom ex-vivo BMDMs experiments, we also approached the polarization of Raw 264.7 cells under similarconditions. However, these polarization experiments did not success; CM from LLC and B16F1 cells did notpromote any change in expression of pro-tumorigenicmarkers in Raw264.7 cells. Although deeper studies areneeded,our findingscorroboratethatADAMTS1canmodulatethe immunecellpopulation,specificallyBMDMs,towardapro-tumorigenicphenotype.IrisRamosHernández,MarinaCortijo,NoeliaMaldonado,MaríaTristán,PedroJusticia,SabinaSánchez,MarcoViteri,KarimBenabdelLah,FranciscoMartín.GENYO. Centre for Genomics and Oncological Research, Pfizer/Universidad de Granada/Junta deAndalucía,Granada18016,Spain.Optimizationofthedeliverymethodfortheknock-ininvitrowiththeCRISPR-Cas9systemAutologousadoptivetherapyofmodifiedTlymphocytestoexpressChimericAntigenReceptors(CARs),combinesgenetic engineering and tumour immunology inorder to improve cancer therapy. Infusionof autologousCAR-Tcells directed against CD19 for the treatment of refractory lymphoma and type-B leukaemia have had greatefficacy,leadingtotheapprovaloftwonewgenetherapydrugs,YescastaandKhymriah.Nevertheless,thereareseveraldrawbacksthatmustbeovercometoapplythistreatmenttoabroaderrangeofpatients.Twoimportantlimitingfactorsare1)thedifficultytoobtainenoughsuitableTcellsfromcancerpatients,and2)theunregulatedexpression of the CARs. Our hypothesis is that the use of CAR-T cells from healthy donors TCR knock-out(preventing Graft versus Host Disease) and that express the CAR physiologically (preventing exhaustion) couldavoidthementionedlimitations.ToachievethesegoalssimultaneouslyweaimtoengineerTcellsthroughgenomeediting(GE) inordertoinserttheCAR’scDNA intotheTCR locus.Firstly, tocheckandoptimizetheGEprocesswedemonstratedtheefficientdisruptionoftheTCRusingourgRNAdirectedtothealphachainoftheTCRlocus.AfterthatwedesignedadonorDNA expressing a reporter gene (eGFP). This system allowed us to analyse the efficiency and safety of the GE
mechanismbycomparingthe levelsofTCR-eGFP+(precisegenomeediting)versusTCR+eGFP+(unspecificdonorinsertions). Our results showed that the incubation of the DNA donor with the preformed ribonucleoparticleduringhalfhourincreasetheefficiencyandspecificityoftheknock-inprocess.WealsoestablishtheimportanceofthedonorDNAdesign regarding the sizeof thehomology arms.As the reduction to300pb inourdesign showworsepreliminaryresultsaboutknock-inefficiencyandspecificity,andworsecellviability.AraceliAguilarGonzález1,2,JuanElíasGonzálezCorrea1,PilarMuñoz1,RosarioMaríaSánchezMartín1,2andFranciscoMartín11Pfizer/Universidad de Granada/Junta de Andalucia Centre for Genomics and Oncological Research(GENYO).PTSGranada–AvenidadelaIlustración,114,18016Granada,Spain.2 Department of Medicinal and Organic Chemistry, Faculty of Pharmacy, University of Granada –CampusCartuja,18071Granada,Spain.GenerationofcellularmodelstostudygenetherapystrategiesforPompediseasePompediseaseisararedisordercausedbymutationsinthelysosomalacidalpha-glucosidaseenzyme(GAA)genethatleadtoaccumulationofglycogeninmultipletissues.ThereplacementenzymetherapyistheonlytherapeuticoptionbutiseffectiveonlyforasubsetofPompepatientsandpartiallyeffective.Asanalternative,genetherapycouldofferadefinitivecurebyrestoringthenormalexpressionoftheGAAgene.Thepurposeofthisprojectwasto generate murine GAA-KO Sol8 cell lines to study efficacy and safety of gene therapy strategies for Pompedisease. Using CRISPR/Cas9 technology targeting the GAA gene, we have generated different murine skeletalmuscle cell lines (Sol8) that resembledifferent genotypespresent in Pompepatientswith a severe formof thedisease, aswell asmutations found in themurine Pompemodel (B6; 129-Gaatm1Rabn/J). Functional analysesshowedabsenceofGAAactivityinallthemodelsgenerated.Interestingly,initialassaystorestoreactivitythroughLV-GAAtransductionatthedifferentmodelsshoweddifferent