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

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=