Abstract Background: Neuroblastoma (NB) is an aggressive pediatric cancer with poor prognosis, particularly in patients with MYCN amplification (MNA). MYCN-driven metabolic plasticity allows NB cell survival even under therapeutic stress. Pharmacologic ascorbate (Asc) has emerged as a potential low-toxicity anticancer agent that generates hydrogen peroxide (H2O2) and induces oxidative stress, selectively killing cancer cells. This study investigated the ROS-mediated metabolic vulnerability of MYCN-amplified and non-amplified NB cells in response to ascorbate. Method: Patient-derived MYCN-amplified (MNA) (COG-N-415) and non-amplified (COG-N-618) neuroblastoma cells were treated with 0-20 mM ascorbate for 24 hours. Cell viability was measured using the CellTiter-Glo® assay. Intracellular ATP levels were quantified by HPLC. H2O2 and catalase were used to assess ROS dependence. Mitochondrial respiration and glycolytic activity were assessed by Seahorse XF analysis after 1-10 mM ascorbate treatment. Result: Ascorbate induced a dose-dependent decrease in cell viability in both MNA and non-MNA neuroblastoma cells, with approximately 90% cell death at 10 mM. COG-N-618 (non-MNA) cells exhibited greater sensitivity than COG-N-415 (MNA), showing significant viability loss at 5 mM ascorbate. ATP levels decreased in both cells upon 5 mM ascorbate treatment. Catalase rescued ascorbate-induced ATP depletion, confirming an H2O2-mediated mechanism. Seahorse analysis demonstrated progressive inhibition in mitochondrial respiration and glycolysis, with COG-N-618 cells showing dose dependent Oxidative Phosphorylation (OCR) and Glycolysis (ECAR) inhibition at 1-10 mM. For the COG-N-415 cells (MNA), both OCR and ECAR decreased at 1 mM of ascorbate, but 5 mM and 10 mM did not cause further decrease, indicating an adaptive mechanism in the MYCN-amplified cells. Conclusion: Pharmacologic ascorbate induces cytotoxicity in neuroblastoma cells and triggers ROS-dependent metabolic disruption and MYCN downregulation. Citation Format: Tasfia Farah, Qi Chen. Pharmacologic ascorbate exerts anticancer activity through ROS-dependent metabolic vulnerability in MYCN-amplified neuroblastoma abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2941.
Farah et al. (Fri,) studied this question.