DDRE-34. RIBONUCLEOTIDE REDUCTASE REGULATORY SUBUNIT M2 AS A DRIVER OF GLIOBLASTOMA TMZ-RESISTANCE THROUGH MODULATION OF dNTP PRODUCTION

Abstract

Abstract Glioblastoma (GBM) remains one of the most resistant and fatal forms of cancer. Previous studies examine pre- and post-tumor recurrence; however, it is incredibly difficult to study tumor evolution during therapy where resistance develops. To investigate this, our lab performed a single-cell RNA-sequencing screen before, during, and after temozolomide-based (TMZ) chemotherapy in a patient-derived xenograft (PDX) model in vivo. Our analysis found 149 genes uniquely expressed during TMZ-therapy compared to pre- and post-therapy (p< 0.0001). Of these, the ribonucleotide reductase (RNR) gene family stood out due to the preferential switch to Ribonucleotide Reductase Regulatory Subunit M2 (RRM2) during therapy. Classically, RRM2, or its isoform RRM2B, forms a complex with RRM1 to create an RNR, mediating deoxynucleoside triphosphate (dNTP) production. Our single-cell data revealed that GBM cells rely on RRM1-RRM2 interaction during therapy, but switch to RRM1-RRM2B in post-therapy recurrent GBM. In vitro, RRM2-knockdown cells increased TMZ susceptibility, whereas RRM1- and RRM2B-knockdowns were more resistant to TMZ (p< 0.001). Immunocytochemistry found elevated yH2AX fluorescence in RRM2-knockdowns after TMZ treatment, signifying reduced DNA repair capacity compared to the control (p< 0.001). To understand the mechanism of RRM2-mediated chemoresistance, targeted metabolomics was applied to quantify dNTP signatures during TMZ-therapy. In response to TMZ, dCTP and dGTP production in GBM cells increased 100-fold and 80-fold respectively (p< 0.001). RRM2-knockdowns produced significantly less dCTP and dGTP (p< 0.0001). By supplementing RRM2-knockdowns with dCTP and dGTP, TMZ-susceptibility was rescued, suggesting that RRM2 drives chemoresistance by promoting production of these two nucleotides. In vivo, following intracranial injection of GBM cells, mice treated with the RRM2 inhibitor Triapine with TMZ survived longer than those treated with TMZ alone, indicating promising clinical opportunities in targeting RRM2 (p< 0.0001). Overall, our data present a novel understanding of how RRM2 activity is altered during therapeutic stress to counteract TMZ-induced DNA damage.