Antitumor Activity of Combined Topoisomerase Inhibition and FLASH Radiotherapy in Head and Neck Carcinoma Biomodels

Locally advanced head and neck carcinoma remains associated with high morbidity and long-term survival below 50%. Treatment commonly relies on cisplatin-based chemoradiotherapy, which is effective but frequently associated with significant acute and chronic systemic toxicities. Therefore, safer and more effective therapeutic strategies are urgently needed. FLASH radiotherapy (RT) has emerged as a promising irradiation modality because of its potential to reduce damage to healthy tissues while preserving antitumor efficacy. Here, we investigated the anticancer activity of the Topoisomerase (Topo)-II inhibitor ARN-24139, alone and combined with FLASHRT, in human papillomavirus-negative SCC-25 head and neck carcinoma biomodels. Antitumor activity was assessed in 2D cell cultures using viability, apoptosis, clonogenic, wound-healing, and γH2AX assays, as well as in SCC-25 3D spheroids and in chorioallantoic membrane (CAM) tumor models. ARN-24139 induced dose-dependent cytotoxicity in SCC-25 cells, with IC₅₀ values of 7.3 ± 0.8 µM at 48 h and 7.2 ± 0.5 µM at 72 h, while showing limited toxicity in healthy HBEpC cells. Sequential low-dose FLASH-RT followed by ARN-24139 enhanced antitumor activity, reducing cell viability at 4 Gy after 8 days and decreasing tumor growth and Ki67 expression in CAM models. These proof-of-concept findings support further investigation in more clinically representative and mechanistically informative HNSCC models.