BIOL-03. PROTEIN TRANSLATION FROM NON-CODING GENOMIC LOCI PRODUCE BIOLOGICALLY-ACTIVE PROTEINS IMPLICATED IN CANCER CELL SURVIVAL IN PEDIATRIC BRAIN TUMORS

Protein translation is both a fundamental cellular process essential for life as well as an oncogenic mechanism employed by tumors to enact cancer cell biology. While protein translation is most readily manifest in the ~20,000 known human protein coding genes, there are, in fact, several thousand additional regions of the cancer genome that are translated and contribute the complexity of the molecular milieu of cancer. Here, we systematically addressed the question of whether such uncharacterized genomic regions encode truly biologically active proteins and applied these findings to pediatric brain tumors. We experimentally interrogated 553 candidates selected from non-canonical open reading frame (ORF) datasets. Of these, 57 induced viability defects when knocked out in a broad array of human cancer cell lines. Upon ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. CRISPR tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We characterized several of these in the context of pediatric brain tumors, where dense CRISPR tiling screens revealed unique functional relevance of dozens of non-canonical ORFs in pediatric brain cancer cell survival. We found that one of these ORFs, ASNSD1 uORF, encodes a well-folded protein whose translation is a selective genetic dependency distinct from the adjacent ASNSD1 annotated protein. In vitro molecular biology assays confirmed the MYC-amplified medulloblastoma cell lines had a heightened dependency on this protein, and that MYC binds to the promoter of this gene, with MYC expression correlating with ASNSD1 in patient tumors. Co-immunoprecipitation assays defined ASNSD1 uORF as a novel member of the prefoldin complex of cytoplasmic protein stability regulators. Overall, our experiments suggest that the abundant protein translation found in the “non-coding” genome may produce biologically active non-canonical ORFs that are potential therapeutic targets.