Currently, androgen deprivation therapy (ADT) is to either reduce androgen production by surgical or chemical castration or inhibit AR activity by blocking androgen binding to the C-terminal ligand-binding domain (LBD) of AR. Although there are new agents of ADT entering clinics, ADT is not curative and only prolongs the relapse of CRPC. However, several constitutively active AR-Vs lacking the LBD have been identified in CRPC cells and patients, which have been associated with the overall low survival rate of patients and the drug resistance to ADT or chemotherapeutics. Therefore, targeting AR-Vs is a critical and unmet need for CRPC treatment.

It is believed that the formation of AR-Vs can occur through two different mechanisms: genomic rearrangement and alternative splicing. Although genomic rearrangement of AR gene has been reported in PCa cell lines and clinical tissues, it remains controversial because data of RNA-seq from SU2C study did not reveal this mechanism. Alternative splicing of AR precursor mRNA is common mechanism for generating a variety of AR-V proteins expressed in PCa cell lines and specimens. More than 90% of human protein-coding genes undergo programmed alternative pre-mRNA splicing to generate protein variants far greater than the number of genes. While this process inherently provides for transcriptome diversity necessary for normal development, aberrant pre-mRNA splicing is implicated in numerous disease conditions, including cancer and neurodegeneration. As such, therapeutic intervention using small-molecule inhibitors (SMIs) to target the pre-mRNA splicing machinery (spliceosome) has gained considerable attention in recent years.

The critical role of AR-Vs in CRPC indicates an opportunity and a paradigm shift in the targeting strategy of ADT because AR-Vs are ligand-independent activation (i.e., constitutively active) and expected to be resistant to any AR antagonists currently used in CRPC therapy. It has been shown that the onset of AR-Vs mainly results from alternative gene splicing. In order to identify potential small-molecule inhibitor (SMI) for AR-V gene splicing, a cell-based model using an AR-minigene containing exon 3, intron 3, and encrypted exon 3 sequences was established for chemical library screening; thailanstatin D (TST-D, spliceostatin A analog) was identified among several positive hits. TST-D can significantly suppress the expression of AR-V7 mRNA and protein but in a less extent on the full-length AR expression. Also, TST-D is an effective growth inhibitor for CRPC-expressing AR-V7 but less potent for other PCa cell lines. Mechanistically, TST-D is able to inhibit AR-V7 gene splicing by interfering the interaction between U2AF65 and SAP155 and preventing them from binding to polypyrimidine tract located between the branch point and the 3’ splice site.

In vivo, TST-D exhibits a potent tumor-inhibitory effect on CRPC xenografts leading to cell apoptosis. Also, TST-D shows some undesirable side effects due to its action on alternative gene splicing of other genes. Thus, we further engineer a PCa-specific targeting agent using small-molecular ligand for prostate specific membrane antigen (PSMA) with high levels in the presence of 95% of PCa to make PSMA-TSTD. We also demonstrated a PCa-specific imaging capability of this agent. By transfecting AR-V7 minigene into PC3-PSMA+ and - cells then treated with PSMA-TSTD, the result indicated that PSMA-TSTD was able to inhibit AR-V7 gene splicing. Also, PSMA-TSTD at the concentration of 3 μM can inhibit 50% growth of CRPC cell-expressing AR-V7; the effective concentration of this compound is still within the range of the drug candidate that can be further developed into therapeutics. Taken together, PSMA-TSTD represents a novel theranostic agent with PCa specificity and interrupting alternative gene splicing of AR gene, which could target therapy-resistant CRPC.

Citation Format: Rey-Chen Pong, Bing Guan, Bin Wang, Guiyang Hao, Xiankai Sun, Yi-Qiang Cheng, Jer-Tsong Hsieh. Targeting androgen receptor (AR) variants in castration-resistant prostate cancer (CRPC) with a cancer-specific theranostic agent [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A090.