Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 3 | Overexpression of CITED2 at the animal level is able to alleviate PH. (A) The results of gene enrichment analysis in normoxia and hypoxia showed that CITED2 was downregulated in hypoxia, showing a negative correlation. (B–D) Expression of CITED2 mRNA and protein in the pulmonary arteries of model mice. (E) Schematic diagram of lentivirus model mouse construction. (F) The decreased cardiopulmonary function of model mice was effectively reversed by overexpression of CITED2. (G) Right ventricular systolic blood pressure and right ventricular specific gravity were improved in the CITED2 overexpression group. (H, I) H&E and Masson staining of tissue sections of mouse pulmonary arterioles. (K) The ex- pression of PCNA was downregulated in the pulmonary arteries of mice in the lentivirus-treated group. (J) Tissue immunofluorescence experiments showed that the expression of Ki67 in small pulmonary vessels decreased significantly after CITED2 overexpression. A: artery. (L, M) The expression of cycle-related proteins in the pulmonary arteries of mice in the lentivirus-treated group was downregulated. LV: lentivirus. (Bar = mean ± S.E.M, **p < 0.01; ***p < 0.001).

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Over Expression, Expressing, Staining, Immunofluorescence

Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 4 | Overexpression of CITED2 can affect the proliferation of smooth muscle cells and hinder cell cycle progression. (A) Western blot re- sults of efficiency detection after overexpression of CITED2. (B) The results of the CCK8 experiment indicated that overexpression of CITED2 could affect the growth and viability of mPASMCs. (C) Overexpression of CITED2 affected the expression of PCNA in cells. (D) After overexpression of CITED2, the proportion of cells in the G2/M and S phase was reduced, which affected cell division. (E, F) The results of the EdU cell fluorescence assay and Ki67 cell fluorescence assay indicated that overexpression of CITED2 could reduce cell proliferation. Red and green represent remark- ably proliferating cells. (G, H) Overexpression of CITED2 at the cellular level can affect the expression levels of multiple proteins in the cell cycle. (Bar = mean ± S.E.M, **p < 0.01; ***p < 0.001).

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Over Expression, Western Blot, Expressing, Fluorescence

Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 6 | FOXJ3 cooperates with SEs to regulate the transcription of CITED2. (A) Schematic representation of overexpression plasmid con- struction and transfection. (B–D) In the dual-luciferase experiment, plasmids containing three SEs motifs were co-transfected with FOXJ3 plasmids into mPASMCs. When both were present, the luciferase activity was the greatest. (Bar = mean ± S.E.M, *p < 0.05).

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Over Expression, Plasmid Preparation, Transfection, Luciferase, Activity Assay

Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 9 | Differential effects of CITED2 on PASMC proliferation under normoxia and hypoxia. Normally, the H3K27ac modification of the SEs and promoter regions of CITED2 is significant. Downstream proteins influenced by CITED2, such as cyclin proteins, CDKs and PCNA, decreased. SMC proliferation is also diminished. However, the phenomenon is reversed under hypoxia.

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Modification

Journal: Cancer discovery

Article Title: Targeting p300/CBP in lethal prostate cancer

doi: 10.1158/2159-8290.CD-20-0751

Figure Lengend Snippet: (a-d) Association between primary prostate cancer (PC) CBP (a), metastatic castration resistant prostate cancer (mCRPC) CBP (b), primary PC p300 (c), and mCRPC p300 (d) expression levels with androgen receptor (AR) signature from either TCGA primary PC expression data (a, c; n=550) or SU2C/PCF mCRPC expression data (b, d; n=120). r-values and p-values are shown and were calculated using Spearman’s correlation. (e-h) Association between primary prostate cancer (PC) CBP (e), metastatic castration resistant prostate cancer (mCRPC) CBP (f), primary PC p300 (g), and mCRPC p300 (h) expression levels with acquired androgen deprivation therapy (ADT) resistance signature from either TCGA primary PC expression data (e, g; n=550) or SU2C/PCF mCRPC expression data (f, h; n=120). r-values and p-values are shown and were calculated using Spearman’s correlation. (i) SU2C/PCF mCRPC transcriptome analyses of RNA-sequencing data from 120 patient biopsies for CBP, p300 and AR expression divided into very high (upper 25% expressed genes), medium high (50%−75% expressed genes), medium low (25%−50% expressed genes) and very low (lower 25% expressed genes). (j) Nuclear protein expression (H-score) of CBP and p300 in 43 matched, same patient, castration sensitive prostate cancer (CSPC; grey) and CRPC (red). Median H-score and interquartile range is shown. p-values were calculated using Wilcoxon matched-pair signed rank test. (k-l) Representative micrographs of CBP and p300 detection by immunohistochemistry in matched, same patient, CSPC and CRPC biopsies. Needle biopsies (NB), prostatectomies (prost), bone biopsies (bone) and lymph node biopsies (LN) are shown. Scale bar represents 50 μm.

