Targeting DRP1 to limit mitochondrial plasticity overcomes radiation resistance in head and neck squamous cell carcinoma

Radioresistance is a primary cause of treatment failure in head and neck squamous cell carcinoma (HNSCC). This study aimed to elucidate the role of Dynamin-related protein 1 (DRP1)-mediated mitochondrial fission in radioresistance and evaluate DRP1 targeting as a therapeutic strategy. RNA sequencing (RNA-seq) transcriptomic profiling was performed comparing parental and radioresistant HNSCC sublines. DRP1 expression and prognostic value were evaluated using public datasets and validated within an independent institutional clinical cohort of patients receiving postoperative adjuvant or definitive radical radiotherapy. DRP1 function was assessed across multiple HNSCC cell models via genetic knockdown and pharmacological inhibition using Mdivi-1 and the selective peptide inhibitor P110. Functional evaluations included clonogenic survival, radiation-induced DNA damage and mitochondrial metabolism. In vivo efficacy was evaluated in subcutaneous xenograft and syngeneic orthotopic mouse models. DRP1 was identified as a top-upregulated gene in radioresistant HNSCC cells. Higher DRP1 expression was associated with poorer overall survival in public HNSCC data and adverse radiotherapy-related findings in an exploratory institutional cohort. Mechanistically, DRP1 knockdown induced mitochondrial hyperfusion and a dramatic surge in radiation-induced reactive oxygen species (ROS), which collapsed antioxidant defenses and increased residual DNA damage after irradiation. The antioxidant N-acetyl-L-cysteine (NAC) attenuated this radiosensitization, supporting a contribution of oxidative stress. Untargeted metabolomics identified changes in metabolite abundances and pathway enrichment related to amino acid metabolism, glutathione synthesis, and bioenergetic homeostasis following DRP1 depletion. Pharmacological targeting with Mdivi-1 and P110 reproduced key phenotypes of DRP1 knockdown in vitro. In vivo, Mdivi-1 enhanced the antitumor effects of radiotherapy in xenografts derived from the acquired radioresistant CAL27 IR-R subline and in a complementary syngeneic orthotopic MOC-1 tongue tumor model. Histological examination of major organs at the study endpoint revealed no overt evidence of treatment-related injury. DRP1 confers radioresistance in HNSCC by maintaining mitochondrial fission dynamics to limit radiation-induced oxidative stress and DNA damage accumulation. These findings provide a preclinical rationale for further investigation of DRP1-targeted radiosensitization strategies in HNSCC.