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Supplementary Figures S1 to S13 Supplementary Figure S1: Summary of clinical samples analyzed for BRD4 protein expression and SPOP mutational status The clinical samples used in this study were from a population of metastatic castration resistant prostate cancer (mCRPC) patients treated at The Royal Marsden Hospital. Patients with sufficient formalin fixed paraffin embedded (FFPE) tissue from diagnostic (archival) hormone sensitive prostate cancer (HSPC) biopsies (n=53) and same patient, matched, CRPC biopsies (n=38) were analyzed for nuclear BRD4 expression by immunohistochemistry (dark grey shading). Clinical correlations were determined as shown (yellow shading). Twenty-seven (of 53) diagnostic (archival) HSPC biopsies and 12 (of 38) mCRPC biopsies had next generation sequencing (NGS) available to determine SPOP mutational analysis (light grey shading). Supplementary Figure S2: BRD4 immunohistochemistry validation Immunohistochemistry (A), immunofluorescence (B) and western blot (C) analysis of BRD4 protein expression in LNCaP95 cells transfected with overexpression plasmid, control siRNA or BRD4 siRNA for 72 hours (magnification 200x; scale bar 50 µm). Supplementary Figure S3: BRD4 protein expression in SPOP mutant prostate cancer Expression (H-score) of nuclear BRD4 expression in 27 (of 53) HSPC and 12 (of 38) CRPC biopsies with next generation sequencing available to determine SPOP mutational analysis. Median H-score and interquartile range is shown. Supplementary Figure S4: AR-V7 and BRD4 protein expression in prostate cancer cell lines AR-FL, AR-V7, C-MYC, BRD4 and GAPDH protein expression determined by western blot analysis of prostate cancer cell lines. Supplementary Figure S5: JQ1 treatment downregulates AR-V7 protein expression in androgen dependent and independent prostate cancer cell lines LNCaP95 (A), VCaP (B) and 22Rv1 (C) were treated with vehicle (DMSO 0.1%) or various concentrations of JQ1 (0.1 µM, 0.5 µM, 1.0 µM, 2.5 µM and 5.0 µM) for 48 hours. The effect of JQ1 treatment on AR-FL, AR-V7 and C-MYC protein expression was determined. Single representative western blot shown from three separate experiments. Supplementary Figure S6: I-BET151 treatment effects on BET family protein expression LNCaP95 (A), VCaP (B) and 22Rv1 (C) were treated with vehicle (DMSO 0.1%) or various concentrations of I-BET151 (0 µM, 0.1µM, 0.5µM, 1.0 µM, 2.5 µM and 5.0 µM) for 48 hours. The effect of I-BET151 treatment on BRD2, BRD3 and BRD4 RNA expression was determined. Mean RNA expression (normalized to B2M and vehicle; defined as 1.0) with standard deviation from three individual experiments is shown (unless otherwise stated). p-values (*p=

Supplementary Methods

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.

Supplementary Figure Legends for Figures S1 to S13

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 0.2 and

Background and objectives Several hundred inherited genetic variants or SNPs that alter the risk of cancer have been identified through genome-wide association studies. In populations of European ancestry, these variants are mostly present at relatively high frequencies. To gain insight into evolutionary origins, we screened a series of genes and SNPs linked to breast or prostate cancer for signatures of historical positive selection. Methodology We took advantage of the availability of the 1000 genome data and we performed genomic scans for positive selection in five different Caucasian populations as well as one African reference population. We then used prostate organoid cultures to provide a possible functional explanation for the interplay between the action of evolutionary forces and the disease risk association. Results Variants in only one gene showed genomic signatures of positive, evolutionary selection within Caucasian populations melanophilin (MLPH). Functional depletion of MLPH in prostate organoids, by CRISPR/Cas9 mutation, impacted lineage commitment of progenitor cells promoting luminal versus basal cell differentiation and on resistance to androgen deprivation. Conclusions and implications The MLPH variants influencing prostate cancer risk may have been historically selected for their adaptive benefit on skin pigmentation but MLPH is highly expressed in the prostate and the derivative, positively selected, alleles decrease the risk of prostate cancer. Our study suggests a potential functional mechanism via which MLPH and its genetic variants could influence risk of prostate cancer, as a serendipitous consequence of prior evolutionary benefits to another tissue. Lay Summary We screened a limited series of genomic variants associated with breast and prostate cancer risk for signatures of historical positive selection. Variants within the melanophilin (MLPH) gene fell into this category. Depletion of MLPH in prostate organoid cultures, suggested a potential functional mechanism for impacting on cancer risk, as a serendipitous consequence of prior evolutionary benefits to another tissue.

