SATURDAY, DECEMBER 7, 2019

POSTER ABSTRACTS SESSION: 5:30 PM-7:30 PM

631.ChronicMyeloidLeukemia:BiologyandPathophysiology,excludingTherapy:PosterI

Saturday,December7,2019:5:30PM-7:30PM

HallB,Level2(OrangeCountyConventionCenter)

1632 The Embryonic Program Activated during Blast Crisis of ChronicMyelogenous Leukemia (CML) Implicates a TCF7L2 and MYC CooperativeChromatinBindingandRepresentsaDruggableTarget

ChristopheDesterke,PhD

Chronic myeloid leukemia (CML) is characterized by an inherent geneticinstability,whichcontributestotheprogressionofthediseasetowardsacceleratedandblastcrisis(BC).Theoccurrenceofthelatterhasbeenhamperedbytheuseoftyrosine kinase inhibitors (TKI),which changed this natural progression, butBCstill occurs in patients resistant to TKI. Several cytogenetic (major and minorroutes) and genomic (TP53 mutation, p16/INK4A deletions, DNA repairabnormalities such as BRCA1, DNA-PKcs, hnRNPmetabolism) events have beenreported in the progression towards BC. Previous data have also suggested theinvolvementofembryonicstemcellprogramactivatedinBCcellssuchasLin28A.Inthiswork,wehavetakenadvantageofthepreviouslyreportedgeneprofilingofBC in a large cohort of patients (Radich et al. 2006) and found a correlationbetweenblastnumbersand the involvementof theTranscriptionFactor7 like2(TCF7L2)inBC.TFC7L2isamemberoftheTCFfamilyofproteinsthatareknownto activateWNT target genes such as Cyclin D1. TCF7L2 has been shown to beoverexpressedinacutemyeloidleukemia(AML)andrepresentsadruggabletarget(Saenz et al Leukemia 2019). The involvement of TCF7L2 in CML-BC and itsinteractionwith the epigenetic regulators has not been studied so far. The genecorrelation study that we have performed using the blast numbers and theexpression of TCF7L2 in CD34+ CML cells was found to be highly significant(Pearsontest,r=0.56,p-value=5.2e-4)(Fig.1A).TCF7L2promoterwasclassedasactive in K562 with ChromHMM Functional genomic analysis. K562 epigeneticspeaksofTCF7L2CHIP-seqwerefoundprincipallymappedinproximalpromoters(39%ofthepeaks, -3000pbupstreamTranscriptionStartingSites(TSS),Fig.1B)and183uniquepeaksmatchedwith promoter of 144unique genes found to becorrelated to the blast number in blood of the CMLpatients duringBC (Fig 1C).This TCF7L2-dependent BC program was characterized to be active becausepromoters were also found positive for H3K27Ac and negative for H3K27Me3histonemarks,andfunctionallyenrichedwithbindingsitesforMYC/MAX

