Novel inhibitors of AR-v7 nuclear import: new therapeutic opportunities for CRPC

Reactivation of androgen receptor (AR) signaling by active splice variants (AR-Vs) is one of key drivers of castration resistant prostate cancer (CRPC). AR-v7 is the most prevalent AR-V and its expression has been clinically associated with poor overall survival, resistance to the AR inhibitors enzalutamide and abiraterone, as well as taxane resistance. Given that treatment with AR inhibitors and taxanes are the only effective therapeutic modalities in CRPC, development of specific AR-v7 inhibitors is urgently needed. Mechanistically, AR-v7 re-activates AR signaling by being constitutively active in the nucleus. While taxane chemotherapy inhibits the nuclear import of AR which is significantly associated with clinical outcomes in CRPC, it has no effect on AR-v7 nuclear localization and activity. Mechanistically, AR-v7 lacks the microtubule-binding domain and—unlike AR—does not utilize the canonical importin-α/β pathway, or RanGTP for nuclear import. Using wheat germ agglutinin to block active protein nuclear uptake resulted in AR-v7 cytoplasmic sequestration, indicating a requirement for an alternative transport receptor. Further, mutation of AR-v7 dimerization domain (D-box) led to its cytoplasmic sequestration, indicating that the D-box is also required for nuclear import. As inhibition of AR nuclear import is a clinically validated therapeutic strategy, we developed a novel drug discovery platform to identify compounds that specifically inhibit AR-v7 nuclear import. Using cells stably expressing inducible AR-v7 in conjunction with an enzyme complementation assay we tested 166,000 compounds by high throughput screening (HTS). The robust HTS performance (Z>0.8) together with subsequent counter screens including confirmation and compound titration, cell toxicity, a tertiary imaging based screen, led to identification of lead compounds that inhibit AR-v7 nuclear import. The lead compounds share structural features, across two main chemotypes, which are amenable to structure-activity relationship studies to identify the most desirable compound for in vivo studies. Using newly synthesized compounds from each of the two chemotypes, we showed specific dose-dependent inhibition of AR-v7 nuclear import. Currently, we are testing these compounds, on inhibition of AR-v7 transcriptional activity across several cell models including enzalutamide-resistant cells as well as inhibition of tumor growth in AR-v7 xenograft models. In parallel we are testing the lead compound for potential direct binding to the AR-v7 dimerization domain or to candidate alternative nuclear transport receptors. Further development of our lead small molecules will yield novel chemotypes, with desirable pharmacological properties that target the unique AR-v7 nuclear import pathway and can be clinically combined with existing AR therapies. Note: This abstract was not presented at the meeting. Citation Format: Seaho Kim, Mohd Azrin Jamalruddin, Eiman Mukhtar, Michael Miller, Leigh Baxt, Stacia Kargman, Andrew Stamford, Peter Meinke, Paraskevi Giannakakou. Novel inhibitors of AR-v7 nuclear import: new therapeutic opportunities for CRPC [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 1017.