Article Snippet: Expression plasmids for CBP (OriGene Technologies, RC219036) and p300 (OriGene Technologies, RC223265) were used for protein re-expression experiments in doxycycline-inducible cell lines developed.

Techniques: Expressing, RNA Sequencing Assay, Immunohistochemistry

Journal: Cancer discovery

Article Title: Targeting p300/CBP in lethal prostate cancer

doi: 10.1158/2159-8290.CD-20-0751

Figure Lengend Snippet: (a-b) 22Rv1 and (c-d) LNCaP95 were treated with vehicle (0nM = DMSO 0.1%) or various concentrations of CCS1477 (1nM, 10nM, 30nM, 100nM, 300nM, 1000nM, 3000nM or 5000nM) for 48-hours (72 hours for b). The effect of each condition on AR-FL, AR-V7, KLK2, KLK3, FKBP5, TMPRSS2, C-MYC, CBP and p300 mRNA expression (22Rv1 a; LNCaP95 c) and AR-FL, AR-V7, C-MYC, and GAPDH protein expression was determined (22Rv1 b; LNCaP95 d). Mean mRNA expression (normalized to an average of B2M/GAPDH/HPRT1 and control siRNA; defined as 1.0) with standard error of mean from three individual experiments is shown. p-values (*p=<0.05, **p=<0.01, ***p=<0.001) were calculated for each treatment condition compared to vehicle using unpaired student t-test. Single representative western blot shown from three separate experiments. (e) 22Rv1 cells were plated in hormone-deficient media for 72 hours. Following which, cells were treated with vehicle (0nM = DMSO 0.1%) or CCS1477 500nM for 8 hours, with 10nM dihydrotestosterone (DHT) being added 3 hours before harvest. Chromatin immunoprecipitation (ChIP) was performed with CBP, p300 and AR-FL antibodies, followed by PCR with primers designed for known AR binding sites whose gene expression was significantly downregulated by CCS1477 treatment (KLK3, TMPRSS2, FKBP5 ANKRD30B and CHRNA2), and to known CBP binding sites (TFF1 and TGFA enhancer, enh). Mean binding as percentage input with standard error of mean from three individual experiments is shown. p-values (*p=<0.05, **p=<0.01, ***p=<0.001) were calculated for vehicle compared to CCS1477 treatment using unpaired student t-test.

Article Snippet: Expression plasmids for CBP (OriGene Technologies, RC219036) and p300 (OriGene Technologies, RC223265) were used for protein re-expression experiments in doxycycline-inducible cell lines developed.

Techniques: Expressing, Western Blot, Chromatin Immunoprecipitation, Binding Assay

Journal: Cancer discovery

Article Title: Targeting p300/CBP in lethal prostate cancer

doi: 10.1158/2159-8290.CD-20-0751

Figure Lengend Snippet: (a-c) 22Rv1 and (d-f) LNCaP95 were transfected with a total of 100nM siRNA with 50nM of either CBP, p300, p300 plus CBP, or C-MYC siRNA (made up to total with Control siRNA) for 72 hours. The effect of each condition on AR-FL, AR-V7, KLK2, KLK3, FKBP5, TMPRSS2, C-MYC, CBP and p300 mRNA expression (22Rv1 a; LNCaP95 d) and AR- FL, AR-V7, C-MYC, KLK3, CBP, p300 and GAPDH protein expression was determined (22Rv1 b; LNCaP95 e). Mean mRNA expression (normalized to an average of B2M/GAPDH/HPRT1 and control siRNA; defined as 1.0) with standard error of mean from three individual experiments is shown. Growth assays by CellTitre-Glo were carried out (22Rv1 c; LNCaP95 f) (normalized to control siRNA; defined as 1.0) with standard deviation of an individual experiment with six replicates 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 t-test. Western blots are shown in triplicate. (g-i) 22Rv1 cells with doxycycline-inducible short hairpin (sh) RNA to Control (shCON), CBP (shCBP) or p300 (shp300) were treated with 10ng/μg doxycycline for 48 hours prior to transfection with empty vector plasmid (EV), CBP plasmid (CBP rescue) or p300 plasmid (p300 rescue). The effect of each condition on CBP, p300, AR-FL, AR-V7, KLK3, FKBP5, TMPRSS2, C-MYC, ODC and CAD mRNA expression (g) and CBP, p300, AR-FL, AR-V7, C-MYC and GAPDH protein expression (h) was determined after 48 hours. Mean mRNA expression (normalized to 18S and shCON:EV; defined as 1.0) with standard error of mean from three individual experiments is shown. p-values (*p=<0.05, **p=<0.01, ***p=<0.001) were calculated for shCBP:EV compared to shCBP:CBP rescue, and shp300:EV compared to shp300:p300 rescue, using unpaired student t-test. Western blots demonstrate single experiment. The effect of each condition on relative growth was determined after 5 days using the PicoGreen dsDNA assay (i). Mean relative growth (normalized to baseline and shCON:EV; defined as 1.0) with standard error of mean from three individual experiments is shown. p-values (*p=<0.05, **p=<0.01, ***p=<0.001) were calculated for shCBP:EV compared to shCBP:CBP rescue, and shp300:EV compared to shp300:p300 rescue, using unpaired student t-test.

