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Supplementary Tables from Targeting Bromodomain and Extra-Terminal (BET) Family Proteins in Castration-Resistant Prostate Cancer (CRPC)

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posted on 2023-03-31, 20:43 authored by Jonathan Welti, Adam Sharp, Wei Yuan, David Dolling, Daniel Nava Rodrigues, Ines Figueiredo, Veronica Gil, Antje Neeb, Matthew Clarke, George Seed, Mateus Crespo, Semini Sumanasuriya, Jian Ning, Eleanor Knight, Jeffrey C. Francis, Ashley Hughes, Wendy S. Halsey, Alec Paschalis, Ram S. Mani, Ganesh V. Raj, Stephen R. Plymate, Suzanne Carreira, Gunther Boysen, Arul M. Chinnaiyan, Amanda Swain, Johann S. de Bono

Supplementary Tables 1 to 10 Supplementary Table 1: Cell lines ATCC - american type culture collection, FBS - fetal bovine serum, CSS - charcoal stripped serum, * - LNCaP95 cells were kindly provided by Drs. Alan K Meeker and Jun Luo (Johns Hopkins University, Baltimore, Maryland, USA), NA - non-applicable, ^ - phenol red free, BPE/EGF - bovine pituitary extract and human recombinant epidermal growth factor. Supplementary Table 2: ON-TARGETplus siRNA pools for gene expression knock down Supplementary Table 3: TaqMan probes for qRT-PCR analysis Supplementary Table 4: Primary antibodies for western blot analysis Supplementary Table 5: AR regulated genes included in AR activity score Supplementary Table 6: Patient baseline characteristics at prostate cancer diagnosis N - number, SD - standard deviation, IQR - interquartile range, NR - not recorded, PSA - prostate specific antigen, NA - non-applicable. ^5 patient without PSA values; one value >2000 (analyzed as 2000) *All cases adenocarcinoma 1Student's t-test 2Fisher's Exact Test 3Wilcoxon Rank-Sum Test Supplementary Table 7: Cellular pathways regulated by I-BET151 treatment Cellular pathways annotated by CellMap (C), Reactome (R), KEGG (K), NCI PID (N), Panther (P) and BioCarta (B) with p value <0.005 and false discovery rate (FDR) <0.05 are shown. Supplementary Table 8: Alternative splicing events in response to I-BET151 treatment SE - skipped exons, A5SS - alternative 5′ splice sites, A3SS - alternative 3′ splice sites, MXE - mutually exclusive exons, RI - retained introns, FDR - false discovery rate, NRCD - normalized reads count difference. All genes with normalized reads count difference (NRCD) >0.2 and <-0.2, and false discovery rate (FDR) <0.05 highlighted in blue. Supplementary Table 9: Patient derived organoids derived from CRPC biopsies harboring AR aberrations. CRPC - castration resistant prostate cancer, AR - androgen receptor, CN - copy number, AR-V7 androgen receptor variant 7, AR-FL - androgen receptor full length, SPOP - substrate-binding adaptor speckle-type POZ protein, IHC - immunohistochemistry H-score. *Mutational, CN analysis or IHC performed on same patient but alternative metastatic biopsy Supplementary Table 10: Patient baseline characteristics at prostate cancer diagnosis. N - number, SE - standard error, NR - not recorded, PSA - prostate specific antigen. *one patient received curative therapy (with oligo-metastatic disease) ^one patient received docetaxel in hormone sensitive setting 1Fisher's Exact Test 2Wilcoxon Rank-Sum Test

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ARTICLE ABSTRACT

Purpose: Persistent androgen receptor (AR) signaling drives castration-resistant prostate cancer (CRPC) and confers resistance to AR-targeting therapies. Novel therapeutic strategies to overcome this are urgently required. We evaluated how bromodomain and extra-terminal (BET) protein inhibitors (BETi) abrogate aberrant AR signaling in CRPC.Experimental Design: We determined associations between BET expression, AR-driven transcription, and patient outcome; and the effect and mechanism by which chemical BETi (JQ1 and GSK1210151A; I-BET151) and BET family protein knockdown regulates AR-V7 expression and AR signaling in prostate cancer models.Results: Nuclear BRD4 protein expression increases significantly (P ≤ 0.01) with castration resistance in same patient treatment-naïve (median H-score; interquartile range: 100; 100–170) and CRPC (150; 110–200) biopsies, with higher expression at diagnosis associating with worse outcome (HR, 3.25; 95% CI, 1.50–7.01; P ≤ 0.001). BRD2, BRD3, and BRD4 RNA expression in CRPC biopsies correlates with AR-driven transcription (all P ≤ 0.001). Chemical BETi, and combined BET family protein knockdown, reduce AR-V7 expression and AR signaling. This was not recapitulated by C-MYC knockdown. In addition, we show that BETi regulates RNA processing thereby reducing alternative splicing and AR-V7 expression. Furthermore, BETi reduce growth of prostate cancer cells and patient-derived organoids with known AR mutations, AR amplification and AR-V7 expression. Finally, BETi, unlike enzalutamide, decreases persistent AR signaling and growth (P ≤ 0.001) of a patient-derived xenograft model of CRPC with AR amplification and AR-V7 expression.Conclusions: BETi merit clinical evaluation as inhibitors of AR splicing and function, with trials demonstrating their blockade in proof-of-mechanism pharmacodynamic studies. Clin Cancer Res; 24(13); 3149–62. ©2018 AACR.

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