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Abstract Osteosarcoma is an aggressive bone malignancy that predominantly affects adolescents and young adults. Despite recent advancements in treatments, the challenges of recurrence and chemoresistance persist. Ferroptosis is a form of regulated cell death driven by iron-dependent lipid peroxidation, and emerging evidence suggests significant roles for NRF2 and GPX4 in iron metabolism, regulating tumor cell functions. In osteosarcoma, however, the molecular mechanisms of the NRF2/GPX4 axis and its therapeutic potential remain unknown. In this study, we first determined GPX4 and xCT expression levels and cellular responses to RSL3, a GPX4 inhibitor, and Erastin, an xCT inhibitor in osteosarcoma cells. A total of seven osteosarcoma cell lines, consisting of five human cell lines (HOS, 143B, Saos-2, MG-63, U2OS) and two canine cell lines (COS31 and DOUG), were used. Our results revealed that the IC50 values ranged from 0. 02 uM to 1. 01 uM in cells treated with RSL3 and from 0. 35 uM to 1. 41 uM in cells treated with Erastin, respectively. These values were proportional to GPX4 and xCT levels across the cell lines. Then, U2OS, as the most sensitive, and Saos-2, the least sensitive based on their IC50 values, were selected to examine the iron regulatory machinery. In U2OS cells, RSL3 treatment significantly decreased NRF2 and GPX4, while increasing ferroportin-1 and ferritin. GPX4 knockdown also led to comparable changes in NRF2, ferroportin-1, and ferritin expression. NRF2 appears to mediate iron metabolism, ferroportin-1 acts as an iron exporter, and ferritin stores intracellular iron. Therefore, our findings suggest that GPX4-mediated ferroptosis is regulated by iron metabolic pathways, potentially to prevent ferroptotic cell death. We also found that Ferrostatin-1, a lipid peroxidation inhibitor showed no changes in ferroportin-1, ferritin, and NRF2. In Saos-2, RSL3 induced a remarkable reduction in GPX4 expression, akin to U2OS. However, no significant change was found in NRF2, ferroportin-1, and ferritin. Instead, Ferrostatin-1 restored the decreased GPX4 and increased NRF2, ferroportin-1, and ferritin levels. In addition, Z-VAD (a pan-caspase inhibitor), Necrosatin-1s (a RIPK1 inhibitor), and Ferrostatin-1 were capable of preventing Saos-2, COS31, and DOUG cells from RSL3-induced cell death at lethal doses. Such protection was undetected in U2OS. These data suggest that metabolic pathways controlling intracellular iron are crucial for NRF2/GPX4-mediated ferroptosis in osteosarcoma cells. Furthermore, we generated RNA-seq and LC-MS/MS data to identify novel molecular targets governing the metabolic pathways of ferroptosis in osteosarcoma. Our ongoing work includes downstream molecular and functional assays following gene modifications in osteosarcoma cells (overexpression and knockout) and assessing metabolic vulnerabilities by targeting iron-mediated ferroptosis in xenografts. Citation Format: Md Abdullah, Donghee Lee, Rong Li, Jong Hyuk Kim. Metabolic liabilities of iron-dependent ferroptosis mediated through NRF2/GPX4 axis in osteosarcoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 4459.
Abdullah et al. (Fri,) studied this question.