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

HepG2 expressing DNAJB1-PRKACA and EGFP have been previously described (Dinh et al., 2019) . HepG2 cells were grown in Dulbecco's Modified Eagle Media (DMEM) containing 1 g/L glucose (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific), 1% GlutaMAX (Thermo Fisher Scientific), 110 mg/L sodium pyruvate, and 1% penicillin-streptomycin (Thermo Fisher Scientific). The DNAJB1-PRKACA K128H kinase-dead mutant was cloned using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent Technologies) using the following primers (5'-CCTTCTGTTTGTCGAGGATATGCATGGCATAGTGGTTCCCG-3' and 5'-CGGGAACCACTATGCCATGCATATCCTCGACAAACAGAAGG-3'). PCR products were cloned into the pCR-Blunt II-TOPO vector (Thermo Fisher Scientific) and subcloned into the pLV-EF1a-IRES-Puro vector (Addgene plasmid #85132, gift from Tobias Meyer). For lentivirus production, HEK293/T17 cells were transfected with DNAJB1-PRKACA K128H plasmid along with psPAX2 (Addgene plasmid #12260, gift from Didier Trono) and pMD2.G (Addgene plasmid #12259, gift from Didier Trono) to produce lentiviral particles, which were concentrated using Lentiviral-X Concentration (Takara Bio USA, Mountain View, CA) per manufacturer's protocol. HepG2 cells were transduced with varying concentrations of lentivirus for 24 hours and selected with 2 µg/mL puromycin (Thermo Fisher Scientific) for 4 days. Selectable cells treated with the lowest concentration of virus were used for further passaging and experiments to obtain a majority of cells with 1 viral integration.