Metabolic reprogramming in diffuse intrinsic pontine gliomas (DIPG): dual inhibition of mitochondrial oxidative phosphorylation and lactate metabolism to enhance anti-tumor and radiosensitizing effects in DIPG cells

Diffuse midline gliomas (DMGs), including diffuse intrinsic pontine gliomas (DIPGs), are universally fatal pediatric brain tumors with no effective treatments. DIPG tumors actively utilize mitochondrial oxidative phosphorylation (OXPHOS). Inhibition of Complex I (a core OXPHOS component) by phenformin radiosensitizes DIPG in vitro and in vivo. However, phenformin’s clinical application is limited by its risk of lactic acidosis. We investigated whether co-administration of the pyruvate-dehydrogenase-kinase (PDK) inhibitor dichloroacetate (DCA) can mitigate phenformin-induced acidosis while enhancing its anti-tumor activity. Patient-derived DIPG cells (SU-DIPG17, HSJD-DIPG007, SU-DIPG-VI) were treated with phenformin (0.625 mM), DCA (25 mM) or both. Mitochondrial and glycolytic flux (oxygen consumption rate (OCR) / extracellular acidification rate (ECAR)), ATP production, Reactive Oxygen Species (ROS), cell-cycle and apoptosis were quantified alongside RNA-seq and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS)/MS metabolomics. Hypoxia was measured in neurospheres using fluorescence. Radiosensitization was assessed by γ-H2AX foci and clonogenic survival. In vivo, HSJD-DIPG007 orthotopic xenografts received phenformin (125 mg/kg/day) and DCA (250 mg/kg/day) by oral gavage for 4 weeks, alone or with focal brain-stem irradiation (2 Gy × 10); tumor immunohistochemistry and survival were recorded. DCA alone shifted glucose metabolism from glycolysis to oxidative phosphorylation (OXPHOS), reducing ECAR and intracellular lactate. When combined with phenformin, DCA significantly suppressed phenformin-induced glycolysis and ECAR while further reducing ATP. In vitro, this combination induced synergistic cell-cycle arrest, apoptosis, and ROS-induced DNA damage. Multi-omics integration revealed coordinated repression of glycolytic/ hypoxia-inducible factor (HIF) programs and diversion of glucose into redox-supportive pentose-phosphate pathways. Hypoxic staining confirmed reduced hypoxia and HIF-1α in 3D neurospheroids. The combination produced additional in vitro radiosensitization, with the phenformin + DCA + radiation triple regimen achieving the greatest γ-H2AX persistence and clonogenic kill. In mice, the triple combination schedule incurred systemic toxicity, reflected from weight loss, and did not extend survival over 4 weeks treatment. DCA effectively counteracts phenformin-induced lactic acidosis in vitro yet heightens metabolic stress and radiosensitization in DIPG cells, providing proof-of-concept that a carefully chosen metabolic partner can boost tumor control. However, weight loss in vivo limited the length of treatment schedule and optimizing dose and schedule, or selecting safer mitochondrial inhibitors with PDK blockade, will be essential next steps before determining in vivo efficacy of this metabolic strategy. Diffuse midline gliomas, including diffuse intrinsic pontine glioma, are aggressive childhood brain tumors with no effective treatments. These tumors depend on energy produced by mitochondria, making mitochondrial metabolism a promising target for new therapies. However, drugs like phenformin, which block mitochondrial metabolism, can cause dangerous lactic acid buildup, limiting their clinical use. To address this, our study combined phenformin with dichloroacetate, a drug that targets glucose metabolism and can reduce lactic acidosis. This combination successfully lowered acid buildup, disrupted tumor cell metabolism, and enhanced cancer cell sensitivity to radiation in vitro, a key treatment for these tumors. However, a pilot in vivo study highlights the challenges that will need to be overcome in dosing and toxicity when combining multiple metabolic drugs with radiation.