Abstract SY04-01: Mapping the functional microproteome in cancer

Abstract In the 20 years since the completion of the Human Genome Project, cancer biology remains rooted in the belief that the human genome encodes ∼20,000 protein-coding genes. This assumption has been fundamentally challenged by the discovery of microproteins, which are encoded across the genome by thousands of non-canonical open reading frames (ORF). Whether these microproteins reflect a previously untapped source of cancer genes may therefore have a profound impact on our understanding of cancer biology. To address this question, we have interrogated the prevalence and impact of non-canonical ORF translation across multiple cancer types. Using ribosome profiling, ORF cDNA libraries, expression profiling and CRISPR/Cas9 screens, we find that thousands of non-canonical ORFs are translated in cancer cells; nearly half (46%) of tested non-canonical ORFs may make a detectable microprotein; nearly 30% induced gene expression changes in cancer cell lines; and 10% induced a cancer cell viability defect when knocked-out. High-resolution CRISPR saturation mutagenesis identified a subset of non-canonical ORFs with exquisite genetic selectivity in about one-third of cases, providing high-confidence genetic evidence for a causal role of the non-canonical ORF in cancer cell viability. To position specific candidates in the context of cancer biology, we have characterized two cancer-associated microproteins encoded by non-canonical ORFs. In breast cancer, we described G029442 - renamed glycine-rich extracellular protein-1 (GREP1) - as a secreted protein that is a preferential genetic dependency in this disease through its regulation of the oncogenic cytokine, GDF15. Moreover, GREP1 expression is prognostic for poor patient outcomes. In childhood medulloblastoma, we found that MYC-driven medulloblastoma cells preferentially upregulated an upstream open reading frame in the ASNSD1 5’ untranslated region, termed ASNSD1-uORF. ASNSD1 uORF was a top genetic vulnerability in multiple models of medulloblastoma and it functions as a component of the prefoldin-like complex, which post-translationally regulates nascent proteins. Together, ASNSD1-uORF and the prefoldin-like complex impacted cancer cell cycle through the proteomic regulation of proteins involved in mitosis and chromosome spindle complexes. In summary, our work supports a generalizable principle that ncORFs commonly encode biologically-active microproteins in diverse malignancies. Ongoing investigation of ncORFs therefore represents a new frontier in cancer research with the potential to define the next generation of therapeutic target genes. Citation Format: John R. Prensner. Mapping the functional microproteome in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_2):Abstract nr SY04-01.