Abstract 5224: Identification of drug-target interactions through rapid selection of drug resistant protein variants generated by CRISPR/Cas9-induced NHEJ

Abstract

Abstract Identification and validation of the cellular target of bioactive hits identified in a phenotypic screen is crucial for their further development into a drug. The gold standard proof for identification of a small molecule's target is the discovery of mutations that confer resistance. However, selection of drug resistance and subsequent deconvolution of the relevant mutations that confer drug resistance remains time consuming. Therefore, a methodology that can accelerate the drug resistance selection process and that can simplify identification of relevant drug resistance mutations would benefit the drug discovery and development process greatly. Here we report a simple method to rapidly identify the mechanism of action of small molecules with anti-cancer activity. The method utilizes the positive selection of in-frame drug resistance mutations in the target protein of a small molecule derived from the localized genetic variation created by non-homologous end joining (NHEJ) repair of CRISPR/Cas9-induced double strand breaks. In brief, we introduced double strand breaks in the target proteins of selinexor, ispinesib and triptolide and treated these cells with the respective drug. Resistance was obtained rapidly and sequencing of resistant colonies revealed known as well as many new drug resistant protein variants. Next, we investigated whether this approach can be applied as a screening strategy on a subset of genes to identify the direct cellular target protein of a small molecule. We therefore, as proof of concept, designed a pooled sgRNA tiling library targeting all PAM sites of multiple genes and applied this to ispinesib. Drug resistant colonies formed rapidly and were enriched with sgRNAs targeting the ispinesib binding site in KIF11, ispinesib’s target protein. Further sequencing of the sgRNA target gene locus revealed drug resistance mutations in KIF11, validating the feasibility of the methodology to uncover the target of a small molecule from a small pool of candidate genes. To conclude, we provide a new method for identification of the cellular target protein and binding site of a small molecule based on positive selection of drug resistant protein variants generated by targeted CRISPR/Cas-induced NHEJ repair. Citation Format: Jasper E. Neggers, Bert Kwanten, Tim Dierckx, Dirk Daelemans. Identification of drug-target interactions through rapid selection of drug resistant protein variants generated by CRISPR/Cas9-induced NHEJ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5224. doi:10.1158/1538-7445.AM2017-5224