Innate Immune Signaling in Fibrolamellar Carcinoma

Fibrolammellar Carcinoma (FLC) is a rare form of liver cancer that afflicts children and young adults. At the time of diagnosis, the cancer is stage IV in ~60% of patients without effective treatment option. For the remaining 40%, liver transplantation is the only curative therapy, providing a suitable donor can be found in time and even

then, with significant risks for short- and long-term complications. Virtually all patients with FLC have a genomic alteration on Chr. 19 resulting in fusion of exon 1 of DNAJB1 of the Hsp40 family to exons 2-10 of PRKACA, a catalytic subunit of PKA. The resulting DNAJB1-PRKACA fusion gene encodes a Dnajb1-Prkaca protein with preserved catalytic activity. Using CRISPR/Cas9 technology

combined with hydrodynamic tail vein injection, we and others have shown conclusively that a syntenic Dnajb1- Prkaca fusion gene on mouse Chr. 8 is sufficient to elicit liver cancer with hallmarks of FLC. FLC is thought to arise from liver stem cells, but the mechanisms whereby Dnajb1-Prkaca promotes oncogenic

transformation are not well understood. To permit functional studies in a developmentally relevant context, we used CRISPR/Cas9 technology to engineer syntenic Dnajb1-Prkaca in mouse liver organoids. We discovered that liver organoids expressing Dnajb1-Prkaca are prone to undergoing lytic cell death. Next, we performed

extensive transcriptome analyses that revealed an innate immune signature prominently featuring constituents of the NLRC4 inflammasome. We moreover determined that Dnajb1-Prkaca expressing liver organoids exhibit significantly elevated levels of cleaved caspase-8 consistent with its potent activation.

In this work, we will first test if the NLRC4 inflammasome is activated by Dnajb1-Prkaca in mouse liver organoids and establish its role in lytic cell death. We will then determine if the Nlrc4 and/or Casp-8 genes are necessary for tumorigenesis elicited by engineering of the Dnajb1-Prkaca fusion gene in the mouse liver. The latter would

represent a major breakthrough in understanding the pathogenesis of FLC.