Extended Data Fig. 8: ET516 specifically repress AR-dependent signaling and inhibits cell proliferation of prostate cancer expressing mutants resistant to antiandrogens.

a. GSEA enrichment plots comparing DHT(1 nM)-stimulated LNCaP cells to vehicle cells showing positive enrichment for gene set corresponding to AR target genes. b. Differential regulated signal pathway in ET516 treated LNCaP cells compared to DHT-stimulated control cells. LNCaP cells were treated with ET516 two hour prior to the DHT stimulation for 10 hours. c. GSEA enrichment plots comparing ET516 treated LNCaP cells to control cells showing no obvious enrichment for gene set corresponding to ER and p53 target genes compared to AR target genes. d. ET516 was subjected to a panel of transcription factor reporter assays. Data are presented as the mean ± SD, n = 3 replicates. e. A panel of condensates assays to test the specificity of ET516. Scale bar, 5 μm. ET516 was added to a series of phase-separated condensates for 2 hours and the puncta number of ET516/vehicle was analyzed. Data are presented as the mean ± SD, n = 4–7 biologically independent samples per group. In d-e, hydrocortisone(10 nM), E2(10 nM) and IL-6(4 ng/ml) were used to activate glucocorticoid receptor, estrogen receptor and Stat3, respectively. YAP condensates were induced with TEAD co-transfection. f. Images of LNCaP cells expressing AR(F877L/T878A) treated with DHT combined with DMSO (vehicle), ET516 and enzalutamide (Enza) for 8 days (Top). Brightfield images of LNCaP cells expressing AR(V7) treated with DMSO (vehicle), ET516 and enzalutamide (Enza) for 8 days (Bottom). Scale bar, 100 μm. g. Representation of hollow fiber assay for evaluating the efficacy of ET516, EPI001 and enzalutamide. h. Efficacy results of hollow fiber assay that treated with (ET516, 30 mg/kg, p.o., BID; EPI001, 30 mg/kg, p.o., BID; Enzalutamide, 30 mg/kg, p.o., QD) for 10 days, Data are presented as the mean ± SD, N = 6 mice each group.

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