Diffuse Intrinsic Pontine Gliomas (DIPGs) are the most devastating of all brain tumours. They mostly affect young children and, as there are no effective treatments, almost all will die of their tumour within 12 months. Our research has shown that targeting mitochondria represents an attractive novel approach to treating DIPG. One of the key proteins responsible for maintaining proper mitochondrial function is adenine nucleotide translocase (ANT). PENAO is a novel compound that targets mitochondrial function by inhibiting ANT and is currently in Phase I/IIa clinical trial in adults with solid tumours. We evaluated the cytotoxicity of PENAO in DIPG neurospheres using alamar blue assays and Annexin/PI staining and assessed its effect on mitochondrial activity through flow cytometric analysis of reactive oxygen species (ROS) production (MitosoxRed stain) and membrane depolarization (JC-1 stain). Treatment of DIPG neurospheres with PENAO profoundly inhibited cellular proliferation with a half maximal inhibitory concentrations (IC50) range of 0.5-3uM. Sensitivity to PENAO was found to be associated with increased apoptotic death while loss of mitochondrial activity was observed with significantly increased cytosolic levels of ROS and depolarised mitochondria. To assess whether the cytotoxic activity of PENAO can be further enhanced with other metabolic inhibitors we performed combination treatments of PENAO with the mTOR inhibitor Temsirolimus and examined cell death by alamar blue and Annexin/PI staining. We found that the combination of mTOR and ANT inhibition had a profound synergistic effect. Co-treatment of DIPG neurospheres with PENAO and Temsirolimus dramatically inhibited cellular proliferation with a logarithmic reduction in IC50 concentrations while western blot analysis indicated the combination therapy acted via synergistic inhibition of PI3K/AKT/mTOR pathway. Our current findings indicate that ANT/mTOR inhibition is a promising therapeutic strategy to treat unresectable and irradiation-resistant DIPG. We are currently investigating the effectiveness of this combination treatment in xenograft models of DIPG.