levelsofGAAactivitydependingonthemutationtype,beeneasiertorestoretheactivityin5’mutationscomparedtothosegeneratingtruncatingproteins.JuanElíasGonzálezCorrea,AraceliAguilar-González1,2,IrisRamos-Hernández1,FranciscoMartín1andPilarMuñoz11:GENyO-CentrodeGenómicaeInvestigaciónOncológica:PfizerUniversidaddeGranada/JuntadeAndalucía2:UniversidaddeGranadaGene-celltherapyforPompediseasePompediseaseisarareautosomalrecessivemultisystemdisordercausedbymutationsinthegenecodingfortheacid alpha-glucosidase enzyme (GAA) that leads to accumulation of glycogen in most tissues, producing tissuedamage,especiallyincardiacandskeletalmuscle.Intheabsenceoftreatment,patientsdieintheirfirstyearsduetocardiacor respiratory failure.Current treatments,basedon intravenous infusionof recombinanthumanGAA(ERT), are only palliative. As an alternative to ERT, gene therapy is becoming a realistic possibility that couldtheoretically cure Pompe patients. In this work we aim to achieve this goal through transplantation ofHematopoietic Stem Cells (HSCs) over-expressingGAA. This strategymust achieve two importantmilestones inorder to be successful: 1) high engraftment into damaged organs and 2) high secretion of GAA that must beuptakenbythesetissues.Therefore,wefirststudiedengraftmentefficacyofHSCs(Lin-andSca1+cells)expressingeGFPinaPompemurinemodel,showingalongtermexpressionanddistribution,reachingseveraltargettissuesincludingthecentralnervoussystem(CNS).Inparallel,wedesignedmodifiedmurineGAAstransgenesinordertoachievehighexpressionlevels,secretionandimproveduptakebytargetcells.Myeloidcells(K562)transducedwithLVs-mGAAoptexpressedand secretedhigh levelsofGAA thatwasefficiently capturedbymurinemuscle targetcellsandperitonealmacrophages fromGAAKOmice.Wehaveperformedcomparativeexperiments inorder tostudy the improvement inexpression, secretionanduptakebetweendifferentdesignsofmGAA,mGAAopt and
IGF2-mGAA. We are currently studying the therapeutic efficacy of our strategy in a murine model of PompediseasebyintravenousinoculationofLin-GAAKOcellstransducedwithLVsexpressingmGAAopt,IGF2-mGAAandmGAA.CarlosBaliñas-Gavira1,2MaríaIsabelRodríguez1,2,ÁlvaroAndrades1,2,MartaCuadros1,3,JuanCarlosÁlvarez-Pérez1,2,ÁngelF.Álvarez-Prado4,VirginiaG.deYébenes4,SabinaSánchez-Hernández5,ElviraFernández-Vigo6, Javier Muñoz6, Francisco Martín5, Almudena R. Ramiro4, José Ángel Martínez-Climent7,8andPedroP.Medina,1,21GeneExpressionRegulationandCancerGroup(CTS-993).GENYO.CentreforGenomicsandOncologicalResearch:Pfizer-UniversityofGranada-AndalusianRegionalGovernment,Granada18016,Spain.2DepartmentofBiochemistryandMolecularBiologyI,UniversityofGranada,Granada18016,Spain.3 Department of Biochemistry and Molecular Biology III and Immunology, University of Granada,Granada18071,Spain.4BCellBiologyLab,CentroNacionaldeInvestigacionesCardiovasculares,Madrid28029,Spain.5 Genomic Medicine Department, GENYO, Centre for Genomics and Oncological Research, Pfizer-UniversityofGranada-AndalusianRegionalGovernment,Granada18016,Spain.6 Proteomics Unit, CNIO – Spanish National Cancer Research Center, Madrid 28029, Spain.ISCIIIProteoRed.7DivisionofHaemato-Oncology,CentreforAppliedMedicalResearch(CIMA),CIBERONC,UniversityofNavarra,Pamplona31008,Navarra,Spain.8HealthResearchInstituteofNavarra(IDISNA),Pamplona31008,Navarra,Spain.Frequentmutationsintheamino-terminaldomainofBCL7AimpairitstumorsuppressorroleinDLBCLMutationsingenesencodingthesubunitsoftheSWI/SNFchromatinremodelingcomplexarefrequentlyfoundindifferenthumancancers.Althoughthetumorsuppressorfunctionofthiscomplexhasbeenwidelyestablishedinsolidtumors,itsroleinhematologicmalignanciesremainstobeexplored.RecurrentpointmutationsinBCL7A,asubunitoftheSWI/SNFcomplex,havebeenreportedindiffuselargeB-celllymphoma(DLBCL)buttheirfunctionalimpactremainsunknown.Inthiswork,there-analysisof largeDLBCLcohortshasrevealedthatmutationsintheamino-terminaldomainofBCL7AarepresentinasubstantialfractionofDLBCLpatientsandwereportapreviouslyunnoticedmutationalhotspotinthesplicedonorsiteofintronone.Furthermore,weprovideevidencethatBCL7Arecurrently suffers biallelic inactivation in DLBCL cell lines and patients, a characteristic pattern of tumorsuppressorgenes. Importantly,ourstudyhasbroughtto lightthatthesplicesitemutations,amongotherBCL7Amutations,arethemostfrequent