Fig 2: AU-15330 inhibits tumour growth in preclinical models of CRPC and synergizes with enzalutamide.a, Tumour volume (measured twice per week using callipers) in the VCaP-CRPC model with AU-15330 alone or in combination with enzalutamide (two-sided t-test). Data are mean ±s.e.m. (vehicle: n = 18; AU-15330: n = 20; enzalutamide: n = 18; AU-15330 + enzalutamide: n = 16). b, Waterfall plot depicting change in tumour volume after 33 days of treatment. Response evaluation criteria in solid tumours (RECIST) was used to stratify tumours: progressive disease (PD), at least a 20% increase in tumour size; stable disease (SD), increase of <20% to a decrease of <30%; partial response (PR), at least a 30% decrease. The vehicle and enzalutamide groups have 100% PD; the AU-15330 group has 61% PD, 33% SD and 6% PR; and the AU-15330 + enzalutamide group has 0% PD, 12% SD and 88% PR. c, Representative haematoxylin and eosin (H&E) staining and immunohistochemistry from the VCaP-CRPC xenograft study (n = 2 tumours per condition). Insets in the H&E images show expanded views of apoptotic cells. d, Tumour volume measurements showing efficacy of AU-15330, enzalutamide or combined treatment in C4-2B-derived CRPC xenografts (n = 20 per condition; two-sided t-test). Data are mean ± s.e.m. e, Tumour volume measurements showing the effect of enzalutamide alone or in combination with AU-15330 in the castration-resistant MDA-PCa-146-12 PDX study (two-sided t-test). Data are mean ± s.e.m. f, Mechanism of action of AU-15330-triggered cytotoxicity in AR–FOXA1-signalling-driven prostate cancer. SWI/SNF ATPase degradation induces a rapid, targeted loss in chromatin accessibility at the core-enhancer circuitry of AR, FOXA1, ERG and MYC, thereby attenuating their cancer-promoting transcriptional programs and tempering the enhancer-wired supra-physiologic expression of driver oncogenes.Source data

Fig 3: TRIM8 interacts with and ubiquitinates EWS/FLI in Ewing sarcoma cells(A) Schematic of the proximity ligation assay (PLA).(B) Histogram showing fluorescence signal from the PLA in TC71 C-dTAG TRIM8 cells. No antibody (neg. ctrl), anti-FLI, and anti-HA alone were used as negative controls. Anti-FLI and anti-HA combination were used to detect interaction between TRIM8 and EWS/FLI.(C) PLA signal TC71 C-dTAG TRIM8 cells treated with either DMSO or dTAGV-1 (1 μM) for 48 h. Data representative of three independent experiments.(D) Schematic of fluorescent proteins expressed in A673-E/F-Halo cell line is shown above. Airyscan super-resolution images of the nucleus (Hoechst stained, blue), endogenous EWS/FLI-Halo (JF646 labeled, red), and transiently expressed TRIM8-EGFP (green) in an A673-E/F-Halo cell.(E) Two-color image showing that endogenous EWS/FLI-Halo hubs enrich for TRIM8-EGFP. The nuclear fluorescence background is subtracted in both channels of the average image (see the STAR Methods for details).(F) Average background-free radial profiles of EWS/FLI-Halo and TRIM8-EGFP at EWS/FLI-Halo hubs (averaged from 6,872 hubs in 15 cells).(G and H) Poly-ubiquitinated proteins in TC71 cells infected with either non-targeting or TRIM8-targeting sgRNA were enriched with tandem ubiquitin binding entities (TUBE). Both input and TUBE-enriched proteins were immunoblotted with the indicated antibodies. EWS/FLI levels were quantified using ImageJ and the poly(ub)-E/F:total-E/F ratio was used to calculate poly-ubiquitinated EWS/FLI levels. Fold change (FC) in poly-ubiquitinated EWS/FLI ±SEM from three independent experiments is shown in (H). ∗∗∗p < 0.001.(I) TC71 C-dTAG TRIM8 cells treated with 1 μM of dTAGV-1 for 6 h in triplicate were lysed and immunoblotted with the indicated antibodies. Six hour pomalidomide (1 μM) treatment served as a positive control for TMT mass spectrometry.(J) Total proteome of cells in (I) was analyzed using TMT quantification mass spectrometry. Non-EWS/FLI proteins that significantly changed in abundance are highlighted in blue and EWS/FLI in red. Significance cutoffs used were p < 0.0001 and fold change (FC) > 1.25 and noted with dotted lines. Note: ERG1 (Q14534) is a squalene monooxygenase and not the ETS family member ERG (P11308).

