A multi-omic dissection of super-enhancer driven oncogenic gene expression programs in ovarian cancer [CRISPRI_SCREEN]

Ovarian cancer is one of the deadliest cancers among women worldwide and is the leading cause of gynecologic-related cancer deaths in the U.S. Enhancers are often repurposed by the tumor cells for the regulation of genes that allow tumor cells to become more aggressive and resistant to therapy. Recent evidence suggests that exploiting transcriptional dependence by targeting oncogenic super-enhancers may be a viable therapeutic avenue. To this end, we leveraged genomic data such as H3K27ac, BRD4, and copy number information to identify putatively oncogenic super-enhancers. We found that copy number amplification of these super-enhancers is predictive of clinical outcome and that the targets genes associated to the copy number events via chromatin eQTL analysis are involved in numerous oncogenic processes. To systematically probe the functions these super-enhancers we designed a CRISPR interference screen (dCas9-KRAB) so specifically inhibit each super-enhancer and measure the consequences on gene expression via RNA-seq. These results show pervasive gene expression changes that underlie the biology of ovarian cancer. Finally, we select two salient super-enhancers for further analysis. CRISPR-based deletion of these two super-enhancers results in in dramatic changes in gene expression and decreased cell proliferation of ovarian cancer cells. Taken together these analyses highlight the importance of BRD4-bound and copy number amplified super-enhancers in ovarian cancer oncogenic regulation. Overall design: For this experiment we used dCas9-KRAB expressing ovarian cancer cells (OVCAR3) to systematically inhibit 86 different super-enhancers and 10 controls in a 96-well plate format. We designed two guide RNAs for each super-enahncer and targeted one different super-enhancer per well of the 96-well plate. After 72hrs after guideRNA transfection and epigentic silencing by dCas9-KRAB, RNA was extracted from each well and the changes in gene expression were measured using the QuantSeq 3' mRNA-Seq Library Prep Kit FWD from Lexogen company. The super-enhancer locations, gRNA locations, and gRNA sequences are located in one of the supplimentary tables from the associated manuscript.