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RESEARCH POSTER PRESENTATION DESIGN © 2011
www.PosterPresentations.com
©2011PosterPresentations.com2117FourthStreet,[email protected]
Christina Murphy1, Berkley Gryder2, Javed Khan2, Jack Shern3
Figure5.PotentialcombinationstrategiesfortargetingthePAX3-FOXO1fusiononcogene. Inadditiontothecompoundsisolatedinthedrugscreen,Bromodomain inhibitorshadpreviouslybeenshownbyourlabtospecificallydownregulatethetranscriptionaloutputofPAX3-FOXO1.WehypothesizethatcouplingBromodomain inhibitorswithourdiscoveredcompoundswillhavesynergistictoxicitytowardfusion-positivecelllines.Potentialcombinationstrategiesinclude:
DrugScreen
RNAIsolation IncuCyte Experiments
Discoveryofcandidatecompounds
A high-throughput drug screen identifies inhibitors of PAX3-FOXO in pediatric Rhabdomyosarcoma
Rhabdomyosarcoma(RMS)isthemostcommonsofttissuesarcomaafflictingchildren,withanincidenceof4.5casesper1millionadolescentsyearly.1 RMSarisesfromskeletalmuscleprecursorcells,thoughitsanatomicsitesoforiginarehighlyvariedandnotrestrictedtostriatedmuscle.Histologicaldifferencesbetweenembryonalandalveolar
RMSsubtypeswereestablishedasabasisforclinicopathological correlation.2Embryonalrhabdomyosarcoma(ERMS)hasearlierageofonsetandbetterprognosis,3 whilealveolarrhabdomyosarcoma(ARMS)isassociatedwithdifferentprimarysites,ismoreaggressive,usuallypresentswithmetastasisandshowsseverelylagging5-yearsurvivalrateswhencomparedtoERMS.1Thedawnofnext-generationsequencingrevealed2distinct
RMSgenotypes,characterizedbypresenceorabsenceofagenefusionresultingfromPAXtranslocationwithFOXO16. Fusion-positivetumorscarrysignificantlyfewergeneticmutationswhencomparedtofusion-negativetumors7.MolecularprofilingofRMShasbecomeinvaluableininitialdiagnosis,asARMSistypicallyidentifiedbyfusion-positivestatus8.ThefusioneventyieldsachimerictranscriptconsistingoftheN-terminusofPAX,whichisaDNA-bindingdomain, andtheC-terminusofFOXO1,anactivatingdomain9,10.TheoncogenicconsequencesofthisfusionproteinincludeheightenedactivationofnormaldownstreamPAX3targetsaswellasalteredgeneexpressionpatterns11.PresenceofPAX-FOXOratherthanhistologyseemstobetheprimarycauseofpooroutcomesassociatedwiththespecificsubtype.
BACKGROUND
PURPOSE
RESULTSFigure4. Developmentofasecondaryscreentoevaluatecandidatecompounds. Toevaluatethetranscriptionaleffectsofthecompoundsdiscoveredintheprimaryscreen,wedevelopedanassaytorapidlyelevatedexpressionchangesofthegenesdownstreamofPAX3-FOXO1.Cellsweretreatedwithallcandidatecompoundsat1uM for6hoursandRNAwasisolated.Geneselection. RNAsequencing datapreviouslygeneratedfromfusion-positiveRhabdomyosarcomatumorswasminedtodiscovergenesdistinctlyupregulatedinP3Ftumors.
RESULTS RESULTSFigure7. PotentialforBET-inhibitor,topoisomerase-inhibitorsynergy.
CONCLUSION
ACKNOWLEDGEMENTSThankstoDr.JackShern andtherestoftheShern lab,includingDr.Fountaine andDr.Sayers.ThanksalsototheNationalInstitutesofHealthfortheirsupportthroughtheSummerInternshipProgram.
1University of Notre Dame, Notre Dame, IN 60040, USA2 Genetics Branch, NCI, NIH, Bethesda, MD, USA
3Pediatric Oncology Branch, Oncogenomics Section, Center for Cancer Research, National Institutes of Health, Gaithersburg, MD, 20877, USA
MechanismAnalysis
Pre-ClinicalValidation ClinicalTrial
FUTUREDIRECTIONS•RNAsequencingtodeterminegeneticprofileofdrug-treatedRH4cells.•Mechanisticinvestigationofmainclassesofdrugspulleddownfromdrugscreen.•OptimizationofalowdoseBET-inhibitor,topoisomerase-inhibitorcombinationtreatmentforclinicaltrial.
Figure1. RH4celllineandpGreenFire Reporterusedfortheprimaryscreen.
ThechemotherapeuticbackbonefortreatingRMShasremainedrelativelyunchangedsince1975,andintensifiedregimenyieldsonlymarginalresultsforthemostaggressiveofRMScases.Significanttoxicityandlong-termmorbidityareespeciallyconcerningintheseyoungpatients.Anenhancedunderstandingofthetumorbiologyanddiscoveryofnewdrugsforprecisiontargetingwillimproveoutcomesforthesechildren.Beingthatfusion-positiveRMSisassociatedwiththecaseshavingsomeofthemostgrimoutcomes,thePAX3-FOXOfusiononcogenewarrantsfurtherinvestigationasatherapeutictarget.