interactions (p=1.15e-6). The analysis of CHIP-sequencing of MYC revealed asignificantoverlappingofTCF7L2epigeneticprogramwithMYC(foldenrichment:20.81,p<2.2e-16).Surprisingly, theTCF7L2programwas found independentofRUNX1 and GATA2 transcriptional program. To determine these potentialinteractions,wehavedesignedexperiments inK562cell lineusing theb-cateninactivator Lithium Chloride (LiCL2) and the Myc/Max dimerization inhibitor10058-F4.K562cellswerecultured in thepresenceofLiCL2(10mM&24hours)and the compound 10058-F4 (64µM & 48hours) and the expression of threeepigenetic targetswas analyzedbyQ-RT-PCR in the presence ofDMSO controls.ThethreetargetschosenwereproteinarginineN-methyltransferase(PRMT1),theATPase/Helicase RUVBL1 and the WD-repeat containing protein WDR77. Asexpected,after culturewithLiCL2, theexpressionofPRMT1was increasedx6.3fold(p=8.49e-13),thatofRUVBL1byx1.66Fold(p=1.67e-6)andthatofWDR77by x 2 fold (p=4.97e) (Fig.1D). On the contrary, the culture of K562 cells in thepresenceofMYC/MAX inhibitor10058-F4,decreased theexpressionof3 targetsascomparedtoDMSOcontrols(x1.6foldforPRMT1,p=6.52e-5;x2foldreductionfor RUVBL1, p-value=2.71e-5; and x 1.4 fold forWDR77, p =0.0000643). TheseresultsshowforthefirsttimeacooperativeroleofTCF7L2andMYCduringblastcrisisofCMLandprovidemechanisticinsightsintotheinteractionsfortheroleofMYC in CML blast crisis. In addition they strengthen previous data showing apossible embryonic footprint in the blast development over the hematopoieticdifferentiationprogramduringprogressionofthediseaseandprovidearationaleforthepharmacologicaltargetingofBCbytheuseofMYC/MAXinhibitorssuchas10058-F4.ExperimentsareunderwaytoevaluatetheroleofthesefactorsandtheMYC/MAXinhibitorsinprimaryCMLsamples.

1633 Shikonin Overcomes Drug Resistance and Induces Necroptosis ByRegulatingtheMir-92a-1-5p/MlklAxisinChronicMyeloidLeukemiaCells

XianboHuang,MD,PhD

BACKGROUND: Resistance to cell death and metabolic reprogramming arecommon featuresof tumorcells.Although the introductionof selectiveBCR/ABLtyrosine kinase inhibitors (TKIs) has dramatically improved the outcomes andsurvival rates of chronic myeloid leukemia (CML) patients, some patients (20-30%)developTKIresistance.Themostaggressiveandtreatment-resistantCMListhe subtype harboring BCR/ABL with the T315I mutation, and this subtype isrefractory tonearly all TKI-induced apoptosis. Thus, alternative approaches thatinduceapoptosis-independentcelldeatharethoughttocompensateforapoptotic-resistantcells.Recently,necroptosis (alsocalledprogrammednecrosis),which isgenerallydrivenbyRIPK1/RIPK3/MLKLactivation,hasbeendemonstratedtobeanewtypeofprogrammedcelldeathmodethatisdifferentfromapoptosis.Thus,adeeperunderstandingofthemolecularmechanismsregulatingnecroptosismightlead to the development of new therapeutic strategies that could remarkablyimprove the treatment-responses and outcomes of patients with TKI-resistantCML.

RESULTS: Shikonin,a compoundpurified fromtraditionalChinesemedicine,hasbeenreportedtoinducecelldeathinvarioustumorcell linesviaawiderangeofmechanisms. Inour current study,we found that shikonin caneffectively inhibitproliferation and induce necrosis-like morphological alterations (Fig. A and B)accompanied by RIPK1/RIPK3/MLKL signaling activation ((Fig. C) in CML celllines, including theT315Imutant type (32Dp210-T315I).Theeffectsof shikonincan be attenuated by the necroptosis-specific inhibitor (essentially a RIPK1inhibitor)Nec-1,butnotbythepan-apoptosisinhibitorz-VAD-fmk,indicatingtheoccurrenceof necroptosis in these cells ((Fig.B andC).Ourdata also show thatshikoninhasinvivoanti-CMLactivityvianecroptosisinductionin32Dp210-T315IcellsxenograftedintoNOD/SCIDmiceviasubcutaneousinjection((Fig.D).

