High-grade gliomas (HGGs), including glioblastoma and diffuse intrinsic pontine glioma, are amongst the most fatal brain tumors. These tumors are associated with a dismal prognosis with a median survival of less than 15 months. Radiotherapy has been the mainstay of treatment of HGGs for decades; however, pronounced radioresistance is the major obstacle towards the successful radiotherapy treatment. Herein, tumor hypoxia is identified as a significant contributor to the radioresistance of HGGs as oxygenation is critical for the effectiveness of radiotherapy. Hypoxia plays a fundamental role in the aggressive and resistant phenotype of all solid tumors, including HGGs, by upregulating hypoxia-inducible factors (HIFs) which stimulate vital enzymes responsible for cancer survival under hypoxic stress. Since current attempts to target tumor hypoxia focus on reducing oxygen demand of tumor cells by decreasing oxygen consumption rate (OCR), an attractive strategy to achieve this is by inhibiting mitochondrial oxidative phosphorylation, as it could decrease OCR, and increase oxygenation, and could therefore improve the radiation response in HGGs. This approach would also help in eradicating the radioresistant glioma stem cells (GSCs) as these predominantly rely on mitochondrial metabolism for survival. Here, we highlight the potential for repurposing anti-parasitic drugs to abolish tumor hypoxia and induce apoptosis of GSCs. Current literature provides compelling evidence that these drugs (atovaquone, ivermectin, proguanil, mefloquine, and quinacrine) could be effective against cancers by mechanisms including inhibition of mitochondrial metabolism and tumor hypoxia and inducing DNA damage. Therefore, combining these drugs with radiotherapy could potentially enhance the radiosensitivity of HGGs. The reported efficacy of these agents against glioblastomas and their ability to penetrate the blood-brain barrier provides further support towards promising results and clinical translation of these agents for HGGs treatment.

Immune checkpoint inhibition with PD-1/PD-L1 blockade is a promising area in the field of anti-cancer therapy. Although clinical data have revealed success of PD-1/PD-L1 blockade as monotherapy or in combination with CTLA-4 or chemotherapy, the combination with radiotherapy could further boost antitumour immunity and enhance clinical outcomes due to the immunostimulatory effects of radiation. However, the synergistic combination of PD-1/PD-L1 blockade and radiotherapy can be challenged by the complex nature of the tumour microenvironment (TME), including the presence of tumour hypoxia. Hypoxia is a major barrier to the effectiveness of both radiotherapy and PD-1/ PD-L1 blockade immunotherapy. Thus, targeting the hypoxic TME is an attractive strategy to enhance the efficacy of the combination. Addition of compounds that directly or indirectly reduce hypoxia, to the combination of PD-1/ PD-L1 inhibitors and radiotherapy may optimize the success of the combination and improve therapeutic outcomes. In this review, we will discuss the synergistic combination of PD-1/PD-L1 blockade and radiotherapy and highlight the role of hypoxic TME in impeding the success of both therapies. In addition, we will address the potential approaches for targeting tumour hypoxia and how exploiting these strategies could benefit the combination of PD-1/PD-L1 blockade and radiotherapy.

Diffuse intrinsic pontine glioma (DIPG) is an incurable pediatric brain tumor with a median survival of 12 months. Current management is limited to radiotherapy; however, the tumor recurs secondary to radioresistance. Tumor hypoxia appears to be one of the major contributors to radioresistance of DIPG, as oxygenation is critical to successful radiotherapy treatment. Therefore, strategies to alleviate hypoxia could enhance the effectiveness of radiotherapy and result in improved survival outcomes of patients with DIPG. Recent approaches to target tumor hypoxia are predicated on inhibiting mitochondrial respiration of the tumors to decrease oxygen consumption rate (OCR) and increase oxygenation. Here, we aimed to identify a safe but potent mitochondrial inhibitor that could decrease OCR and hypoxia, and improve the radiosensitivity of DIPG. A subset of anti-parasitic drugs (atovaquone, ivermectin, quinacrine, mefloquine and proguanil) which are known mitochondrial inhibitors were studied against a panel of patient-derived DIPG cell lines. We assessed their antiproliferative effects, OCR inhibition and radiosensitising efficacy using cell proliferation, extracellular flux and colony formation assays. Among the five tested drug candidates, atovaquone was found to be the most potent OCR inhibitor with minimal antiproliferative effects on DIPG cultures. It also decreased hypoxia in 3-dimensional DIPG neurospheres, reduced the expression of hypoxia-inducible factor-1α and improved the radiosensitivity of neurospheres of DIPG. Its anti-mitochondrial role was further confirmed by inhibition of various mitochondrial parameters and increase in reactive oxygen species. Overall, these results provide promising in vitro evidence of atovaquone as a hypoxia modifier and radiosensitiser in DIPG and pave a way for rapid translation to in vivo studies.

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. Here, our strategy is 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. Specifically, we performed a high-throughput screening to identify potent OCR inhibitors using 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. 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.