DDDR-11. INHIBITING OXIDATIVE PHOSPHORYLATION AND HYPOXIA WITH ATOVAQUONE – A STRATEGY TO RADIOSENSITIZE DIFFUSE MIDLINE GLIOMA

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Abstract Diffuse midline glioma (DMG) is a uniformly fatal paediatric brainstem tumour with median survival of less than 1 year. Radiotherapy has been the only effective treatment for decades, but most DMGs recur within several months due to radioresistance. The hypoxic tumour microenvironment is a main feature of solid tumours including gliomas and a major contributor to radioresistance. Therefore, alleviating tumour hypoxia to enhance the effectiveness of radiotherapy is a potential therapeutic strategy to improve the survival outcomes of DMG patients. We found evidence suggesting increased mitochondrial oxidative phosphorylation (OXPHOS) in DMG. Hence, we aimed to target OXPHOS by decreasing the oxygen consumption rate (OCR) of DMG cells, which in turn will alleviate hypoxia by sparing more oxygen and subsequently improve the radiosensitivity of DMG cells. We performed a high-throughput screening to identify potent OCR inhibitors from a library of 1963 FDA-approved drugs. The most promising OCR inhibitor identified was atovaquone, a drug used for treatment of pneumocystis pneumonia and malaria. We found that atovaquone inhibited mitochondrial metabolism of DMG cells by specifically targeting the mitochondrial complex III. Increased mitochondrial reactive oxygen species after exposure to atovaquone suggested that it increases oxidative stress. The treatment alleviated hypoxia and decreased the expression of hypoxia-inducible factor-1a in 3-dimensional DMG neurospheres and improved the radiosensitivity in several DMG cultures. To overcome the poor bioavailability of commercially available atovaquone that results in low therapeutically effective brain concentrations, we tested its efficacy against the amorphous solid dispersion (ASD) atovaquone formulation which appears to enhance atovaquone levels in the brain. We found that both the formulations inhibited OCR and hypoxia at similar doses and enhanced DMG radiosensitivity. The combination of ASD atovaquone and radiation significantly improved survival in orthotopic DMG mouse model. Collectively, this data provides evidence of atovaquone as a potential radiosensitizer for DMG.