miRNAs play an important role in tumorigenesis mainly via regulation of geneexpression.Ournextgenerationsequencing-basedmicroRNAexpressionprofilingshowedsignificantdysregulationofmiR-92a-1-5pexpressioninashikonin-treatedCML cell line (K562) (Fig. E). We then measured the miR-92a-1-5p expressionlevels in bone marrow samples from CML patients and patients withnonhematologic malignant diseases. The data showed that the miR-92a-1-5pexpressionlevelwashigherinprimarycellsobtainedfromCML-BCpatientsthaninthosefromnon-CML-CPpatients,suggestingthatmiR-92a-1-5pupregulationiscorrelated with poor outcomes (Fig. F). Bioinformatics analyses and a dualluciferase reporter gene assay proved that MLKL, a downstream factor in thenecroptosispathwaythatusuallyactsasthenecroptosisexecutor,isadirecttargetofmiR-92a-1-5p(Fig.G).OverexpressionofmiR-92a-1-5pinvitroledtodecreasedMLKLproteinabundanceinCMLcells(Fig.G).InhibitionofmiR-92a-1-5pviauseofaspecificantago-miRNAcouldinhibitCMLxenografttumorgrowthandinduce

necroptosis viaMLKLupregulation in vivo (Fig.H).Hence,we believe thatmiR-92a-1-5p plays a role in promoting the proliferation and survival of CML viadownregulatingtheabundanceofMLKL,thenecroptosisexecutor.

CONCLUSIONS: In conclusion, our studyproves that shikonin canovercomeTKIresistanceandinducenecroptosisinCMLcells,mainlyviaamechanisminvolvingRIPK1/RIPK3/MLKL activation. Our study also suggests that miR-92a-1-5p isfrequently overexpressed in CML patients with poor outcomes and that it canpromote tumor survival by inhibiting MLKL expression. For the first time, wedemonstrated thatmiR-92a-1-5p inhibition via antago-miRNA canpotentially beused to treat CML via necroptosis induction. Since necroptosis has not yet beenconsideredtobeatherapeuticstrategyfortumortreatment,ourresearchconfirmsthatitmightindeedserveasanewmodalitytobettercontroldrug-resistantCML.

1634SplenicCD24lowRedPulpMacrophagesProvideanAlternateNicheforChronicMyeloidLeukemiaStemCells

MichaelAAmrein,MD,PhD

Chronicmyeloid leukemia (CML) isa typical stem-celldrivenmalignancy,drivenby leukemia stemcells (LSCs). LSCsare resistant to conventional therapies.Thisresistance is mediated by cell-intrinsic mechanisms and interactions with themicroenvironment.LSCsdependonsignalsfromaspecializedmicroenvironment,a so called niche, to maintain their stem cell characteristics. In CML the bonemarrow(BM)asanicheiswell-investigatedandseveraltherapeutictargets,whichaim at LSCs by interrupting their interaction with the BM-niche are underinvestigation. However, even though splenomegaly is a hallmark of CML thecontributionof the splenicmicroenvironment toCMLdevelopmenthasnotbeenstudied so far. This project aims to investigate the role of the splenicmicroenvironmentasanindependentsecondaryLSCnicheanditscontributiontodiseasedevelopment.

To induce a CML-like disease in mice we retrovirally transduced FACS-sortedLineage-Sca-1+cKit+BMcellswithpMSCV-p210BCR/ABL-IRES-GFPandinjectedthetransducedcellsintonon-irradiatedmice.

To findout if the spleen contributes todiseasedevelopmentwe inducedCML insplenectomized and sham operated mice. Splenectomized mice survivedsignificantly longercompared toshamoperatedcontrols (mediansurvival31vs.22days;p=0.0006)with20%ofthesplenectomizedmicesurvivinglongerthan90days. Moreover, the number of LSCs in the BM of splenectomized mice wasreduced3.7-fold(p=0.002).

