LAUSR

Purpose: Despite aggressive multimodality treatments, the median survival of glioblastoma (GBM) remains within the range of 12-15 months after diagnosis with standard-of-care surgery, radiation therapy (RT) and temozolomide (TMZ). Resistance frequently arises, exacerbating the clinical difficulty of treating GBM. Therefore, new or enhanced therapeutic strategies are critically needed. Radiation treatment induces DNA damage that cells must repair for their survival, and DNA repair requires a constant and balanced supply of dNTPs. The ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the production of dNTPs. Therefore, we hypothesize that inhibition of the RRM2 subunit of RNR sensitizes GBM to radiation. Experimental Design: We analyzed RRM2 expression in GBM via mRNA expression analysis and assessed its correlation with GBM patient clinical outcomes using different public datasets. We examined radiosensitization effects of RRM2 inhibition by either small interfering RNA (siRNA) or RRM2 inhibitor (3-aminopyridine-2-carboxaldehyde thiosemicarbazone; Triapine) known to cross the blood-brain barrier using clonogenic assays in U87MG/EGFRVIII and LN229 GBM cell lines. Human tumor xenografts were generated to explore the radiosensitization effect of Triapine on GBM tumors in vivo. Radiation-induced DNA damage was evaluated via γ-H2AX foci assays and expression levels by western blotting. We further investigated whether CHK1 inhibition with LY2603618-Rabusertib could abrogate the replication stress induced expression of RRM2. Results: RRM2 is significantly higher expressed in GBM compared to non-tumor tissues (p<0.0001 in both TCGA and Rembrandt datasets), and higher RRM2 expression is significantly associated with higher tumor grade (p<0.0001 in CGGA and p=0.001 in Rembrandt datasets) and worse overall survival (p=0.014 in CGGA dataset). Interestingly, RRM2 inhibition sensitizes GBM cells to radiation treatment in vitro and leads to reductions in tumor growth and significantly increases mice survival in vivo, with acceptable levels of toxicity. Mechanistically, RRM2 inhibition-mediated radiosensitization is associated with higher levels of γ-H2AX expression by westerns, and higher foci counts, indicating higher levels of unresolved DNA damage after radiation. Moreover, we confirmed that Triapine inhibition of RRM2 leads to replication stress which in turn upregulates RRM2 in a CHK1 mediated mechanism, as inhibition of CHK1 abrogated the Triapine induction of RRM2. Conclusions: These results indicate that RRM2 is upregulated and important for GBM survival after radiation. Moreover, RRM2 inhibition with triapine effectively sensitizes GBM tumors to radiation in vitro and in vivo. Our findings suggest that triapine warrants clinical testing with radiation. Citation Format: Sergio Corrales-Guerrero, Tiantian Cui, Veronica Castro-Aceituno, Linlin Yang, Haihua Feng, Sindhu Nair, Changxian Shen, Monica Venere, Terence M. Williams. Targeting ribonucleotide reductase subunit 2 (RRM2) to radio-sensitize glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2407.