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