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

卵巢癌是全球女性致死率最高的恶性肿瘤之一，亦是美国妇科恶性肿瘤相关死亡的首要病因。肿瘤细胞常会重利用增强子序列，以调控促进肿瘤侵袭性增强及治疗抵抗相关基因的表达。近期研究证据表明，靶向致癌性超级增强子（super-enhancer）以利用其转录依赖性，或是一条可行的治疗途径。为此，我们依托组蛋白H3赖氨酸27乙酰化（H3K27ac）、溴结构域蛋白4（BRD4）及拷贝数信息等基因组数据，鉴定出疑似致癌性的超级增强子。我们发现，这类超级增强子的拷贝数扩增可预测患者临床结局，且通过染色质表达数量性状基因座（chromatin eQTL）分析与该拷贝数事件关联的靶基因，参与了众多致癌生物学过程。
为系统探究这类超级增强子的功能，我们设计了CRISPR干扰筛选（CRISPR interference screen）实验（采用失活型Cas9结合KRAB结构域（dCas9-KRAB）系统），以特异性抑制每个超级增强子，并通过RNA测序（RNA-seq）检测基因表达的变化。结果显示，广泛的基因表达变化构成了卵巢癌生物学特性的基础。最后，我们选取两个关键的超级增强子开展后续分析。基于CRISPR技术的这两个超级增强子敲除，可引发显著的基因表达变化，并降低卵巢癌细胞的增殖能力。综上，本系列分析凸显了结合BRD4且发生拷贝数扩增的超级增强子，在卵巢癌致癌调控中的重要作用。
实验整体设计：本实验采用表达dCas9-KRAB的卵巢癌细胞系OVCAR3，在96孔板体系中系统性抑制86个不同的超级增强子及10个阴性对照。我们为每个超级增强子设计两条向导RNA（gRNA），并在96孔板的每一个孔中靶向一个独立的超级增强子。在向导RNA转染及dCas9-KRAB介导的表观遗传沉默72小时后，从每孔中提取总RNA，并使用Lexogen公司出品的QuantSeq 3'端mRNA测序文库制备正向试剂盒（QuantSeq 3' mRNA-Seq Library Prep Kit FWD）检测基因表达变化。超级增强子位点、向导RNA位点及向导RNA序列可参见相关论文的其中一份补充表格。