inDLBCLbutbeyondthenumbers,wehavealsodemonstratedthatthesplicesitemutationshaveafunctionalimpact.ThesplicesitemutationsrenderamutantBCL7A,lackingaportionoftheaminoterminaldomain,andwild-typeBCL7Aexpressionrestorationhasatumorsuppressor-likephenotypebothinvitroand invivo.Ourdatasupport thatsplicesitemutationsblock theBCL7Atumorsuppressor functionandpreventitsbindingtotheSWI/SNFcomplex.Inaddition,wereportthattheSWI/SNFcomplexsubunitsaccumulatemutations inhighpercentageofDLBCLtumors.AberrantSWI/SNFcomplexformationmightrepresentageneralmechanismoflymphomagenesisandtargetingthispathwayisapromisingtoolinthefightagainstcancer.Overall,these findingshighlight the tumorsuppressor roleofBCL7A inDLBCL,andsuggest that theSWI/SNFcomplex isinvolvedinDLBCLpathogenesis.PabloTristánRamos1,2,AlejandroRubio-Roldan1,5,GuillermoPeris1,3,5,LauraSánchez1,5,SuyapaAmador-Cubero1,SebastienViollet4,GaelCristofari4,SaraR.Heras1,21GENYO, Centre for Genomics and Oncological Research: Pfizer/University of Granada/AndalusianRegionalGovernment.PTSGranada,Av.delaIlustración,114,18016Granada,Spain.
2Dept. Biochemistry and Molecular Biology II, Faculty of Pharmacy, University of Granada, CampusUniversitariodeCartuja,18071Granada,Spain3Dept.ofComputerLanguagesandSystems,UniversitatJaumeI,CastellóndelaPlana,12071,Spain.4UniversityCoted’Azur,Inserm,CNRS,IRCAN,Nice,France5Theseauthorscontributedequallytothiswork.ThetumorsuppressormicroRNAlet-7inhibitshumanLINE-1retrotranspositionMorethanhalfofthehumangenomeismadeoftransposableelements(TEs)whoseongoingactivitycontinuestoimpact its structureand function.Among them,Long INterspersedElementclass1 retrotransposons (LINE-1sorL1s)aretheonlyautonomouslyactiveTEsinhumans.ActiveL1sare6kblongandencodetheenzymaticmachineryrequiredfortheirmobilization(L1-ORF1pandL1-ORF2p).Inmosthumansomaticcells,theexpressionofactiveL1elementsissilencedbyseveralmechanisms.However,L1sareexpressedandcanmobilizeinmanyhumantumortypes, generating mutagenic insertions that could affect tumor’s malignancy. On the other hand, microRNAs(miRNAs)areendogenous22ntRNAsthatplaycrucialgene-regulatoryrolesineukaryotesbypairingtomRNAsofprotein-codinggenestodirecttheirpost-transcriptionalrepression.Amongthem,tumorsuppressormiRNAssuchas the let-7 family regulate theexpressionofoncogenesandare frequentlydownregulated incancer.However,whether they exert any influence in L1mobilization is not completely understood. Interestingly, our analysis ofwhole genome and miRNA-sequencing data from lung tumour samples shows that downregulation of severalmembersofthelet-7familycorrelateswithincreasedsomaticL1retrotransposition.Indeed,let-7overexpressionordepletiondecreasesandincreases,respectively,engineeredL1retrotranspositioninapanelofculturedhumancells,includinglungcancercells.Mechanistically,usingmolecularandbiochemicalassaysweshowthatlet-7bindstotheL1mRNAandimpairsthetranslationofL1-ORF2p,whichisindispensableforretrotransposition,reducingitsmobilization.Overall,ourdatauncoversanewroleforthetumorsuppressor let-7miRNAs,which istomaintaingenomeintegritybyrestrictingL1retrotransposition.MaríaDoloresMoyaGarzón1,CristinaMartin-Higueras2,BárbaraE.Rodríguez-Rodríguez2,FranciscoFranco-Montalban1,AngelL.Pey3,JoséA.Gomez-Vidal1,EduardoSalido-Ruiz2,MónicaDiaz-Gavilan11MedicinalandOrganicChemistryDepartment,FacultyofPharmacy,UniversityofGranada,Granada18071,Spain2PathologyDepartment,FacultyofMedicine,UniversityofLaLaguna,Tenerife38320,Spain3DepartmentofPhysicalChemistry,FacultyofSciences,UniversityofGranada,Granada18071,SpainDesign,synthesisandevaluationofsalicylatesasdoubleGO/LDHAinhibitorswithapplication inthetreatmentofprimaryhyperoxaluriatype1Primaryhyperoxaluria type1 (PH1) is a rare genetic disease related todisorders in glyoxylatemetabolism. Thisinbornerroroflivermetabolismiscausedbyadeficitintheenzymealanineglyoxylateaminotransferase(AGT)duetodifferentmutationsaffectingtheAGXTgene.Underthesecircumstances,glyoxylateisoxidizedintooxalatebyenzymeslikeglycolateoxidase(GO)andlactatedehydrogenaseA(LDHA).Sinceoxalateconstitutesanend-productof metabolism, it precipitates as insoluble calcium