Abstract 3624: Non-canonical proteins are cancer cell vulnerabilities in diverse malignancies

Abstract In the 20 years since the completion of the Human Genome Project, cancer biology remains rooted in the assumption that the human genome encodes ~20,000 protein-coding genes. Yet, I and others have shown that thousands of “non-canonical” open reading frames (ncORFs) populate the human genome, potentially representing a dramatic expansion of the cancer proteome. Despite their abundance, little is known about the role of ncORFs as cancer driver genes. We developed functional genomics approaches to pursue this question across human cancers. To determine whether ncORFs represent biologically active proteins, we experimentally interrogated 553 candidates selected from ncORF datasets. Of these, 257 (46%) showed evidence of stable protein expression using multiple assays, and 401 (72%) induced gene expression changes when expressed in cancer cell lines. The bioactivity of ncORFs was dependent on their ability to translate a protein: mutation of the ORF start codon prevented induction of gene expression changes observed with the wild type ncORF in 48 of 51 (94%) cases. Using custom CRISPR/Cas9 knock-out screens targeting >2,000 ncORFs in 20 cancer cell lines, we found that genomic knock-out of approximately 10% of ncORFs induced viability defects in cancer cells. We focused on two candidates for functional studies. In breast cancer, we described G029442 - renamed glycine-rich extracellular protein-1 (GREP1) - as a secreted protein that is highly expressed and prognostic for poor patient outcomes. Knock-out of GREP1 in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. In medulloblastoma, we found that MYC-driven medulloblastoma cells are enriched for bioactive upstream ORFs (uORFs) that are encoded within the 5’ untranslated regions of mRNAs. We validated the ASNSD1 uORF as a top genetic vulnerability in multiple models of medulloblastoma, and its overexpression is sufficient to increase neural stem cell proliferation. Mechanistically, ASNSD1 uORF promotes a MYC-associated cellular program and interacts with the multiprotein prefoldin complex, which is required for tumors to maintain post-transcriptional regulation. Our work supports a generalizable principle that ncORFs commonly encode biologically-active proteins 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, Ian Yannuzzi, Karl Clauser, Karsten Krug, Oana Enache, Adam Brown, Amy Goodale, David E. Root, Pratiti Bandopadhayay, Todd Golub. Non-canonical proteins are cancer cell vulnerabilities in diverse malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3624.