Cell lines
VCaP, LNCaP, 22Rv1, DU145, PC-3, MCF-7, HepG2, A549, K562, HL-60, U937, HEK293, WPMY-1, U-87 MG, BJAB, SK-ES-1, RD-ES, A204, PBMC, NCI-H526, AU565, MM1.S, SU-DHL-8, and NCI-H211 were purchased from the American Type Culture Collection (ATCC). MOLT-16, NALM6, REH, and TALL-1 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). CHLA-99, CHLA-258, CHLA-32, CB-AGPN, COG-E-352, and TC-205 were obtained from the Children’s Oncology Group (COG) cell line and xenograft repository. GM24694 was obtained from the Coriell Institute for Medical Research. PNT2 and MFM223 were obtained from Sigma-Aldrich. The Kelly cell line was obtained from the European Collection of Authenticated Cell Cultures (ECACC). CADOES1 was obtained from the Cell Lines Service (CLS). LK2 was obtained from the Japan Research Bioresources Collection (JCRB) cell bank. NB-1 was obtained from Sekisui XenoTech.
CWR-R1 cells (Sigma-Aldrich, SCC118) were provided by D. Vander Griend. SU-DHL-5 cells (ATCC CRL-2958/DSMZ ACC 571) were provided by the R. Ryan laboratory. TC-32 cells (COG Cell Line and Xenograft Repository) were provided by the ER Lawlor laboratory. T47D cells (ATCC HTB-133) were provided by the C. Speers laboratory. SUM185PE cells (Asterand Bioscience) were provided by the Rae laboratory. WA72 is an in-house PDX-derived cell line established at the Michigan Center for Translational Pathology (MCTP), University of Michigan, and is not commercially available. All cell lines were cultured under conditions recommended by the original repository or supplier laboratory, as appropriate.
Two cell lines used in this study, BJAB and U937, are listed in the International Cell Line Authentication Committee (ICLAC) Registry of Misidentified Cell Lines. Both entries reflect historical contamination reports that have since been resolved. The U-87 MG (obtained from ATCC) is also listed in the ICLAC registry (ICLAC-00535). This cell line was included at the specific request of a peer reviewer, to test the activity of the compound in an AR+cellular context of non-prostate cancer, given reports that AR functions as an oncogenic driver in this model. This experiment aimed to provide broader context on compound selectivity in AR+ types of tumors other than prostate cancer, and not to draw conclusions specifically regarding the biology of the tissues of glioblastoma origin. No other cell lines used in this study appear in the ICLAC registry.
All human cell lines were authenticated by short tandem repeat (STR) genetic testing. All cell lines were tested every two weeks to be free of mycoplasma contamination.
Chemistry
Detailed summary and chemical data for all designed DALTAC molecules and negative controls are provided in the Supplementary Methods.
Antibody
For immunoblotting, the following antibodies were used: p300 (Invitrogen: MA1-16608, 1:500); AR (Abcam: ab133273, 1:1000); H2BK20ac (Cell Signaling Technology: 34156S, 1:1000); H2BK16ac (Abcam: ab177427, 1:1000); H3K27ac (Cell Signaling Technology: 8173S, 1:1000); H2B (Active Pattern: 39210, 1:1000); H3 (Cell Signaling Technology: 3638S, 1:1000); MYC (Cell Signaling Technology: 9402S, 1:1000); KLK3/PSA (Dako: A0562, 1:1000); NKX3-1 (Cell Signaling Technology: 83700S, 1:1000); CCND1 (Abcam: ab16663, 1:1000); vinculin (Cell Signaling Technology: 18799S, 1:1,000); GAPDH (Santa Cruz Biotechnology: sc-47724, 1:1000); BRD4 (Bethyl Laboratories: A700-004CF, 1:1,000); AR-V7 (Cell Signaling Technology: 19672S, 1:1000); HA label (Cell Signaling Technology: 3724S, 1:1000); CBP (Abcam: ab253202, 1:1000); and V5 (Invitrogen, R960-25, 1:1000). Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Bio-Rad; 1:10,000) and goat anti-mouse (Bio-Rad; 1:5,000) secondary antibodies were used.
For ChIP-seq, the following antibodies were used: H3K27ac (Diagenode: C15410196, 1 µg per million cells); p300 (Active Motif: 61401, 2 µg per million cells); H2BK20ac (cell signaling technology: 34156S, 1 µg per million cells); AR (Abcam: ab108341, 2 µg per million cells); FOXA1 (Thermo Fisher Scientific: PA5-27157, 2 µg per million cells); BRD4 (Diagenode: C15410337, 2 µg per million cells); ERG (Cell Signaling Technology: 97249S, 2 µg per million cells); RNA Pol II (Diagenode: C15200253, 1 µg per million cells); CBP (Abcam: ab253202, 2 µg per million cells); SRC2 (Cell Signaling Technology: 96687S, 4 µl per million cells) and PR (Cell Signaling Technology: 8757S, 4 µl per million cells).
For immunoprecipitation, an acetylated lysine antibody (Cell Signaling Technology: 9441S) was used at 1 μg per 100 μg of lysate.