oxalate crystals in the renal system, causing kidneymalfunctioninganddeterioration,andcombinedkidney-livertransplantation is theonlyhealingtreatment.Withthe aim of developing a useful pharmacological approach for the treatment of PH1, and following substratereductiontherapy(SRT)asourmainstrategy,weidentifiedfurylsalicylatesasmoderateGOinhibitorsandefficientagentsreducingoxalateoutputonAgxt1-/-mousehepatocytesculture.However,forsomeofourfurylsalicylatesbehavingasmodestGOinhibitors,wefoundasignificantreductioninoxalateproductioninculture,andtherefore,the possibility of additional targets for these compounds was considered. Both GO and LDHA are able tometabolizethesamesubstrate,glyoxylate.Infact,ithasbeenrecentlypublishedthatsiRNA-mediatedLDHAandGOinhibitionachieveefficientoxalatereductionandpreventsthedepositionofcalciumoxalatecrystalsinanimalmodels.Thus,wecontemplatedthatourinhibitorscouldalsobeabletobindtheactivesiteofLDHA,whichwould
leadtodualGO/LDHAinhibition.Inordertoverifythishypothesis,thosecompoundssuspectedtohaveadditionalbiological targetswere tested for LDHA inhibition.The resultsobtainedconfirm that someofourGO inhibitors,which resulted excellent agents decreasing oxalate production on Agxt1-/- hepatocytes, also present a notableinhibitoryactivityagainstLDHA.Additionally,dockingstudiesonGOandLDHAarebeingconductedtostudytheinteractionsbetweenourinhibitorsandtheseenzymes.Antonio Delgado González1,2,RosarioM. Sanchez-Martin1,2, Garry P. Nolan3,Wendy J. Fantl4, andJuanJ.Diaz-Mochon1,21 GENYO, Pfizer-Universidad de Granada-Junta de Andalucia Centre for Genomics and OncologicalResearch,Granada18016,Spain2DepartmentofMedicinalandOrganicChemistry,FacultyofPharmacy,UniversityofGranada,Granada18071,Spain3 Baxter Laboratory for Stem Cell Biology, Department of Microbiology & Immunology, StanfordUniversitySchoolofMedicine,Stanford,CA94305,USA4 Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, CA94305,USAMetallofluorescent nanoparticles as mass-tag barcodes for multiparametric mass-tag cellularbarcodingofovariancancercellsbymasscytometryMassCytometryallowsthedetectionofnearly40,andupto52,markerswithincells,withsinglecellresolution.Inmasscytometry,isotopesofrareearthelementsorotherseveralelements,whicharenotnaturallypresentedincells,aredetected.Asaconsequence,mass-tagcellularbarcodingcanbeachievedusingmass-tagreagents, likesmall molecules and barcoding agents based on antibodies, such as CD45. Nevertheless, small molecules areusuallytoxicforcells,whilstbarcodingagentsbasedonantibodiesaredependentoncellexpressionofantibodies.Alternatively, we have developed novel non-toxic metallofluorescent nanoparticles carrying a fluorophore andspecificpalladiumisotopesasmass-tagbarcodesformass-tagcellularbarcoding.Ournanoparticlescircumventthedrawbacks associated to conventional mass-tag barcodes as they are innocuous for cells and independent ofcellularprocesses.Therefore,thesenanoparticlescanbeusedasuniversalmass-tagbarcodesformass-tagcellularbarcoding of a myriad of cell types. In fact, polystyrene nanoparticles have been reported to be easily andefficiently internalizedwithinadherentcells,suspensioncells,stemcells,andprimarycells.Bearingthis inmind,three metallofluorescent nanoparticles containing different palladium isotopes (106Pd, 110Pd, and 106-110Pd)wereusedasmass-tagbarcodesforthespecificbarcodingofthreeovariancancercell lines(TYK-nu,Kuramochi,and OVCAR-4). Simultaneously, the cell lines were stained with a set of specific ovarian cancer cell panel ofantibodies for both surface and intracellular markers. The nanoparticles-based barcoding allowed the specificidentification of each barcoded cell line among a mixture of barcoded cells. Furthermore, specific proteinexpressionpatternanalysisof these threeovarian cancer cell lineshad confirmed that thisproposedbarcodingnanodevicedoesnotaffecttheirproteinexpression.Then,eachbarcodedcell linepatternmatchedwiththatofthe non-barcoded cell line. Therefore, a multiparametric and non-toxic mass-tag cellular barcoding usingmetallofluorescentnanoparticleswassuccessfullyaccomplished.
