Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC-family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets. [Display omitted] •Ribo-seq reveals widespread translation of non-canonical ORFs in medulloblastoma•High-resolution CRISPR tiling reveals uORF functions in medulloblastoma•The ASNSD1-uORF microprotein controls downstream pathways with the prefoldin-like complex•The ASNSD1-uORF microprotein is necessary for medulloblastoma cell survival Hofman et al. reveal a novel survival mechanism in high-risk childhood medulloblastoma through the translation of non-canonical open reading frames (ORFs). The study uncovers the critical role of these often-overlooked ORFs, particularly the ASNSD1-uORF, in medulloblastoma cell survival, offering new avenues for targeted therapies in pediatric cancer.