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Anti-cancer uses of non-oncology drugs have occasionally been found, but such discoveries have been serendipitous. We sought to create a public resource containing the growth inhibitory activity of 4,518 drugs tested across 578 human cancer cell lines. We used PRISM, a molecular barcoding method, to screen drugs against cell lines in pools. An unexpectedly large number of non-oncology drugs selectively inhibited subsets of cancer cell lines in a manner predictable from the cell lines' molecular features. Our findings include compounds that killed by inducing PDE3A-SLFN12 complex formation; vanadium-containing compounds whose killing depended on the sulfate transporter SLC26A2; the alcohol dependence drug disulfiram, which killed cells with low expression of metallothioneins; and the anti-inflammatory drug tepoxalin, which killed via the multi-drug resistance protein ABCB1. The PRISM drug repurposing resource (https://depmap.org/repurposing) is a starting point to develop new oncology therapeutics, and more rarely, for potential direct clinical translation.

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.

Drugs targeting adenosine receptors were originally developed for the treatment of Parkinson's disease and are now being tested in immuno-oncology clinical trials in combination with checkpoint inhibitors. We recently reported the killing activity of 4,518 drugs against 578 diverse cancer cell lines determined using the PRISM molecular barcoding approach. Surprisingly, three established adenosine receptor antagonists (CGS-15943, MRS-1220, and SCH-58261) showed potent and selective killing of FOXA1-high cancer cell lines without the need for immune cells. FOXA1 is a lineage-restricted transcription factor in luminal breast cancer, hepatocellular carcinoma, and prostate cancer without known small molecule inhibitors. We find that cytotoxic activity is limited to adenosine antagonists with a three-member aromatic core bound to a furan group, thus indicating a potential off-target mechanism of action. To identify genomic modulators of drug response, we performed genome-wide CRISPR/Cas9 knockout modifier screens. Killing by CGS-15943 and MRS-1220 was rescued by knockout of the aryl hydrocarbon receptor (AHR) and its nuclear partner ARNT. In confirmatory studies, knockout of AHR completely rescued killing by CGS-15943 in multiple cell types. Co-treatment with an AHR small molecule antagonist also rescued cell viability. Knockout of adenosine receptors did not alter drug response. Given that AHR is a known transcriptional regulator, we performed global mRNA sequencing to assess transcriptional changes induced by CGS-15943. The top two genes induced were the p450 enzymes CYP1A1 and CYP1B1. To determine sufficiency, we overexpressed CYP1A1 in a resistant cell line. Ectopic CYP1A1 expression sensitized to CGS-15943-mediated killing. Mass spectrometry revealed covalent trapping of a reactive metabolite by glutathione and potassium cyanide following in vitro incubation with liver microsomes. In addition, treatment of breast cancer cells with CGS-15943 for 24 hours resulted in increased γ-H2AX phosphorylation by western blot, indicative of DNA double stranded breaks. In summary, we identified off-target anti-cancer activity of multiple established adenosine receptor antagonists mediated by activation of AHR. Future studies will evaluate the functional contribution of FOXA1 and activity in vivo. Starting from a phenotypic screening hit, we leverage functional genomics to unlock the underlying mechanism of action. This project will pave the way for developing more effective therapies for biomarker-selected cancers, with potential to improve the care of patients with liver, breast, and prostate cancer. Citation Format: Steven M. Corsello, Ryan D. Spangler, Ranad Humeidi, Caitlin N. Harrington, Rohith T. Nagari, Ritu Singh, Vickie Wang, Mustafa Kocak, Jordan Rossen, Amael Madec, Nancy Dumont, Todd R. Golub. Adenosine receptor antagonists exhibit potent and selective off-target killing of FOXA1-high cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3400.