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 children and 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.

Keywords: Genomic Engineering; Liver Cancer; Mouse Model; PKA; Protein Kinase A.

Figure 1. Generation of CRISPR/Cas9 reagents that engineer the Dnajb1–Prkaca fusion in vitro and in adult mouse liver

(A) In the mouse genome, Dnajb1 and Prkaca are located on chromosome 8 in a region syntenic to human chromosome19. The target sequences and locations of the gRNA pair used to engineer the Dnajb1–Prkaca fusion are shown. (B) Ability of the gRNA pair shown in (A) to engineer the Dnajb1–Prkaca fusion in transfected Neuro-2a cells. (Left) Schematic of the Dnajb1–Prkaca genomic fusion and location of primers (arrows) used to amplify the breakpoint. Sanger sequencing chromatogram and sequences for various fusion breakpoints are shown. “Predicted” sequence indicates gene fusion without any indel mutagenesis. (Right) IDAA profile showing the frequency of the various breakpoint amplicons. (C) Ability of the gRNA pair shown in (A) to engineer the Dnajb1–Prkaca fusion in the liver of hydrodynamically tail vein injected mice. (Left) Dnajb1–Prkaca specific PCR from cDNA derived from the liver of mice injected with the gRNA pair (Right) Schematic of the fusion transcript and location of primers (arrows) used to amplify the fusion breakpoint. The Sanger sequencing chromatogram demonstrates an in-frame fusion transcript.

Figure 2. The Dnajb1–Prkaca fusion induces FL-HCC in mice

Representative macroscopic and microscopic images of mouse FL-HCC elicited by Dnajb1–Prkaca. (A) Macroscopic image of a tumor (arrow). (B) Whole-scan H&E image and magnification showing tumor (Tu)-non-tumor (N) border. (C) Microscopic H&E image of tumor area showing trabeculae of tumor cells separated by variably dilated sinusoids. (Upper inset) Detail of an “oncocytic” tumor cell with granular eosinophilic cytoplasm and large nucleus with prominent nucleolus. (Lower inset) Detail of “pale body” and hyaline globulus. (D) Transmission electron micrograph of tumor cell showing cytoplasm packed with mitochondria (arrows) and nucleus with prominent nucleolus and coarse chromatin. (E) Periodic acid-Schiff staining of hyaline globules. (F) Small neoplastic lesion with large oncocytic cells with granular eosinophilic cytoplasm and prominent nucleoli (black arrow) and leukocyte infiltration (white arrow).

Figure 3. Histological characterization of Dnajb1–Prkaca elicited mouse FL-HCC

(A) PicroSirius red staining showing mild collagen fibrosis between tumor cells. (B) Tumor cells express HepPar1. (Inset) Detail of a tumor cell expressing HepPar1 with mitochondrial localization and illustrating the mitochondria-rich cytoplasm. (C) Carcinoembryonic antigen (CEA) staining showing canalicular distribution in tumor area (Tu), as well as in normal liver area (N). (D) Cytokeratin 7 expression in tumor area. (E) Cytokeratin 19 expression in tumor area. (F) Broad expression of glutamine synthetase by tumor cells (Tu), but not by hepatocytes of the normal liver area (N).

Figure 4. Molecular analysis of Dnajb1–Prkaca elicited mouse FL-HCC

(A) Sashimi plot of RNA-seq read coverage for Dnajb1 and Prkaca in normal liver and mouse FL-HCC. Peaks depict reads per kilobase per million reads mapped (RPKM). Arcs depict reads spanning splice junctions. (B) Venn Diagram showing overlap of differentially expressed genes between human (Simon et al.) and mouse FL-HCC. Differentially expressed genes for mouse FL-HCC were defined by a BaseMean value >25 and log2FC >1.5 over normal liver. Gene Ontology Biological Process (MSigDB, c5.bp) is shown for the 145 common differentially expressed genes, showing enrichment of mitosis and proliferation genes. (C) Immunoblotting of tissue lysates from mouse FL-HCC tumor (Tu), adjacent normal liver (N) or livers from control mice. PKAc: immunoblotting using an antibody raised against the catalytic subunit alpha of PKA. Vinculin is used as loading control. (D) Ki67-staining of tumor cells (Tu) and adjacent normal tissue (N). Bars represent quantification of Ki67 positive cells/number of hepatocytes.