CRISPR/Cas9 Engineering of Adult Mouse Liver Demonstrates That the Dnajb1–Prkaca Gene Fusion Is Sufficient to Induce Tumors Resembling Fibrolamellar Hepatocellular Carcinoma

Abstract

Background & Aims Fibrolamellar hepatocellular carcinoma (FL-HCC) is a primary liver cancer that predominantly affects young adults with no underlying liver disease. A somatic, 400 Kb deletion on chromosome 19 that fuses part of the DnaJ heat shock protein family (Hsp40) member B1 gene (DNAJB1) to the protein kinase cAMP-activated catalytic subunit alpha gene (PRKACA) has been repeatedly identified in patients with FL-HCC. However, the DNAJB1–PRKACA gene fusion has not been shown to induce liver tumorigenesis. We used the CRISPR/Cas9 technique to delete in mice the syntenic region on chromosome 8 to create a Dnajb1–Prkaca fusion and monitored the mice for liver tumor development. Methods We delivered CRISPR/Cas9 vectors designed to juxtapose exon 1 of Dnajb1 with exon 2 of Prkaca to create the Dnajb1–Prkaca gene fusion associated with FL-HCC, or control Cas9 vector, via hydrodynamic tail vein injection to livers of 8 week-old female FVB/N mice. These mice did not have any other engineered genetic alterations and were not exposed to liver toxins or carcinogens. Liver tissues were collected 14 months after delivery; genomic DNA was analyzed by PCR to detect the Dnajb1–Prkaca fusion, and tissues were characterized by histology, immunohistochemistry, RNA sequencing, and whole-exome sequencing. Results Livers from 12 of the 15 mice given the vectors to induce the Dnajb1–Prkaca gene fusion, but none of the 11 mice given the control vector, developed neoplasms. The tumors contained the Dnajb1–Prkaca gene fusion and had histologic and cytologic features of human FL-HCCs: large polygonal cells with granular, eosinophilic, and mitochondria-rich cytoplasm, prominent nucleoli, and markers of hepatocytes and cholangiocytes. In comparing expression levels of genes between the mouse tumor and non-tumor liver cells, we identified changes similar to those detected in human FL-HCC, which included genes that affect cell cycle and mitosis regulation. Genomic analysis of mouse neoplasms induced by the Dnajb1–Prkaca fusion revealed a lack of mutations in genes commonly associated with liver cancers, as observed in human FL-HCC. Conclusions Using CRISPR/Cas9 technology, we found generation of the Dnajb1–Prkaca fusion gene in wild-type mice to be sufficient to initiate formation of tumors that have many features of human FL-HCC. Strategies to block DNAJB1–PRKACA might be developed as therapeutics for this form of liver cancer.

DNAJB1-PRKACAfusion kinase interacts with β-catenin and the liver regenerative response to drive fibrolamellar hepatocellular carcinoma

“…Thus, induction of an endogenous Dnajb1-Prkaca fusion through intrachromosomal deletion drives tumors with features of FL-HCC in mice. Independent work from others recently demonstrated an FL-HCC phenotype of lesions in asymptomatic mice using similar methods (22).…”

Structures of the PKA RIα holoenzyme with the FLHCC driver J-PKAcα or wild type PKAcα

“…It does not respond well to chemotherapy and the overall five year survival rate of FLHCC patients is only 30-45% (El-Serag and Davila, 2004; Kakar et al, 2005; Katzenstein et al, 2003; Lim et al, 2014; Mavros et al, 2012; Weeda et al, 2013). The chimeric gene DNAJB1-PRKACA , ubiquitously and exclusively found in almost all FLHCC patients, is the result of a ~400 kb deletion in one copy of chromosome 19 (Darcy et al, 2015; Engelholm et al, 2017; Honeyman et al, 2014; Kastenhuber et al, 2017; Oikawa et al, 2015; Riggle et al, 2016a, 2016b; Simon et al, 2015). This produces an enzymatically active chimeric protein J-PKAcα.…”

DNAJB1-PRKACA fusion induces liver inflammation in zebrafish model of Fibrolamellar Hepatocellular Carcinoma

“…1A). Expression of the DNAJB1-PRKACA fusion transcript is sufficient to drive tumorigenesis in vivo in murine models (Engelholm et al, 2017; Kastenhuber et al, 2017) . Zebrafish has two homologous genes for dnajb1, dnajb1a (ENSDARG00000099383) and dnajb1b (ENSDARG00000041394), located in chromosomes 3 and 1 respectively.…”

“…There is only one identified molecular target responsible for driving the disease, resulting from a 400kb deletion on chromosome 19, the DNAJB1-PRKACA fusion transcript (Honeyman et al, 2014; Simon et al, 2015). Recently developed murine models using CRISPR/Cas9 or overexpression of this fusion gene support the idea that the fused DNAJB1-PRKACA gene alone is sufficient to drive tumorigenesis in vivo (Engelholm et al, 2017; Kastenhuber et al, 2017) .…”