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SynergisticTargetingofAndrogenReceptorCircuitsinMetastaticProstateCancer

HOXB13

KiranMahajanPh.D.TBandGUDepartments

EpigeneticRegulationofHOXB13inCastrationResistantProstateCancer

Learner’sObjectives

1.  TheAndrogenReceptor(AR)isexpressedandfuncFonalinamajorityoflethalcastraFonresistantprostatecancers(CRPCs).

2.  AnimportantARco-regulatorinCRPCsisthehomeodomaincontainingsequencespecifictranscripFonfactor,HOXB13.

3.  TheexpressionofHOXB13isepigeneFcallyregulatedbytheBETbromodomainprotein,BRD4.

Introduction

•   HOXB13iscriFcalforthedifferenFaFonoftheventrallobeoftheprostateglandinmice.

•   Inhumancancers,HOXB13expressioncorrelateswithadvancedpTstage,highGleasongrade,posiFvelymphnodestatus,highpre-operaFvePSAlevels,TMPRSS2:ERGfusion,PTENdeleFons,ARexpression,cellproliferaFonandearlyPSArecurrence.

•   Co-expressionanalysisidenFfiedasubsetoftumorswithhighHOXB13andARbutlowPSAexpressionthathadaparFcularlypoorprognosis.

•   TheepigeneFcmechanismbywhichHOXB13isregulatedinARposiFveprostatecancersisnotknown.

MechanismofActionofHOXB13

Norrisetal,MolecularCell,2009

HOXB13-AcriticalregulatorofCRPCgrowth

C4-2B

HOXB13 pKO

C4-2B

Intact

Intact

Castrated

HOXB13p KO Castrated

HOXB13

Actin

Parental HOXB13pKO

C4-2BHOXB130.0

0.5

1.0

1.5

2.0g

en

e/A

ctin

mR

NA

ex

pre

ss

ion

C4-2B C4-2B HOXB13 pKO 1C4-2B C4-2B HOXB13 pKO 2

C4-2B

HOXB13 pKO

C4-2B

HOXB13 pKO

0

1000

2000

Tum

or w

eigh

ts (m

gs)

****

****

Castrated Intact

**

HOXB13directtargetsinPC

HOTPAM9-Stratificationofprostateadenocarcinomasfrommetastaticprostate

cancers

ValidationofHOTPAM9inMetastaticProstateCancer

CIT

BUB1

TRPM8

AURKA

NCAPG

MK

I67

NE

K2

AU

RK

AB

UB

1C

ITH

SP

B8

NC

AP

GTR

PM

8N

UF2

HO

XB

13c-

Myc

0.0

0.2

0.4

0.6

0.8

1.045678

GEN

E/AC

TIN

mR

NA

C4-2B Control siRNA

C4-2B HOXB13 siRNA

BRD4epigeneticallyregulatesHOXB13expressioninPC

- - + - + - + BET inhibitor

EGF IGF HRG

IP: HOXB13IB: AR

IB: HOXB13

IB: AR

IB: ActinHOXB13

0.0

0.5

1.0

1.5Control siRNA

HOXB13 siRNA

BRD4 siRNA

HO

XB13

/AC

TIN

mR

NA

DMSO

JQ1

HOXB13 gene

Exon1 Exon2

ChIP anti-BRD4

ChIP anti-IgG

0

2

4

6

HO

XB13

/AC

TIN

mR

NA

*

** ****

DMSO 0.1 0.5 1 2.5 uM

JQ1

DM

SO

JQ1

MA4

ENZA

ABR

0.0

0.1

0.2

0.3

C4-2B

% o

f In

put BRD4 ChIP

IgG ChIP

DetectionofHOXB13inCTCs

VCAPcellsNoAbcontrol VCAPcellsplus5ug/mlHOXB13AlexaFluor488Ab

VCAPcellsplus5ug/mlHOXB13FITCAb VCAPcellsplus5ug/mlAlexaFluor488IgG1Ab

DAPI/CK-PE CK-PE DAPI CD45-APC HOXB13FITC DAPI/CK-PE CK-PE DAPI CD45-APCHOXB13AF488

DAPI/CK-PE CK-PE DAPI CD45-APC HOXB13FITC DAPI/CK-PE CK-PE DAPI CD45-APC IgG1AF488

A. B.

C. D.

Non-invasiveDetectionofHOXB13CTCs

HOXB13+ HOXB13-

CondiLon

TotalEvents

Unassignedevents

CTCstotal(n)

