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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.

BackgroundUnderstanding the pathways that drive adrenocortical carcinoma (ACC) is essential to the development of more effective therapies. This study investigates the role of the transcription factor HOXB9 and other HOX factors in ACC and its treatment.MethodsWe used transgenic mouse models to determine the role ofHoxb9in adrenal tumour development. Patient transcriptomic data was analysed for the expression of HOX genes and their association with disease. Drug response studies on various adrenocortical models were done to establish novel therapeutic options.ResultsOur human ACC dataset analyses showed high expression ofHOXB9, and other HOX factors, are associated with poorer prognosis. Transgenic overexpression ofHoxb9in the adrenal cortex of mice with activatedCtnnb1led to larger adrenal tumours. This phenotype was preferentially observed in male mice and was characterised by more proliferating cells and an increase in the expression of cell cycle genes, includingCcne1. Adrenal tumour cells were found to be dependent on HOX function for survival and were sensitive to a specific peptide inhibitor.ConclusionsThese studies showHoxb9can promote adrenal tumour progression in a sex-dependent manner and have identified HOX factors as potential drug targets, leading to novel therapeutic approaches in ACC.

// Jeffrey C. Francis 1 , Amy Capper 1 , Jian Ning 2 , Eleanor Knight 2 , Johann de Bono 3 and Amanda Swain 1, 2 1 Division of Cancer Biology, The Institute of Cancer Research, London SW3 6JB, UK 2 Tumour Profiling Unit, The Institute of Cancer Research, London SW3 6JB, UK 3 Division of Clinical Studies, The Institute of Cancer Research, London SM2 5NG, UK Correspondence to: Amanda Swain, email: amanda.swain@icr.ac.uk Keywords: aggressive invasive prostate cancer; prostate cancer mouse models; cell lineage plasticity; epithelial to mesenchymal transition Received: May 18, 2017 Accepted: July 03, 2017 Published: January 10, 2018 ABSTRACT Aggressive lethal prostate cancer is characterised by tumour invasion, metastasis and androgen resistance. Understanding the mechanisms by which localised disease progresses to advanced lethal stages is key to the development of effective therapies. Here we have identified a novel role for the transcription factor, SOX9, as a driver of aggressive invasive prostate cancer. Using genetically modified mouse models, we show that increased Sox9 expression in the prostate epithelia of animals with Pten loss leads to a highly invasive phenotype and metastasis. In depth analysis of these mice and related in vitro models reveals that SOX9 acts a key regulator of various processes that together promote tumour progression. We show that this factor promotes cell lineage plasticity with cells acquiring properties of basal stem cells and an increase in proliferation. In addition, increased SOX9 leads to changes in cytoskeleton and adhesion, deposition of extracellular matrix and epithelia to mesenchyme transition, properties of highly invasive cells. Analysis of castrated mice showed that the invasive phenotype driven by SOX9 is independent of androgen levels. Our study has identified a novel driver of prostate cancer progression and highlighted the cellular and molecular processes that are regulated by Sox9 to achieve invasive disease.

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.

Germline mutations in the tumour suppressor BRCA2 predispose to breast, ovarian and a number of other human cancers. Brca2-deficient mouse models are used for preclinical studies but the pattern of genomic alterations in these tumours has not yet been described in detail. We have performed whole-exome DNA sequencing analysis of mouse mammary tumours from Blg-Cre Brca2(f/f) Trp53(f/f) animals, a model of BRCA2-deficient human cancer. We also used the sequencing data to estimate DNA copy number alterations in these tumours and identified a recurrent copy number gain in Met, which has been found amplified in other mouse mammary cancer models. Through a comparative genomic analysis, we identified several mouse Blg-Cre Brca2(f/f) Trp53(f/f) mammary tumour somatic mutations in genes that are also mutated in human cancer, but few of these genes have been found frequently mutated in human breast cancer. A more detailed analysis of these somatic mutations revealed a set of genes that are mutated in human BRCA2 mutant breast and ovarian tumours and that are also mutated in mouse Brca2-null, Trp53-null mammary tumours. Finally, a DNA deletion surrounded by microhomology signature found in human BRCA1/2-deficient cancers was not common in the genome of these mouse tumours. Although a useful model, there are some differences in the genomic landscape of tumours arising in Blg-Cre Brca2(f/f) Trp53(f/f) mice compared to human BRCA-mutated breast cancers. Therefore, this needs to be taken into account in the use of this model.

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.