The mammalian prostate arises from the urogenital sinus under the influence of testicular androgens. Few factors have been identified to be important in the early stages of prostate development. Here we review the role of the transcription factor Sox9 in prostate development. Sox9 is a member of the Sox gene family that plays an important role during embryogenesis in the cellular differentiation of various tissues, including testicular Sertoli cells, neural crest cells and chondrocytes. This gene is expressed in the epithelia of all mouse prostatic lobes from the initial stages of their development. Mice with a prostate specific deletion of Sox9 showed a lack of ventral prostate development and abnormal anterior prostate differentiation. In depth analysis of these mutant animals suggested that Sox9 is required for the early differentiation of the prostate bud epithelia, consistent with the function of this factor in other developmental processes. These studies also revealed different phases of prostate bud development. These phases were characterized by being dependent on different molecular pathways and having lobe specific properties. Future studies on the identification of pathways regulated by Sox9 will provide insight into the molecular networks required for prostate epithelia differentiation.

The Notch family of receptors is involved in a wide variety of developmental processes, including cell fate specification, cell proliferation, and cell survival decisions during cell differentiation and tissue morphogenesis. Notch1 and Notch ligands are expressed in the developing limbs, and Notch signalling has been implicated in the formation of a variety of tissues that comprise the limb, such as the skeleton, musculature, and vasculature. Notch signalling has also been implicated in regulating overall limb size. We have used a conditional allele of Notch1 in combination with two different Cre transgenic lines to delete Notch1 function either in the limb mesenchyme or in the apical ectodermal ridge (AER) and limb ectoderm. We demonstrate that Notch signalling, involving Notch1 and Jagged2, is required to regulate the number of Fgf8-expressing cells that comprise the AER and that regulation of the levels of fibroblast growth factor signalling is important for the freeing of the digits during normal limb formation. Regulation of the extent of the AER is achieved by Notch signalling positively regulating apoptosis in the AER. We also demonstrate that Notch1 is not required for proper formation of all the derivatives of the limb mesenchyme.

Gene expression and functional studies have indicated that the molecular programmes involved in prostate development are also active in prostate cancer. PTEN has been implicated in human prostate cancer and is frequently mutated in this disease. Here, using the Nkx3.1:Cre mouse strain and a genetic deletion approach, we investigate the role of Pten specifically in the developing mouse prostate epithelia. In contrast to its role in other developing organs, this gene is dispensable for the initial developmental processes such as budding and branching. However, as cytodifferentiation progresses, abnormal luminal cells fill the ductal lumens together with augmented epithelial proliferation. This phenotype resembles the hyperplasia seen in postnatal Pten deletion models that develop neoplasia at later stages. Consistent with this, gene expression analysis showed a number of genes affected that are shared with Pten mutant prostate cancer models, including a decrease in androgen receptor regulated genes. In depth analysis of the phenotype of these mice during development revealed that loss of Pten leads to the precocious differentiation of epithelial cells towards a luminal cell fate. This study provides novel insight into the role of Pten in prostate development as part of the process of coordinating the differentiation and proliferation of cell types in time and space to form a functional organ.

Epidemiological studies have shown that one of the strongest risk factors for prostate cancer is a family history of the disease, suggesting that inherited factors play a major role in prostate cancer susceptibility. Germline mutations in BRCA2 predispose to breast and ovarian cancer with its predominant tumour suppressor function thought to be the repair of DNA double-strand breaks. BRCA2 has also been implicated in prostate cancer etiology, but it is unclear the impact that mutations in this gene have on prostate tumourigenesis. Here we have undertaken a genetic analysis in the mouse to determine the role of Brca2 in the adult prostate. We show that deletion of Brca2 specifically in prostate epithelia results in focal hyperplasia and low-grade prostate intraepithelial neoplasia (PIN) in animals over 12 months of age. Simultaneous deletion of Brca2 and the tumour suppressor Trp53 in prostate epithelia gave rise to focal hyperplasia and atypical cells at 6 months, leading to high-grade PIN in animals from 12 months. Epithelial cells in these lesions show an increase in DNA damage and have higher levels of proliferation, but also elevated apoptosis. Castration of Brca2;Trp53 mutant animals led to regression of PIN lesions, but atypical cells persisted that continued to proliferate and express nuclear androgen receptor. This study provides evidence that Brca2 can act as a tumour suppressor in the prostate, and the model we describe should prove useful in the development of new therapeutic approaches., Author Summary In Western countries, prostate cancer is the most common male cancer and the second biggest cause of cancer-related deaths in men. Men with a familial history of either breast or ovarian cancer have an elevated predisposition to prostate cancer, suggesting there is a genetic element to this disease. Indeed, the inheritance of a mutated form of the breast cancer susceptibility gene BRCA2 has been linked to the development of prostate cancer, although the precise role that BRCA2 dysfunction plays in the development of prostate cancer is unclear. To address this, we have generated an animal model in which the mouse Brca2 gene is specifically deleted in the adult prostate. These mice develop precancerous prostate lesions, which progress in severity and incidence with the loss-of-function of an additional tumour suppressor, Trp53. Importantly, blocking male steroidal hormone production by castration leads to partial regression of the prostate lesions, however cells continue to proliferate after androgen withdrawal. This suggests human BRCA2 mutant prostate tumours, like the majority of prostate cancers, will respond to hormone therapy, but will relapse, as frequently occurs in this disease. In summary, our model suggests that BRCA2 acts as a tumour suppressor in the prostate and provides a pre-invasive model to test novel therapeutics.