Cancer stem cells (CSCs) virtually present in all tumors albeit in small numbers are primarily responsible for driving cancer progression, metastasis, drug resistance, and recurrence. Prostate cancer (PCa) is the second most frequent cancer in men worldwide, and castration resistant prostate cancer (CRPC) remains a major challenge despite the tremendous advancements in medicine. Currently, none of the available treatment options are effective in treating CRPC. We earlier reported that VNPP433‐3β, the lead next‐generation galeterone analog is effective in treating preclinical in vivo models of CRPC. In this study using RNA‐seq, cytological, and biochemical methods, we report that VNPP433‐3β inhibits prostate CSCs by targeting key pathways critical to stemness and epithelial–mesenchymal transition. VNPP433‐3β inhibits CSCs in PCa, presumably by degrading the androgen receptor (AR) thereby decreasing the AR‐mediated transcription of several stem cell markers including BMI1 and KLF4. Transcriptome analyses by RNA‐seq, Ingenuity Pathway Analysis, and Gene Set Enrichment Analysis demonstrate that VNPP433‐3β inhibits transcription of several genes and functional pathways critical to the prostate CSCs thereby inhibiting CSCs in PCa besides targeting the bulk of the tumor.

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Targeted protein degradation is a fast-evolving therapeutic strategy to target even the traditionally undruggable target proteins. Contrary to the traditional small-molecule inhibitors of enzyme or receptor antagonists that bind the active site pockets in the target protein, molecular glue degraders facilitate interaction of target proteins with E3 ubiquitin ligases by stabilizing the ternary complex and induce physical proximity, thereby triggering ubiquitination and subsequent proteasomal degradation. AR plays a key role in all stages of prostate cancer. It is activated by the binding of androgenic hormones and transcriptionally regulates multiple genes including the ones that regulate cell cycle. Using HiBiT CRISPR cell line, biochemical methods, and RNA sequencing, we report the potential role of VNPP433-3β, the next generation galeterone analog as molecular glue that brings together AR, the key driver of prostate cancer and MDM2, an E3 ubiquitin ligase leading to ubiquitination and subsequent degradation of f-AR and AR-V7 in prostate cancer cells.

Prostate cancer (PCa) relies in part on AR-signaling for disease development and progression. Earlier, we developed drug candidate galeterone, which advanced through phase 2-clinical trials in treating castration-resistant PCa (CRPC). Subsequently, we designed, synthesized, and evaluated next-generation galeterone-analogs including VNPP433-3β which is potently efficacious against pre-clinical models of PCa. This study describes the mechanism of action of VNPP433-3β that promotes degradation of full-length AR (fAR) and its splice variant AR-V7 besides depleting MNK1/2 in in vitro and in vivo CRPC models that stably overexpresses fAR. VNPP433-3β directly engages AR within the cell and promotes proteasomal degradation of fAR and its splice variant AR-V7 by enhancing the interaction of AR with E3 ligases MDM2/CHIP but disrupting AR-HSP90 binding. Next, VNPP433-3β decreases phosphorylation of 4EBP1 and abates binding of eIF4E and eIF4G to 5′ cap of mRNA by depleting MNK1/2 with consequent depletion of phosphorylated eIF4E. Finally, RNA-seq demonstrates modulation of multiple pathways that synergistically contribute to PCa inhibition. Therefore, VNPP433-3β exerts its antitumor effect by imposing 1) transcriptional regulation of AR and AR-responsive oncogenes 2) translational regulation by disrupting mRNA-5′cap-dependent translation initiation, 3) reducing AR half-life through enhanced proteasomal degradation in vitro and AR-overexpressing tumor xenografts in vivo.

Introduction Metastatic castration-resistant prostate cancer (mCRPC) remains the second leading cause of cancer-related death in men in the United States and the second most frequently diagnosed cancer among males worldwide. Dysregulated androgen receptor (AR) is associated with nearly all known cases of prostate cancer (PCa) and its expression level has been correlated with poor survival. This study describes the mechanism of action of VNPP433-3β, a next generation galeterone analog (NGGA) in inhibiting PCa. Methods PCa cell lines CWR22Rv1 and LNCaP were treated with VNPP433-3β and followed up using cellular thermal shift assay (CETSA), RNA seq, mRNA 5’ cap-binding assay, translation and proteasome inhibition assays. Co-immunoprecipitation, molecular docking and fluorescence spectroscopy were used to study the interactions. Results VNPP433-3β, the lead next generation galeterone analog (NGGA) directly interacts with AR and inhibits metastatic castration resistant PCa (mCRPC) by enhancing degradation of full-length AR (fAR) and its splice variant AR-V7 in a dose-dependent manner. Moreover, VNPP433-3β drives the proteasomal degradation of fAR and AR-V7 by augmenting the interaction between AR and E3 ligases MDM2 & CHIP but disrupting HSP90 binding to AR. VNPP433-3β also impedes eIF4E phosphorylation by depleting MNK1/2 besides disrupting binding of eIF4E and eIF4G to mRNA 5’ cap thereby impeding translational activity of the cancer cells. Finally, the transcriptome analyses by RNA-seq reveal that VNPP433-3β modulates transcription of several genes and activates more than 20 cellular pathways, each of which synergistically contribute to PCa inhibition. Hence, VNPP433-3β obstructs PCa by invoking 1) transcriptional regulation of AR-responsive oncogenes via degrading AR, 2) translational regulation by depleting MNK1/2 thereby impeding phosphorylation of eIF4E and subsequent mRNA 5’cap-dependent translation initiation and 3) affecting AR half-life through enhanced proteasomal degradation. Conclusion As VNPP433-3β promotes degradation of fAR and AR-V7, concurrently depletes Mnk1/2, prevents eIF4E phosphorylation and modulates several cellular pathways to halt PCa, it could potentially be developed for clinical trials in mCRPC patients. Citation Format: Elizabeth Thomas, Retheesh S Thankan, Puranik Purushottamachar, Vincent C.O. Njar. Mechanistic insights on the effects of the lead next generation galeterone analog, VNPP433-3β in castration resistant prostate cancer [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 LBA027.