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CDOT • First hire Oct 2011 (now ~30 scientists) • Collaborations are key to program success • Outsourcing of some essential activities
Approach • Establish tools to take on undruggable therapeutic targets • Commitment to comprehensive approach
Goals: Make next generation impactful therapies.
Pursue deep understanding of target biology and disease systems.
Apply learnings in an integrated, open and collaborative drug discovery environment.
Confiden'al
CenterforDevelopmentofTherapeu'cs,BroadIns'tute
MedullaryCys'cKidneyDisease1
BiologyoftheMUC1DiseaseMuta'on&
Therapeu'csDiscoveryandDevelopment
TheMCKD1ProjectTeam
3
✪
✪
✪
AnthonyBleyer,M.D.NephrologyandGene'cRenalDiseaseWakeForestSchoolofMedicine
StanislavKmoch,Ph.D.CenterforAppliedGenomics1stSchoolofMedicine,Prague
✪
CarlosSlimHealthInst.
SethAlper,M.D.,Ph.D.RenalDivisionBIDeaconessMedicalCenter
Broad
• ThebiologyunderlyingtheroleofmutantMUC1inkidneydiseaseisunknown.
• OurleadinghypothesisistheframeshiQedmutantMUC1isatoxicproteinandthatdecreasingproteinlevelswillbenefitpa5ents.
• Torapidlydrivetowardnewtreatments,weini'atedan“at-risk”therapeu'csprogramtargetedondecreasingMUC1levelswhilebeginningparallelresearchonbiologyofthedisease.
MCKD1:BiologyandTherapeu'cs
Biology Therapeu/cs
NovelTreatment
MUC1iscausalgeneforMCKD1
Amuta'oninMUC1causestheproteintoincorrectlylocalize
HealthyMUC1oncellsurface
MCKD1MUC1stuckinsidecell
Nuclei
CrossSec/onofcells
MUC1
MUC1
• +Cmuta'onpredictsneo-sequenceandtrunca'on
• Truncatedneopep'dehaspIof12
N/start
signal VNTR SEA TM
C/stop
extracellular intracellular
frameshift-created stop codon
+C frameshift
Muta'onproducesmanycopiesofalteredVNTRunit
VTSAPDTRPAPGSTAPPAHG CHLGPGHQAGPGLHRPPSPR
MUC1expressedindistalconvolutedtubuleandcollec'ngduct
MUC1inkidney
• Muc1globalKOmicehavesubtlephenotypes
• Increasedsensi'vitytobacteriainfec'ons• Reducedintes'nalmucusinCFTR-/-cross• Preliminarydatasupportsaroleinwoundhealing
inkidney
• MUC1overexpressedinsomecancers• Pro-survivalandan'-apopto'cac'vi'es• C-terminusmodulates:
• b-catenin,p120catenin,p53andER-a• EGFreceptorac'vity,Srcfamilykinases,GSK-3β,PKCδ
N
C
N-li
nked
gly
cans
O
-link
ed g
lyca
ns
membrane
Whatisknownaboutthefunc'onofMUC1?
MucinRepeatsTargetproteintoApicalMembrane
modifiedfromHughey,RP
KufeD,2013
CleavedextracellulardomaincanremainassociatedwithMUC1-C
SKINBREAST
KIDNEY
CourtesyofStanKmoch
MUC1Extra-renalInvolvement• +Cfsmuc1isexpressedinall'ssues• Mammary,Respiratory,GItractfunc'onarenormal
HowdoesfsMUC1causedisease?
• Currentmodel:dominantgain-of-func'ontoxiceffect.Whatothermodel(s)issupportedbythedata?
• WhydoesfsMUC1,normallyexpressedindistalnephron
epithelialcells,leadtopathologynotonlyinthosecellsbutalsoininters''umandeventuallyinproximaltubuleandglomerulus?
• WhydoesthewidelyexpressedmutantMUC1presentclinicallyina'ssue-specificmanner?
• invivomodels–mousetransgenicsongoing• Zebrafish?Others?
Complexbiologicalques'onsremain.
ExploringMuc1biologyandfspathophysiology
Kidneydifferen'a'onandfunc'onalendpoints
Morphology&woundhealing
Proteinexpression&trafficking Stress,
pro-autophagicorapopto'cresponses
Migra'on
Iontransport
Cystogenesis
Genetranscrip'on
SIgnaling
BUTFIRSTGENERATETHEREAGENTS!!
Whatdoweneed?
• MCKD1pa'ent-derivedcelllines– hTERT/SV40LgTandhTERTalone
• fsmuc1an'bodydevelopment– Mul'plealempts–pep'desandphagedisplay
• ReliablesequencingtechnologyforVNTR– Manyitera'onsw/PacBiothewinner
• PerfectcDNAexpressionconstructsforWTandfs–round2!
