Epigenetic Regulation of HOXB13 in Castration Resistant Prostate Cancer

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1 Epigenetic Regulation of HOXB13 in Castration Resistant Prostate Cancer ì Synergistic Targeting of Androgen Receptor Circuits in Metastatic Prostate Cancer Kiran Mahajan Ph.D. TB and GU Departments HOXB13

2 Learner s Objectives 1. The Androgen Receptor (AR) is expressed and funcfonal in a majority of lethal castrafon resistant prostate cancers (CRPCs). 2. An important AR co-regulator in CRPCs is the homeodomain containing sequence specific transcripfon factor, HOXB The expression of HOXB13 is epigenefcally regulated by the BET bromodomain protein, BRD4.

3 Introduction HOXB13 is crifcal for the differenfafon of the ventral lobe of the prostate gland in mice. In human cancers, HOXB13 expression correlates with advanced pt stage, high Gleason grade, posifve lymph node status, high pre-operafve PSA levels, TMPRSS2:ERG fusion, PTEN delefons, AR expression, cell proliferafon and early PSA recurrence. Co-expression analysis idenffied a subset of tumors with high HOXB13 and AR but low PSA expression that had a parfcularly poor prognosis. The epigenefc mechanism by which HOXB13 is regulated in AR posifve prostate cancers is not known.

4 Mechanism of Action of HOXB13 Norris et al, Molecular Cell, 2009

5 gene/actin mrna expression HOXB13-A critical regulator of CRPC growth 2.0 C4-2B HOXB13 pko C4-2B C4-2B HOXB13 pko HOXB13 Actin C4-2B HOXB13 * *** 2000 **** 1000 C4-2B Intact HOXB13 pko Intact 0 Castrated HOXB13p KO Castrated O C4-2B pk 2B O XB H 13 O XB H Castrated C 4- pk 2B O ** C Tumor weights (mgs) Parental HOXB13pKO C4-2B Intact

6 HOXB13 direct targets in PC

7 HOTPAM9-Stratification of prostate adenocarcinomas from metastatic prostate cancers

8 Validation of HOTPAM9 in Metastatic Prostate Cancer GENE/ACTIN mrna C4-2B Control sirna C4-2B HOXB13 sirna CIT BUB1 0.0 MKI67 NEK2 AURKA BUB1 CIT HSPB8 NCAPG TRPM8 NUF2 HOXB13 c-myc TRPM8 AURKA NCAPG

9 BRD4 epigenetically regulates HOXB13 expression in PC % of Input HOXB13 gene ChIP anti-brd4 DMSO JQ1 BRD4 ChIP IgG ChIP R AB EN ZA M A4 Exon1 JQ SO D M Exon ChIP anti-igg C4-2B EGF HOXB13/ACTIN mrna HOXB13/ACTIN mrna 6 * 2 0 DMSO 0.1 ** ** JQ1 ** 2.5 um Control sirna HOXB13 sirna 1.0 BRD4 sirna IGF - + HRG - + BET inhibitor IP: HOXB13 IB: AR IB: HOXB13 IB: AR IB: Actin HOXB13

10 Detection of HOXB13 in CTCs A. DAPI/CK-PE C. DAPI/CK-PE B. VCAP cells No Ab control CK-PE DAPI CD45-APC HOXB13 FITC VCAP cells plus 5ug/ml HOXB13 FITC Ab CK-PE DAPI CD45-APC HOXB13 FITC VCAP cells plus 5ug/ml HOXB13 Alexa Fluor 488 Ab DAPI/CK-PE D. CK-PE DAPI CD45-APC HOXB13 AF488 VCAP cells plus 5ug/ml Alexa Fluor 488 IgG1 Ab DAPI/CK-PE CK-PE DAPI CD45-APC IgG1 AF488

11 Non-invasive Detection of HOXB13 CTCs HOXB13 + HOXB13 - CondiLo n Total Events Unassigne d events CTCs total (n) % of total events n % n % % Total Events DMSO JQ1 DMSO % DMSO JQ1 DMSO JQ1 %CTCs %HOXB13+ve JQ %

12 Summary ì 1. BRD4, is an epigenefc regulator of HOXB13 expression. 2. HOXB13 expression can be targeted in prostate cancers with BET bromodomain inhibitors such as JQ1. 3. We idenffied a subgroup of mitofc kinases as key transcripfonal targets of HOXB13-a target gene signature termed as HOTPAM9 (HOXB13 Target genes separafng Prostate Adenocarcinomas from Metastasis) in CRPCs..

13 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, PC CDMRP, Department of Defense Moffitt Support Account Administration Rose Reyes Kimberly Heffner

14 References 1. Pomerantz, M. M. et al. The androgen receptor cistrome is extensively reprogrammed in human prostate tumorigenesis. Nature gene,cs 47, , doi: /ng.3419 (2015). 2. Frieling, J. S., Basanta, D. & Lynch, C. C. Current and emerging therapies for bone metastafc castrafon-resistant prostate cancer. Cancer control : journal of the Moffi7 Cancer Center 22, (2015). 3. Grasso, C. S. et al. The mutafonal landscape of lethal castrafon-resistant prostate cancer. Nature 487, , doi: /nature11125 (2012). 4. Mallo, M. & Alonso, C. R. The regulafon of Hox gene expression during animal development. Development 140, , doi: /dev (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, , doi: / can (2013) Ylitalo, E. B. et al. Subgroups of CastraFon-resistant Prostate Cancer Bone Metastases Defined Through an Inverse RelaFonship Between Androgen Receptor AcFvity and Immune Response. European urology, doi: /j.eururo (2016).

15 ì Thank you for your ajenfon!

16 Post-Test/Questions Which is a key epigenefc 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