Genome-wide CRISPR-mediated Gene Disruption Presents a Shortcut to Acquired Resistance that Reveals Small Molecule Mechanism of Action

Recorded On: 02/06/2018

Phenotypic screening in small molecule drug discovery presents the opportunity to discover novel therapies, but thorough identification of a small molecule target remains an obstacle. To address this challenge we applied whole-genome pooled CRISPR screening as a Shortcut To Acquired Resistance in Search of mechanism (STAR-Search). This strategy uses CRISPR to generate a population where individual cells each possess a distinct targeted mutation. This comprehensive pool of mutations is then subjected to positive selection, which enriches cells that acquire resistance to compound treatment. The resistance is caused by targeted mutations that are readily identified by sequencing the stably integrated targeting construct. We hypothesize that the identity of gene disruptions underlying resistance can reveal mechanism of action or factors proximal to the direct target. Our group has successfully applied STAR-Search to multiple phenotypic screening hits, thus demonstrating its strong potential as a tool in target identification/validation. Our application of STAR-Search examined three small molecules that each elicits cytotoxic effects against a unique spectrum of cancer lines. CGS-18, which preferentially induces apoptosis in breast cancer lines, was dosed onto MDA-MB-468 cells stably transduced with the Brunello CRISPR gRNA library. Cells that survived CGS-18 selection showed enrichment of gRNAs targeting a single gene SULT1A1. MDA-MB-468 cells also undergo apoptosis in response to CGS-59 treatment, so this positive selection was performed in parallel with the previous screen. In this selected population, gRNAs targeting MGST1 were the most highly enriched. Validation experiments have confirmed that individual disruption of SULT1A1 or MGST1 confers resistance to CGS-18 or CGS-59, respectively. The third small molecule, CGS-85, displayed selective killing of multiple myeloma cell lines. This compound was profiled in the BioMap Diversity+ Panel where its phenotypic effects showed strong correlation to the reference database profile generated by the oxidative phosphorylation inhibitor oligomycin. LP-1 cells transduced with the CRISPR library that survived either CGS-85 or oligomycin selection showed enrichment of gRNAs targeting a large number of genes, but this group converged on a common mechanism: mitochondrial oxidative phosphorylation. Despite substantial overlap between the majority of screening hits, prominent differences suggested distinct direct molecular targets. Subsequent enzyme assays showed CGS-85 potently inhibits isolated mitochondrial complex I, whereas oligomycin confirmed as an inhibitor of complex V. Together these examples illustrate the potential of STAR-Search to reveal small molecule mechanisms of action and specifically uncover novel biological connections due to the comprehensive and systematic nature of the genome-wide CRISPR targeted disruptions.

Jon Oyer

Abbvie

Jon Oyer works within the Target Identification & Validation group of the Genomic Research Center at AbbVie. This group specializes in applying a diverse stack of technologies and methods to the challenge of small molecule characterization. A partial list of these collaborative efforts include transcriptome analysis, proteomics, phage display, cellular thermal shift assays, and functional genomic approaches. Jon received his undergraduate degree from University of Washington before completing his graduate studies in Molecular & Medical Genetics at Oregon Health & Science University. Prior to joining AbbVie, Jon also conducted postdoctoral research studying epigenetic regulation in embryonic stem cells with Gail Mandel at Oregon Health & Science University and epigenetic disruptions that drive hematological malignancies with Jonathan Licht at Northwestern University.

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