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. 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 step-wise approach to employ multiple CRISPR-Cas9 screens to elucidate functional non-canonical ORFs implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream open reading frames (uORFs) exhibited selective functionality independent of the main coding sequence. One of these, ASNSD1-uORF or ASDURF, was upregulated, associated with the MYC family oncogenes, and was required for 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 cancer genomics studies seeking to define new cancer targets.

In the decades following the discovery that genes encode proteins, scientists have tried to exhaustively and comprehensively characterize the human genome. Recent advances in computational methods along with transcriptomic and proteomic techniques have now shown that historically noncoding genomic regions may contain non-canonical open reading frames (ncORFs), which may encode functional miniproteins or otherwise exert regulatory activity through coding-independent functions. Increasingly, it is clear that these ncORFs may play critical roles in major human diseases such as cancer. In this review, we summarize the history and current progress of ncORF research and explore the known functions of ncORFs and the miniproteins they may encode. We particularly highlight the emerging body of evidence supporting a role for ncORFs and miniproteins contributions in cancer. Finally, we provide a blueprint for high-priority areas of future research for ncORFs in cancer, focusing on ncORF detection, functional characterization, and therapeutic intervention.

High-risk medulloblastoma is one of the most recalcitrant pediatric cancers, and children with MYC-amplified disease frequently succumb to relapsed disease. Extensive analyses of the coding genome in this disease have characterized additional somatic events in some subsets of patients, though most tumors lack targetable mutations and do not yield insights regarding their aggressive behavior. At the same time, medulloblastoma is known to exhibit extensive rewiring of translational control in MYC-driven tumors, consistent with recent genetic evidence that the impact of this transcription factor on control of mRNA translation may be the most critical aspect of its function during tumorigenesis. Therefore, to propose previously unknown mechanisms for this disease, we have investigated the functional impact of translation of non-canonical open reading frames (ORFs) across medulloblastoma model systems. We demonstrate that these ORFs are commonly translated in medulloblastoma model systems and patient tumors, correlating with disease subtype. Using genome-wide CRISPR/Cas9 screens, we found that ORFs are frequently essential for cell survival in medulloblastoma and describe widespread reliance on upstream open reading frames (uORFs) in particular. From these, we identify a uORF in the ASNSD1 gene that is selectively upregulated and required for maintenance of cell survival by coordinating the function of the prefoldin-like complex, a poorly understood complex implicated in post-translational control. Together, our findings provide a blueprint for oncogenic uORFs as critical disease mediators both in medulloblastoma and, by extension, human cancers more broadly.

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.