Fig 2: Unique OSU-ERb-12 plus Enza cistrome-transciptome dependent target genes significantly associate with high androgen and low neuroendocine score tumors in the SU2C cohort.The Zscore data frame from the SU2C cohort was filtered for 192 unique genes that were defined by; a loss of AR binding and upregulation by OSU-ERb-12 plus Enza; a loss of MYC binding and downregulation by OSU-ERb-12 plus Enza; a loss of H3K27ac binding and downregulation by OSU-ERb-12 plus Enza. These relationships were visualized with a heatmap (pheatmap) and tumors clustered by expression, and the significance of the relationship between tumor cluster id and either neuroendocrine score (NE range) or androgen score (AR range), as define previously(45), was measured with a chi-sq test.

Fig 3: OSU-ERb-12 plus Enza induces a unique transcriptome associated with MYC repression in LNCaP, LNCaP C4-2 and 22 Rv1 cells.A. LNCaP, LNCaP C4-2 and 22Rv1 cells in triplicate were treated with either vehicle, OSU-ERb-12 alone (100 nM) or E2 alone (***), Enza (1 μM) or the combinations for 12h and RNA-Seq was undertaken. FASTQ files were QC processed, aligned to hg38 (Rsubread), and processed with edgeR workflow to identify significant differentially enriched genes (DEGs) (logPV > 1 & absFC > .26) of the drug combination compared to the combination of the average of the individual treatments, and are illustrated by Volcano plots with top DEGs labelled. B. GSEA analyses (Hallmarks, and Chemical and Genetic Perturbations) was undertaken and enrichment terms were word frequency analyzed which identified that terms including Breast, Prostate, Nuclear Receptors and MYC were amongst the most frequent, and these terms were filtered for conditions where the NES was in the opposite direction, and the delta calculated, with top 40 NES terms illustrated. C. NES plots for the indicated treatments with either OSU-ERb-12 or E2 co-treatments. D. Western immunoblot measurements of MYC after treatment with OSU-ERb-12 plus Enza (time 24 h).

Fig 4: Cistrome-transciptome relationships impacted by OSU-ERb-12 plus Enza.A. Unique AR and MYC cistromes in response to OSU-ERb-12/Enza were overlapped using GIGGLE with the CistromeDB collection (> 10,000 total ChIP-seq datasets, across > 1100 factors). The logPV of the FDR-corrected pval is illustrated for prostate specific TFs and coregulators. B. Each peak was annotated to genes within 100 kb and filtered as to whether the gene was a DEG from the respective RNA-Seq and the score.absFC calculated for peak:gene relationships that were also DEGs, and a t-test used to assess whether these values were significantly different between the groups where the CUT&RUN peak enrichment was reduced (D) or increased (U) and the annotated gene expression what down-regulated (D) or up-regulated (u). C. IGV image of the AR, MYC and H3K27ac at the ZBTB16 gene. Triplicate BAM files were merged and converted to bigwig files and visualized at the ZBTB16 gene as an example of a gene that displays significantly reduced enrichment by OSU-ERb-12/Enza for H3K27ac and MYC, and is downregulated at the mRNA level.

Fig 5: Inhibition of colony formation by OSU-ERb-12 combinations and regulation of differentiation markers in 22Rv1 cells.A. Representative images of 22Rv1 colonies treated with OSU-ERb-12 (100 nM) with either Enza (1 μM) or FK228 (1nM) or the combinations with quantification of colony number. B. Western immunoblot measurements of AR, CK5 and CK8 after treatment with OSU-ERb-12 plus Enza (time 7 days) with quantification (C).