Abstract C022: Large scale phenotypic screening using PRISM, integrated with functional genomic screening and transcriptional profiling accelerates target identification of cytotoxic small molecules
Authors: Larpenteur Kevin , Waal Lucian de , Wang Li , Kocak Mustafa , Bryan Jordan , Bender Samantha , Barkho Sulyman , Kukurugya Matt , Goodale Amy , Paolella Brenton , Cho Min , McFarland James , Rothberg Michael , Zhu Cong , Trepiccio Colin , Tsherniak Aviad , Root David , Bennett Bryson , Piccioni Federica , Bittker Josh , Rong James , Mader Christopher , Stokoe David , Firestone Ari , Golub Todd R , Roth Jennifer 
Published: March 2020

Abstract
A comprehensive analysis of FDA approved drugs has shown that drug discovery through phenotypic screening yields a higher percentage of first-in-class compounds, compared to canonical target-based drug discovery. This has led to a renewed interest in phenotypic screening to identify novel mechanisms of action. The PRISM technology of adding a unique DNA barcode to cancer cell lines has been able to reduce the cost and time associated with phenotypic viability screening. Using the PRISM method, we profiled the cytotoxic activity of over 4,100 bioactive compounds in 578 barcoded cancer cell lines at one dose, followed by 8-point dose of 1100 active compounds. The use of genomically characterized cell lines from the Cancer Cell Line Encyclopedia (CCLE) allows us to identify baseline features associated with sensitivity to a particular compound. One of the compounds from the screen that we selected for follow up was BRD-K01353379, which killed 28 cancer cell lines of diverse lineages and showed a correlation between high expression of Ethanolamine Kinase 1 (ETNK1) and reduction of cell viability. Interestingly, BRD-3379 showed a similar PRISM cytotoxic profile to 2,3-DCPE, that was screened in our Repurposing effort. 2,3-DCPE is structurally similar to BRD-3379 and has been reported to induce cytotoxicity through upregulation of p21 and downregulation of Bcl-XL. To elucidate the mechanism of action of this compound we performed a genome-wide CRISPR-Cas9 resistance screen to identify molecular targets that are required for sensitivity to BRD-3379. Here, we found that loss of expression of the PRISM biomarker ETNK1 conferred resistance in a BRD-3379 sensitive cell line, JHH-7. Subsequently, we profiled metabolites in a cell line sensitive to BRD-3379 and found that the compound was phosphorylated. We confirmed in a biochemical assay that BRD-3379 is in fact a substrate of recombinant ETNK1, which phosphorylates the ethanolamine moiety of BRD-3379. Next, we transcriptionally profiled the effect of BRD-3379 by bulk RNAseq in a sensitive and non-sensitive cell line, as well as in a pool of 100 cell lines using single cell RNAseq [McFarland et al., manuscript in preparation]. Both bulk RNAseq and single cell transcriptional analysis showed that upregulation of both p21 and p57 is strongly associated with sensitivity to BRD-3379. This mechanism of action is similar to that of 2,3-DCPE. Here, we have demonstrated that by combining large-scale cell line viability screening, genomic characterization of these cell lines, genome wide CRISPR-Cas9 resistance screens, transcriptional profiling, and biochemical assays, we can facilitate rapid identification of molecular targets of small molecules and their respective mechanism of action by which they induce cytotoxicity.
Citation Format: Kevin Larpenteur, Lucian de Waal, Li Wang, Mustafa Kocak, Jordan Bryan, Samantha Bender, Sulyman Barkho, Matt Kukurugya, Amy Goodale, Brenton Paolella, Min Cho, James McFarland, Michael Rothberg, Cong Zhu, Colin Trepiccio, Aviad Tsherniak, David Root, Bryson Bennett, Federica Piccioni, Josh Bittker, James Rong, Christopher Mader, David Stokoe, Ari Firestone, Todd R Golub, Jennifer Roth. Large scale phenotypic screening using PRISM, integrated with functional genomic screening and transcriptional profiling accelerates target identification of cytotoxic small molecules [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C022. doi:10.1158/1535-7163.TARG-19-C022