Abstract 6405: Fosciclopirox suppresses growth of high-grade urothelial cancer by targeting Notch signaling

Ciclopirox (CPX) is a FDA-approved topical antifungal agent that has demonstrated preclinical anticancer activity in solid and hematologic malignancies. It's clinical utility as an anticancer agent, however, is limited by poor oral bioavailability, gastrointestinal toxicity, and poor water solubility. Fosciclopirox, the phosphoryloxymethyl ester of CPX (Ciclopirox Prodrug, CPX-POM), is rapidly and completely metabolized to CPX, the active metabolite, which subsequently undergoes renal elimination resulting in urine concentrations of CPX that exceed in vitro IC50's several-fold. We characterized the activity of CPX-POM and its major metabolites in vitro utilizing authenticated human T24, HT-1376, and UM-UC-3 high-grade urothelial cancer cell lines. CPX inhibited cell proliferation, clonogenicity, and spheroid formation, and increased cell cycle arrest at S and G0/G1 phases. Mechanistically, CPX suppressed activation of Notch signaling, which was partially rescued by ectopic expression of the intracellular domain of Notch1. Molecular modeling and cellular thermal shift assays demonstrated CPX binding to γ-secretase complex proteins Presenilin1 and Nicastrin, which are essential for Notch activation. Interrogation of The Cancer Genome Atlas (TCGA) database demonstrated that both proteins were upregulated in bladder tumor tissue, and that higher levels of Presenilin1 and Nicastrin were significantly associated with lower overall survival in muscle invasive bladder cancer (MIBC) patients. To establish in vivo preclinical proof of principle, we tested fosciclopirox in the validated N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) mouse bladder cancer model in two separate studies. Intraperitoneal (IP) administration of CPX-POM once daily for four weeks at doses ranging from 25 to 200 mg/kg significantly decreased bladder weight and resulted in a migration to lower stage tumors in CPX-POM treated animals compared to untreated animals. This was coupled with a reduction in proliferation index, as well as reductions in Presenilin1 and Hey1 expression in bladder tumor tissues in CPX-POM treated animals. A similar anti-tumor response was observed following once daily versus three times weekly IP CPX-POM in this chemical carcinogen mouse model of bladder cancer. The safety, dose tolerance, pharmacokinetics and pharmacodynamics of intravenous (IV) CPX-POM were characterized in a US multi-center, First-in-Human, Phase 1, open-label, dose escalation study (NCT03348514). Eight