Fig 4: The canonical SWI/SNF complex is the primary cofactor of enhancer-binding transcription factors and is essential for enabling their oncogenic gene programs.(a, c) Genome-wide ChIP-seq read-density heatmaps and Venn diagrams for CTCF in VCaP (a) or LNCaP (c) cells treated with either DMSO or AU-15330 (1 μM) for 6h. Vinculin is the loading control probed on a representative immunoblot. (b, d) Immunoblots of indicated proteins in VCaP (b) or LNCaP (d) cells treated with AU-15330 (1 μM) for increasing time durations. Total histone H3 is the loading control probed on all immunoblots. This experiment was repeated independently twice. (e) GSEA plots for ERG, FOXA1, and MYC-regulated genes using the fold change rank-ordered gene signature from AU-15330-treated (1 nM, 24h) VCaP cells. NES, net enrichment score; adj P, adjusted p-value; DEGs, differentially expressed genes. (f, g) GSEA of FOXA1, MYC, or ARID1A-regulated genes (see Methods for gene sets) in the fold change rank-ordered AU-15330 gene signature in indicated PCa cells. DEGs, differentially expressed genes. (n = 2 biological replicates, GSEA enrichment test) (h, i) Expression of indicated genes (qPCR) in VCaP (h) or LNCaP (i) cells upon treatment with DMSO, AU-15330, dBRD7 (BRD7 degrader), or dBRD7/9 (dual BRD7 and BRD9 degrader) at 1 μM for 24h. Data are presented as mean +/− SD (n = 3, technical replicates) from one-of-two independent experiments. (j) Immunoblots for indicated proteins in LNCaP and VCaP cells treated with AU-15330, dBRD9 (BRD9 degrader), or VZ185 (BRD7/9 degrader) at indicated concentrations. Vinculin is the loading control probed on all immunoblots. This experiment was repeated independently twice. Source data

Fig 5: Female adipose EC exhibit elevated proliferation rates compared to male EC(A and B) Female and male mice were fed an HFD for 7 weeks. EdU was injected two sequential days prior to tissue extraction.(A) Whole-mount confocal imaging of white visceral adipose tissue to detect proliferating EC. Green (Griffonia simplicifolia-FITC), capillaries; Blue (DAPI), nuclei; Red (ERG), Endothelial cell nuclei; Yellow (EdU), proliferating cells. Arrows point to EdU-positive endothelial cell nuclei. Scale bar = 25 μm.(B) Quantification of EdU-positive EC (ERG+) relative to the total number of EC per field of view. Data points represent individual fields of view from 4 female and 3 male mice. Bars represent mean ± SEM ∗∗p = 0.0037, two-tailed unpaired Student’s t test.(C and D) RNA was extracted from visceral white adipose tissue of the EdU cohort of female and male mice (n = 4 and 3 respectively). Expression levels of Vegfa (C) and Vegfb (D) relative to Hprt1 were quantified by qPCR. Also, refer to Figure S3.(E) Adipose EC from young, healthy female and male mice were isolated and cultured under growth conditions. Proliferation was assessed by EdU incorporation. EdU-positive cells were quantified as a percentage of total EC number. n = 7 independent experiments/sex; ∗p = 0.015, two-tailed unpaired Student’s t test.