Article Snippet: Expression plasmids for CBP (OriGene Technologies, RC219036) and p300 (OriGene Technologies, RC223265) were used for protein re-expression experiments in doxycycline-inducible cell lines developed.

Techniques: Transfection, Expressing, Standard Deviation, Concentration Assay, Western Blot, Plasmid Preparation, Picogreen Assay

Journal: Cancer discovery

Article Title: Targeting p300/CBP in lethal prostate cancer

doi: 10.1158/2159-8290.CD-20-0751

Figure Lengend Snippet: (a-f) Patient 1 (a-c) and patient 2 (d-f) were treated with CCS1477 within an ongoing Phase I clinical trial (NCT03568656). Patient 1 (a-c) was treated orally with 50mg twice daily (BD) on a 3- days-on-and-4 days-off-schedule. Blood KLK3 levels and timing of pre-dose (after 4-days off treatment) and post-dose (4-hours post-CCS1477 dose) biopsies are shown (a). The patient remains on trial. Quantification (b) and representative micrographs for haematoxylin and eosin (H and E), AR-FL, AR-V7, C-MYC, KLK3, CBP, p300 and Ki67 immunohistochemistry pre-dose and post-dose are shown (c). Scale bar represents 200 μm. Patient 2 (d-f) was treated orally with 25mg BD continuously. Blood KLK3 levels and timing of pre-dose (prior to starting treatment) and post-dose (3 hours post CCS1477 dose) biopsies are shown (d). The patient has stopped treatment. Quantification (e) and representative micrographs for H and E, AR-FL, AR-V7, C-MYC, KLK3, CBP, p300 and Ki67 immunohistochemistry pre-dose and post-dose are shown (f). Scale bar represents 200 μm.

Article Snippet: Expression plasmids for CBP (OriGene Technologies, RC219036) and p300 (OriGene Technologies, RC223265) were used for protein re-expression experiments in doxycycline-inducible cell lines developed.

Techniques: Immunohistochemistry

Journal: bioRxiv

Article Title: p300/CBP degradation is required to disable the active AR enhanceosome in prostate cancer