MatildeOrtizGonzález 1,2,JoséMaceira2,FernandoReyes3,JuanCantizani3,NuriadePedro3,IgnacioPérez-Victoria3MiguelSoriano4,FranciscadeVicente3,JoséA.GarcíaSalcedo1*1UnidaddeEnfermedadesInfecciosasyMicrobiología,InstitutodeInvestigaciónBiosanitariaibs,Granada.ComplejoHospitalarioUniversidaddeGranada/Granada.2GENYO.CentrodeGenómicae InvestigaciónOncológica:Pfizer/UniversidaddeGranada/ JuntadeAndalucía,Granada3 Fundación Centro de Excelencia en Investigación de Medicamentos Innovadores en Andalucía,MEDINA,Granada4CenterforIntensiveMediterraneanAgrosystemsAnewmemberofCurvicollidefamilykillsAfricantrypanosomesbyinhibitingtranscription.Africantrypanosomiasisisaserioushealthproblemwithanaddedsocio-economicimpactinsub-SaharanAfrica,due todirect infection inbothhumansand theirdomestic livestock.There isnovaccineavailableagainst theseAfricantrypanosomesandthemainreasonistheabilityoftheparasitetochangethemajorsurfaceglycoprotein(VSG) avoiding the antibody-mediated response. Most of the current drugs used to treat the disease presentresistance, toxicity and specificity problems. Therefore, there is a clear need of novel, safe, and affordabletreatments. In a search for new molecules with trypanocidal activity, we have performed a highthroughputscreeningof2000microbialextractsfromafungiandactinomycetesnaturalproductslibrary.Severalknownactivemoleculeswereidentified,includingCordycepin,CurvicollideA-C,Chaetocin,11-DeoxyverticillinA&VerticillinandalsoanewmemberofcurvicollidefamilywithanunreportedmolecularstructurethatwehavenamedCurvicollideD. The new compound showed an effective concentration 50 (EC50) of 1 uM, 16-fold lower than in humanhepatomaG2cells.TreatmentwithCurvicollideDinducedcellcyclearrest,causinganaccumulationofcellsintheG2/M phase. The new compound also induces morphological changes and at intracellular level, Curvicollide Dcausedthedisruptionofthenucleolarstructure.AnalyzingthenucleolusfunctionrevealedthatRNApolymeraseI(Pol I) transcription was inhibited at the ribosomal locus, but also at the locus where the variant surfaceglycoprotein isexpressed.WenextstudytheeffectonRNAPol II transcription, findingthat itwas inhibitedtoo.Finally, FID assay demonstrated that curvicollide D is an intercalator of duplex DNA. Taken together, this datademonstratethatCurvicollideDbindsDNAandinhibitstranscription,causingcelldeath.Inaddition,theseresultsprovideforthefirsttimeaninsightonthemechanismofactionofthemembersoftheCurvicollidefamily.
