Abstract 4431: RRM2-driven dNTP biosynthesis: a therapeutic vulnerability in temozolomide-resistant glioblastoma

Abstract Glioblastoma (GBM) remains one of the most aggressive and fatal cancers, with a median survival of only 12-18 months despite standard treatments, including surgical resection, temozolomide (TMZ) chemotherapy, and radiotherapy. Tumor recurrence and therapy resistance are significant barriers to improving patient outcomes. To address this, our lab investigated the molecular mechanisms driving resistance during TMZ therapy using a single-cell RNA-sequencing screen on a patient-derived xenograft (PDX) model. This approach enabled us to study tumor evolution pre-, during, and post-therapy. Our analysis identified 149 unique genes expressed during TMZ therapy, with the ribonucleotide reductase (RNR) family, particularly Ribonucleotide Reductase Regulatory Subunit 2 (RRM2), emerging as critical players. During TMZ treatment, GBM cells preferentially utilize RRM1-RRM2 interaction to support deoxynucleoside triphosphate (dNTP) biosynthesis, whereas post-therapy recurrent GBM shifts to RRM1-RRM2B complexes. Functional studies demonstrated that RRM2 knockdown sensitized GBM cells to TMZ, reducing DNA repair capacity, as evidenced by increased yH2AX fluorescence, while RRM1 or RRM2B knockdown conferred resistance (p<0.001). Metabolomic profiling revealed that RRM2 mediates dCTP and dGTP production during TMZ treatment, critical for chemoresistance. Supplementation with these dNTPs rescued TMZ susceptibility in RRM2-deficient cells, highlighting its pivotal role in DNA repair and therapeutic resistance. Recognizing the limitations of first-generation RNR inhibitors, we explored the second-generation inhibitor Triapine (3-AP). Preclinical studies showed that 3-AP effectively inhibits RRM2 activity, enhancing TMZ sensitivity and extending survival in PDX models (p<0.0001). Mice treated with a combination of 3-AP and TMZ demonstrated significantly longer survival compared to TMZ alone. These findings underscore the potential of targeting RRM2-mediated dNTP biosynthesis to overcome chemoresistance in GBM. We are conducting a Phase 1/1b clinical trial to assess the safety, toxicity, and maximum tolerated dose (MTD) of combining 3-AP with TMZ in recurrent GBM patients. Citation Format: Atique U. Ahmed, Karan Dixit, Ella N Perrault, Jack M Shireman, Priya Kumthekar, Roger Stupp. David C James. RRM2-driven dNTP biosynthesis: a therapeutic vulnerability in temozolomide-resistant glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4431.