Abstract B091: Small-molecule inhibitors targeting a specific metabolic pathway for precision therapy of advanced castrate-resistant prostate cancer
Authors: Basu Hirak , Wilganowski Nathaniel , Lieblich Jessica , Wu Grace , Fokt Izabela , Ramachandran Sumankalai , Yu Jiaquin , Titus Mark , Priebe Waldemar , Beebe David J. , Wilding George 
Published: February 2019

Introduction: The mechanism of prostate cancer (PCa) progression to the mostly lethal, metastatic, castrate-resistant stage (mCRPC) remains generally unknown. This prevents identifying patients likely to progress to mCRPC and designing new therapies for their treatment. A transcription factor JunD is overexpressed in PCa, but not in normal prostate epithelial cells. It complexes with androgen receptor (AR) to induce spermidine/spermine acetyl transferase (SSAT) [3]. SSAT initiates polyamine oxidation that generates copious amounts of reactive oxygen species (ROS) production in polyamine-rich PCa cells. ROS activate NF-κB. NF-κB can also induce SSAT and activate AR. This sets up a feed-forward loop for PCa growth at low androgen.

Here, we present one mechanism of PCa progression to CRPC as well as its invasion and metastasis that may provide predictive biomarkers to identify patients with potentially lethal PCa at an early stage. Effects of an agent that can block both AR-JunD and AR-NF-κB interactions and prevent growth of PCa cells in vitro as well as in vivo xenografts will also be presented.

Method: We have used a novel microscale device to separate invading from noninvading PCa cells ex vivo in a bone microenvironment. Immunocytochemistry (ICC), proteomic and metabolomic techniques have been employed to analyze the separated cells to understand the mechanism of invasion. Gaussia luciferase reconstitution assay has been used in a high-throughput screen (HTS) to identify inhibitors of AR-JunD and AR-NF-κB interactions. State-of-the-art in vitro cell growth assay along with in vivo pharmacokinetic (PK), maximum tolerated dose (MTD) and enzalutamide-resistant CRPC cell xenograft growth in nude mice are used to optimize the lead.

Summary of Unpublished Results: Immunocytochemistry (ICC) data show that in cultured enzalutamide-resistant C4-2 and -sensitive LNCaP cells as well as in some patient PCa cells, more SSAT-positive cells are in the migratory than in the stationary section of the microscale device. Metabolomic analysis show a decrease in 3-phospho-glycerate levels in C4-2 cells, suggesting a decrease in GAPDH enzymatic activity that is related to an enhanced oxidation of GAPDH protein.

Our published data of screening of 27,000+ compounds from two different chemical libraries detected a single compound that specifically inhibits both AR-JunD and AR-NF-κB2 (p52) interactions. We have synthesized several of its analogs. Lead analogs inhibit growth of AR-positive C4-2 and LNCaP cells, but have little effect on the growth of AR-negative PC-3 and SSAT-silent LNCaP siSSAT cells in culture. Co-immunoprecipitation (co-IP) assay shows its efficacy in inhibiting AR-JunD and AR-p52 interactions in LNCaP cells. It has been formulated in 25% ethanol:water and administered to nude mice orally. It is well tolerated at 50 mg/kg p.o. daily for more than 28 days and has a serum Cmax of ~ 3 μM within 30 minutes and a plasma t1/2 of ~1 h after a single oral dose of 100 mg/kg. Twenty-four hours after dosing it is found in the flank tumor (1.5 ng/mg tissue; ~450 nM) and in the prostate tissues (10 ng/mg tissue; ~3 μM) in addition to liver and kidney. Its effects on the growth of C4-2 xenografts in nude mice will be presented.

Conclusion: A mechanism of PCa progression to CRPC and then to mCRPC provides a rational basis for targeted drug design that prevents PCa progression and metastasis. This should open up a new avenue of precision therapy of potentially lethal PCa at an early stage.

Citation Format: Hirak Basu, Nathaniel Wilganowski, Jessica Lieblich, Grace Wu, Izabela Fokt, Sumankalai Ramachandran, Jiaquin Yu, Mark Titus, Waldemar Priebe, David J. Beebe, George Wilding. Small-molecule inhibitors targeting a specific metabolic pathway for precision therapy of advanced castrate-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B091.