FlowcytometricanalysisofthespleenandBMcompartmentsofCMLbearingmicerevealedthatthemajorityoftheleukemicstemandprogenitorcells(LSPCs)werelocated in the spleen (19-foldmore LSCs in the spleen; p =0.007).MoreoverwefoundtheleukemiccompartmentinthespleentobeenrichedforLSPCscomparedtotheBM(20%spleenvs.10%BM;%LSPCsoftotalleukemiccells;p=0.01).ToconfirmthisphenotypicobservationfunctionallyweperformedalimitingdilutiontransplantationofleukemiccellsfromspleenandBM.InlinewiththephenotypicobservationwefoundahigherfrequencyofLSCsinthespleencomparedtotheBM(1/41'703vs.1/432'594;p=0.02).WenextanalyzedthegeneexpressionofLSPCsfromspleenandBM.We found that thegeneexpressionprofileof splenicLSPCsshowed higher expression of stemness-related genes and reduced expression ofmyeloiddifferentiationgenescomparedtoBMLSPCs,indicatingthatthespleenismoresupportiveofprimitiveLSPCs.

Knowing that the spleen contributes to disease development by providing analternatenicheforLSCswenextanalyzedthespleensusingconfocalmicroscopy.WefoundthattheLSCsresidedexclusivelyintheredpulp.PreviousstudieshaveshownthatHSCsresideindirectcontactwithredpulpmacrophages(RPMs)

during extramedullary hematopoiesis (Dutta et al., JEM, 2015). In addition wefound that in spleens from human CML patients CD34+ leukemia cells localizedtogetherwithmacrophages(p=0.001).FurthermorewecouldshowthatRPMsarecapableofproducingbothSCFandG-CSF.TotesttheroleofRPMsasapotentialnichecomponentinvitroweco-incubatedLSCsandRPMsovernightbeforeplatingtheLSCsinacolonyformationassay.Wefoundthattheco-incubationwithRPMsimprovedthecolonyformationcapacityofLSCs(CFUs166vs.138;p=0.0356).Totest the role of RPMs in vivo we depleted macrophages in CML mice usingclodronateliposomes.Thisresultedinsignificantlyreducedsplenomegaly(867mgvs. 249mg; p<0.0001) and reduced numbers of splenic LSCs (23-fold; p=0.001).ThiswasfurtherconfirmedinageneticallyengineeredmousemodellackingRPMs(Spic-/-)(735mgvs.450mg;p=0.0112and22-foldfewerLSCs;p<0.0001).Finally,while in naïve mice RPMs can be differentiated into a CD24low and CD24highpopulation,theCD24highpopulationislostinCMLbearingmice.

In summary, we found that the spleen provides an alternate niche for LSCs,therebycontributingtoCMLdevelopment.ComparedtotheBMnichethesplenicnicheismoresupportiveofprimitiveLSPCs,asshownbythehigherfrequencyofLSCSsfoundinthespleenandthehigherexpressionofstemnessrelatedgenesinsplenic LSCs compared toBMLSCs.Moreoverwe identifiedCD24lowRPMs as aunique and central component of the splenic LSC niche. Even though we couldshowthatRPMsarecapableofproducingSCFandG-CSFtheexactmechanismsbywhichRPMssupportLSCsremainstobeinvestigated.

1635 Mechanistic Insights into the Inhibition of T Regulatory Cells ByDasatinibMayPredictImmunostimulatoryEffectsinCMLPatients

PatrickHarrington,MRCP,FRCPath

Background:Dasatinibpotently inhibits theSrc familykinaseLckat therapeuticconcentrations.LckplaysacriticalroleinsignallingfromtheTcellreceptorwithimmediate downstream targets including ZAP70 and LAT. The cell surfacescaffoldingproteinLAThasbeenpreviouslyshowntoplayacentralroleinnormalTregdevelopment.STAT5,thedownstreamtargetofIL-2,alsoplaysacriticalroleinTregdifferentiationandmaintenanceofFOXP3expressionthroughitsbindingofthepromoterregionoftheFOXP3gene.

The proportion of Tregs has been shown to inversely correlate with improvedmolecular response in patients on dasatinib and a subset of patients takingdasatinibwill develop a large granular lymphocytosis (LGL)which is associatedwith inflammatory toxicity and improved outcome (Mustjoki et al. Leukaemia2009).