• StablecelllinesforWTandfs–round2!• WellcharacterizedshRNAandCRISPRvectors
BuildingbelermodelstostudybiologyandforRxDx
Source:aconsentedMCKD1pa'ent&anormalhumankidney
Singlerenalepithelialcells
Infec'onwithhTERT+/-SV40TAg
25+Pa'entclonalstablelinesestablished
• ConfirmedfsMUC1presenceandepithelialorigin
14
Collabora/onwithA.BleyerandSAlper
xy
yzWildtypeMUC1
Tamm-Horsfallprotein–UMOD(TALHmarker)
DAPI
hTERT-immortalizedMCKD1pa'entcellspolarizeinvitro
xy
yzNKCC2(TALHmarker)
NK-APTase(baso-lateralmembrane)DAPI
Clone1A8 JaneHsu,JuanGu'errez,SavithriKota,SethAlper
fsmuc1inpa'entcelllinesisintracellular
MCKD1pa/entderivedcells Normalkidneyderivedcells
hTERT+SV40LgTlinesNewphagedisplayAb#3
DougDanielsandJuanGu'errez
Blue=nucleiGreen=fsmuc1
MCKD1MouseModelDevelopment
• Pursuingthreemodels• EachmodelwillusethefullhumanMUC1geneincludingextensive
promoterregions
• HPRTlocustarge'ngwithMCKD1mutantfullhumangene+promoterregion
– Createamouseharboringasingleaddi'onalhumancopyofgeneontheXchromosome
• Knock-inofMCKD1mutantfullhumangene+promoterregionintomouseMuc1locus
– Replacestheendogenousmousegene+promoterregionwiththehumanlocus
• Randominser'onmodel(SethAlper)– Furtheralong(avoidedre-cloningbolleneck)
17
Therapeu'csDiscoveryandDevelopment
• Therapeu'cstrategyfocusedondominantgain-of-func'onhypothesis– HTStoiden'fycompoundsbasedonthehypothesisthatreducing
MUC1proteinlevelscouldsignificantlyreduceordelaydiseaseprogression
• Pursuingbiologicalmechanismofdiseasestudiesinparallel• DevelopingmousemodelsusingWTandfsMUC1inparallel
withotherstudies– Firstrandomtransgenicfoundersiden'fiedthisweek;characteriza'on
beginning– Twoothermodels,includingaknock-inreplacementmodel,inprogress
Therapeu'cprogramgoals
Stage1(complete)• Iden'fydiseasegene• Proveconceptthatreduc'onofMUC1expressionisfeasible
Stage2• ExpandourknowledgeofMUC1roleinnormalkidneydevelopmentandfunc'on
• ExploretheroleoffsMUC1inMCKD1• Develophigh-fidelitydiseasemodelstoassessfsrole
– MCKD1pa'entderivedcelllines– animal(ratandmurine)models
• Ini'ateatherapeu'csprogramforMCKD1. 19
HCSHTSMCKD1pa'entline100KBioA/DOS/ML
QPCRHTSMCKD1pa'entline100KBioA/DOS/ML
L1000 screen MCF7
20KBioA/DOS
3MUC1focusedHTScompleted
40BioAretested15cpdshitviaQPCR15impactproteinlevelsviaIF
1200cpdsretestedatdoseVerylowretestrate
OverlapwithL1000dataseen HTSCompleteRetestsongoing
MCKD1kidneylinesinHTS
Hits identified via LINCS data troll in MCF7 • 8 known classes of drugs • All 8 classes also identified as hits in QPCR HTS and further validated • 4 classes validated to inhibit MUC1 protein via IF based dose response
HTShits:Bioac'vesiden'fiedviaL1000
BRD-K01436366-001-10-7
-6 -4 -2 0 20.0
0.2
0.4
0.6
0.8
1.0
Frac
tion
MU
C1
+ve
Cel
l
log [concentration uM]
BRD-K01436366-001-10-7
log [concentration uM]
Frac
tion
of V
iabl
e C
ell
-6 -4 -2 0 20.0
0.5
1.0
1.5
QPCRMUC1mRNAlevels IFMUC1cellsurfaceprotein cellviabilityDAPI
DAPI
MUC1
EC50=50nM
(uM)
Immunofluorescentdetec'onofsurfaceWTMUC1
4inhibitorclassesreduceMUC1proteinlevels
5μM 0.5μM 0.05μM 0.005μM 0.0005μM DMSO
BRDXX29Class1
wt-MUC1
BRDXX16Class2
wt-MUC1
βAc'n
BRDXX88Class3
BRD6929Class4
fs-MUC1
5μM 0.5μM 0.05μM 0.005μM 0.0005μM DMSO
BRDXX19Class4
fs-MUC1
βAc'n
Clinicalgenomicdata>Discovery>Transla/on>Therapy
23
Collaborators/AdvisorsTonyBleyerStanKmochMarkDalyMar'nPollakRamnikXavier
SLIM INITIATIVE FOR GENOMIC MEDICINE
5th SAB MEETING Dec 13rd 2012
Biology/NephrologyCollaboratorsSethAlperJohannesSchlondorffSavithriKotaDavidDoroquezBroadAndiGnirkeDaveJaffeChadNusbaumIainMacCallumAravindSubramanianCoreyFlynnJohnDoenchMelanieDonahue
StephenMossNature2008
BroadJuanGu'errezMarkRothEleanorHoweBrianHubbardLucienneRoncoNicolaTollidayShomitSenguptaJaneHsuMikeSerranoWuPatrickMcCarrenPatrickFaloonJosePerezMichellePalmerLeighCarmodyDougDanielsLizCulybaPaulClemonsSteveCarrEricKuhnToddCarterEricLander
MUC1HumanandMouseGeneStructures
24
• Widerangeofspliceformsreported• Exon2glycosylatedrepeatisvariable• Exon2isveryGCrich
• Onetranscriptreported• Exon2glycosylatedrepeatisnotvariable• Exon2islessGCrichthanhuman• Equivalentmuta5oninmouseTRwouldnotresultin
extendednovelprotein
Human
Mouse