Fig 2: The importance of noncanonical AR-targeted genes in ENZ-resistant growth of CRPC cells.a Western blot showing the indicated proteins expression in C4-2CON and C4-2ENZ-R cell lines. RT-qPCR analysis shows the indicated genes expression in C4-2CON and C4-2ENZ-R cells (b) or in control and AR knockdown C4-2ENZ-R cells (c). Data shown means ± s.d., (n = 3 replicates/group). d Western blot analysis of the indicated proteins in C4-2ENZ-R cells infected with the indicated lentivirus expressing gene-specific shRNAs for 96 h. e–g Cell proliferation analysis of C4-2CON and C4-2ENZ-R cells infected by the specific shRNAs. Data shown means ± s.d., (n = 6 replicates/group). h RT-qPCR analysis of expression of ID1, PFN2, and ID3 in C4-2ENZ-R cells transfected control or AR, CXXC5, or TET2-specific shRNAs. Data shown as means ± s.d. (n = 3 replicates/group). i ChIP-qPCR analysis of CXXC5, TET2, and AR occupancy at genomic loci of ID1, PFN2, and ID3 in C4-2ENZ-R cells transfected control or AR, CXXC5, or TET2-specific shRNAs. Data shown as means ± s.d. (n = 3 replicates/group). j Genomic DNA purified from 293T cells overexpressing wild-type (WT) or mutant TET2 (H1881R) was stained with methylene blue or blotted using anti-5hmC (left). Co-IP analysis of interaction of TET2-WT and TET2-H1881R with CXXC5 and AR in 293T cells (right). k His-tag pulldown analysis of His-tagged CXXC5 interaction with AR and TET2 proteins produced by in vitro transcription/translation. l Western blot showing the indicated proteins expression in C4-2ENZ-R cells infected with lentivirus expressing indicated shRNAs or expression vectors. m RT-qPCR analysis of expression of ID1, PFN2, and ID3 in C4-2ENZ-R cells infected with lentivirus as in l. Data shown as means ± s.d. (n = 3 replicates/group). n Cell proliferation analysis of C4-2ENZ-R cells with knockdown of endogenous TET2 and restored expression of TET2-WT or TET2-H1881R. Data shown as means ± s.d. (n = 6 replicates/group). Statistical significance was determined by unpaired two-tailed Student’s t tests in b, c, h, i, m and by two-way ANOVA in e, f, g, n. Experiments in a, d, j, k were repeated twice.

Fig 3: Identification of upregulated nonconanical AR target genes in ENZ-resistant CRPC cells.a Heatmap shows RNA-seq read intensity of the upregulated genes (see the list of genes in Supplementary Data 1) at the ARBS-gained-CpGi loci in C4-2ENZ-R cells. b GSEA analysis of upregulated signature genes associated with the ARBS-gained-CpGi sites in ENZ-resistant CRPC cells. c UCSC tracks (integration of all replicates) show profiles of RNA-seq signals, ChIP-seq signals of IgG, AR, CXXC5, TET2, FOXA1, and H3K27ac at ARBS-gained-CpGi gene loci (ID1, PFN2, ID3) or ARBS-lost gene loci (KLK3, TMPRSS2, and NKX3.1). d Kaplan–Meier survival analyses of the association of CXXC5 expression with the overall survival of SU2C CRPC patients treated with AR signaling inhibitors (including ENZ and ABI). For overall survival analysis, log-rank (Mantel–Cox) test was used to determine the statistical difference between stratified groups. e Kaplan–Meier survival analyses of the association of noncanonical AR gene expression score (see Methods) with the overall survival of SU2C CRPC patients treated with AR signaling inhibitors (including ENZ and ABI). For overall survival analysis, log-rank (Mantel–Cox) test was used to determine the statistical difference between stratified groups. f GSEA plot showing the enrichment of noncanonical AR genes in the indicated groups with favorable or unfavorable outcome of SU2C CRPC patients treated with AR signaling inhibitors (including ENZ and ABI). 44 patients unfavorable versus 27 favorable patients; Statistical significance was determined by two-sided Kolmogorov–Smirnov test. g Expression of ID1 and PFN2 genes in the indicated groups with favorable (n = 27) or unfavorable (n = 44) outcome of SU2C CRPC patients treated with AR signaling inhibitors (including ENZ and ABI). Data shown as means ± s.d. Statistical significance was determined by unpaired two-tailed Student’s t tests. h Kaplan–Meier survival analyses of the association of noncanonical AR gene (ID1 and PFN2) and canonical AR gene (KLK3 and TMPRSS2) expression with the overall survival of SU2C CRPC patients treated with AR signaling inhibitors (including ENZ and ABI). For overall survival analysis, log-rank (Mantel–Cox) test was used to determine the statistical difference between stratified groups.

Fig 4: ENZ-resistant CRPC cells are sensitive to BET and CBP/p300 inhibitors in vitro and in vivo.a Heatmap shows the sensitivity of C4-2CON and C4-2ENZ-R cells to the indicated pathway inhibitors. b Cell proliferation analysis shows the inhibitory effect of BET-CPB/p300 dual inhibitors NEO1132 and NEO2734 on C4-2CON and C4-2ENZ-R cell proliferation. Data shown as means ± s.d. (n = 3 replicates/group). Statistical significance was determined by two-way ANOVA. c Cell proliferation analysis of control and ENZ-R LNCaP, VCaP, and LAPC4 cells treated with the indicated concentration of NEO2734. Data shown as means ± s.d. (n = 6 replicates/group). Statistical significance was determined by two-way ANOVA. d Western blot analysis of AR, CXXC5, ID1, ID3, PFN2, TET2, BRD4, and p300 in C4-2CON and C4-2ENZ-R cells treated with NEO2734 at the indicated concentrations. ERK2 was used as a loading control; Experiments were repeated twice. e–g Effect of the indicated inhibitors on C4-2ENZ-R xenograft tumor volume and weight. Data are represented as means ± s.d. (n = 10 replicates/group). Statistical significance was determined by two-way ANOVA for tumor volume and by unpaired two-tailed Student’s t tests for tumor weight. h Effect of the indicated inhibitors on mouse body weight. Data shown as means ± s.d. (n = 10 replicates/group). i IHC of AR, CXXC5, ID1, PFN2, Ki-67, and cleaved-caspase3 in tumors treated for 28 days with vehicle, ENZ, or NEO2734. Representative images (scale bar, 100 µm) and quantified data are shown in upper and lower panels, respectively. Data shown as means ± s.d. (n = 5 replicates/group). Statistical significance was determined by unpaired two-tailed Student’s t tests.