WehypothesisedthatareductioninTregfrequencyandfunctionwouldcorrelatewiththeexpansionofaclonalLGLpopulationincertainpatientstakingdasatinib.To investigate this,weperformedex-vivoanalysisof thephosphorylationofkeycell signalling proteins and intracellular cytokine production in Tregs and TeffectorsfromCMLpatientsandhealthycontrols.

Methods:WefirstperformedphosphoflowcytometryinTregsandTeffectorstoassess the effect of dasatinib on signalling downstream from the TCR, includingactivated/phosphorylatedZAP70(pZAP70),LAT(pLAT)andSTAT5(pSTAT5).11-colourflowcytometrywasperformedaftercellswereactivatedwithH2O2for15minutes,duetoitsactivityasapotentphosphataseinhibitor.AgatingstrategyofCD3+/CD4+/CD25+/FOXP3+/CD127locellswasusedforidentificationofTregs.

Wethenperformed10-colourintracellularflowcytometryassessingtheimpactofdasatiniboncellularcytokineproductionincludingTNF,IFNgamma,IL-2,IL-4andIL-10afterstimulationwithOKT3.

Results: 8patientswithCMLand2healthy controlswere recruited.Of theCMLpatients5weretakingdasatinib,100mgdaily, twoweretaking imatinibandonenilotinib.PatientsondasatinibhadlowerproportionofTregscomparedwiththenondasatinibgroupwithameanproportionofCD3+cellsof1.4vs2.1.

Patients on dasatinib had significantly reduced phosphorylation of ZAP70compared with the non dasatinib group in CD3+ cells, CD4+ cells and Tregs,followingH2O2stimulation.Themean increase inmedian fluorescence intensity(MFI)ofpZAP70was1.7vs3.6inCD3+cells,1.4vs3.5inCD4+cellsand1.5vs2.8inTregs(p=0.006,p=0.004,p=0.045).Similarly,pLATshowedalowerincreaseinphosphorylationinthedasatinibgroupinallTcellsubsetsevaluated,withmeanincreaseinMFIof4.3vs11.9inCD3+cells,4vs12.3inCD4+cellsand4.3vs8.2inTregs.STAT5alsoshowedreducedphosphorylationinthedasatinibgroupwithameanincreaseinMFIof6.4vs21.2inCD3+cells,5.7vs20.3inCD4+cellsand5.9vs19.7inTregs(p=0.007,p=0.003,p=0.01).

TwopatientsondasatinibhadreversalofnormalCD4:CD8ratio,withoneofthesealsohavingabsolute lymphocytosis, inkeepingwith likelydevelopmentofclonalLGLpopulations.Thesepatientshad lower increase inMFIofpZAP70,pLATandpSTAT5 following stimulation within isolated Tregs compared to patients ondasatinibwithnormalCD4:CD8(Figure1A).

WithinCD4+cellsthemeanproportionalincreaseinIL-2productionwaslowerinthe dasatinib group at 0.4 vs 5 (p=0.003). Using an additional panel, a singlepatient was also evaluated and shown to have strikingly reduced production ofTNFwithin isolatedTregsandIl-2withinCD4+cellscomparedtocontrol(Figure1B).

Conclusion:DasatinibinhibitsphosphorylationofZAP70,LATandSTAT5withinT cell subsets including Tregs, with the strongest effect seen against STAT5. Inaddition, dasatinib causes a reduction in pro-inflammatory cytokine productionwithin CD4+ cells, with the most significant inhibitory effect seen against IL-2.Tregs have abundant expression of the IL-2 receptor on the cell surface andbindingleadstoSTAT5signallingresultingintranscriptionofFOXP3.

A more pronounced inhibition of phosphorylation of ZAP70, LAT and STAT5 isseenintwopatientsondasatinibwithreversalofCD4:CD8whencomparedwithotherpatientsondasatinib,suggestiveofamorepotentreductionofTregfunction.ReductioninnumberandfunctionofTregsmightexplaintheimmunostimulatoryeffectsseeninpatientswhodevelopamonoclonalLGLpopulation.

