CDK7 is a Novel Therapeutic Vulnerability in Fibrolamellar Carcinoma

Abstract

Fibrolamellar carcinoma (FLC) is a rare and lethal cancer that afflicts young individuals. The tumor arises in the background of a healthy liver, and patients typically present with advanced cancer at the time of diagnosis. Unfortunately, for these patients with advanced or recurrent cancer, no proven systemic therapies exist resulting in only 30-45% of patients surviving to 5 years. Investigations into the molecular underpinning of FLC have revealed a unique gene fusion between heat shock protein 40 (DNAJB1) and the catalytic subunit alpha of protein kinase A (PRKACA), leading to the formation of an oncoprotein (DNAJ-PKAc) that retains kinase activity and is a proven tumor-causing event in FLC. To uncover potential therapeutic targets, we engineered an FLC cell line by introducing the DNAJB1-PRKACA oncogene rearrangement into human hepatocellular cells using CRISPR/Cas9. We identified aberrant cell cycle progression, and follow-up molecular analysis revealed evidence of enhanced cyclin dependent kinase 7 (CDK7) activation in the DNAJB1-PRKACA expressing FLC cells. These findings were confirmed in human samples of FLC. In turn, targeting CDK7 with selective inhibitors demonstrated efficacy in several patient-derived models of FLC, with minimal toxicity to normal liver. Collectively, this work uncovers a novel candidate therapeutic vulnerability in FLC.

DNAJB1-PRKACA fusion protein-regulated LINC00473 promotes tumor growth and alters mitochondrial fitness in fibrolamellar carcinoma

“…A major limitation of this model for evaluating the primate-specific LINC00473 is its murine origin. In more recent years, at least two other human cancer lines were engineered to express the fusion oncogene, both of which we confirm to exhibit elevated levels of LINC00473 52,70 . While it has been shown that the exogenous introduction of LINC00473 in mouse neurons can modulate synaptic activity, it is unknown whether the transcriptional programs and signaling pathways critical for LINC00473mediated cancer phenotypes are conserved in non-primate contexts 55,56 .…”

“…HepG2-DP and HepG2-Ctl: HepG2-DP cells were previously described and were a generous gift from Dr. Sean Ronnekleiv Kelly 70 . Briefly, the DNAJB1-PRKACA expressing HepG2 cells (HepG2-DP) were generated using a mammalian dual-guide RNA-CRISPR-CAS9-EGFP vector (VectorBuilder, Chicago, IL) with guide RNA sequences targeting the first intronic regions of DNAJB1 and PRKACA (gRNA1 (DNAJB1) 5'-CAGGAGCCGACCCCGTTCGT-3', gRNA2 (PRKACA): 5'-GTAGACGCGGTTGCGCTAAG-3').…”

DNAJB1-PRKACA fusion protein-regulated LINC00473 promotes tumor growth and alters mitochondrial fitness in fibrolamellar carcinoma

“…A major limitation of this model for evaluating the primate-specific LINC00473 is its murine origin. In more recent years, at least two other human cancer lines were engineered to express the fusion oncogene, both of which we confirm to exhibit elevated levels of LINC00473 [ 52 , 70 ]. While it has been shown that the exogenous introduction of LINC00473 in mouse neurons can modulate synaptic activity, it is unknown whether the transcriptional programs and signaling pathways critical for LINC00473-mediated cancer phenotypes are conserved in non-primate contexts [ 55 , 56 ].…”

“…HepG2-DP and HepG2-Ctl : HepG2-DP cells were previously described using the HepG2 cell line (RRID: CVCL_0027) and were a generous gift from Dr. Sean Ronnekleiv Kelly [ 70 ]. Briefly, the DNAJB1-PRKACA expressing HepG2 cells (HepG2-DP) were generated using a mammalian dual-guide RNA-CRISPR-CAS9-EGFP vector (VectorBuilder, Chicago, IL) with guide RNA sequences targeting the first intronic regions of DNAJB1 and PRKACA (gRNA1 (DNAJB1) 5’-CAGGAGCCGACCCCGTTCGT-3’, gRNA2 (PRKACA): 5’-GTAGACGCGGTTGCGCTAAG-3’).…”

“…Briefly, the DNAJB1-PRKACA expressing HepG2 cells (HepG2-DP) were generated using a mammalian dual-guide RNA-CRISPR-CAS9-EGFP vector (VectorBuilder, Chicago, IL) with guide RNA sequences targeting the first intronic regions of DNAJB1 and PRKACA (gRNA1 (DNAJB1) 5’-CAGGAGCCGACCCCGTTCGT-3’, gRNA2 (PRKACA): 5’-GTAGACGCGGTTGCGCTAAG-3’). Genomic DNA and mRNA were used to confirm the presence of DNAJB1-PRKACA fusion, as previously described [ 70 ]. Cells were grown in DMEM complete media (DMEM, high glucose, Gibco, 11965092; 10% FBS, heat-treated at 56C for 30 minutes, Gibco, 26140079; 5% penicillin-streptomycin 10,000U/mL, Gibco, 15140122; 5% Glutamax 100X, Gibco, 35050061).…”