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A key question in genome research is whether biologically active proteins are restricted to the ∼20,000 canonical, well-annotated genes, or rather extend to the many non-canonical open reading frames (ORFs) predicted by genomic analyses. To address this, we experimentally interrogated 553 ORFs nominated in ribosome profiling datasets. Of these 553 ORFs, 57 (10%) induced a viability defect when the endogenous ORF was knocked out using CRISPR/Cas9 in 8 human cancer cell lines, 257 (46%) showed evidence of protein translation when ectopically expressed in HEK293T cells, and 401 (73%) induced gene expression changes measured by transcriptional profiling following ectopic expression across 4 cell types. CRISPR tiling and start codon mutagenesis indicated that the biological effects of these non-canonical ORFs required their translation as opposed to RNA-mediated effects. We selected one of these ORFs,G029442--renamedGREP1(Glycine-Rich Extracellular Protein-1)--for further characterization. We found thatGREP1encodes a secreted protein highly expressed in breast cancer, and its knock-out in 263 cancer cell lines showed preferential essentiality in breast cancer derived lines. Analysis of the secretome of GREP1-expressing cells showed increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth inhibitory effect ofGREP1knock-out. Taken together, these experiments suggest that the non-canonical ORFeome is surprisingly rich in biologically active proteins and potential cancer therapeutic targets deserving of further study.

Anti-cancer uses of non-oncology drugs have been found on occasion, but such discoveries have been serendipitous and rare. We sought to create a public resource containing the growth inhibitory activity of 4,518 drugs tested across 578 human cancer cell lines. To accomplish this, we used PRISM, which involves drug treatment of molecularly barcoded cell lines in pools. Relative barcode abundance following treatment thus reflects cell line viability. We found that an unexpectedly large number of non-oncology drugs selectively inhibited subsets of cancer cell lines. Moreover, the killing activity of the majority of these drugs was predictable based on the molecular features of the cell lines. Follow-up of several of these compounds revealed novel mechanisms. For example, compounds that kill by inducing PDE3A-SLFN12 complex formation; vanadium-containing compounds whose killing is dependent on the sulfate transporter SLC26A2; the alcohol dependence drug disulfiram, which kills cells with low expression of metallothioneins; and the anti-inflammatory drug tepoxalin, whose killing is dependent on high expression of the multi-drug resistance gene ABCB1. These results illustrate the potential of the PRISM drug repurposing resource as a starting point for new oncology therapeutic development. The resource is available at https://depmap.org.

The brain is the foremost non-gonadal tissue for expression of non-coding RNAs of unclear function. Yet, whether such transcripts are truly non-coding or rather the source of non-canonical protein translation is unknown. Here, we used functional genomic screens to establish the cellular bioactivity of non-canonical proteins located in putative non-coding RNAs or untranslated regions of protein-coding genes. We experimentally interrogated 553 open reading frames (ORFs) identified by ribosome profiling for three major phenotypes: 257 (46%) demonstrated protein translation when ectopically expressed in HEK293T cells, 401 (73%) induced gene expression changes following ectopic expression across 4 cancer cell types, and 57 (10%) induced a viability defect when the endogenous ORF was knocked out using CRISPR/Cas9 in 8 human cancer cell lines. CRISPR tiling and start codon mutagenesis indicated that the biological impact of these non-canonical ORFs required their translation as opposed to RNA-mediated effects. We functionally characterized one of these ORFs, G029442—renamed GREP1 (Glycine-Rich Extracellular Protein-1)—as a cancer-implicated gene with high expression in multiple cancer types, such as gliomas. GREP1 knockout in >200 cancer cell lines reduced cell viability in multiple cancer types, including glioblastoma, in a cell-autonomous manner and produced cell cycle arrest via single-cell RNA sequencing. Analysis of the secretome of GREP1-expressing cells showed increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth inhibitory effect of GREP1 knock-out. Taken together, these experiments suggest that the non-canonical ORFeome is surprisingly rich in biologically active proteins and potential cancer therapeutic targets deserving of further study.