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

Our results thus far suggest that MAPK signaling regulates FLC pathogenesis (Fig. 3G, 5C,D) . To confirm that MAPK signaling is overactive in FLC, we measured the levels of phosphorylated MEK and ERK in WT and Dnajb1-Prkaca-expressing AML12 cells (Fig. 7A,B) . As expected, we observed dramatically increased phospho-MEK and phospho-ERK in cells expressing the fusion compared to WT cells. Treatment of AML12 cells expressing Dnajb1-Prkaca with the MEK inhibitor cobimetinib resulted in a dose-responsive decrease in cell viability (Fig. 7C-F) . Both SLC16A14 and CA12 (Car12), which we identified as prominent FLC-enhancer-hotspot associated genes, have been implicated in drug resistance in other cancers (Doyen et al., 2013; Januchowski et al., 2014; Kopecka et al., 2016) , and CA12 has been reported previously as a mediator of the effects of the MAPK pathway (Hsieh et al., 2010) . Knockdown of Slc16a14 by siRNA dramatically reduced viability of AML12 cells expressing Dnajb1-Prkaca, and also increased the potency of cobimetinib (Fig. 7C,D) . Knockdown of Car12 did not have much of an effect on its own, but in combination with cobimetinib did significantly reduce cell viability compared to cobimetinib alone (Fig. 7E,F, Fig. S6A ).