Abstract C074: Dual NR4A1/2 ligands inhibit rhabdomyosarcoma cell growth and trigger ferroptosis by activating CD71

Abstract Background: Rhabdomyosarcoma (RMS) is the most prevalent soft tissue sarcoma in children and adolescents, with a poor prognosis in metastatic or treatment-resistant cases. Conventional therapies often fail to eliminate aggressive tumor populations. Ferroptosis, a regulated form of cell death driven by iron accumulation and lipid peroxidation, has emerged as a promising therapeutic strategy for targeting RMS cells. Objective: This study investigates the potential of bis-indole-derived C-DIM compounds to induce ferroptosis in RMS cells by targeting the NR4A1/2–Sp1–CD71 axis, which regulates iron uptake and oxidative stress. Methods: Human RMS cell lines (RD and RH30) were treated with C-DIM compounds, and cell viability was assessed using the XTT assay. Intracellular ROS levels and lipid peroxidation were evaluated using H2DCFDA and BODIPY™ 581/591 staining, respectively, while malondialdehyde (MDA) assays quantified oxidative damage. CD71 mRNA and protein levels were measured by qPCR and Western blot. siRNA-mediated knockdowns of NR4A1, NR4A2, and Sp1 were performed to examine their roles in regulating CD71 expression and ferroptosis. A CD71 promoter luciferase assay was used to assess Sp1-dependent transcriptional activity. Results: C-DIM treatment significantly reduced RMS cell viability by enhancing ferroptosis, as evidenced by increased ROS, MDA production, and lipid peroxidation. CD71 expression was markedly upregulated following treatment. Knockdown of NR4A1/2 or Sp1 led to a decrease in CD71 expression and protected RMS cells from ferroptosis. CD71 promoter assays confirmed that Sp1 regulates its transcription in response to C-DIMs. Conclusions: C-DIM compounds promote ferroptosis in RMS cells through an NR4A1/2–Sp1-mediated upregulation of CD71. These findings highlight a novel therapeutic strategy targeting the NR4A1/2–Sp1–CD71 axis to induce ferroptosis and overcome treatment resistance in rhabdomyosarcoma. Citation Format: Arafat Rahman Oany, Srijana Upadhaya, Stephen Safe. Dual NR4A1/2 ligands inhibit rhabdomyosarcoma cell growth and trigger ferroptosis by activating CD71 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C074.