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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