EXPERIMENTALDESIGN
Figure2. P3F-dependentenhancersequenceusedinthereporterconstruct.
RNAseq espression ofselectedgenesinprimaryRhabdomyosarcomatumorsandnormaltissues.Log2FPKMvalueofthegenesselectedforinclusioninNanostringassaydesignwereobtainedfromRNAseq dataonapaneloftumors(left)andnormaltissuesamples(right).
Secondaryscreenofcandidatecompounds:Nanostring
ExpressionAssay
Figure3. PrimaryPAX3-FOXO1inhibitorcompoundscreen.
Figure6.Real-timequantitativelive-cellanalysisbyIncuCyte ofRH4cellline48hfollowingdrugtreatment.
Thishigh-throughputdrugscreenprovidedmanypotentialleadstowardprojectsinvestigatingPAX3-FOXOtargetfordevelopmentoffuturetherapies.TheNanostring assaywillelucidatethedifferentmechanismsofpotentiallyspecific,lesstoxiccompoundstobeincludedinanewgenerationoffusion-positiveRMSchemotherapeutics.BETinhibitorsinpaststudiesinourresearchgrouphaveshowntoinhibitPAX3-FOXO1-dependenttranscription.Topoisomeraseinhibitorswerearecurringclassofdrugspulleddowninthecompoundscreen.OurunderstandingofthePAX3-FOXOsuperenhancerleadsustobelievethattopoisomerasesareespeciallycrucialtoPAX3-FOXO-driventranscriptionduetothetorsionalstrainaccruedfromextremechromatinremodeling.TopoisomeraseswereeffectiveintreatingRH4cells,evenatlownanomolardoses.WhencombinedwithBET-inhibitors,apotentiallysynergisticeffectwasobservedthatwarrantsfurtherinvestigation.Acombinationtreatmentofthesetwoinhibitorydrugscouldpotentiallyprovideanewlow-doseoptionforchemotherapeutictreatment.Bothteniposide andPLX2arecommerciallyavailableandcouldberolledintoclinicaltrialafterfurtherexperimentationandpre-clinicalvalidation.
TopoisomeraseInhibitors
20 2 0.2 0.02
0.002
16
32
64
128
256
Concentration
Nor
mal
ized
Luc
ifera
se
CMVALKXTT
Tenipo
side
20 2 0.2 0.02
0.002
8
16
32
64
128
256
512
1024
Concentration
Nor
mal
ized
Luc
ifera
se
CMVALKXTT
Amon
afide
Amsacrine
20 2 0.2 0.02
0.002
8
16
32
64
128
Concentration
Nor
mal
ized
Luc
ifera
se
CMVALKXTT
OtherHDACInhibitors
1-alaninechlam
ydocin
20 2 0.2 0.02
0.002
2
4
8
16
32
64
128
256
512
Concentration
Nor
mal
ized
Luc
ifera
se
CMVALKXTT
20 2 0.2 0.02
0.002
16
32
64
128
256
Concentration
Nor
mal
ized
Luc
ifera
se
CMVALKXTT
Camptothe
cinde
rivative
Men
ogaril
20 2 0.2 0.02
0.002
4
8
16
32
64
128
256
Concentration
Nor
mal
ized
Luc
ifera
se
CMVALKXTT
Psam
maplin
A
Tetro
carcinA
20 2 0.2 0.02
0.002
0.1250.250.5
1248
163264
128
Concentration
Nor
mal
ized
Luc
ifera
se
CMVALKXTT
20 2 0.2 0.02
0.002
0.03125
1
32
1024
Concentration
Nor
mal
ized
Luc
ifera
se
CMVALKXTT
Midostaurin
20 2 0.2 0.02
0.002
16
32
64
128
256
512
Concentration
Nor
mal
ized
Luc
ifera
se
CMVALKXTT
PD-407
824
20 2 0.2 0.02
0.002
16
32
64
128
256
512
Concentration
Nor
mal
ized
Luc
ifera
se
CMVALKXTT
0 50 1000
50
100
Hours
% C
onflu
ence
RH4 PLX2 dose response
10uM PLX2 +Teniposide5uM PLX2 + 100nM Teniposide
2.5uM PLX2 +Teniposide1.25uM PLX2 + Teniposide
0.625uM PLX2 +Teniposide0.312uM PLX2 +Teniposide0.156uM PLX2 + Teniposide0.078uM PLX2 + Teniposide0.039uM PLX2 + Teniposide
DMSO + 100nM Teniposide10uM PLX25uM PLX22.5uM PLX21.25uM PLX20.625uM PLX20.312uM PLX20.156uM PLX20.078uM PLX20.039uM PLX2DMSO alone
DoseResponsecurvesofnormalizedluciferasevaluesoftheALKenhancerconstruct,aCMVonlypromotorconstructandviability(DTTassay).
Bromodomain inhibitor + TOPO1 inhibitor Bromodomain inhibitor + HDAC inhibitor
Bromodomain inhibitor + TOPO2 inhibitor Bromodomain inhibitor + Kinase inhibitor (Midostaurin)
Fusion Positive Tumors Normal Tissue Samples
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