Poster Presentation NSW State Cancer Conference 2023

Atovaquone Radiosensitises Diffuse Midline Gliomas by Inhibiting Mitochondrial Metabolism and Hypoxia (#318)

Faiqa Miss Mudassar 1 2 , Prunella Ms Ing 1 , Cecilia Ms Chang 3 , Sandy Ms Nguyen 1 , Kristina Dr M Cook 2 4 , Zachary Dr N Warnken 5 , Geraldine Prof O'Neill 6 7 , Han Dr Shen 1 2 , Eric A/Prof Hau 1 2 8 9
  1. Translational Radiation Biology and Oncology Group, Centre for Cancer Research, The Westmead Institute for Medical Research, Sydney, NSW, Australia
  2. Sydney Medical School, The University of Sydney, Sydney, NSW, Australia
  3. The Kinghorn Cancer Centre and Cancer Research Division, Garvan Institute of Medical Research, Sydney, NSW, Australia
  4. Charles Perkins Centre, The University of Sydney, Sydney, NSW, Australia
  5. Via Therapeutics LLC, Austin, Texas, United States
  6. Children’s Cancer Research Unit, The Children’s Hospital at Westmead, Sydney, NSW, Australia
  7. Children’s Hospital at Westmead Clinical School, Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia
  8. Department of Radiation Oncology, Crown Princess Mary Cancer Centre, Westmead Hospital, Sydney, NSW, Australia
  9. Blacktown Cancer and Haematology Centre, Blacktown Hospital, Sydney, NSW, Australia

Background: 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, a main feature of solid tumours including gliomas, is a major contributor to radioresistance and impedes the efficacy of radiotherapy. Therefore, alleviating tumour hypoxia to enhance the effectiveness of radiotherapy is a therapeutic strategy to improve survival outcomes of DMG patients.

Aims: Here, we aimed to decrease the oxygen consumption rate (OCR) of DMG cells by targeting their mitochondria, which in turn will alleviate hypoxia by sparing more oxygen and subsequently improve the radiosensitivity of DMG cells.

Methods: Specifically, we performed a high-throughput screening to identify potent OCR inhibitors using a library of 1963 FDA-approved drugs. Top candidates were studied against a panel of patient-derived DMG cell lines using proliferation assays, seahorse extracellular flux assays, colony formation assays, western blots, confocal microscopy and flow cytometry.

Results and Discussion: 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. It induced the formation of mitochondrial reactive oxygen species suggesting that it increases oxidative stress. It alleviated hypoxia and decreased the expression of hypoxia-inducible factor-1a in several 3-dimensional DMG neurospheres and improved the radiosensitivity of a range of DMG cultures. To overcome the issues of poor bioavailability of commercially available atovaquone resulting in low therapeutically effective brain concentrations, we tested its efficacy against the amorphous solid dispersion (ASD) atovaquone formulation which appears to enhance the atovaquone levels in the brain. We found that both the formulations inhibited OCR and hypoxia at similar doses and improved the radiosensitivity of DMG. With these promising findings, our further work is assessing the in vivo efficacies of commercially available atovaquone and ASD atovaquone formulation using orthotopic DMG models.