Childhood brain cancers arise in the context of the developing human brain and have become the leading causes of cancer-related mortality in children. The golden age of sequencing has provided a wealth of knowledge regarding the genetic drivers of brain tumors. This knowledge has sparked several clinical trials in a variety of pediatric tumors such as medulloblastomas, high-grade gliomas and embryonal tumors. However, therapeutic leads and clinical trials to combat childhood ependymomas have lagged far behind. Therefore, there is an urgent and unmet need to develop effective therapies for children with ependymomas. Recent molecular studies have revealed the complex biology of childhood ependymomas influenced by both the anatomic compartment from which they arise and distinct genetic/ epigenetic tumors drivers. This has led to a recent molecular classification system that takes both parameters into account. We propose to advanced precision-medicine for ependymomas by tackling childhood ependymomas that occur in the hemispheric/ supratentorial region of the brain that exhibit the highly recurrent ZFTA-RELA fusion in 80% of cases. Our strong in vitro and in vivo data demonstrate that the ZFTA-RELA fusion tumor cells are reliant on glutamine to not only support their proliferation, but also help in survival by expanding the capacity of tumor cells to withstand oxidative insults. Our central hypothesis is that glutamine is a critical metabolic substrate that, via generation of glutathione, enables maintenance of redox balance in ZFTA-RELA bearing ependymomas. Consequently, inhibition of glutamine metabolism will be therapeutic by increasing oxidative stress to kill ZFTA- RELA tumor cells. Accordingly, we propose a research program to define the role of glutamine in ZFTA-RELA ependymomas. In specific aim 1 we will define the molecular mechanisms by which ZFTA-RELA enhances glutamine metabolism. We will determine the epigenetic mechanism and the role of fusion partners ZFTA and RELA in upregulating glutamine metabolism. In specific aim 2, We will define molecular mechanisms by which glutamine enhances redox resistance in ZFTA-RELA cells. We will also combine glutamine-based positron emission tomography imaging with carbon-13 isotope labeling to map the fate of glutamine carbons in vivo in ZFTA-RELA ependymoma animal models. In specific aim 2, we will determine the ability of suppressing glutamine metabolism as a therapeutic target in vivo. We will also determine if combining inhibition of glutamine metabolism with standard-of-care radiation therapy enhances therapeutic efficacy. These aims together will advance the field by defining the molecular mechanisms by which ZFTA-RELA drives cancer in childhood ependymomas, develop non-invasive, metabolic imaging based biomarkers, and define therapeutic strategies to combat these deadly pediatric brain tumors.