Background: Androgen receptor (AR) signaling is a main driver of prostate cancer progression and remains a crucial target for therapeutic intervention even in late stages of the disease. While current anti-androgen therapies targeting directly or indirectly the AR ligand binding domain (LBD) are initially effective, resistance ultimately develops, and new methods of inhibiting the AR pathway are needed. The selective targeting of the N-terminal domain (NTD) of the AR represents a novel method of blocking AR signaling to by-pass LBD-related resistance. EPI-7386 is a potent and metabolically stable NTD inhibitor (aniten) currently in a phase 1 dose-escalation study in mCRPC patients (NCT04421222). Here we further characterized the binding to AR NTD and the mechanism of action of EPI-7386. Methods: Target engagement was measured by Cellular Thermal Shift Assay (CETSA) and two-dimensional Nuclear Magnetic Resonance (2D NMR) spectroscopy. The potency and selectivity of EPI-7386 was determined in cellular models expressing different forms of AR using reporter and cell viability assays. qPCR, NanoString, and RNA sequencing were used to explore the activity of EPI-7386 on the AR transcriptome. To determine the effect of EPI-7386 on AR genomic occupancy, Chromatin immunoprecipitation sequencing (ChIP-seq) was performed. Results: We confirmed target engagement of EPI-7386 with an LBD truncated AR variant by CETSA using a cell line which expresses only AR-V567es, suggesting the interaction of EPI-7386 with AR NTD. In the same cell line, AR antagonist enzalutamide that binds to AR LBD showed no target engagement with AR-V567es. Furthermore, 2D NMR study results demonstrate an interaction of EPI-7386 with amino acid residues located in the transcription activation unit 5 (Tau-5) region of the AR NTD, a region which has been described to be involved in interactions with transcriptional cofactors such as CBP/p300. EPI-7386 strongly impaired the transcriptional activity and gene expression driven exclusively by LBD truncated AR variants including AR-V567es and AR-V7 and decreased cell viability. EPI-7386 has been shown to suppress the AR regulated transcriptome and the combination of EPI-7386 with lutamides resulted in broader and deeper inhibition of AR-regulated gene expression. The analysis of the AR cistrome by ChIP-seq showed that EPI-7386 displaces genome-wide androgen induced AR binding and the combination with enzalutamide completely abrogated AR binding. Conclusion: EPI-7386 is a potent AR NTD inhibitor that has the capacity to by-pass AR LBD resistance mechanisms to current anti-androgen therapies by uniquely inhibiting AR-mediated signaling. The agent has the potential for providing clinical benefit as a single agent in patients whose tumors are progressing on anti-androgens or in combination with current anti-androgens in earlier line patients.

Citation Format: Nan Hyung Hong, Shihua Sun, Peter Virsik, Alessandra Cesano, Elahe A. Mostaghel, Stephen R. Plymate, Berenger Biannic, Han-Jie Zhou, Ronan Le Moigne. Comprehensive preclinical characterization of the mechanism of action of EPI-7386, an androgen receptor N-terminal domain inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P192.