Abstract 4751: The antiviral, Daclatasvir, downregulates Tribbles 2 pseudokinase and reverses enzalutamide resistance in prostate cancer
Authors: Monga Jitender , Guddeti Rohith , Rogers Craig , Gadgeel Shirish , Chitale Dhananjay , Ghosh Jagadananda 
Published: March 2024

Abstract
Background: FDA-approved enzalutamide is commonly prescribed for advanced prostate cancer. However, enzalutamide-resistant prostate cancer (ERPC) invariably develops, which leads to aggressive, lethal disease. Recently, we found that the Tribbles 2 (TRIB2) pseudokinase is overexpressed in ERPC cells and confers resistance to enzalutamide by promoting lineage plasticity to neuroendocrine differentiation. Though TRIB2 emerged as an excellent molecular target for ERPC, suitable inhibitors are not commercially available for effective targeting.
Methods: Compounds were tested using a luciferase-tagged TRIB2 fusion protein-based assay system. Binding of drugs with TRIB2 protein was analyzed by thermal shift assays and by advanced computer-based homology modeling. Degradation of TRIB2 protein was measured by Western blot. Drug effects on re-sensitization of ERPC cells and synergy with enzalutamide were determined through cell viability and apoptosis assays. To gauge the in vivo effects of Daclatasvir (DCV), ERPC tumor xenograft-bearing mice were treated with varying doses of DCV via oral gavage. Tumor growth was calculated by measuring volumes and molecular markers in tumors were analyzed by immunohistochemistry.
Results: By designing a luciferase-tagged TRIB2 fusion protein-based assay system, we screened a library of about 1,600 FDA-approved compounds and found that DCV, effectively inhibits the TRIB2-luciferase activity (more than 70% inhibition in 24 hours at 10 micromolar) but does not inhibit the activity of free luciferase. Notably, DCV directly binds to pure TRIB2 protein, leading to its destabilization and a reduction in the half-maximal melting temperature (Tm) from 41oC to 37oC, as confirmed by thermal shift assays. Interestingly, we found that DCV degrades TRIB2 proteins via activation of proteasomes and re-sensitizes ERPC cells to enzalutamide. DCV downregulates the master neuronal transcription factor, BRN2, and the stemness factor, SOX2, and synergizes with enzalutamide at lower, sub-lethal doses to decrease the viability of prostate cancer cells by inducing apoptosis. Finally, DCV was found to effectively inhibit the growth of ERPC tumors and decrease the protein level of TRIB2 in mice when delivered via oral gavage.
Conclusion: These findings indicate that DCV effectively downregulates TRIB2 both in vitro and in vivo and suggest that further testing of DCV may help design an innovative therapeutic approach for management of enzalutamide-resistant, aggressive, lethal prostate cancer.
Citation Format: Jitender Monga, Rohith Guddeti, Craig Rogers, Shirish Gadgeel, Dhananjay Chitale, Jagadananda Ghosh. The antiviral, Daclatasvir, downregulates Tribbles 2 pseudokinase and reverses enzalutamide resistance in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4751.