A pooled CRISPR/Cas9 screen to identify genes synthetically lethal with epigenetic drugs in non-small cell lung cancer cells

Lung cancer is the most common cause of cancer-related death worldwide, resulting in 1.3 million deaths per year. Most lung tumors are non-small cell lung cancers (NSCLC), and approximately 11 % of NSCLC harbor a mutation in the oncogenic KRAS gene. The clinical success of epigenetic drugs has been minimal in NSCLC, with no exception for the KRAS mutant NSCLC subtype. This project aimed to identify genes that act synthetically lethal with epigenetic drugs to better understand the resistance mechanisms to these compounds in NSCLC cells, focusing on KRAS mutant cells. Target genes were identified using a pooled CRISPR/Cas9 screening approach, which was established in this project. Four genes synthetically lethal with the histone methyltransferase inhibitor DZNep were identified in KRAS mutant H2030 cells, and two genes were identified as synthetically lethal with the histone deacetylase inhibitor Entinostat in the same cell line. The epigenetic regulators CHD8 and EP300 were selected among the target genes for further characterization. In silico analysis of CHD8 and EP300 in the TCGA NSCLC dataset revealed a strong positive correlation of the expression of both genes and indicated functional overlaps. The synthetically lethal effect of CHD8 or EP300 depletion with DZNep treatment was validated in the KRAS-mutant cell line H1944 but lacking in EGFR-mutant H1975 cells. Flow cytometry analysis confirmed elevated apoptosis in H2030, but not H1975 cells, after DZNep treatment in conjunction with CHD8 or EP300 knockdown. Gene expression profiling and functional annotation of deregulated genes after DZNep treatment in combination with CHD8 or EP300 knockdown verified a high number of affected cell-cycle and cell viability-related genes. The cytokine CCL20 was identified as upregulated by DZNep but downregulated upon knockdown of CHD8 or EP300, suggesting a role in conferring CHD8 or EP300-mediated resistance to DZNep treatment in H2030 cells. Proof of concept experiments showed that CHD8 is critical for the upregulation of CCL20 upon DZNep treatment in mutant H2030 cells and that a knockdown of CCL20 mimics the synthetically lethal effect of CHD8 depletion with DZNep treatment