Medulloblastoma (MB) is the most common malignant paediatric brain tumor. Among distinct MB subtypes, Group 3 represents one of the most aggressive subtypes, with high relapse rate, frequent metastasis, and mortality. These malignant traits are largely driven by MB stem cells (MBSCs), a subpopulation with enhanced self-renewal capacity and resistance to standard therapies, making them prime targets for innovative treatment strategies.

Group 3 MB human cell lines were treated with 0.3 µM Brefeldin A (BFA), a potent inducer of endoplasmic reticulum stress. Proteomic analyses via nano-LC-MS/MS were employed to investigate structural alterations in CD133, a key cell surface and stemness-associated receptor. Finally, computational screening was performed to identify Food and Drug Administration (FDA) and European Medicines Agency (EMA)-approved compounds, with improved pharmacokinetic and pharmacodynamic profiles that mimic BFA’s mechanism of action.

We demonstrate that BFA induced structural disruption of CD133 and compromised MBSCs stem-like phenotype. This loss of stem-like properties correlates with diminished clonogenic potential, downregulation of the oncogenic PI3K/AKT/mTOR signaling axis, and impaired intercellular communication. Importantly, we identified clinically approved compounds capable of recapitulating the biological effects associated with BFA treatment.

This study uncovers a previously unrecognized mechanism of CD133 structural modulation and highlights the existence of clinically approved compounds that may impair MBSC function, offering a promising possible avenue for targeting therapy-resistant tumor-initiating cells in aggressive Group 3 MB.