Abstract B017: Aberrant translation of microproteins as a source of new cancer dependencies in medulloblastoma

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation.Dysregulation of ribosome activity not only influences known protein-coding genes, but also unannotated small open reading frames (sORFs) as well.We have recently shown that sORFs may produce functional microproteins in medulloblastoma.Here, we performed an integrative analysis of medulloblastoma patient samples and cell lines.We used ribosome profiling, RNAseq, and mass spectrometry to delineate dysregulation of microprotein production.We employed cell models of MYC overexpression to determine the role of MYC in selective translation of microproteins in medulloblastoma, finding specific associations with upstream open reading frames (uORFs) located in gene leader sequences.We then designed a step-wise approach to define functional microproteins in medulloblastoma through the use of CRISPR/Cas9 screens, saturation mutagenesis, and base editing.We observed distinctive signatures of microprotein bioactivity based on the molecular drivers of medulloblastoma according to disease subtypes.In the Group 3 subtype, we observed an enrichment for bioactive uORFs relative to other disease settings.We then characterized several key microprotein dependencies resulting from uORFs including a molecular mechanism for one candidate, ASNSD1-uORF, in mediating protein-RNA dyssynchrony in Group 3 medulloblastoma.Protein-RNA dyssynchrony is the divergent regulation of protein abundance from RNA abundance, and is a hallmark of Group 3 medulloblastoma.We probed the clinical and therapeutic implications of protein-RNA dyssynchrony and determined that this process may be associated with patterns of drug sensitivity and resistance in medulloblastoma.With this insight, we have now used ASNSD1-uORF to stratify medulloblastoma resistance patterns to inhibitors of RNA transcription.We determine that an interplay between RNA translational control and RNA transcriptional control, while driving resistance in some settings, may also reveal additional drug targets, such as cyclin-dependent kinases.Our findings underscore the fundamental importance of sORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets and therapeutic insights. Citation Format: John Prensner, Pratiti Bandopadhayay, Soumik Saha, Leslie Lupien, Seung H. Choi. Aberrant translation of microproteins as a source of new cancer dependencies in medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B017.