For immunofluorescent staining (COMET-mIF) and PLA testing, the following antibodies were used: p300 (Invitrogen: 33-7600, 1:150); CBP (Invitrogen: PA5-27369, 1:150); AR (Abcam: ab133273, 1:400); H3K27ac (Cell Signaling Technology: 8173S, 1:100); Ki-67 (Abcam: ab243878, 1:300); H2BK20ac (Abcam: ab177430, 1:250); CK8 (Abcam: ab53280, 1:50); ERG (Abcam: ab92513, 1:200); PSA (cell brand: 324M-16, 1:50); and MYC (Abcam: ab32072, 1:50).
For ELISA, the following antibodies were used: p300 (Cell Signaling Technology: 86377S, 1:1,000); and AR (Thermo Fisher Scientific: MA5-13426, 1:50).
Cell viability test
VCaP cells were seeded at a density of 10,000 cells per well, and all other prostate cancer cell lines were seeded at 3,000 cells per well in poly-d-96-well plates coated with lysine (Corning) and maintained at 37°C with 5% CO2. After 24 h, cells were treated with a serial dilution of the tested compounds, with 6 wells replicated for each concentration. After 120 h of incubation, cell viability was determined using the CellTiter-Glo Luminescent Cell Viability Assay (Promega) according to the manufacturer’s instructions. Luminescence was measured on an Infinite M1000 Pro plate reader (Tecan) and data were analyzed using GraphPad Prism (v10; GraphPad software).
Incucocyte proliferation tests
A total of 4,000 VCaP cells or 1,500 LNCaP cells per well were seeded into poly-d-96-well plates coated with lysine. After 24 h of incubation, cells were treated with a fixed concentration (100 nM) or serial dilutions of compounds and incubated for 4 h. The medium was then removed and the cells were washed three times with phosphate-buffered saline (PBS) before adding fresh medium containing either vehicle or compounds. Phase contrast images were collected every 4 hours and cell proliferation was quantified by measuring the percentage of confluence (phase object area).
Immunoblotting
Cells were lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with 1× Halt protease inhibitor cocktail (Thermo Fisher Scientific) and denatured in 1× NuPAGE LDS sample buffer containing reducing agent (Invitrogen) by incubation at 70°C for 15 min. Protein concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of proteins (15 to 30 μg) were resolved on NuPAGE 3 to 8% Tris-acetate or 4 to 12% Bis-Tris protein gels (Thermo Fisher Scientific) and transferred to 0.45 μm nitrocellulose membranes (Thermo Fisher Scientific) using the iBlot 3 dry transfer system (Thermo Fisher Scientific). Membranes were blocked in TBS-T (Tris-buffered saline with 0.1% Tween-20) containing 5% nonfat milk powder for 1 h at room temperature and incubated overnight at 4°C with primary antibodies. After washing, membranes were incubated with HRP-conjugated secondary antibodies and imaged using an Odyssey CLx imager (LI-COR Biosciences). Supplementary Figure 1 shows full-length, uncropped blots with molecular weight markers for all immunoblot experiments.
Fluorescence polarization binding assays
The binding affinities of the tested compounds with AR and CBP/p300 were measured using competitive binding assays based on fluorescence polarization. AR binding was assessed using the PolarScreen Androgen Receptor Competitor Assay Kit (Thermo Fisher Scientific, A15880) following the manufacturer’s instructions.
CBP/p300 fluorescence polarization assays were performed in 96-well plates (MTP96VPWB-EP, Eppendorf) using a CLARIOstar microplate reader (BMG Labtech). Serial dilutions of the test compounds were incubated with fluorescein-labeled tracer (2 nM final) and CBP/p300 protein (20 nM final) in assay buffer (PBS pH 7.4 supplemented with 0.01% BGG, 0.01% Tween-20, and 2 mM DTT) to a final reaction volume of 100 μl per well. Plates were mixed for 15 minutes and incubated at room temperature for 1 hour to reach equilibrium. Fluorescence polarization (mP units) was measured at excitation of 485 nm and emission of 530 nm.
For AR and CBP/p300 assays, data analysis was performed in GraphPad Prism 10 using nonlinear regression. IC50 values were determined by fitting the competition curves and the inhibition constant (KI) competitive inhibitor values were derived using nonlinear regression based on Kd of the tracer and the concentrations of tracer and protein in the reaction. All fluorescence polarization experiments were conducted in duplicate across three independent biological replicates.
Cellular thermal shift test
VCaP or LNCaP cells were seeded in poly-d-100 mm culture dishes coated with lysine (Corning) and incubated overnight. Cells were incubated with control DMSO or indicated compounds for 4 h at 37°C before being collected and washed once with PBS. The cell suspension was then dispensed into PCR tubes (50 μl per tube) and exposed to a predefined temperature gradient for 3 min using a Veriti thermal cycler (Applied Biosystems). After equilibration at room temperature, cells were lysed by three freeze-thaw cycles in liquid nitrogen. Insoluble protein aggregates were pelleted by centrifugation at 20,000g for 20 min at 4 °C, and the resulting supernatants containing soluble proteins were collected for Western blot analysis.
Computer modeling
All modeling and simulations were carried out with Schrödinger 2025-2 (Schrödinger). Because the co-crystal…
Gn Health