CarlosPerisTorres,MaríadelCarmenPlaza-Calonge,JuanCarlosRodríguez-ManzanequeGENYO, Centre for Genomics and Oncological Research: Pfizer/Universidad de Granada/Junta deAndalucía,Granada18016,SpainExtracellularproteaseADAMTS1isrequiredforuvealmelanomadevelopmentbyinducingstemnessandendothelial-likefeaturesontumorcells.Tumormicroenvironmentcompositionand itsremodelingbyextracellularmatrixproteaseshavebeenremarkedaskeyplayersduringtumorgrowth.Particularly,extracellularproteaseADAMTS1hasbeenassociatedwithtumordevelopmentbyinducingstemnessfeaturesandendothelial-likephenotypesintumorcells.Inthisworkweaimed
toevaluatetheroleofADAMTS1inhumanmelanoma.First,wecharacterizedandclassifiedeightmelanomacelllines according to their endothelial-like phenotype. We observed a positive correlation of ADAMTS1 geneexpressionandendothelial-likephenotypes,suggestingtheircloserelationship.ThenweeditedADAMTS1insomeof them with CRISPR-Cas9 and observed that ADAMTS1 deficiency affected their endothelial signature andphenotype. Remarkably, in vivo experiments with NOD scid gamma mice revealed that ADAMTS1-KO tumorsshowed a clear impairment of the vasculature and a significant down-regulation of endothelial and stemnessgenes,aswellasasubstantialreductionintumorgrowth.Likewise,immunohistologicalanalysesrevealedthattheclose stemness-endothelial spatial relationship of wild-type tumors was lost in ADAMTS1-KO ones. Moreover,TCGA public data allowed us to identify several of our genes of interest as poor prognosis factors in uvealmelanoma, including other ADAMTS proteases and endothelial-related genes. Focusing on the impairment ofstemnesscapacitieswhenADAMTS1 is inhibited,weevaluatedmelanomasphereformationcapacityofourcells,revealingthatonlywild-typecellswereabletogeneratethem.Furthermore,theyshowedaclearenrichmentonstemnessmarkers,alsocorrelatingwithanupregulationofADAMTS1andendothelial-relatedCDH5.Additionally,recombinant human ADAMTS1 recovered the sphere formation capacity in ADAMTS1-KO cells, highlighting theimportant roleofADAMTS1 in the initial stepsof tumorgrowth.Taking together,ourdatadescribe for the firsttimetheprotumorigenicroleofADAMTS1anditsrequirementforhumanuvealmelanomadevelopmentasmainplayer in the complex crosstalkbetween stemness features, endothelial-likephenotypeandextracellularmatrixregulation.María Tristán Manzano 1, Noelia Maldonado-Pérez1, Pedro Justicia1, Sabina Sánchez-Hernández1,Marina Cortijo-Gutierrez1, Pilar Muñoz1, Kristina Pavlovic2, MDolores Carmona2, ConcepciónMarañón1,ManelJuan3,ConchaHerrera2,KarimBenabdellah1andFranciscoMartin11:GENyO-CentrodeGenómicaeInvestigaciónOncológica:PfizerUniversidaddeGranada/JuntadeAndalucía(Spain)2:HospitalClínicoUniversitarioReinaSofía-UnidadTerapiaCelular(Spain).3:DepartmentofImmunology,HospitalClínicdeBarcelona(Spain).Developmentoflentiviralvectorsforafine-tunedregulatedimmunotherapy.T cells expressing Chimeric Antigen Receptors (CAR-T cells) have emerged to boost immunotherapy againstrefractorycancers.Anti-CD19-CAR-Tcellshaveresultedremarkablysuccessfulbutstillencloses important issuesthat compromise the safety (cytokine release syndrome, neurologic toxicity, B-cell aplasia) and efficacy of thetherapy(poorlyregulatedCAR-Tcellsleadtoexhaustionandrelapsing).Toovercomethesechallenges,weaimtodeveloplentiviralvectors(LVs)thatcanregulateCARexpressionlevelsinordertocontrolCAR-Tcellactivity.Wepursuedtwodifferentapproaches:1)ControltheCARexpressionbyexternalfactorsand2)Achieveaphysiological(TCR-like)CARexpression.Forthefirststrategy,weshowedthattheLentOnPlus-Is2-LV,previouslygeneratedbyour group, is a good platform to regulate transgene expression in T cells in vitro and in vivo without therequirement of transactivators. The system exhibited very low leaking and good sensitivity to Doxycycline(<1ng/mlinvitroand<1ug/mlinvivo).Basedonthat,weproducedinducible3rdgeneration-CAR-Tcells(iCARIs2-Tcells),thatlysedspecificallyCD19+cellsonlyinthepresenceofDox.Forthesecondapproach,wefirstanalyzedthe expression kinetic of different LVs in T cells after activation and compared themwith the TCRpattern.WefoundthatEF1-alphapromoter,themostusedinclinics,increasedtransgeneexpression,unlikethephysiologicaldownregulation of TCR. Interestingly, LVs harboring a chimeric promoter derived from theWAS locus (AWE), aproteinimplicatedinTCRsignaling,wasapromisingcandidateforTCR-likeCAR-Tcells.Wethereforeconstructedclinical LVs-expressing an anti-CD19-CAR under the control of WAS promoter(AWARI) that lysed specificallyCD19+cells lines in vitro and in vivo with similar efficacy compared to EF1alpha-CAR-LVs, but using a morephysiologicalCARkinetics.WearecurrentlyanalyzingexhaustionmarkersandpersistencetoinvestigatewhetherWAS-CAR-TcellsaremoreefficientcomparedtoEF1-alpha-CAR-Tcellsinvivo.
