DDDR-28. DNA DAMAGE SENSING KINASES ATR AND ATM-DEPENDENT PHOSPHORYLATION OF RRM2 PROMOTES PROTEIN STABILIZATION AND CHEMO-ADAPTATION

Abstract

Abstract Glioblastoma (GBM) is a highly heterogeneous, primary malignant brain tumor in adults that shows limited response to standard-of-care therapies, such as chemotherapy and radiation. This is due, in part, to the development of therapeutic resistance. To characterize the adaptive mechanisms GBM cells use to evade the standard chemotherapy, temozolomide (TMZ), single-cell RNA sequencing analysis was conducted on an in vivo patient-derived xenograft (PDX) model of GBM, before, during, and following TMZ-based therapy. Our data revealed 149 genes to be uniquely expressed in the during-TMZ condition (p< 0.0001), including the rate-limiting regulatory enzyme of dNTP metabolism, ribonucleotide reductase (RNR) enzyme. Network analysis of pathways enriched during therapy revealed an interaction between the (RNR) enzyme, RRM2, and DNA damage response genes in the ATM and ATR kinase-signaling pathways. RRM2-dependent deoxynucleotide (dNTP) production is required for DNA repair and cell survival following TMZ treatment. Therefore, we investigated whether ATR and ATM kinases directly interact with RRM2 to promote its stability. TMZ treatment triggered the activation of DNA damage response machinery and protein kinases, ATM, ATR, CHK1, and CHK2, which physically interacted with RRM2 across multiple GBM PDX cell lines. Co-IP and immunoblot analyses along with cycloheximide chase analysis showed increased phosphorylation of the threonine residue on RRM2 during therapy, preventing its degradation. As such, we hypothesize that early DNA damage sensing by ATM and ATR kinases, and their subsequent phosphorylation of RRM2, promotes the maintenance of purine nucleotide pools needed for DNA repair and development of chemo-resistance. This novel and targetable TMZ response axis presents a promising clinical opportunity for GBM therapy, using FDA-approved inhibitors of these targets to block the purine metabolic adaptations that drive resistance and sensitize GBM cells to TMZ therapy.