Fig 2: BET inhibitor alone or in combination with Enzalutamide downregulates DLX1 expression and mitigates its oncogenic properties.a ChIP-Seq data (GSE55064) showing ERG enrichment on DLX1 promoter in VCaP cells treated with JQ1 or vehicle control for 24 h. H3K27Ac represents active promoter in untreated cells. PLAU used as a positive control. b ChIP-qPCR data showing relative ERG enrichment on DLX1 promoter in VCaP cells treated with JQ1 (0.5 µM) for 48 h (P < 0.0001). c Q-PCR data showing relative expression of target genes in VCaP cells treated with Enza (10 µM), JQ1 (0.5 µM) alone or in combination for 48 h. KLK3 used as a positive control for JQ1 treatment. d Same as c except immunoblot. ß-actin was used as loading control. e Same as c except 22RV1 cells. f Same as e except immunoblot (P < 0.0001). g Cell proliferation assay in VCaP cells treated with drug conditions as mentioned in c. h Same as g except 22RV1 cells (P < 0.0001). i Same as g except 42D ENZR cells (P < 0.0001). j Boyden Chamber Matrigel migration assay in VCaP cells using same treatment conditions as c. Inset shows representative image of the migrated cells (scale bar 30 µm). k Same as j except 22RV1 cells (scale bar 30 µm, P < 0.0001). l Same as j except Enza-resistant, 42D ENZR cells (scale bar 30 µm, P < 0.0001). m Fluorescence intensity of the catalyzed ALDH substrate in VCaP cells under same treatment conditions as c. Marked windows show ALDH1+ percent cell population. n Same as m except 22RV1 cells. o Same as m except 42D ENZR cells. p Bar plot showing number of spheres formed in prostatosphere assay using VCaP under same treatment conditions as c. Inset shows representative image of the spheres formed (scale bar 100 µm). q Q-PCR data for the DLX1 expression using RNA isolated from VCaP prostatospheres. Data shown from three biologically independent samples (n = 3). Data represent mean ± SEM. For panels b, c, e two-way ANOVA, Tukey’s multiple comparison test; g, h, i One-way ANOVA, Dunnett’s multiple comparison test; j–l, p, q One-way ANOVA, Tukey’s multiple comparison was applied. Source data are provided as a Source Data file.

Fig 3: LLU-206 suppresses the growth of 22RV1 and LNCaP-ENZR xenografts in vivo. (A) LNCaP ENZR cells were generated by culturing the LNCaP cells in continuous exposure of ENZ for three months in vitro. (B) Mice bearing LNCaP-ENZR xenografts were administered with LLU-206 (10, 25 mg/kg) via tail vein and tumor volumes were measured every other day (n = 6). (C) Tumor weight of each group. (D) PSA levels of plasma were measured by ELISA kit. (E) Representative IHC staining of Ki-67. (F) Representative H&E staining of tumors. (G) The ARfl, ARv7, PSA protein levels in the tumors were detected by Western blot. (H) Representative bioluminescence images of 22RV1 xenografts in the prostate. (I) Tumor growth changes in mice bearing 22RV1 orthotropic tumors were analyzed by average fluorescence values (n = 6). (J) The relative tumor weight was measured at the end of the experiment in each group. (K) Representative H&E staining of 22RV1 tumor tissues were presented as well as IHC analysis of Ki-67. (L) Ki-67 expression and tumor necrotic were quantified using Image J and Image-Pro Plus 6.0. (M, N) The ARfl, ARv7, and PSA protein levels in the tumors were detected by Western blot and quantified. Scale bars, 50 μm, 100 μm, 200 μm ∗P < 0.05, ∗∗∗P < 0.001. n.s., not significant.

Fig 4: Screening and identification of a novel small molecule inhibitor targeting AR. (A) Pyramid chart of the screening inhibitors of ARfl and ARv7 from the small molecule compounds library. (B) Three parental compounds were obtained by computer-aided drug design. (C) Effects of the top 8 compounds on cell viability in 22RV1 cells (n = 3). (D–F) LNCaP (ARfl positive cells), 22RV1 (both ARfl and ARv7 positive cells) and PC3 (transfected with ARfl and ARv7 expressing plasmids) cells were administrated with the top eight compounds (10 µmol/L, 48h). Moreover, the protein levels of ARfl, ARv7 and PSA were analyzed by Western blot. (G) The chemical structure of LLU-206. (H) Binding model of LLU-206 in AR-LBD binding pocket. (I) The docking prediction of LLU-206 (purple) with AR-LBD (blue). (J) The interaction between AR-LBD protein and LLU-206 was measured by biacore. Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001. n.s., not significant.

Fig 5: VNPT-178 and VNLG-74A antagonize the fAR and promote its depletion(A) Androgen-dependent luciferase expression was determined in LNCaP and 22Rv1 cells transiently co-transfected with ARR2-Luc and pRL-null reporters. Plot represents mean RLU fold-change ± SEM of three independent experiments. (B) Western blots of AR and PSA expression in LNCaP and 22Rv1 cells 48 hours after treatment with indicated compounds. (C) LNCaP and (D) 22Rv1 cells treated with increasing doses of VNPT-178 or VNLG-74A for 24 hours exhibit differences in AR, PSA, and cleaved caspase-3 expression. Densitometry of total AR and PSA immunoblots are plotted as the mean fold-change ± SEM of three replicate assays. (E) VNPT-178 and VNLG-74A uniquely promote PARP cleavage in LNCaP cells after 48 hours.