1636The InteractionofTumorCellsandMyeloid-DerivedSuppressorCellsinChronicMyelogenousLeukemia

HuiXu

Chronic myelogenous leukemia (CML) is a malignant myeloproliferative diseasecharacterized by the formation of the BCR-ABL fusion gene. At present, basicstudies of the pathogenesis of relapse after stopping tyrosine kinase inhibitors(TKIs) treatment have mainly concentrated on two main aspects: the leukemiastemcells(LSCs)andthetumormicroenvironment.However,whetherrelapseornon-relapsepatientswhodiscontinuedTKIstherapy,LSCsarestillexist.Amongavariety of factors that compose the CML microenvironment, myeloid-derivedsuppressor cells (MDSC) are considered to be a strong contributor to theimmunosuppressive tumor microenvironment. Here, we designed the study toinvestigatethepotentialrelationbetweentumorcellsandMDSCinCMLandfindriskfactorsforrelapseafterdiscontinuation.

We detected the percentage of MDSC and the BCR-ABL (IS) transcript levels inbonemarrowof50CMLpatientsinchronicphaseatourcenter.ThedataindicatedthatthefrequencyofMDSChadsignificantpositivecorrelationwithBCR-ABL(IS)transcript levels (Figure 1A). In addition, the counts of MDSC had significantdifference at different response stages (Figure 1B), especially the M-MDSC, asubtypeofMDSC.ThepercentageofM-MDSCwassignificantlyhigher inpatientswith newly diagnosed or complete hematological response (CHR) or majormolecular response (MMR) compared with those of CML patients obtainedcompletemolecularresponse(CMR)(Figure1C).WhenK562cellsorCD34+cellswere cocultured with M-MDSC at a 1:10 ratio, K562 cells or CD34+ cellsproliferated significantly at day 3 (Figure 1D and E). K562 subcutaneous tumorformationinBALB/cnodemiceconfirmedthattumorsweightandvolumeofthecoculturegroupwerehigherthancontrol.

Then, we further investigated whether tumor cells have an impact on MDSCthrough microvesicles (MV). After adding K562-MV to peripheral bloodmononuclear cells (PBMCs) from healthy donors, MDSC counts appearedsignificantly elevated in the different K562 cells counts group compared to thecontrol group (Figure 1F).To analyze the roles of K562-MV collected before andafterTKIsdiscontinuationonMDSC,weestablishedaTKIsdiscontinuationmodelusing the K562 cell, which emulates the cessation of TKIs treatment of CMLpatients in some extent. The results showed that regardless of the Imatinib orDasatinib treatment, a significant increase was observed in the proportion ofMDSC after TKIs treatment cessation comparedwith the TKIs treatment groups(Figure1G).Experiments invivoalsoprovedK562-MVafterdifferenttreatmentspromotedtheproliferationofMDSC(Figure1HandI).

Inconclusion,ourstudyintroducesthenotionoftheroleofMDSCasmediatorsinthe cross talk between tumor cells and the microenvironment. MDSC wouldprovideanovelandusefulmodel topredict therelapseofCMLbyestablishingatypeofnewriskstratificationsystem.MDSCcouldbealsoactasapromisingtargetintherelapseofCML.Inaddition,wefoundamutualpromotionofproliferationoftumor cells andMDSC, this bidirectional interaction results in a vicious cycle byproviding a protective niche against immune attacks. Therapeutic interventionsmodulating this interaction might accelerate the success of treatment-freeremission.