doi: 10.1101/2024.03.29.587346

Figure Lengend Snippet: p300 is a determinant cofactor of the activated AR enhanceosome in prostate cancer. a . Immunoblot analysis of key histone marks in four pairs of matched prostate cancer (T) and benign adjacent tissues (N). Quantitation and fold change (FC) of the respective histone marks is provided to the right. PTMs, post-translational modifications. b . Representative multiplex Immunofluorescence (IF) images of H2BK5ac (red)/KRT8 (green), and H2BK20ac (red)/KRT8 (green) staining in patient-matched adjacent benign and tumor tissues. Magnification: 200x. Scalebar = 50 µm. c . H2BK5ac and H2BK20ac IF mean intensity per field (integrated optical density – Fiji is just ImageJ) from images in panel b. n=5 (two-sided t-test). d . Cumulative DepMap CRISPR knockout essentiality scores of histone acetyltransferases and deacetylases in AR-positive prostate cancer cell lines. Aggregated z-scores for each gene derived from LNCaP and VCaP DepMap screens are shown, with p300 and CBP highlighted in red. e . Proportion of FOXA1, SMARCA4, p300, and BRD4 ChIP-seq peaks that map to the top quartile of AR ChIP-seq peaks located in non-promoter regions in VCaP cells. f . Venn diagrams illustrating overlaps of genome-wide p300 and AR ChIP-seq peaks in VCaP cells. g . ChIP-seq and ATAC-seq read-density heatmaps at AR/p300 co-bound and AR only binding sites in VCaP cells. Transcription factors (TFs), transcription cofactors, RNA Pol II, and respective histone marks indicated. h . Ranked H3K27ac ChIP-seq signal of super-enhancers (SEs) on AR/p300 co-bound sites and AR only sites in VCaP cells identified by HOMER. Bottom panel: Activity scores of super-enhancers comprising either AR/p300 co-bound or AR only sites (two-sided t-test). i . Immunoblot analysis of p300, CBP, and the indicated histone marks in LNCaP WT (wild-type), p300 KO (knock-out), CBP KO, and p300 KO with siCBP (small interfering RNA) cells. j . Incucyte live-cell analysis of LNCaP and 22Rv1 cells with respective CRISPR KO and siRNAs indicated. siNT, non-targeting siRNA. Data are presented as mean +/− SD (n = 6). (two-sided t-test)

Article Snippet: Short guide RNAs (sgRNAs) targeting the exons of human p300 or CBP were designed by Benchling ( https://www.benchling.com/ ).

Techniques: Western Blot, Quantitation Assay, Multiplex Assay, Immunofluorescence, Staining, CRISPR, Knock-Out, Derivative Assay, ChIP-sequencing, Genome Wide, Binding Assay, Activity Assay, Small Interfering RNA

Journal: bioRxiv

Article Title: p300/CBP degradation is required to disable the active AR enhanceosome in prostate cancer

doi: 10.1101/2024.03.29.587346

Figure Lengend Snippet: Degradation of p300/CBP, but not inhibition of their reader bromodomains, abolishes histone acetylation activity at AR enhancer elements. a. Immunoblot analysis of indicated histone acetylation marks in VCaP cells treated with DMSO, 1 µM GNE-049, or 1 µM CCS1477 for 24 hours. b. Structure of CBPD-409 and schematic of p300 and CBP domains. CBPD-409-targeted bromodomain (BRD) is highlighted by the arrow. Key domains of p300/CBP proteins are noted. Taz1: TAZ zinc finger domain; Kix: Kinase-inducible domain (KID) interacting domain; PHD: Plant homeodomain; HAT: Histone acetyltransferase domain; iBiD: N-terminal interferon-binding domain. CRBN, cereblon. c. Immunoblot analysis of p300 and CBP in VCaP cells treated with 100 nM CBPD-409 for the indicated durations. d. TMT (tandem mass tag) mass spectrometry assay to evaluate effects of CBPD-409 (100 nM, 4 hours) on the proteome of VCaP cells. Data are plotted as log2 of the fold change (FC) versus DMSO control against –log2 of the p-value per protein from n = 3 independent experiments. All t-tests performed were two-tailed t-tests assuming equal variances. e. Immunoblot analysis of labeled H2B N-terminus acetylation in VCaP cells treated with the indicated concentration of CBPD-409, GNE-049, or CCS1477 for 4 hrs. f. Acetyl-lysine proteomics analysis of VCaP cells. Fold change heatmap illustrating alterations in acetyl-lysine levels of core histone proteins (H2A, H2B, H3, and H4) in VCaP cells treated with 100 nM CBPD-409 at indicated time points, compared to DMSO vehicle treated cells. Data plotted from n=3 independent samples. g. Venn diagrams of genome-wide changes of H2BK5ac and H2BK20ac ChIP-seq peaks after CBPD-409 (100 nM 4 hours) or GNE-049 (1 µM 4 hours) treatment of VCaP cells. h. ChIP-seq read-density heatmaps of H3K27ac, H2BK5ac, and H2BK20ac at AR cis -regulatory elements in VCaP cells with 4 hours of 100 nM CBPD-409 or 1 µM GNE-049 treatment. i. Normalized ChIP-seq read density of H3K27ac, H2BK5ac, and H2BK20ac at super-enhancers associated with AR binding sites, based on AR ChIP-seq data in VCaP cells with 4 hours of 100 nM CBPD-409 or 1 µM GNE-049 treatment (Wilcox test). j. Gene set enrichment analysis (GSEA) plots for AR, Myc, E2F, and G2M checkpoint pathway-related genes using the fold change rank-ordered gene signature from the CBPD-409 treated VCaP cells. NES, net enrichment score; adj P, adjusted p-value; DEGS, differentially expressed genes. n=2 biological replicates. k. Immunoblot analysis of indicated proteins and histone marks in VCaP cells treated with 100 nM CBPD-409 for the indicated times. l. RNA-seq heatmaps for classical AR target genes (defined as upregulated by 1 nM R1881 in 12 hours) in LNCaP cells treated with 100 nM CBPD-409 or 1 µM GNE-049. Cells were cultured in medium with 10% charcoal-stripped FBS (CSS) overnight, then pre-treated with CBPD-409 or GNE-049 for 1 hour and subsequently stimulated with 1 nM R1881 for 12 hours. m. ChIP-seq tracks of AR, H3K27ac, H2BK20ac, and RNA Pol II within the TMPRSS2 gene loci in VCaP cells treated with or without 100 nM CBPD-409 for 4 hours. n. Volcano plots of coding genes nascent RNA expression (left panel) and enhancer RNAs (eRNAs) expression (right panel) in VCaP cells treated with 100 nM CBPD-409 for 4 hours. The AR target genes which were significantly suppressed by CBPD-409 are highlighted by red in the left panel. The AR-associated eRNAs which were significantly suppressed by CBPD-409 are highlighted by blue in the right panel. The names of key AR target genes (left panel) or eRNAs associated target genes (right panel) are labeled. The data were generated from the 5-ethynyluridine (EU) labeled nascent RNA-seq. The volcano plots represent the log2 fold change (FC) of CBPD-409 relative to DMSO plotted against the – log10 p-value for each gene, based on n=2 independent experiments. Statistical tests were two-tailed t-tests with the assumption of equal variances.

