Abstract A025: Dual NR4A1/2 ligands inhibit PAX3-FOXO1 and promote ferroptosis in Rhabdomyosarcoma
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and adolescents, with poor outcomes in metastatic and treatment-resistant disease. The alveolar subtype is frequently driven by the PAX3-FOXO1 fusion oncoprotein, which promotes tumor progression and therapeutic resistance. Ferroptosis, an iron-dependent regulated cell death process characterized by lipid peroxidation and oxidative stress, represents a promising therapeutic vulnerability in RMS. This study investigated whether bis-indole-derived dual NR4A1/2 ligands (C-DIM compounds) suppress PAX3-FOXO1 signaling and induce ferroptosis through regulation of iron metabolism and antioxidant defense pathways in RMS cells. Human RMS cell lines (RD and RH30) were treated with C-DIM compounds. Cell viability was assessed using XTT assays. Intracellular ROS and lipid peroxidation were measured using H2DCFDA and BODIPY™ 581/591 staining, and oxidative damage was quantified by malondialdehyde (MDA) assays. Expression of ferroptosis-related markers, including CD71, GPX4, SLC7A11, and FSP1, was analyzed by qPCR and Western blotting. NR4A1, NR4A2, and Sp1, Sp4 were silenced using siRNA to determine their functional roles. C-DIM treatment significantly reduced RMS cell viability and suppressed PAX3-FOXO1 expression and downstream oncogenic signaling. These effects were associated with increased ROS production, lipid peroxidation, and MDA accumulation, confirming ferroptosis induction. Mechanistically, dual NR4A1/2 ligands triggered early and dose-dependent induction of CD71, suggesting enhanced iron uptake as an initiating event. In parallel, key ferroptosis-protective regulators, including GPX4, SLC7A11, and FSP1, were significantly downregulated. Knockdown of NR4A1/2 or Sp1/4 down regulates GPX4, SLC7A11, and FSP1and reduced ferroptotic cell death, indicating a critical role for the NR4A1/2–Sp1/4 axis. Dual NR4A1/2 ligands suppress PAX3-FOXO1 signaling and promote ferroptosis through coordinated modulation of iron uptake and antioxidant defense pathways. Early CD71 induction combined with suppression of GPX4, SLC7A11, and FSP1 highlights a multi-level ferroptotic mechanism in RMS. Further studies are needed to elucidate the precise regulatory mechanisms underlying the suppression of ferroptosis-related genes. Targeting NR4A1/2 may represent a promising therapeutic strategy for overcoming resistance in rhabdomyosarcoma. Arafat Rahman Oany, Stephen Safe. Dual NR4A1/2 ligands inhibit PAX3-FOXO1 and promote ferroptosis in Rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Breaking Barriers in the Fight against Rare Cancers; 2026 Jul 18-20; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(14_Suppl):Abstract nr A025.