Increased glucocorticoid receptor (GR) expression and activity following androgen blockade can contribute to castration-resistant prostate cancer (CRPC) progression. Therefore, we hypothesized that GR antagonism will have therapeutic benefit in CRPC. However, the FDA-approved nonselective, steroidal GR antagonist, mifepristone, lacks GR specificity, reducing its therapeutic potential. Here we report that two novel non-steroidal and highly selective GR modulators (SGRMs), CORT118335 and CORT108297, have the ability to block GR activity in prostate cancer (PC) and slow CRPC progression. In contrast to mifepristone, these novel SGRMs did not affect AR signaling, but potently inhibited GR transcriptional activity. Importantly, SGRMs decreased GR-mediated tumor cell viability following AR blockade. In vivo, SGRMs significantly inhibited CRPC progression in high GR-expressing, but not in low GR-expressing xenograft models. Transcriptome analysis following AR blockade and GR activation revealed that these SGRMs block GR-mediated proliferative gene expression pathways. Furthermore, GR-regulated proliferation-associated genes AKAP12, FKBP5, SGK1, CEBPD, and ZBTB16 are inhibited by CORT108297 treatment in vivo. Together, these data suggest that GR-selective non-steroidal SGRMs potently inhibit GR activity and PC growth despite AR pathway inhibition demonstrating the therapeutic potential of SGRMs in GR-expressing CRPC.

Corticotropin-releasing hormone (CRH) and vasopressin are the most important hypothalamic factors regulating adrenocorticotropic hormone (ACTH) secretion. In this study we have investigated the responsiveness of the pituitary-adrenocortical axis to intravenous administration of CRH or lysine vasopressin (LVP) in 16 control dogs, 22 dogs with pituitary-dependent hyperadrenocorticism and five dogs with hyperadrenocorticism due to an adrenocortical tumor, using doses of CRH and LVP that caused equivalent ACTH responses in the control dogs. After CRH administration, the increment in plasma ACTH was significantly (p < 0.05) lower in dogs with pituitary-dependent hyperadrenocorticism (221 +/- 53 ng/l) than that in control dogs (279 +/- 41 ng/l). In the dogs with pituitary-dependent hyperadrenocorticism, the relative increases in ACTH after CRH were significantly (p < 0.05) lower than those after LVP. Despite the absence of an increase in ACTH following LVP administration in dogs with hyperadrenocorticism due to an adrenocortical tumor, there was a significant increase in plasma cortisol, the increment (790 +/- 238 nmol/l) being not statistically different from that in the control dogs (412 +/- 37 nmol/l). We conclude that in spite of the changes inherent to pituitary-dependent hyperadrenocorticism, i.e. neoplastic transformation of corticotropic cells and hypercortisolism, there is persistence of responsiveness to hypophysiotropic hormones. The ACTH secretion by corticotropic cells in pituitary-dependent hyperadrenocorticism was relatively less sensitive to stimulation with CRH than with LVP. Adrenocortical tumors develop an aberrant sensitivity to LVP.

Expression of the constitutively active androgen receptor splice variant AR-V7 is associated with resistance to anti-androgen therapies and worse clinical outcomes in prostate cancer. Dimerization is a requirement for AR-V7 transcriptional activity, but whether that occurs through homodimerization or heterodimerization remains poorly understood. Because NR3C-family nuclear hormone receptors rely on a highly conserved D-box motif within the DNA-binding domain to mediate dimerization, we hypothesized that AR-V7 can heterodimerize with other NR3C-family nuclear hormone receptors, with a particular interest in the glucocorticoid receptor (GR). Like AR-V7, GR is upregulated in prostate cancer following androgen blockade, regulates canonical AR target genes, and is associated with resistance to anti-androgen therapies. Using NanoBRET technology in HEK293 cells, we found that AR-V7 interacts with liganded NR3C-family nuclear hormone receptors, including the full-length androgen receptor, the progesterone receptor, the mineralocorticoid receptor, and, most notably, GR. Using co-immunoprecipitation and proximity ligation assays, we demonstrate that AR-V7 associates with GR following androgen blockade in both the VCaP cell line and the CWR-22RV1 cell line, two models of prostate cancer that express endogenous AR-V7 and GR. Preliminary ChIP-Seq experiments in these cell lines suggest AR-V7 and GR chromatin occupancy at the same genomic sites in a ligand-dependent manner. Taken altogether, this data suggests AR-V7 can utilize GR as an alternative binding partner to initiate heterodimerization and transcriptional activation. Citation Format: Eric Bueter, David Hosfield, Erin McAuley, Phillip Selman, Geoffrey Greene, Suzanne D. Conzen, Russell Z. Szmulewitz. The androgen receptor splice variant AR-V7 associates with the glucocorticoid receptor following androgen blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1016.

<p>Supplemental Tables 1-2</p>

<p>Supplemental Figures 1-8</p>