The Switch/Sucrose Non-Fermentable (SWI/SNF) DNA remodelling complexes regulate gene expression and aberrant SWI/SNF function is associated with epigenetic dysregulation underlying cancer. Recurrent mutations in genes encoding SWI/SNF subunits have been described in >20% of all human malignancies. Lung cancer is the fifth most common cancer diagnosed annually and the highest cause of cancer related death. Lung adenocarcinoma (LUAD) makes up to 40% of all lung cancer patients and has a 5-year survival of ~20%, due to the poor efficiency of the current treatment options, including chemotherapy, radiotherapy and immunotherapy. This highlights the urgent need of more targeted therapies. SMARCA4 encodes the core ATPase subunit of the SWI/SNF complex and is mutated in ~8% of LUAD patients. Additionally, a total 37% of LUAD patients exhibit a mutation in one of the 31 SWI/SNF genes, and these are largely mutually exclusive from each other.
To understand the mechanism of SWI/SNF-mediated epigenetic dysregulation in LUAD, we performed an in vitro CRISPR knockout screen using an epigenetic-focused gRNA library in SMARCA4 wildtype (NCI-H358) and SMARCA4-null (A549) LUAD cell lines, as well as a A549 isogenic cell line in which SMARCA4 was reconstituted (SMARCA4 overexpression). We transduced Cas9-expressing LUAD lines with the EpicK library (1,252 genes, 9-11 gRNAs per gene, total of 11,227gRNAs) at an MOI of 0.3 and a minimum 1000x representation. Cells were collected after 28 days of culture, under 4ug/mL of puromycin selection, for library preparation and next-generation sequencing. Comparisons of gRNA abundance between SMARCA4 wildtype, SMARCA4-null, and SMARCA4 overexpression LUAD cell lines were used to reveal synthetic lethality with SMARCA4-deficiency.
We revealed 18 genetic dependencies in the absence of SMARCA4. SMARCA2, a SMARCA4 paralog and a known synthetic lethal relationship with SMARCA4, was the top dependency, demonstrating high confidence in the screen. Currently, we are further validating the other 17 targets. Collectively, this data will provide insight into the mechanism of action of the SWI/SNF complexes and elucidate possible targets for treatment.