Supplementary Figures from Targeting Bromodomain and Extra-Terminal (BET) Family Proteins in Castration-Resistant Prostate Cancer (CRPC)
Authors: Welti Jonathan , Sharp Adam , Yuan Wei , Dolling David , Nava Rodrigues Daniel , Figueiredo Ines , Gil Veronica , Neeb Antje , Clarke Matthew , Seed George , Crespo Mateus , Sumanasuriya Semini , Ning Jian , Knight Eleanor , Francis Jeffrey C. , Hughes Ashley , Halsey Wendy S. , Paschalis Alec , Mani Ram S. , Raj Ganesh V. , Plymate Stephen R. , Carreira Suzanne , Boysen Gunther , Chinnaiyan Arul M. , Swain Amanda , de Bono Johann S. 
Published: April 2023

<p>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 Âµm). 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 ÂµM, 0.5 ÂµM, 1.0 ÂµM, 2.5 ÂµM and 5.0 ÂµM) 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 ÂµM, 0.1ÂµM, 0.5ÂµM, 1.0 ÂµM, 2.5 ÂµM and 5.0 ÂµM) 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=<0.05, **p=<0.01, ***p=<0.001) were calculated for each I-BET151 dose compared to vehicle (DMSO 0.1%) using unpaired student's t-test. Supplementary Figure S7: BET family protein and C-MYC knockdown effects on BET family protein expression LNCaP95 (A-B) and VCaP (C-D) were transfected with 50 nM control, BRD2, BRD3, BRD4 or C-MYC siRNA (LNCaP95 A; VCaP C); or 150 nM control or 150 nM (50 nM each) BRD2, BRD3 and BRD4 siRNA with I-BET151 (0.5 ÂµM) or without (vehicle; DMSO 0.1%) for 72 hours (LNCaP95 B; VCaP D). The effect of each condition on BRD2, BRD3 and BRD4 RNA expression was determined. Mean RNA expression (normalized to B2M and control siRNA at equivalent concentration; defined as 1.0) with standard deviation from three individual experiments is shown. p-values (*p=<0.05, **p=<0.01, ***p=<0.001) were calculated for each condition compared to control siRNA (at equivalent concentration) using unpaired student's t-test. Supplementary Figure S8: RNA-seq analysis confirms I-BET151 regulation of AR-V7 and AR signaling in androgen independent cancer cell lines LNCaP95 cells were treated with vehicle (DMSO 0.1%) or I-BET151 (0.5 ÂµM or 2.0 ÂµM) for 48 hours prior to qRT-PCR (A) and RNA-seq (B-C) analysis being performed. The effect of I-BET151 on AR-FL, AR-V7, PSA and TMPRSS2 (qRT-PCR; A) and AR-FL, AR-V7, C-MYC, PSA, TMPRSS2, BRD2, BRD3 and BRD4 (RNA-seq; B-C) RNA expression was determined. RNA expression (normalized to vehicle; defined as 1.0) with standard deviation from a single experiment performed in duplicate. p-values (*p=<0.05, **p=<0.01, ***p=<0.001) were calculated for each I-BET151 dose compared to vehicle (DMSO 0.1%) using unpaired student's t-test. Supplementary Figure S9: BET family protein knockdown and BET family protein expression LNCaP95 (A-B) and VCaP (C-D) were transfected with 50 nM control, BRD2, BRD3, BRD4 or C-MYC siRNA; or 150 nM control or 150 nM (50 nM each) BRD2, BRD3 and BRD4 siRNA with I-BET151 (0.5 ÂµM) or without (vehicle; DMSO 0.1%) I-BET151 for 72 hours (growth determined at 7 days). The effect of each condition on BRD2, BRD3, BRD4 and C-MYC RNA expression was determined (A-D). Mean RNA expression (normalized to B2M and control siRNA at same concentration) with standard deviation from four individual experiments is shown (unless otherwise stated). p-values (*p=<0.05, **p=<0.01, ***p=<0.001) were calculated for each condition compared to control siRNA (at same concentration) using unpaired student's t-test. Supplementary Figure S10: AR-FL and AR-V7 expression in organoid parental biopsies Representative micrographs of AR-V7, AR-FL and BRD4 detection by immunohistochemistry (IHC) in metastatic (lymph node - LN; BMT - Bone Marrow Trephine) CRPC parental biopsies for each organoid (1-9) (x200 magnification; scale bar 50 Âµm). *IHC performed on same patient but alternative metastatic biopsy. Supplementary Figure S11: Development and characterization of prostate cancer patient derived models (A) Metastatic biopsies from prostate cancer patients with castration resistant disease were used to develop patient derived xenograft (PDX), patient derived organoids (PDO) and patient derived xenograft-organoids (PDX-O). (B) PDX CP50 was developed from lymph node biopsy from a patient who had progressed through all standard of care treatments for CRPC. (C) Copy number (CN) comparison of lymph node biopsy (performed 6 months prior to the PDX parental biopsy) and PDX CP50. From outside to inside; chromosome, CN change in parental biopsy, CN change in PDX CP50, somatic mutations in tumor, somatic mutations from tumor seen in PDX CP50. Supplementary Figure S12: Overview of PDX CP50 experimental design Supplementary Figure S13: I-BET151 decreases AR-V7, but not AR-FL, protein expression in prostate cancer patient derived models Immunohistochemistry images of AR-V (A) and AR-V7 (B) detection in PDX CP50 either left intact (n=2) or castrated (n=8) for 7 days prior to being treated with either vehicle (2 intact mice and 4 castrate mice) or 15 mg/kg I-BET151 (4 castrate mice) for 11 days (x200 magnification; scale bar 50 Âµm).</p>