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

As LINC00473 is a primate-specific lncRNA (Reitmair et al., 2012) , we used an alternative non-murine model to study its regulation. Specifically, we stably over-expressed the fusion in the HepG2 human hepatoma cell line using a lentiviral system. DNAJB1-PRKACA expression dramatically increased LINC00473 expression compared to an enhanced green fluorescent protein (EGFP) control (Fig. 6F) . WT PRKACA also increased LINC00473 expression. However, the magnitude of LINC00473 induction was significantly larger with the fusion compared to WT PRKACA, indicating that something other than canonical PKA activity (possibly the DNAJB1 domain) is relevant for robust induction of LINC00473. Importantly, stable expression of a kinase-dead mutant of DNAJB1-PRKACA (K128H) did not increase LINC00473 expression (Fig. 6F) , indicating that the kinase activity of the fusion is necessary for induction of expression. To determine if LINC00473 might be responsive to PKA fusions in other contexts, we examined a cholangiocarcinoma dataset (Nakamura et al., 2015) that characterized tumors with fusions involving PRKACA or PRKACB with ATPase Na+/K+ transporting subunit beta 1 (ATP1B1). Interestingly, the exons of PRKACA retained in the ATP1B1-PRKACA fusion are the same as in DNAJB1-PRKACA. Using RNA-seq data generated for this dataset, we examined the relationship between PKA fusions and the expression of LINC00473. Tumors with PKA fusions demonstrated significantly higher expression of LINC00473 than tumors without PKA fusions (Fig. 6G) , indicating that LINC00473 is responsive to PKA activity in alternative fusion events. Our results suggest that DNAJB1-PRKACA is sufficient to perturb the expression of CA12, SLC16A14, VCAN, RPS6KA2, and LINC00473 in the specific disease models we used and that this regulation is dependent upon (at least for LINC00473) the kinase activity of DNAJB1-PRKACA. Top genes associated with FLCspecific enhancer hotspots (n=47) The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.01.18.911297 doi: bioRxiv preprint