PROJECT SUMMARY To advance our understanding of therapeutic resistance in Glioblastoma (GBM), it is essential to characterize the individual cell during therapy those fuel tumor recurrence in GBM. However, it is challenging to study the GBM during conventional radio- and chemotherapy due to limited accessibility to patient samples during this time period. Our lab performed a single-cell RNA sequencing screen in the patient-derived xenograft model of GBM during temozolomide (TMZ) therapy. Our analysis revealed that the Ribonucleotide Reductase Regulatory Subunit 2 (RRM2) mediates deoxynucleoside triphosphates (dNTPs) production necessary for proper DNA replication stable cell growth, promotes metabolic adaptation to TMZ therapy, and initiate recurrence. We have identified a novel mechanism where RRM2-mediated dCTP and dGTP can enhance the DNA repair in response to TMZ and promotes resistance to therapy. Based on this, we hypothesize that RRM2-mediated RNR activity is critical for chemoresistance in GBM. To investigate this hypothesis, we intend to elucidate the RNR-mediated chemoresistance in GBM (Aim 1). Next, we will evaluate a blood-brain permeable RRM2 inhibitor to prevent RNR-mediated chemoresistance in GBM (Aim 2). We established collaboration with Nanopharmaceutic, which holds the patent for producing clinical-grade 3-AP and will provide us with clinical-grade 3-AP to test its efficacy further and advance our understanding of the mechanism of action by which it can be used to treat GBM patients. Finally, we intend to investigate the mechanism of therapeutic resistance by specific nucleotides produced by the RRM2-mediated de novo pathway (Aim 3). Collectively, our studies will provide novel insights regarding changes in dNTP synthesis that are associated with GBM adaptation and resistance during chemotherapy. This information, in turn, is expected to reveal novel approaches for delaying, if not preventing, tumor recurrence.