POSTDOCTORALSESSIONS Juan Carlos Alvarez Pérez1,3, Juan Sanjuan3, Alberto Arenas1,3, Carlos Baliñas1,3, María IsabelRodríguez1,3,ÁlvaroAndrade1,3,MartaCuadros2,3,andPedroP.Medina*1,3.1UniversidaddeGranada,DepartmentofBiochemistryandMolecularBiologyI,Granada,Spain.2 Universidad de Granada, Department of Biochemistry and Molecular Biology III and Immunology,Granada,Spain.3GeneExpressionRegulationandCancerGroup(CTS-993),CenterforGenomicsandOncologyResearch(GenyO),Granada,Spain.Targeting of KRAS oncogenic mutations with CRISPR-Cas9 reduces the tumorigenic potential ofhumanizedMEFs.CancerisageneticdiseasewheresomecellswithDNAdamagedbegintodividewithoutstoppingandspreadintosurrounding tissues. Interestingly, some tumors rely onmutations in key oncogenes (drivermutations) to drivecarcinogenesis and maintain the tumor phenotype. Suggestively, if we can disrupt these mutations, we candamageanestablished tumoralphenotype.We seekout forusingCrispr-Cas9 technology to targetKRASdrivermutations and evaluate its therapeutic value on humanized MEFs. Mutated RAS genes represent the mostfrequentlymutatedoncogenefamilyandarepresentinapproximately25%ofhumantumorsincludingpancreaticductal adenocarcinomas (PDACs), colorectal adenocarcinomas (CRCs), lung adenocarcinomas, and gastricadenocarcinomas.MutationsthatactivateKRASaccountfor30%ofthe1.6millionannualcasesoflungcancer,thedeadliest type of cancer. Most of these KRAS mutations are located on codon 12 being KRAS G12C thepredominantmutation subtype in smokers,whileG12D themost common subtypeof KRASmutations inneversmokers. We designed gRNA-guided-CRISPR-Cas9 to specifically target the two single-nucleotide missensemutationsonKRAScodon-12,whileleavingKRASWTuntouched.DisruptionofKRASG12DgeneusingitsspecificgRNAdecreases the tumorogenicpotential of KRASG12D-MEFs,measuredby lossof contact growth inhibition,withoutmodifyingtheirproliferativeability.
Maria Belen Lopez-Millan1, Diego Sanchéz-Martínez1 Heleia Roca-Ho1 Francisco Gutiérrez-Agüera1Oscar Molina1 Rafael Diaz de la Guardia1 Raúl Torres-Ruiz1,2 Jose Luís Fuster3 Paola Ballerini4 UteSuessbier5,CesarNombela-Arrieta,5ClaraBueno1,6,PabloMenéndez1,6,71 Department of Biomedicine, School of Medicine, Josep Carreras Leukemia Research Institute,UniversityofBarcelona,Barcelona,Spain2MolecularCytogeneticsGroup,HumanCancerGeneticsProgram,CentroNacionaldeInvestigacionesOncológicas(CNIO),Madrid,Spain3PediatricHematologyandOncologySection,HospitalClínicoVirgendelaArrixaca,Murcia,Spain4PediatricHematology,ArmandTrousseauHospital,Paris,France5HematologyDepartment,UniversityHospital-UniversityofZurich,Zurich,Switzerland6CentrodeInvestigacionBiomedicaenRed-Oncología(CIBERONC),Zurich,Switzerland7InstituciòCatalanadeRecercaiEstudisAvançats(ICREA),Barcelona,SpainNG2antigenisatherapeutictargetforMLL-rearrangedB-cellacutelymphoblasticleukemia.B cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer, with cure rates of 80%.MLLrearranged (MLLr) B-ALL (MLLr-B-ALL) has, however, an unfavorable prognosis with common therapyrefractorinessandearlyrelapse,andthereforenewtherapeutictargetsareneededforrelapsed/refractoryMLLr-B-ALL. MLLr leukemias are characterized by the specific expression of chondroitin sulfate proteoglycan-4, alsoknown as neuron-glial antigen-2 (NG2). NG2was recently shown involved in leukemia invasiveness and centralnervous system infiltration in MLLr-B-ALL, and correlated with lower event-free survival (EFS). We here
