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 hypothesized that inhibition of CA12 in FLC cells may mitigate the intrinsic drug resistance because of the results in the AML12 cells expressing Dnajb1-Prkaca (Fig. 7E) and because an effective CA12 inhibitor, SLC-0111, is currently in clinical trials as combination therapy for metastatic pancreatic cancer. We found that pharmacological suppression of CA12 with SLC-0111 in FLC cells significantly enhances the potency of cobimetinib at the 500 nM dose (Fig. 7H, Fig. S6B ). Again, this effect was confirmed across three distinct derivations of the FLC cell line. We quantified the interaction between cobimetinib and SLC-0111 using the combination index (CI). SLC-0111 and cobimetinib combination treatment resulted in a synergistic response (CI = 0.00686 (0.5 µM cobimetinib + 100 µM SLC-0111) and 0.000791 (0.5 µM cobimetinib + 200 µM SLC-0111) in FLC2, where CI < 1 indicates synergy). To determine whether CA12 interacts with the MAPK signaling pathway, we inhibited either MEK or CA12. Cobimetinib treatment resulted in decreased ERK phosphorylation as expected, but had no effect on CA12 expression (Fig. 7G) . However, treatment with SLC-0111 reduced phosphorylated MEK and ERK levels (Fig. 7J) , suggesting that CA12 may function, at least in part, upstream of the MAPK signaling pathway. Taken together, our results suggest that the MAPK signaling pathway is dysregulated in FLC and inhibition of this pathway in combination with pharmacological suppression of CA12 or inhibition of SLC16A14 represent exciting candidate molecular therapeutic approaches.