Diffuse intrinsic Pontine Glioma (DIPG) is a childhood high-grade glioma originating in the pons. It has a dismal survival rate of less than 5% at 2 years, and is a leading cause of death in children with brain tumors (1). DIPG remains incurable despite over 250 clinical trials in past 3 decades. Novel effective combination therapies are needed to achieve cure. We have established a national DIPG autopsy study to harvest tumour tissue and successfully established DIPG neurosphere cultures. We performed a high-throughput drug screen (HTS) with over 3500 FDA-approved compounds against neurospheres. Only 1% of compounds demonstrated significant cytotoxic activity and fenretinide-a synthetic retinoid was one the most active drugs identified. Fenretinide has been used in the treatment of adult and pediatric cancers such as breast cancer and neuroblastoma. It causes accumulation of reactive oxygen species (ROS) and lipid second messengers, resulting in cell death through apoptosis (2). In-vitro cytotoxicity assay showed fenretinide had potent activity against DIPG neurospheres with an IC50 of 1-2uM. In comparison, other retinoids were ineffective. In order to develop a rational combination therapy, we assessed whether fenretinide enhanced the efficacy of other pro-apoptotic agents. Gene expression analysis of DIPG cells showed aberrant activity of the receptor tyrosine kinase (RTK)-Ras-phosphoinositide 3-kinase (PI3K) signaling pathway and over-expression of PDGFRA and EGFR. Ponatinib is a multi-targeted tyrosine-kinase inhibitor which showed significant cytotoxic activity in a HTS performed with the NCI oncology drug set. In-vitro cytotoxicity assay confirmed this single agent activity of ponatinib, with an IC50 of 2 uM. The combination of fenretinide and ponatinib had a significant synergistic cytotoxic effect against neurospheres with increased apoptotic death and caspase 3/7 induction. In summary, we have identified a rational combination strategy that represents a novel and potentially effective therapy for DIPG.