Article Snippet: Short guide RNAs (sgRNAs) targeting the exons of human p300 or CBP were designed by Benchling ( https://www.benchling.com/ ).

Techniques: Inhibition, Activity Assay, Western Blot, Binding Assay, Mass Spectrometry, Two Tailed Test, Labeling, Concentration Assay, Genome Wide, ChIP-sequencing, RNA Sequencing Assay, Cell Culture, RNA Expression, Expressing, Generated

Journal: bioRxiv

Article Title: p300/CBP degradation is required to disable the active AR enhanceosome in prostate cancer

doi: 10.1101/2024.03.29.587346

Figure Lengend Snippet: p300/CBP degradation compared to bromodomain inhibition leads to stronger suppression of oncogenic gene programs. a. Volcano plot of gene expression in VCaP cells treated for 24 hours with either 100 nM CBPD-409 or 1 µM GNE-049. A set of genes, including CCND1 , CITED2 , and NKX3-1 , are exclusively repressed by CBPD-409. The data represent the log2 fold change (FC) of CBPD-409 relative to GNE-049 plotted against the –log10 p-value for each gene. Based on n=2 independent experiments. Statistical tests were two-tailed t-tests with the assumption of equal variances. b. Uniquely down-regulated genes in CBPD-409 relative to GNE-409 treated VCaP cells analyzed for overlap with MSigDB hallmark gene sets. The top five hallmark gene sets (ranked by p-value) are highlighted in red. c. Immunoblot analysis of indicated proteins in VCaP cells treated with 100 nM CBPD-409 or 1 µM GNE-049 for indicated times. d. Immunoblot analysis of labeled proteins and histone marks in VCaP cells pre-treated with different concentrations of thalidomide (Thali) for 1 hour, then treated with CBPD-409 at indicated concentrations for 4 hours. e. Quantitative-PCR (qPCR) of NKX3-1 , CCND1 , CITED2 , and MYC expression in VCaP cells pre-treated with 100 µM thalidomide for 1 hour, then treated with CBPD-409 or GNE-409 for 4 hours. f. Immunoblot analysis of indicated proteins and histone marks in VCaP cells treated with CBPD-409 or CBPD-409-Me (inactive analogue) for indicated times. g. Dose-response curves and IC50 of prostate cancer cells treated with CBPD-409 and GNE-049. Data are presented as mean +/− SD (n = 6). h. Rank-order plot of IC50 values for CBPD-409 across 131 human normal and cancer cell lines following 5 days of treatment. Models of AR-positive prostate cancer, AR-negative prostate cancer, non-neoplastic prostatic cells, multiple myeloma, and neuroblastoma are highlighted in specific colors. The originating tissue lineages are indicated below. PNET, primitive neuroectodermal tumor; EMRS, embryonal rhabdomyosarcoma.

