LAUSR

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the synthesis of deoxyribonucleoside triphosphates (dNTPs) and is required for DNA replication and the repair of DNA damage. Multiple types of cancer, including Ewing sarcoma tumors, are sensitive to RNR inhibitors or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. However, the polypharmacology and off-target effects of RNR inhibitors has complicated the identification of the precise mechanisms that regulate sensitivity and resistance to this class of drugs. Consequently, we used a conditional knockout (CRISPR/Cas9) and rescue approach to target RRM1 in Ewing sarcoma cells and identified that loss of RRM1 results in upregulation of the expression of multiple members of the activator protein-1 (AP-1) transcription factor complex, including c-Jun and c-Fos. This upregulation of AP-1 is mediated, in part, by SLFN11, which is expressed at high levels in Ewing sarcoma tumor and other pediatric sarcomas. Notably, the inducible expression of c-Jun and c-Fos in Ewing sarcoma cells impairs cell growth, downregulates the expression of the stem cell factor Sox2, and upregulates markers of cellular differentiation. In addition, small-molecule inhibitors of RNR, including gemcitabine and hydroxyurea, and HDAC inhibitors, which reduce the level of the RRM1 protein, also activate this AP-1 signaling pathway in Ewing sarcoma cells. Overall, these results provide novel insight into the critical pathways activated by loss of RNR activity and the mechanisms of action of inhibitors of RNR. Citation Format: David Gordon, Emma Croushore, Stacia Koppenhafer, Kelli Goss, Elizabeth Geary. Activator protein-1 (AP-1) signaling inhibits the growth of Ewing sarcoma cells in response to DNA replication stress. [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 3532.