Arsenic trioxide triggers ferroptosis in neuroblastoma via USF1/GPX4 axis

Neuroblastoma (NB) is an extracranial solid tumor arising from impaired differentiation of primitive neural crest cells. High-risk NB (HR-NB), which comprises roughly 40% of cases, has a dismal prognosis. Considering the high iron demand of NB cells and the vulnerability of therapy-resistant cells to ferroptosis, ferroptosis represents a promising therapeutic strategy. Arsenic trioxide(ATO), a clinically approved anti-tumor agent, has shown efficacy across various malignancies. Our previous clinical study provided preliminary evidence of ATO's therapeutic efficacy in high-risk NB, particularly in relapsed and refractory cases. In this study, we confirmed that ATO induces ferroptosis in NB cells and elucidated the underlying molecular mechanism. ATO significantly reduced NB cell viability, and only the ferroptosis inhibitor deferoxamine specifically reversed this cytotoxicity. Proteomic analysis revealed significant enrichment in the ferroptosis-related pathway. Consistently, ATO increased intracellular levels of ROS, Fe²⁺, and malondialdehyde while reducing glutathione levels in NB cells. Mechanistically, ATO downregulated GPX4 expression by suppressing its upstream transcription factor USF1, rather than by enhancing ubiquitin-mediated degradation. USF1 knockdown decreased GPX4 expression and enhanced ferroptosis, whereas USF1 overexpression partially rescued the ferroptosis phenotypes induced by ATO. ChIP-PCR further confirmed the direct binding of USF1 to the GPX4 promoter. Moreover, ATO combined with GSTP1 inhibitor TLK199 exhibited synergistic cytotoxicity against MYCN-amplified SK-N-BE(2) cells. This study demonstrates that ATO triggers ferroptosis in NB cells via the USF1/GPX4 axis, providing a mechanistic rationale for the broader therapeutic application of ATO in HR-NB treatment.