Abstract 1637: GPX4-mediated Pathway Regulates Iron Efflux to Prevent Ferroptotic Cell Death and Promote Immunosuppressive Signals in Osteosarcoma Cells

Abstract Osteosarcoma is a malignant bone sarcoma prevalent in children, adolescents, and young adults, with limited treatment options. Targeting ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation, presents a potential therapeutic strategy for cancers. However, the molecular mechanisms that regulate iron metabolism in ferroptosis remain unclear. In this study, we explored cell defense mechanisms against ferroptosis mediated by intracellular iron regulation and immune signaling pathway in osteosarcoma cells. First, we found that xCT and GPX4 protein were detected with variable expressions across five osteosarcoma cell lines. Erastin, an xCT inhibitor, and RSL3, a GPX4 inhibitor, induced ferroptotic cell death in these cells. Specifically, higher inhibitory dose of RSL3 induced a significant reduction in GPX4 levels in U2OS and MG-63 cells in a dose- and time-dependent manner. In contrast, xCT expression increased in these cells, suggesting an adaptive mechanism to counteract oxidative stress following GPX4 depletion. RSL3 treatment elevated lipid peroxidation levels in U2OS and MG-63 cells, which were restored by Ferostatin-1. Our RNA-seq analysis identified 18 differentially expressed genes in response to RSL3 treatment in U2OS cells, with the highest expression in AKR1C1 gene, which plays a significant role in oxidative stress. We confirmed that GPX4 inhibition upregulated AKR1C1 gene expression as well as other oxidative stress markers, including NRF2, HMOX1, and NQO1.Our data also showed that osteosarcoma cells modulated intracellular iron homeostasis in response to GPX4-mediated ferroptotic stress. GPX4 inhibition significantly enhanced FPN and FTH1 expression, which are essential for iron efflux and storage, in U2OS cells, while MG-63 cells only increased FTH1 with minimal change in FPN. Furthermore, macrophage stimulating protein receptor (MSPR) protein expression was significantly increased by GPX4 inhibitor using Proteome Profiler analysis. We observed that combination treatment with dual MSPR and MET inhibitor, BMS-777607, showed synergistic effects with GPX4 inhibition. Altogether, our findings demonstrate that ferroptotic cell death is inducible in osteosarcoma cells, and the potential resistance mechanism to ferroptosis is mediated by oxidative stress mechanism and intracellular iron regulation. We also identified novel target, MSPR, regulated by GPX4 inhibition, which potentially contribute to the activation of immunosuppressive signals from osteosarcoma. In conclusion, this study underscores that targeting the ferroptotic pathway holds a great promise for therapeutic potential in osteosarcoma by regulating both intracellular pathway and the immunosuppressive tumor microenvironment. Citation Format: Md Abdullah, Donghee Lee, Rong Li, Eslenur Nipa, Jong Hyuk Kim. GPX4-mediated Pathway Regulates Iron Efflux to Prevent Ferroptotic Cell Death and Promote Immunosuppressive Signals in Osteosarcoma Cells [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 1637.