Atovaquone-induced activation of the PERK/eIF2α signaling axis mitigates metabolic radiosensitisation

Background Hypoxia, a key feature of most solid tumours, including head and neck cancer, reduces radiotherapy efficacy by promoting radiation resistance through micro-environmental and genomic alterations. Addressing these resistance mechanisms is crucial, as radiotherapy remains central to managing locally advanced disease. Atovaquone, a mitochondrial electron transport chain complex III inhibitor, is reported to reduce tumour hypoxia in preclinical models, however, this response does not consistently enhance radiation sensitivity. This work examines the potential of atovaquone to modify the hypoxic response in models of head and neck squamous cell carcinoma (HNSCC), uncovering an adaptive resistance mechanism driven by integrated stress response (ISR) signaling that limits the radiosensitising potential of this approach. Methods The bioenergetic response of HNSCC cells to atovaquone was assessed using the Seahorse XFe96 Analyzer with the XF Cell Mito Stress Test. Radiation dose modifying effects of atovaquone were tested by clonogenic survival assays, while ROS yields were analysed by flow cytometry. Western blotting and quantitative reverse transcription-PCR were employed to study activation of ISR signaling and the overall influence of atovaquone on the hypoxic response. Finally, the role of the ISR activation in modulating radiosensitivity was investigated using both siRNA and pharmacological inhibition of eIF2 alpha, a central regulator of the ISR. Results Herein we report that atovaquone significantly disrupts mitochondrial respiration, triggering phosphorylation of eIF2 alpha, a pivotal regulator of the ISR, and a master regulator of protein synthesis. Notably, atovaquone also increased the autophagic load under hypoxia, while autophagy inhibition significantly enhanced apoptosis, improving radiation sensitivity. Combined eIF2 alpha inhibition and atovaquone promotes cell cycle redistribution and significantly enhances mitochondrial ROS production and compared to atovaquone alone, restoring atovaquone mediated radiosensitisation. Conclusions Our data highlight dual counter opposing impacts of atovaquone, serving as a hypoxic radiosensitiser though oxidative phosphorylation (OXPHOS) inhibition, but also in promoting stress induced ISR signaling, conferring resistance to radiation treatment. Importantly, if ISR activation is impeded, the metabolic radiosensitising properties of atovaquone is restored. These data provide a new insight to a molecular response that could help counteract hypoxia-induced radioresistance.