Fig. 5: Non-canonical activity of EZH2 is limited to PRAD models.

A Scatter plot of differentially expressed genes in PRAD and NEPC models in RNA-Seq data as described in Fig. 1A (n = 3/condition/model); blue, significantly downregulated genes upon tazemetostat treatment (log₂FoldChange < 0.5; p-value < 0.05); red, significantly upregulated genes upon tazemetostat treatment (log₂FoldChange > 0.5; p-value < 0.05). B Scatter plot of differentially expressed genes in WCM154-sgEZH2 #1-dTAG-N-EZH2 rescue model in RNA-Seq data as described in Fig. 4H upon treatment with vehicle or dTAGv-1 treatment for 8 h (n = 3/condition; log₂FoldChange < 0.5; p-value < 0.05). C Box plot summary of cumulative z-score of FPKM values (from Fig. 3A; n = 3) of 56 genes co-activated by EZH2 reported in Xu et al.²⁰; white, vehicle treatment; red, tazemetostat treatment; bars, center of box represents median, the lower and upper hinges correspond to the 25th and 75th percentiles, the upper whisker extends from the hinge to the largest value no further than 1.5 times inter-quartile range (IQR), the lower whisker extends from the hinge to the smallest value at most 1.5 times IQR of the hinge, data beyond the end of the whiskers are outliers and are plotted individually; –, p-value < 0.05; *, p-value < 0.05, all statistical analyses used Wilcoxon two-sided tests. D Cells or organoids of PRAD (pink) or NEPC (burgundy) models were treated as indicated in Fig. 3A (n = 3/condition) and RNA was isolated and processed for RNA-seq. GSEA analysis was performed and normalized enrichment score are plotted for the indicated pathway; gray-black gradient, FDR values. E FPKM values of CCNA2, CCNB1, and CCNB2 from the RNA-seq (Fig. 3A; n = 3/condition) in vehicle or tazemetostat treated conditions in indicated models. Columns, means of values (n = 3/condition/model); white, vehicle treatment; red, tazemetostat treatment; bars, SEM values; –, p-value < 0.05; *, p-value < 0.05, all statistical analyses used Wilcoxon two-sided tests. F Western blot analysis of LNCaP and LNCaP-abl samples treated with vehicle (DMSO) or 5 μM tazemetostat for 6 days as described in Fig. 1A (n = 3/condition), showing downregulation of indicated cyclins along with reduced levels of H3K27me3. Blots were reprobed for β-actin and total H3 as loading controls. Representative western blot from three independent experiments is shown. G IC₅₀ dose response curves in PRAD (pale pink) and NEPC (burgundy) using increasing dose of CIR7-2512. dots, means of values (n = 5); bars, SEM values. H NEPC (burgundy) models were treated with vehicle (Veh; DMSO) or 500 nM CIR7-2512 for indicated number of days and cell viability was performed. dots, means of values (n = 5); grey, vehicle treatment; blue, tazemetostat treatment; bars, SEM values; –, p-value < 0.05; *, p-value < 0.05, all statistical analyses used Wilcoxon two-sided tests. I Prostate cancer models (Left panel – AR-driven PRAD; right panel – AR-indifferent NEPC) were treated with vehicle (Veh; DMSO) or 5 μM of tazemetostat (Taz) or 500 nM CIR7-2512 or in combination. CellTiter-Glo® luminescent cell viability assay was performed after 6 days of treatment. Columns, means of values (n = 5; WCM155, Taz treatment; n = 4); white, vehicle treatment; red, tazemetostat treatment; blue, CIR7-2512 treatment; green, combination treatment; bars, SEM values; –, p-value > 0.05 (not significant); *, p-value < 0.05, all statistical analyses used Wilcoxon two-sided tests. J Schematic of the differences in canonical and non-canonical function of EZH2 in prostate adenocarcinoma compared to neuroendocrine prostate cancer. Figure 5/panel J Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.