MDB-07. Nortriptyline triggers apoptosis in group 3 medulloblastoma by inducing mitochondrial dysfunction

Abstract Medulloblastoma (MB), the most common malignant pediatric brain tumor, is classified into four subgroups, with Group 3 (G3MB) being the most aggressive and comprising 25% of cases. G3MB is associated with poor survival rates (50% at 5 years) compared to other subgroups (70–95%). Current treatments, including surgery, irradiation, and chemotherapy, are limited by significant toxicity and late effects, which are particularly problematic in G3MB due to its high recurrence rates and aggressive progression. Despite advances in targeted therapies for other MB subgroups, G3MB remains challenging to treat, necessitating novel approaches. Our study uses patient-derived RNA-sequencing data to identify repositionable drugs for G3MB. Differential gene expression analysis of publicly available datasets (GSE148389, GSE164677) generated a G3MB-specific gene signature, which was analyzed against the LINCS database. Candidate drugs were filtered for blood-brain barrier permeability and FDA approval. Functional assays in G3MB cell lines (HDMB03 and D425) evaluated cytotoxicity, clonogenicity, wound healing, cell cycle, and apoptosis. Mechanistic insights were gained through RNA sequencing of treated cells and mitochondrial function assays (Seahorse, MitoSOX, TMRM). Eighty-one candidates were identified, with antidepressants emerging as the top drug class. Nortriptyline (NT), a tricyclic antidepressant, was the lead candidate, exhibiting IC50 values of ∼7 µM (HDMB03) and ∼11 µM (D425). NT inhibited wound healing, colony formation, and medullosphere generation while inducing apoptosis. Mechanistically, NT disrupted oxidative phosphorylation without a compensatory glycolytic response, increased mitochondrial superoxide, and decreased mitochondrial membrane potential. SynergyFinder analysis revealed that NT synergizes with cisplatin, a G3MB standard of care. Our pipeline identified nortriptyline as a repurposable drug with significant cytotoxic activity against G3MB, offering a potential shift toward targeted, less toxic treatments for high-risk G3MB to improve outcomes and quality of life.