hypothesizedthatblockingNG2maysynergizewithestablishedinductiontherapyforB-ALLbasedonvincristine,glucocorticoids, and L-asparaginase (VxL). Using robust patient-derived xenograft (PDX) models, we found thatNG2iscrucial forMLLr-B-ALLengraftmentupon intravenous(i.v.) transplantation. InvivoblockadeofNG2usingeither chondroitinase-ABC or an anti-NG2-specific monoclonal antibody (MoAb) resulted in a significantmobilizationofMLLr-B-ALLblastsfrombonemarrow(BM)toperipheralblood(PB)asdemonstratedbycytometricand 3D confocal imaging analysis.When combinedwith either NG2 antagonist, VxL treatment achieved higherratesofcompleteremission,andconsequentlyhigherEFSanddelayedtimetorelapse.Mechanistically,anti-NG2MoAbinducesneitherantibody-dependentcell-mediatednotcomplement-dependentcytotoxicity.NG2blockaderather overrides BM stroma-mediated chemoprotection through PB mobilization of MLLr-BALL blasts, thusbecomingmoreaccessible tochemotherapy.Weprovideaproofofconcept forNG2asa therapeutic target forMLLr-B-ALL.BelénRubioRuiz1,2,MaríaSancho-Albero3,AnaMaríaPérez-López1,VíctorSebastián3,PilarMartínDuque3,ManuelArruebo3,JesúsSantamaría3,AsierUnciti-Broceta11CancerResearchUKEdinburghCentre,MRCInstituteofGenetics&MolecularMedicine,UniversityofEdinburgh,CreweRoadSouth,EdinburghEH42XR,UK.2Pfizer-UniversityofGranada-AndalusianRegionalGovernmentCentreforGenomicsandOncologicalResearch(GENYO)andDepartmentofMedicinal&OrganicChemistry,FacultyofPharmacy,UniversityofGranada,CampusdeCartujas/n,18071Granada,Spain.3DepartmentofChemicalEngineering,AragonInstituteofNanoscience(INA),UniversityofZaragoza,Zaragoza,Spain.DevelopmentofPd-loadedexosomesfortargetedbioorthogonalcatalysis.Palladium (Pd) catalysts have played a prominent role in the growing subfield of bioorthogonal catalysis byproducing xenobiotics and uncaging biomolecules in living systems [1]. Although effective intracellular catalystsbased on abioticmetals have been reported in the literature [2], the development of devices able not only todisplaybioorthogonalcatalyticactivitybutalsobepreciselydeliveredtospecificanatomicallocationsisstillinitsinfancy. Exosomes are membrane-enclosed vesicles released by cells to the extracellular space to mediateintercellularexchangeofbiomolecules.Thebiochemical compositionof theexosomalmembrane is reflectiveofthedonorcell,thusinvestingexosomeswithselectivetropismforcellsthatareparentedtotheircelloforigin[3].Basedonthisproperty,weenvisagedthatexosomescouldbeideallysuitedtoprotectanddeliveracatalyticcargointospecificcancercells.Herein,wepresentthedevelopmentofabio-artificialdeviceconsistingofA549cancer-derivedexosomesloadedwithultrathinPdnanosheets.Theresultingbioartificialvesicles(Pd-ExoA549)arehighlycatalyticunderphysiologicalconditions,capableofselectivelydeliveringtheircatalyticcargototheir targetcellsandactivateanovelprodrugoftherecentlyapprovedanticancerdrugpanobinostatinsidethembyPd-mediateddealkylation.
POSTERS MASTERSTUDENT
1. Study of the role of ADAMTS1 to modulate macrophage polarization toward a protumorigenic. RitaCaracuelPernos,Genyo.
2. Optimization of the deliverymethod for the knock-in in vitro with the CRISPR-Cas9 system. Iris RamosHernández,Genyo.
3. Validación de hPSCs con expresión de NUP98-JARID1A como un modelo in vitro de leucemia mieloideagudainfantil.NatividadAlcortaSevillano,Genyo.
8. Magnetoliposomesproductionandevaluationashyperthermiaagents.AbelGarcíaDíaz,Genyo.9. Generación de modelos celulares de Síndrome de Bernard-Soulier mediante edición con CRISPR/Cas9.
MaríaMartínezManzano,Genyo.10. Analysis of the reliability of the imputation of rare variation through the use of genotyped common
13. Generation of cellular models to study gene therapy strategies for Pompe disease. Araceli AguilarGonzález,Genyo.
14. Gene-celltherapyforpompedisease.JuanElíasGonzálezCorrea,Genyo.15. Development of a pipeline for identifying non-coding drivermutations in lung adenocarcinoma. Álvaro
