ON-TARGETplus siRNA pools for gene expression knockdown.

List of the 44 3D structures of HSC70 (HSPA8) in complex with the BAG domain of BAG-1 used in comparative structural analyses.

Antibodies used for western blot analyses.

Thio-2 reduces genome-wide androgen receptor binding in LNCaP prostate cancer cells.

Synthego BAG-1 sgRNA for BAG-1 CRISPR knockout clones.

Development and characterization of BAG-1 knockout mice.

Development of castration resistant prostate cancer patient derived xenograft organoids.

Mouse organ hematoxylin and eosin, and BAG-1 immunohistochemistry, in BAG-1 knockout mice.

Mouse organ hematoxylin and eosin, and BAG-1 immunohistochemistry, in BAG-1 knockout mice.

TaqMan probes for qRT-PCR analyses.

Thio-2 downregulates C-MYC expression through a BAG-1 independent mechanism.

Treatment with Thio-2 inhibits transactivation of the unstimulated and stimulated androgen receptor and the constitutively active androgen receptor splice variant-7.

Thio-2, may bind the N-terminus of the androgen receptor through a similar binding mode to EPI-001.

Description of Materials and Methods not contained within the main Materials and Methods section

Protocols and antibodies for immunohistochemical analyses.

In-vivo treatment of patient derived model of lethal prostate cancer demonstrates Thio-2 to be well tolerated with associated impact on AR signaling and growth.

Thio-2 downregulates androgen receptor signaling and inhibits growth of prostate cancer cell lines through a BAG-1 independent mechanism.

Abstract Therapies that abrogate persistent androgen receptor (AR) signaling in castration-resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain of the AR that drives transcriptional activity in CRPC remains a challenging therapeutic target. Herein we demonstrate that BCL-2–associated athanogene-1 (BAG-1) mRNA is highly expressed and associates with signaling pathways, including AR signaling, that are implicated in the development and progression of CRPC. In addition, interrogation of geometric and physiochemical properties of the BAG domain of BAG-1 isoforms identifies it to be a tractable but challenging drug target. Furthermore, through BAG-1 isoform mouse knockout studies, we confirm that BAG-1 isoforms regulate hormone physiology and that therapies targeting the BAG domain will be associated with limited “on-target” toxicity. Importantly, the postulated inhibitor of BAG-1 isoforms, Thio-2, suppressed AR signaling and other important pathways implicated in the development and progression of CRPC to reduce the growth of treatment-resistant prostate cancer cell lines and patient-derived models. However, the mechanism by which Thio-2 elicits the observed phenotype needs further elucidation as the genomic abrogation of BAG-1 isoforms was unable to recapitulate the Thio-2–mediated phenotype. Overall, these data support the interrogation of related compounds with improved drug-like properties as a novel therapeutic approach in CRPC, and further highlight the clinical potential of treatments that block persistent AR signaling which are currently undergoing clinical evaluation in CRPC.

Therapies that abrogate persistent androgen receptor (AR) signaling in castration resistant prostate cancer (CRPC) remain an unmet clinical need. The N-terminal domain (NTD) of the AR drives transcriptional activity in CRPC but is intrinsically disordered and remains a challenging therapeutic target. Therefore, inhibiting critical co-chaperones, such as BAG-1L, is an attractive alternative strategy. We performed druggability analyses demonstrating the BAG domain to be a challenging drug target. Thio-2, a tool compound, has been reported to bind the BAG domain of BAG-1L and inhibit BAG-1L-mediated AR transactivation. However, despite these data, the mechanism of action of Thio-2 is poorly understood and the BAG domain which is present in all BAG-1 isoforms has not been validated as a therapeutic target. Herein, we demonstrate growth inhibiting activity of Thio-2 in CRPC cell lines and patient derived models with decreased AR genomic binding and AR signaling independent of BAG-1 isoform function. Furthermore, genomic abrogation of BAG-1 isoforms did not recapitulate the described Thio-2 phenotype, and NMR studies suggest that Thio-2 may bind the AR NTD, uncovering a potential alternative mechanism of action, although in the context of low compound solubility. Furthermore, BAG-1 isoform knockout mice are viable and fertile, in contrast to previous studies, and when crossed with prostate cancer mouse models, BAG-1 deletion does not significantly impact prostate cancer development and growth. Overall, these data demonstrate that Thio-2 inhibits AR signaling and growth in CRPC independent of BAG-1 isoforms, and unlike previous studies of the activated AR, therapeutic targeting of the BAG domain requires further validation before being considered a therapeutic strategy for the treatment of CRPC.