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

WT AML12 cells and AML12 cells expressing the DNAJB1-PRKACA fusion have been previously described (Dinh et al., 2019; . They were cultured in DMEM/F12 supplemented with 10% FBS, 0.04 µg/mL dexamethasone, 0.1% gentamicin, 1 µg/mL recombinant human insulin, 0.55 µg/mL human transferrin, and 0.5 ng/mL sodium selenite. FLC cells were previously described (Dinh et al., 2019) . Lines 3 and 18 were grown in complete F medium according to a previously published protocol (Liu et al., 2017) . Line 2 was grown similarly to lines 3 and 18 with three minor modifications. First, F media was conditioned by irradiated mouse embryonic fibroblasts for 3 days prior to use. FLC cells were cultured in conditioned F media without irradiated fibroblasts. Second, R-spondin conditioned media was added to complete F media to 10% volume. R-spondin conditioned media was produced by culturing HEK293T cells expressing murine Rspo1 (gift from Alexander Nikitin lab) in conditioning media (DMEM supplemented with 1% GlutaMAX, 1% HEPES, and 1% penicillin-streptomycin) for 10 days. The supernatant was collected and filtered through a 0.22 µm filter prior to use. Third, the ROCK inhibitor Y-27632 was used a final concentration of 20 µM, increased from the original concentration of 10 µM (Liu et al., 2017) . We detected the presence of murine cells in all three derived cell lines by RT-qPCR. All cell lines were cultured in 5% CO2 at 37°C.