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

SLC16A14, CA12, LINC00473, RPS6KA2, and VCAN are responsive to DNAJB1-PRKACA While the genes we selected are over-transcribed (Fig. 6B ) and overexpressed ( Fig. 6C ) in FLC relative to NML, it is unclear whether this dysregulation is directly caused by the DNAJB1-PRKACA fusion. To determine whether the DNAJB1-PRKACA fusion is sufficient to perturb these genes of interest, we took advantage of two murine models of FLC. In the first model, a transposon expressing human DNAJB1-PRKACA is introduced into the livers of C57BL/6 mice by hydrodynamic tail vein injection, forming FLC-like liver tumors (Kastenhuber et al., 2017) . We examined the expression of our genes of interest in the resulting liver tumors compared to livers from mice injected with an empty vector control. Car12 (the mouse homolog of human CA12) and Slc16a14 displayed significantly higher expression of samples expressing DNAJB1-PRKACA compared to control (Fig. 6D ). The second model is the AML12 cell line that has undergone CRISPR/Cas9 gene editing, resulting in a heterozygous deletion analogous to the endogenous event in humans, leading to the formation of the Dnajb1-Prkaca fusion (Dinh et al., 2019; Turnham et al., 2019) . When we examined the expression of the genes of interest in AML12 cells expressing Dnajb1-Prkaca, we found that Car12 and Slc16a14, as well as Rps6ka2 and Vcan, are significantly elevated compared to wild-type (WT) controls (Fig. 6E) .