Characterizing neddylation as a potential vulnerability in MYC-amplified medulloblastoma

Background: Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Children with MYC-amplified MB almost universally fail current treatments, highlighting a critical need for targeted therapeutic strategies. Our previous data revealed an enrichment of protein synthesis pathways in MYC-amplified MB, pointing toward protein turnover as a potential target. Neddylation is a post-translational modification process that plays a role in protein turnover through proteasomal degradation. We hypothesize that characterizing neddylation in MYC-amplified MB will identify novel therapeutic vulnerabilities, and that targeting neddylation is a rational therapeutic strategy for MYC-amplified MB. Methods: Cell viability assay was conducted in four MYC-amplified MB cell lines and normal human astrocyte (NHA) control after 96hr treatment of neddylation inhibitor MLN4924 or TAS4464. Western blot was performed in TAS4464-treated MYC-amplified MB cell lines to evaluate MYC protein dynamics. To characterize neddylation proteome in MB, Co-Immunoprecipitations (Co-IP) were performed using lysates from MB or NHA models with NEDD8 or IgG (negative control) antibodies. Protein identification and quantification were carried out via mass spectrometry (MS), and the resulting dataset was subjected to STRING-based pathway enrichment analysis. Results: All MYC-amplified MB cell lines exhibited significant sensitivity to TAS4464 compared to NHA. Western blot analysis revealed a transient accumulation of MYC followed by a sustained depletion, consistent with the disruption of neddylation-dependent turnover mechanisms. Nearly 1,000 conserved neddylation substrates were identified across MB cell lines, with the majority representing previously unidentified targets. MB-specific high-confidence targets are enriched in components comprising the mRNA splicing pathway and spliceosome complex. Further functional validation using distinct RNA splicing inhibitors induced potent, dose-dependent cytotoxicity in MYC-amplified MB, with markedly reduced sensitivity observed in NHA. Conclusion: These findings establish neddylation as a critical dependency in MYC-amplified MB, essential for maintaining MYC protein stability and tumor cell survival. The potency of TAS4464 provides preclinical validation for targeting neddylation pathway. Our proteomics data unveiled a previously unrecognized dimension of neddylation in MB, identifying mRNA splicing machinery as a targetable vulnerability. This study represents the first link between neddylation driving splicing in MYC-amplified MB, suggesting a previously undescribed critical and actionable vulnerability that provides a mechanistic rationale for clinical investigation of neddylation inhibitors in very high-risk MYC-amplified MB.