Article Snippet: Short guide RNAs (sgRNAs) targeting the exons of human p300 or CBP were designed by Benchling ( https://www.benchling.com/ ).

Techniques: Inhibition, Expressing, Two Tailed Test, Western Blot, Labeling, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: p300/CBP degradation is required to disable the active AR enhanceosome in prostate cancer

doi: 10.1101/2024.03.29.587346

Figure Lengend Snippet: p300/CBP degradation inhibits CRPC tumor growth and synergizes with enzalutamide without apparent toxicities. a. Schematic of the CBPD-409 in vivo efficacy study utilizing the VCaP-CRPC xenograft model. VCaP cells were subcutaneously implanted in SCID mice, which underwent castration two weeks post-implantation once the tumors reached 200 mm³ to induce disease regression. Subsequently, castration-resistant tumors re-grew to 200 mm³, at which point the treatments commenced. mpk, mg/kg. b. Graph depicting the tumor volume curves and tumor weights in the VCaP-CRPC model, measured biweekly using calipers. Treatments included vehicle, enzalutamide (Enza, 10 mg/kg, administered orally five days per week), CBPD-409 (3 mg/kg, administered orally three days per week), and CBPD-409 in combination with Enza. Statistical analysis was performed using a two-sided t-test. Data are presented as mean ± SEM. Sample sizes are as follows: vehicle, n = 18; CBPD-409, n = 18; Enza, n = 18; Enza + CBPD-409, n = 18. c. Waterfall plot illustrating the change in tumor volume after 33 days of treatment from the VCaP-CRPC study. d. Representative immunohistochemistry images from the VCaP-CRPC xenograft study for the indicated protein (n = 4 tumors per treatment). Scalebar = 50 µm. e. Representative immunohistochemistry images showing staining of murine CBP in specified organs of CD-1 IGS mice treated with CBPD-409 (3 mg/kg, administered orally three days per week). n=10 per treatment. Scalebar = 50 µm. f. CD1 mice body weight (%) measurements throughout the treatment period from vehicle and CBPD-409 treated groups (two-sided t-test). Data are presented as mean +/− SEM (vehicle: n = 10, CBPD-409: n=10). g. Representative alcian blue staining images from the large intestinal tract harvested from CD1 mice (n = 10/treatment group). Right, quantification of goblet:epithelial cell (GC/EC ratio) densities in the colon (two-sided t-test). Data are presented as mean +/− SEM. h. VCaP cells were treated with noted concentrations of CBPD-409 and/or Enza to evaluate drug synergism using the Bliss Independence method. The average synergy score of CBPD-409 and Enza is 23.876 (>10 indicates drug synergism). Data representation includes the mean of four replicates. i. Dose-response curves and IC50 of LNCaP parental and LNCaP enzalutamide-resistant (Enza-R) cells treated with CBPD-409. Data are presented as mean +/− SD (n = 6). j. Graph depicting tumor volume curves and tumor weights in the MDA-PCa-146-12 CRPC PDX model, measured biweekly by calipers. Treatments included vehicle, Enza, and combination Enza with CBPD-409. Statistical analysis was performed using a two-sided t-test. Data are presented as mean ± SEM. Sample sizes are as follows: vehicle, n = 10; Enza, n = 12; Enza + CBPD-409, n = 14. k. Tumor volume curve in the intact WA-74 PDX model, measured biweekly using calipers. Treatments included vehicle, Enza, and combination of Enza and CBPD-409. Statistical analysis was conducted by two-sided t-test. Data are presented as mean ± SEM. Sample sizes are as follows: vehicle, n = 12; Enza, n = 12; Enza + CBPD-409, n = 14. l. Kaplan-Meier survival plot illustrating the survival rates of mice bearing WA-74 CRPC PDX tumors, treated with vehicle, Enza, and combination of Enza and CBPD-409. Survival is measured up to the point where tumor volume reaches 2000 mm³. m. Mechanism of action of p300/CBP degrader versus reader bromodomain inhibitor (rBRDi) in disrupting activated AR enhanceosome in prostate cancer cells. Transient degradation of p300/CBP triggers selective loss of histone marks, followed by dislodgement of RNA Pol II. Conversely, inhibition of p300/CBP bromodomain only partially represses histone marks without affecting RNA Pol II loading.

Article Snippet: Short guide RNAs (sgRNAs) targeting the exons of human p300 or CBP were designed by Benchling ( https://www.benchling.com/ ).

Techniques: In Vivo, Immunohistochemistry, Staining, Inhibition