%oftotalevents

n % n %

DMSO 1987 1310 677 34% 184 27 493 73

JQ1 1386 1323 63 5% 9 14 54 86

DMSO JQ1 DMSO JQ1

0

10

20

30

40

% T

ota

l E

ve

nts

DMSO JQ1

%CTCs %HOXB13+ve

ìSummary

1.BRD4,isanepigeneFcregulatorofHOXB13expression.2.HOXB13expressioncanbetargetedinprostatecancerswithBETbromodomaininhibitorssuchasJQ1.3.WeidenFfiedasubgroupofmitoFckinasesaskeytranscripFonaltargetsofHOXB13-atargetgenesignaturetermedasHOTPAM9(HOXB13TargetgenesseparaFngProstateAdenocarcinomasfromMetastasis)inCRPCs..

Acknowledgements

K. Mahajan lab Neha Agarwal Duy Nguyen Past members Niveditha N. Ami Patel John Vicente (SPARK) Samina Ismail (SPARK) Drug Discovery Brent Kuenzi Ernst Schonbrunn Nick Lawrence Steven Gunawan Uwe Rix Chemical Biology Core Harshani Lawrence Mohammed Ayaz Bioinformatics and

Biostats Yunyun Chen William Ma Youngchul Kim Jiqiang Yao Jamie Teer Comparative Medicine Devon DeLoach Department of Anatomic Pathology Jasreman Dhillon Domenico Coppola Anthony Magliocco Translational Core John Puskas Drug Discovery

Brent Kuenzi Ernst Schonbrunn Nick Lawrence Harshani Lawrence Mohammed Ayaz Steven Gunawan Uwe Rix Nupam Mahajan GU Physicians Jingsong Zhang Michael Poch Sanford Burnham Prebys Institute Ranjan Perera Subbu S Alexey Eroshkin Special Thanks

Dr. Sellers Dr. Cleveland Dr. Chellappan Dr. Julio Pow-Sang Dr. Conor Lynch Funding PC141124, PC141298 CDMRP, Department of Defense Moffitt Support Account Administration Rose Reyes Kimberly Heffner

References1. Pomerantz,M.M.etal.Theandrogenreceptorcistromeisextensivelyreprogrammedinhumanprostatetumorigenesis.Naturegene,cs47,1346-1351,doi:10.1038/ng.3419(2015).2. Frieling,J.S.,Basanta,D.&Lynch,C.C.CurrentandemergingtherapiesforbonemetastaFccastraFon-resistantprostatecancer.Cancercontrol:journaloftheMoffi7CancerCenter22,109-120(2015).3. Grasso,C.S.etal.ThemutaFonallandscapeoflethalcastraFon-resistantprostatecancer.Nature487,239-243,doi:10.1038/nature11125(2012).4. Mallo,M.&Alonso,C.R.TheregulaFonofHoxgeneexpressionduringanimaldevelopment.Development140,3951-3963,doi:10.1242/dev.068346(2013).5. Shah,N.etal.HOXB13mediatestamoxifenresistanceandinvasivenessinhumanbreastcancerbysuppressingERalphaandinducingIL-6expression.Cancerresearch73,5449-5458,doi:10.1158/0008-5472.CAN-13-1178(2013).6. 12 Ylitalo,E.B.etal.SubgroupsofCastraFon-resistantProstateCancerBoneMetastasesDefinedThroughanInverseRelaFonshipBetweenAndrogenReceptorAcFvityandImmuneResponse.Europeanurology,doi:10.1016/j.eururo.2016.07.033(2016).

ì  ThankyouforyourajenFon!

Post-Test/Questions

WhichisakeyepigeneFcregulatorofHOXB13expressioninprostatecancercellsthatcanbetargetedwiththeprototypeinhibitor,JQ1?1.   AKT2.   BRD43.   c-MYC4.   FOXA1