Characterizing the MYC-induced immunopeptidome

Medulloblastoma, the most common malignant brain tumor typically found in children ages 0-9, comprises four subgroups, each with distinct molecular characteristics. Children with MYC or MYCN amplifications, which occur in Group 3 and Sonic Hedgehog subgroups, respectively, face poor survival rates and high metastasis. MYC and MYCN, both DNA-binding transcription factors, drive oncogenesis by promoting rapid cell division. This study investigates how MYC(N) upregulation stimulates the production of immunogenic neoantigens from non-canonical peptides, which may serve as novel targets for immunotherapy. Prior work shows post-transcriptional control is a critical component of MYC biology, with Group 3 tumors exhibiting significant protein-RNA dyssynchrony. Intriguingly, MYC preferentially drives the translation of upstream open reading frames (uORFs), which are non-canonical peptides that are largely unstable but exhibit robust presentation on the MHC-I antigen system.To test this, we engineered UW228 and ONS76 medulloblastoma cell lines to express MYC or MYCN cDNAs using GFP as a control and verified overexpression via western blotting and qPCR. We assessed HLA-I protein uniformity via flow cytometry. Ribosome and RNA sequencing were used to identify actively translated genomic regions and mRNA transcript levels. HLA-I immunopeptidomics was completed to identify non-canonical peptides with increased surface presence due to MYC(N) expression.We successfully verified MYC(N) overexpression and demonstrated the required HLA-I expression for immunopeptidomic analysis. The sequencing data confirmed that MYC(N) drives the translation of specific non-canonical peptides uORFs, showing increased immunopeptidome complexity in high-risk medulloblastoma models. These results are broadly supportive of MYC(N)'s role in driving the flux of non-productive peptides.Completion of this research will identify novel antigens linked to MYC(N), offering critical information for the development of medulloblastoma-specific immunotherapies to improve outcomes for affected patients. Future research will investigate MYC(N)'s impact on proteasome function and the BAG6 complex, which is responsible for regulating proteasomal degradation of non-canonical translation products. Pathway analyses will also be conducted to compare gene family expression and identify upregulated and downregulated biological functions.