Target identification is a critical step in drug discovery, but the process has many challenges including non-specific reagents, limited ability to test numerous models, and incomplete target inhibition. Pooled screening with CRISPR/Cas9 permits the quick and accurate examination of proliferation effects across many genes and many cell lines. To determine the specific dependencies of cell lines on epigenetic pathways, we designed a CRISPR/Cas9 library to target 640 epigenetic genes and screened more than 200 cell lines covering a variety of oncology indications, including breast, lung, and renal cell carcinoma (RCC). We find that CRISPR pooled screening is a highly effective approach for target identification and provides robust, highly reproducible data as long as a sufficient number of small guide RNAs are used. We identify known pan-essential genes, including in the transcription (CDK9), translation (EIF4A1 and EIF4A3) and splicing (SRSF2) machinery. We additionally identify many novel pan-essential genes across a variety of epigenetic pathways, including histone acetylases and deacetylases, chromatin remodeling factors, helicases and others. We also investigated epigenetic synthetic lethal interactions that have been previously reported. For example, it has been reported that the SWI/SNF family displays paralog synthetic lethality for SMARCA2 in the context of SMARCA4 mutations, and for ARID1B in the context of ARID1A mutations. While we do see that some of the same trends hold, the synthetic lethal relationship appears to be more complex than previously realized, including the need to examine mRNA levels in addition to mutation type. Most importantly, we identify more than 100 epigenetic genes which show selective sensitivity, i.e. where knockout shows an anti-proliferative effect in only a subset of the cell lines. These are the most promising targets for further drug discovery programs. We have used additionally CRISPR/Cas9-domain based screening to identify the functionally relevant sites for many of these genes. Furthermore, we can overlay gene expression and mutation data to identify novel synthetic lethal relationships. One gene that displays selective sensitivity is EGLN1, the prolyl hydroxylase for the hypoxia-inducible factor, HIF1α. We find that EGLN1 is required for proliferation only in RCC cell lines which retain wild-type VHL, another component of the hypoxia response pathway, which is frequently lost in RCC. As such, EGLN1 loss is synthetically lethal in the presence of wild-type VHL in RCC cells. Thus this approach not only identifies an enzymatic drug target but also a patient stratification method. Other novel synthetic lethal interactions have also been identified. Our data demonstrates that CRISPR pooled screening is a powerful technique for identification of epigenetic synthetic lethal interactions.

Citation Format: Alexandra R. Grassian, Darren Harvey, Julian Fowler, Allison E. Drew, Igor Feldman, Richard Chesworth, Robert Copeland, Jesse J. Smith, Scott Ribich. CRISPR pooled screening of hundreds of cancer cell lines identifies differential dependencies on epigenetic pathways and synthetic lethal relationships [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 406. doi:10.1158/1538-7445.AM2017-406