1637 Pioglitazone Did Not Affect PPAR-Γ, STAT5, HIF2α and CITED2 GeneExpression in Chronic Myeloid Leukemia Patients with Deep MolecularResponse

AnaBeatrizPascoalLopes

Preliminary reports demonstrated that pioglitazone, an antidiabetic drug that isagonistofperoxisomeproliferator-activatedreceptorgamma(PPAR-γ)wasabletoreduceexpressionofSTAT5anditsdownstreamtargetsHIF2αandCITED2,whichare key guardians of the quiescence and stemness of chronicmyeloid leukemia(CML) leukemiastemcells (LSCs).LeavingquiescencewouldturntheLSCsmoresensitivetoimatinib(IM)andcauseanerosionoftheLSCs.Thiswasdemonstratedin vitro and in vivo in CML patients that achieved completemolecular responseafterpioglitazoneuse.Thiswas therational for thedesignofEDI-PIO trial (PilotStudyofImatinibDiscontinuationinPatientswithChronicMyeloidLeukemiawithDeep Molecular Response – Evaluation of Pioglitazone in Treatment-FreeRemission) (NCT02852486). In this trial, pioglitazone was given in associationwith IM, with the aim to pull out the LSCs from the quiescence and sensitizingthemtoIMeffect,increasingtreatment-freeremission(TFR)ratesaftertreatmentinterruption.

Aims:toevaluatePPAR-γ,STAT5,HIF2αandCITED2geneexpressionbeforeandafterpioglitazoneuseinCMLpatientswithcriteriaforIMdiscontinuation

Patients andmethods: EDI-PIO is a prospective, phase II trial. Inclusion criteria:CML in chronic phase, treated with IM for at least 3 years, with stable deepmolecularresponse(MR4.5)foratleast2years.Patientsreceivedpioglitazone30mg/day, orally, for 3 months before IM discontinuation. BCR-ABL levels weremeasured by real-time quantitative PCR monthly in the first year afterdiscontinuation, every twomonths in the secondyear, and thenevery3monthsduring the subsequent follow-up. Imatinib was reinitiated at molecular relapse(loss of major molecular response or confirmed loss of MR4.0). Total RNA wasextracted from peripheral blood leukocytes, pre and post pioglitazone, and at 3and6monthsafterIMdiscontinuation.AftercDNAsynthesis,analiquotwasusedfor gene expression analysis by reverse transcriptase quantitative polymerasechain reaction (RT-qPCR), using specific primers for PPAR-γ, STAT5, HIF2α andCITED2.Therelativegeneexpressionwascalculatedusingtheequation,2-ΔΔCT.GAPDHwasusedascontrolgene.StatisticalanalysiswasperformedusingANOVA.Treatment-free remission (TFR) was calculated from IM discontinuation untilmolecular relapse, reintroduction of IM by any cause, progression to advancedphasesordeathtoanycause.

Results:Thestudyisclosedforenrollment.BetweenJune2016andJanuary2019,32chronicphaseCMLpatientswererecruited,ofwhich30patientswereincludedin gene expression analysis. Median age was 55 years at trial initiation; 56.7%were men, 50% low risk Sokal and the median time of IM treatment was 117months(41-191).Themedianfollow-uptimewas20months.TFRwas60%at24

months. Eleven patients relapsed and IMwas reintroduced, but none presentedhematologicrelapseorprogressiontoadvancedphases.Therewasnosignificantdifference in STAT5, PPAR-γ, HIF2α and CITED2 expression pre and postpioglitazone,at3and6monthsafterIMdiscontinuation.Nodifferencewasfoundinthecomparisonoftherelapsedvs.non-relapsedgroup.

Conclusions:pioglitazonedidnotaffectSTAT5,PPAR-γ,HIF2αandCITED2geneexpression in this group of pts with deep molecular response. The ACTIM trialdemonstrated a reduction in STAT5 expression in bonemarrow cells 6monthsafterpioglitazoneexposure,butpioglitazonewasgiventoptswithMMR,withoutMR4.0.Therewasnodifferenceingeneexpressioninthegroupswithorwithoutmolecular relapse. TFR rates remains similar to those reported in otherdiscontinuationtrials.