CRISPR-Enabled Profiling of Enhancer RNA-Producing Super-Enhancers in Triple-Negative Breast Cancer Identifies Drivers of Tumor Progression [Hi-C]
Triple-negative breast cancer (TNBC) is the most therapeutically recalcitrant form of breast cancer. Pioneering genomic studies have identified the repertoire of expressed genes in the distinct molecular subtypes of breast cancer and have been instrumental in providing diagnostic, prognostic, and therapeutic guidance. Yet breast cancer remains the second leading cause of cancer mortality in women. This is due, in part, to the paucity of targeted therapies for triple-negative breast cancer (TNBC). We posit that a systematic analysis of regulatory elements that extend beyond protein coding genes will lead to novel avenues for therapeutic intervention. To this end, we analyzed the regulatory mechanisms of TNBC-specific transcriptional enhancers together with their non-coding enhancer RNA (eRNA) transcripts. We systematically probed the functions of the top 30 eRNA-producing super-enhancers using a high-throughput CRISPR-interference (dCas9-KRAB) assay coupled to RNA-seq that enabled unbiased detection of target genes genome-wide. Utilizing Hi-C, we constructed 3D chromatin interaction maps to annotate direct target genes for each super-enhancer and highlight their proclivity for genes that portend worse clinical outcomes in TNBC patients.  To illustrate the utility of this dataset, we selected a novel super-enhancer controlling the nearby PODXL gene and demonstrate that deletion of the super-enhancer (via Cas9 deletion) or specific degradation of its enhancer RNAs (via Cas13 RNA cleavage) leads to profound effects on target gene expression, cell proliferation, and migration. Furthermore, complete knockout of the PODXL super-enhancer leads to a significant decrease in tumor growth and metastatic potential in mouse xenograft experiments. Collectively, this work examines several fundamental questions about how regulatory information encoded into eRNA-producing super-enhancers drives gene expression networks that underlie the biology of triple-negative breast cancer. Overall design: We identified several super-enhancers and their target genes through a CRISPR interference-based screen linked with RNA-seq. Using these results, we identified direct contact relationships and confirmed this using a Hi-C seq experiment to confirm physical interaction of these candidates in wildtype cells.