Genome-wide CRISPR/Cas9 screen to uncover effectors of PRC2 inhibition

The methyltransferase Polycomb Repressive Complex 2 (PRC2), composed of EZH2, SUZ12, and EED subunits, is associated with transcriptional repression via tri-methylation of histone H3 on lysine 27 residue (H3K27me3). PRC2 is a validated drug target, as the EZH2 gain-of-function mutations identified in patient samples drive tumorigenesis. PRC2 inhibitors have been discovered and demonstrated anti-cancer efficacy in clinic. However, their pharmacological mechanisms are poorly understood. MAK683 is a potent EED inhibitor in clinical development. The overall goal of our study is to understand the molecular events leading to tumor regression after PRC2 inhibition. Our study revealed that BMP-ACVR1 signaling pathway as a critical component for the anti-lymphoma efficacy of PRC2 inhibitor. Overall design: To systematically identify the critical functional effectors that contribute to the anti-lymphoma activity of PRC2 inhibitors, we performed a proliferation-based genome-wide CRISPR/Cas9 knockout screen in the sensitive lymphoma cell WSU-DLCL2.We constructed Cas9 stable-expression cell clone and transduced it with the human genome-scale CRISPR knockout (GeCKO v2) lentiviral pooled libraries at a low multiplicity of infection (MOI = 0.2) to ensure transduction with only one sgRNA per cell. Then, WSU-DLCL2 cells were cultured in the presence of puromycin for 7 days followed by DMSO or MAK683 (200 nM) treatment for 14 more days. The survival cells were sequenced for the distribution of sgRNAs at days 0 and 14 after drug treatment.