Hotspots of aberrant enhancer activity in fibrolamellar carcinoma reveal molecular mechanisms of oncogenesis and intrinsic drug resistance

Timothy A. Dinh; Ramja Sritharan; F. Donelson Smith; Adam B. Francisco; Rosanna K. Ma; Rodica P. Bunaciu; Matt Kanke; Charles G. Danko; Andrew P. Massa; John D. Scott; Praveen Sethupathy

We have computationally identified gene-enhancer interactions in this study. However, future experiments will be important to confirm these interactions experimentally and carefully dissect the 3-D regulatory interactions in FLC. For example, methods based on chromosome conformation capture, such as Hi-C, can identify and confirm global chromosomal interactions, while methods based on luciferase assays, such as self-transcribing active regulatory region sequencing (STARR-seq) or other massively parallel reporter assays, can validate the regulatory activity of identified enhancers. More refined dissection of individual or subsets of enhancers using CRISPR/Cas9 genome editing will confirm functional gene-enhancer links and identify the specific nucleotides within enhancers that are essential for such regulation. Furthermore, our ChRO-seq experiments were conducted with primary tumors, which contain a heterogenous mixture of cells. Our results therefore represent the aggregate results across all cell types present. Going forward, single cell experiments will be necessary to dissect the role of transcriptional regulatory networks in tumor, immune, parenchymal, and other cell types within primary tumors.