ABSTRACT Glioblastomas rank among the most lethal of all human cancers. Current standard-of-care therapy for patients afflicted with glioblastoma offers only palliation. Treatment failure derives from numerous causes, including the presence of stem-like tumor cells, called glioblastoma stem cells (GSCs). GSCs contribute to radioresistance, chemoresistance, invasion, immune escape, and angiogenesis. Previously, we reported that critical nodes in methyl donor metabolism and methyl utilization ranked among the most consistently overexpressed pathways in glioblastoma relative to normal brain. Targeting methyl donor metabolism expression reduced cellular proliferation, self-renewal, and in vivo tumor growth of GSCs. Thus, methyl donor metabolism is a promising GSC-specific therapeutic target in glioblastoma that would result in disrupting oncogenic DNA hypomethylation. In preliminary studies, we have extended our efforts to bridge metabolic reprogramming in glioblastoma with maintenance of stemness through regulation of epitranscriptomics to identify metabolic and molecular targets that are preferentially active in GSCs. Leveraging a combination of genetic and pharmacologic inhibitors, we have identified key regulators that manifests as altered epitranscriptomic methylation events to maintain GSCs. In the proposed studies, we will interrogate the functional contributions of selected metabolic enzymes in oncogenic metabolite production and reprogramming of the tumor cell state to maintain stemness. We will investigate the metabolic control of cell state through the metabolites generated or lost in GSCs and then define the specific molecular regulators responsible, including a focus on stemness mediators. In preliminary studies, we find that altered metabolism in GSCs induces alterations in the post-transcriptional regulation of mRNAs that shift the RNA profiles towards a stem-like state. We now seek to understand the metabolic and epitranscriptional regulator underlying these observations to determine the molecular regulation of highly malignant tumor cell populations and support the development of better therapeutic interventions. Moreover, epitranscriptomics may serve as a pharmacodynamic measure of selected targeted therapeutics and that target metabolically regulated epigenetic modulators. To translate these efforts into proof-of-principle novel preclinical paradigms, we are using agents that target metabolic targets and epitranscriptomics. These small molecule inhibitors can potentially be combined with other therapies to create therapeutic paradigms for glioblastoma. To generate the most effective therapeutic model, we will interrogate the preclinical utility of novel targeted therapies that disrupt the metabolic and epigenetic reprogramming with potential to accentuate the efficacy of conventional therapy. Collectively, the proposed studies will lay the foundation for improved understanding of metabolic reprogramming in cancer stem cell biology ...