Superenhancers are master regulators of genes involved in cellular differentiation and tumorigenesis. While it is now well established that typical enhancers are key mediators of alleles associated with a mild risk of polygenic diseases, including cancer, the role of superenhancers is less understood. We evaluated the role of superenhancers in mediating risk of epithelial ovarian cancer (EOC). Using FunciVar software and superenhancers defined using H3K27ac chromatin immunoprecipitation followed by next generation sequencing, performed in cell lines and tissues, we characterized the superenhancer-risk SNP intersect and identified a series of risk-associated loci where multiple candidate causal alleles overlap with ovarian superenhancers. In particular, locus 3q25, the locus most associated with epithelial ovarian cancer (EOC) risk from GWAS studies, has >70 candidate causal risk SNPs, all of which coincide with superenhancers detected in ovarian cancer precursor cells (ovarian and fallopian tube epithelia) but not in ovarian cancers. We are proposing that the presence of one or more SNPs occurring at this locus leads to a higher expression of one or more genes by modulating activation of the superenhancer region. We tested the effect of decreasing the binding affinity of BRD4, a protein that binds superenhancer regions, using a small molecule inhibitor on the expression of genes in this locus. We identified three candidate genes—LEKR1, SSR3, and TiPARP—that demonstrated a decrease expression level as a result of BRD4 binding inhibition.
Preliminary results show that partial knockdown of either SSR3 or TiPARP decreases normal ovarian and fallopian cell proliferation, and knocking down both SSR3 and TiPARP simultaneously shows an enhanced proliferation decrease compared with knockdown of a single gene. We then created stable knockout (KO) models using the CRISPR-Cas9 system to fully KO one, two or all three of our candidate causal genes: LEKR1, SSR3, and TiPARP. Functional evaluation of these KO models is currently underway to test the hypothesis that risk SNPs may regulate more than one gene at this locus. In all we identify a potential role for superenhancers at a subset of EOC risk loci, and present a functional pipeline for identification of the target gene/genes and evaluation of their role in neoplastic transformation.
Citation Format: Kevin C. Vavra, Simon Coetzee, Janet M. Lee, Paul Pharoah, Dennis J. Hazelett, Kate Lawrenson, Simon A. Gayther. Functional evaluation of superenhancers as mediators of epithelial ovarian cancer risk [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5502. doi:10.1158/1538-7445.AM2017-5502