Improving The Radiosensitivity of Diffuse Intrinsic Pontine Gliomas By Modulating Bioenergetic Pathways

Abstract

Diffuse intrinsic pontine glioma (DIPG) is the leading cause of brain tumor-related death in children and radiotherapy (RT) is the only form of treatment that offers a transient benefit. Following completion of RT, almost all DIPG recur locally secondary to radioresistance. Therefore, the identification of therapeutic targets that modulate the radiosensitivity of DIPG cells offers a pathway to the development of effective therapies. In the present study, anti-diabetic biguanides (metformin and phenformin) and a pyruvate dehydrogenase kinase inhibitor (DCA) have been tested in combination with RT on patient-derived DIPG cultures. As a single agent, a superior anti-proliferative activity was observed from phenformin, which reduced the half maximal inhibitory concentration (IC50) values to 20- to 30-fold compared to metformin. When combined with DCA, the co-treatment led to a consistent synergistic inhibition, with a broad range of combinatorial doses showing combination index (CI) less than 1. DIPG cells treated with phenformin and/or DCA were more sensitive to RT showing significantly less clonogenicity when triple combination was applied. Mechanistically, apoptosis was induced by the combination, evidenced by the upregulation of apoptotic markers (cleaved PARP and cleaved caspase 3) and downregulation of anti-apoptotic marker MCL-1. Additionally, the combination synergistically induced higher level of Reactive Oxygen Species, which further upregulated gamma-H2AX, a hallmark of DNA double-strand breaks. Moreover, PARP, a key enzyme that is responsible for single-strand breaks, was also inhibited by the combinatorial treatment, indicating another layer of radiosensitizing machinery. Taken together, this study provides the proof of concept that dual-blockade of bioenergetic pathways can more effectively improve the radiosensitivity of DIPG cells. We are currently evaluating the efficacy of triple combination with phenformin, DCA and RT on a cohort of patient-derived xenograft models of DIPG to validate our in vitro findings.