Fig 2: Model for GR-mediated enzalutamide resistance in TLE3KO prostate cancer cells.In the presence of androgens, TLE3 expression is repressed and enhancers are active. AR regulates target gene expression, including repression of the GR locus (top panel). Upon enzalutamide treatment, TLE3 is upregulated and enhancers are inactive. TLE3 represses expression of AR target genes including GR (middle panel). Enzalutamide treatment in the context of TLE3 loss leads to upregulation of GR which is able to substitute for AR at active enhancers, leading to drug resistance (bottom panel).

Fig 3: ChIP-seq analyses for transcription factor binding at differentially expressed genes in control cells compared to TLE3KO cells cultured in the presence of 10 μM enzalutamide.(A) Coverage profiles for TLE3 and AR at the loci of two genes (RND3 and GNAI1). (B) Heatmap of AR, TLE3 and FOXA1. (co-)binding at genes differentially expressed in TLE3KO compared to control cells treated with enzalutamide are shown. The binding of AR, TLE3 and FOXA1 at these sites is shown for androgen-depleted or R1881-stimulated (4 hr) conditions in parental LNCaP cells. (C) ChIP-seq peak enrichment near the Transcription Start Sites (TSS) of differentially expressed (DE) genes and a random set of genes. The fraction of genes with a peak for TLE3, AR or both transcription factors at indicated distance from the TSS is shown for both genesets.

Fig 4: TLE3 and GR expression in tumors of prostate cancer patients.(A–B) RNA-seq analysis showing the correlation between TLE3 and GR expression in tumor samples from prostate cancer patients. (C) Kaplan-Meier curve showing the biochemical recurrence of prostate cancer patients from the TCGA dataset, only patients receiving anti-hormonal therapy were included (65 patients) using an optimal cut-off for high versus low TLE3 expression. (D) Immunohistochemistry for H and E, TLE3 and GR in tumor biopsy samples collected from two CRPC patients pre- and post-enzalutamide treatment.

Fig 5: GR inhibition resensitizes TLE3KO cells to enzalutamide treatment.(A) Western blot showing protein expression levels of TLE3 and GR in control and TLE3KO cells cultured vehicle or enzalutamide (B) Coverage profiles for TLE3 and AR binding at the GR locus. (C) Long-term growth assay (14 days) showing the drug resistance phenotype of control and TLE3KO cells with and without GR knockdown in the presence of vehicle or enzalutamide. (D) Western blot analysis for TLE3 protein levels in control and TLE3KO cells shown in C, using GAPDH as a loading control. (E) mRNA levels for GR in control and TLE3KO cells carrying shSCR or shGR constructs, shown in C. (F) Long-term growth assay (14 days) for cells harboring a control sgRNA or TLE3-targeting sgRNA cultured in the presence of vehicle, enzalutamide, mifepristone or the combination at indicated concentrations. (G) ChIP-qPCR showing GR occupancy at enhancers proximal to indicated genes. All samples were cultured in the presence of 10 µM enzalutamide with or without 1 µg/ml hydrocortisone (HC) as indicated.