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

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: In this study, two super-enhancers were identified as being oncogenic in ovarian cancer. The genomic coordinates for super-enhancer 60 (SE60) are chr20:52352051-52368920 using the Hg19 human genome reference. The genomic coordinates for super-enhancer 14 (SE14) are chr1:16493671-16516686 using the Hg19 human genome reference. Each super enhancer was deleted using CRISPR-Cas9 in the ovarian cancer cell line OVCAR3. The guides RNAs used for deletion and the genotyping results are described in the associated publication. For each super enhancer, two independent knockout clones were generated (clone 1 and clone 2). Each clone represents a biological replicate. RNA was purified from asynchronous cells for each clone. Library prep was done using the Illumina TruSeq mRNA Library Kit following the manufacturer's protocol. RNA was sequenced at 150 cycles, paired end, on an Illumina NextSeq. The super-enhancer locations, gRNA locations, and gRNA sequences are located in one of the supplimentary tables from the associated manuscript.