Epigenetic Regulation of HOXB13 in ì Castration Resistant Prostate Cancer

Synergistic Targeting of Androgen Circuits in Metastatic Prostate Cancer

Kiran Mahajan Ph.D. TB and GU Departments

HOXB13 Learner’s Objectives

1. The (AR) is expressed and funconal in a majority of lethal castraon resistant prostate cancers (CRPCs). 2. An important AR co-regulator in CRPCs is the homeodomain containing sequence specific transcripon factor, HOXB13. 3. The expression of HOXB13 is epigenecally regulated by the BET bromodomain , BRD4. Introduction

• HOXB13 is crical for the differenaon of the ventral lobe of the prostate gland in mice. • In human cancers, HOXB13 expression correlates with advanced pT stage, high Gleason grade, posive lymph node status, high pre-operave PSA levels, TMPRSS2:ERG fusion, PTEN deleons, AR expression, cell proliferaon and early PSA recurrence. • Co-expression analysis idenfied a subset of tumors with high HOXB13 and AR but low PSA expression that had a parcularly poor prognosis. • The epigenec mechanism by which HOXB13 is regulated in AR posive prostate cancers is not known. Mechanism of Action of HOXB13

Norris et al, Molecular Cell, 2009 HOXB13-A critical regulator of CRPC growth

n 2.0 Parental HOXB13pKO io C4-2B

ress C4-2B HOXB13 pKO 1

p 1.5 C4-2B ex HOXB13 C4-2B HOXB13 pKO 2 1.0 RNA m in t 0.5 c Actin A / e n

e 0.0 g HOXB13 C4-2B

* ***

2000 **** C4-2B Intact

1000 HOXB13 pKO Intact

0 Tumor weights (mgs) weights Tumor ** C4-2B Castrated

C4-2B C4-2B HOXB13p KO Castrated HOXB13 pKO HOXB13 pKO Castrated Intact HOXB13 direct targets in PC HOTPAM9-Stratification of prostate adenocarcinomas from metastatic prostate cancers Validation of HOTPAM9 in Metastatic Prostate Cancer

C4-2B Control siRNA 8 7 6 C4-2B HOXB13 siRNA 5 4 CIT 1.0 0.8 0.6

0.4 BUB1

GENE/ACTIN mRNA 0.2 0.0 CIT NUF2 NEK2 BUB1 c- MKI67 HSPB8 TRPM8 AURKA NCAPG HOXB13 TRPM8

AURKA

NCAPG BRD4 epigenetically regulates HOXB13 expression in PC

HOXB13 BRD4 ChIP t 0.3 IgG ChIP ChIP npu

anti-BRD4 I f

o 0.2

DMSO % 0.1 JQ1 ChIP 0.0 anti-IgG 1 4 A R JQ MA AB Exon2 Exon1 DMSO ENZ C4-2B 6

A EGF IGF HRG 1.5 - - + - + - + BET inhibitor 4 Control siRNA A IP: HOXB13 HOXB13 siRNA 1.0 IB: AR * BRD4 siRNA 2 IB: HOXB13

0.5 IB: AR HOXB13/ACTIN mRN ** ** **

0 HOXB13/ACTIN mRN DMSO 0.1 0.5 1 2.5 uM 0.0 IB: Actin HOXB13 JQ1 Detection of HOXB13 in CTCs

A. VCAP cells No Ab control B. VCAP cells plus 5ug/ml HOXB13 Alexa Fluor 488 Ab DAPI/CK-PE CK-PE DAPI CD45-APC HOXB13 FITC DAPI/CK-PE CK-PE DAPI CD45-APC HOXB13 AF488

C. VCAP cells plus 5ug/ml HOXB13 FITC Ab D. VCAP cells plus 5ug/ml Alexa Fluor 488 IgG1 Ab

DAPI/CK-PE CK-PE DAPI CD45-APC HOXB13 FITC DAPI/CK-PE CK-PE DAPI CD45-APC IgG1 AF488 Non-invasive Detection of HOXB13 CTCs

HOXB13+ HOXB13-

40 DMSO CTCs % of Condio Total Unassigne JQ1 s

total total n % n % t 30

n Events d events n (n) events ve E

l 20 a t To

% 10

0 DMSO 1987 1310 677 34% 184 27 493 73 DMSO JQ1 DMSO JQ1 %CTCs %HOXB13+ve

JQ1 1386 1323 63 5% 9 14 54 86 ì Summary

1. BRD4, is an epigenec regulator of HOXB13 expression. 2. HOXB13 expression can be targeted in prostate cancers with BET bromodomain inhibitors such as JQ1. 3. We idenfied a subgroup of mitoc kinases as key transcriponal targets of HOXB13-a target gene signature termed as HOTPAM9 (HOXB13 Target separang Prostate Adenocarcinomas from Metastasis) in CRPCs. .

Acknowledgements

K. Mahajan lab Biostats Brent Kuenzi Dr. Sellers Neha Agarwal Yunyun Chen Ernst Schonbrunn Dr. Cleveland Duy Nguyen William Ma Nick Lawrence Dr. Chellappan Past members Youngchul Kim Harshani Lawrence Dr. Julio Pow-Sang Niveditha N. Jiqiang Yao Mohammed Ayaz Dr. Conor Lynch Ami Patel Jamie Teer Steven Gunawan John Vicente (SPARK) Comparative Medicine Uwe Rix Funding Samina Ismail (SPARK) Devon DeLoach Nupam Mahajan PC141124, PC141298 CDMRP, Department of Drug Discovery Department of Anatomic GU Physicians Defense Brent Kuenzi Pathology Jingsong Zhang Moffitt Support Account Ernst Schonbrunn Jasreman Dhillon Michael Poch Nick Lawrence Domenico Coppola Administration Steven Gunawan Anthony Magliocco Rose Reyes Uwe Rix Sanford Burnham Kimberly Heffner Translational Core Prebys Institute Chemical Biology Core John Puskas Ranjan Perera Harshani Lawrence Subbu S Mohammed Ayaz Alexey Eroshkin Drug Discovery Bioinformatics and Special Thanks References

1. Pomerantz, M. M. et al. The androgen receptor cistrome is extensively reprogrammed in human prostate tumorigenesis. Nature genecs 47, 1346-1351, doi: 10.1038/ng.3419 (2015). 2. Frieling, J. S., Basanta, D. & Lynch, C. C. Current and emerging therapies for bone metastac castraon-resistant prostate cancer. Cancer control : journal of the Moffi Cancer Center 22, 109-120 (2015). 3. Grasso, C. S. et al. The mutaonal landscape of lethal castraon-resistant prostate cancer. Nature 487, 239-243, doi:10.1038/nature11125 (2012). 4. Mallo, M. & Alonso, C. R. The regulaon of expression during animal development. Development 140, 3951-3963, doi:10.1242/dev.068346 (2013). 5. Shah, N. et al. HOXB13 mediates tamoxifen resistance and invasiveness in human breast cancer by suppressing ERalpha and inducing IL-6 expression. Cancer research 73, 5449-5458, doi:10.1158/0008-5472.CAN-13-1178 (2013). 6. 12 Ylitalo, E. B. et al. Subgroups of Castraon-resistant Prostate Cancer Bone Metastases Defined Through an Inverse Relaonship Between Androgen Receptor Acvity and Immune Response. European urology, doi:10.1016/j.eururo.2016.07.033 (2016). ì Thank you for your aenon! Post-Test/Questions

Which is a key epigenec regulator of HOXB13 expression in prostate cancer cells that can be targeted with the prototype inhibitor, JQ1?

1. AKT 2. BRD4 3. c-MYC 4. FOXA1