Abstract 425: Selective ubiquitination of androgen receptor and its splice variants inhibits castration resistant prostate cancer

Abstract

Abstract Introduction: The primary driver of castration-resistant prostate cancer (CRPC) is the Androgen receptor (AR). Drugs such as Abiraterone or Enzalutamide that target the Ligand Binding Domain (LBD) of AR are largely ineffective on AR splice variants (AR-SVs) that lack LBD and retain the N-terminal Domain (NTD) of AR. AR continues to function through other stimuli (ex: growth factors), leading to disease relapse and progression. Moreover, AR-SVs are known to heterodimerize with AR full length (AR-FL). Hence, treatment strategies that target both AR-FL and AR-NTD are needed for the complete remission of CRPC. Through structure-activity relationship (SAR) studies based on naturally occurring Urolithin A (UroA) structure, we have developed a novel small molecule UroBoc that directly binds to the NTD and inhibits CRPC growth. Methods: To analyze the binding of UroBoc to AR-NTD, we performed differential scanning fluorimetry and measured saturation transfer difference (STD) in NMR spectroscopic studies. In addition, we investigated the effect of UroBoc on CRPC by western blotting, immunofluorescence, Immunoprecipitation, and performed Xenograft studies in castrated/non-castrated mice models. Results: UroBoc treatment shifted the melt temperature (ΔTm) of purified NTD by 12-15 °C (median of 13.5 °C) in differential scanning fluorimetry. Moreover, STD effects observed were 30-18%, indicating a significant binding to NTD in our NMR studies. UroBoc inhibits the growth of AR+ CRPC, including enzalutamide resistant cells, at 800nM concentration, which is 40 times lower than that of the parent compound, UroA. At this concentration, UroBoc is non-toxic to normal prostate epithelial cells or other cancer cells. Interestingly, UroBoc also inhibited AR, AR-SV and PSA expressions in AR-FL (LNCaP, C42B) and AR-V7 (22-RV1) CRPC cell lines. Molecular studies suggested that UroBoc selectively promotes ubiquitination and subsequent degradation of AR and AR-V7 by proteasome activation. Remarkably, UroBoc failed to inhibit AKT, mTOR, PTEN and hormonal receptors such as estrogen and progesterone receptors suggesting AR could be a direct target for UroBoc. Further, in vivo studies revealed that oral administration of UroBoc inhibits tumor growth in CRPC in both castrated and non-castrated mice models. Conclusion: Our results suggest that the novel UroBoc selectively degrades AR, AR-SV and overcomes enzalutamide resistance in preclinical CRPC models. We believe performing PK/PD studies will confirm the translation potential of UroBoc as a therapy for CRPC. Citation Format: Ashish Tyagi, Balaji Chandrasekaran, Uttara Saran, Murali K. Ankem, Nikolay Dokholyan, Jyh M. Lin, Arun K. Sharma, Chendil Damodaran. Selective ubiquitination of androgen receptor and its splice variants inhibits castration resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 425.