Funding outcome

Introduction: Dedifferentiated endometrial carcinoma (DDEC) is an aggressive gynecologic malignancy characterized by SWI/SNF-inactivation. Current standard-of-care platinum and taxane-based chemotherapy is insuffcient in suppressing tumor progression. To gain therapeutic insights, we developed patient-derived ex vivo 3D spheroid models of DDEC and subjected them to highthroughput drug repurposing screen; this identified digoxin as an effective anti-cancer agent with efficacy confirmed in patient-derived xenograft tumor models (PMID: 37556934, 38970843). Herein, we evaluate for potential additive/synergistic drug combination and explore the mechanism of action of digoxin in DDEC. Methods: Gene expression profiling, flow cytometry, mass spectrometry, western blot and PCR were performed to investigate mechanisms. A drug combination screen was conducted with low-dose digoxin (below upper serum concentration) and identified combinations were further evaluated in vitro and in vivo. Results: Gene expression profiling and flow cytometry showed enrichment of apoptotic pathways in digoxin treated cells (p <0.0001). Molecular profiling revealed that DDEC express lower levels of Na⁺/K⁺- ATPase isoforms α1 (p <0.0001), β1 (p <0.0001) and β3 (p <0.05) (targets of digoxin) versus non- DDEC. Drug combination screen identified paclitaxel and other compounds as potential candidates. Further in vitro evaluation confirmed the additive nature between digoxin, carboplatin and paclitaxel, while an antagonistic effect between digoxin and doxorubicin. Subsequent in vivo studies confirmed that digoxin, carboplatin and paclitaxel treatment resulted in significantly greater tumor growth inhibition compared to carboplatin and paclitaxel (90% versus 63%, p =0.0124 and p =0.0032). Conclusion/Implications: Digoxin enhances the efficacy of standard-of-care chemotherapy in DDEC. Digoxin increases apoptosis and this susceptibility appears to relate to the Na⁺/K⁺-ATPase expression profile.