FCF Funded Projects

Kinase fusion function investigation

Status: Completed

Timeframe: 2016 – 2018

Goal: Investigate DNAJB1-PRKACA fusion kinase function in new fibrolamellar carcinoma models

Principal Investigator: Dr. Yi Guo, PhD

Study overview: The DNAJB1-PRKACA fusion kinase is found in nearly 100% of FLC cases. While this novel fusion had previously been shown to promote tumors in mice, defining the mechanistic function of DNAJB1-PRKACA and the pathways it controls is critical for the development of targeted therapeutics. This project investigated the function of the DNAJB1-PRKACA fusion in FLC tumor formation using both Drosophila melanogaster (fruit fly) and mice models. The team had previously established a DNAJB1-PRKACA transgenic Drosophila model, in which the fusion is expressed in the eye where phenotypes are easily visible. This model demonstrates abnormal phenotypes affecting both proliferation and differentiation of Drosophila eyes. The study team also exploited CRISPR/Cas9 genome-engineering technology in murine cultured hepatocytes to recreate the chromosomal deletion found in FLC patients.

The goals of the study were to:

  1. Characterize the oncogenic and fibrogenic activities of genetic engineered murine hepatocytes in vitro and in vivo; and
  2. Screen potential therapeutics using the DNAJB1-PRKACA over-expression model in Drosophila, to provide essential resources and knowledge for future development of new FLC therapeutics.

Key Findings: In this study, the researchers used CRISPR to generate chromosomal deletions corresponding to the FLC fusion protein in mouse hepatocytes, thereby creating mDNAJB1-PRKACA clones. These hepatocyte cell lines carrying the fusion proteins showed increased levels of intracellular PKA activity (phosphoPKA substrates) and grew faster than control cells plated at the identical density. These clones were then injected into mice liver and assessed for tumor formation. While the “normal” control did not have any tumor growth after 4 months, the engineered mDNAJB1-PRKACA-bearing mice showed entirely tumorous liver, as well as severe ascites. This mice model is being assessed for mechanistic studies of FLC.

In the second part of the study, the investigators developed a FLC model using the common fruitfly, Drosophila melanogaster. The DNAJB1-PRKACA fusion protein from humans was over-expressed in the fly eye and caused a visible transformation (small size and rough eye surface) of the fly eye. This transformation from hDNAJB1-PRKACA over-expression is caused by aberrant PKA signaling which interferes with cell proliferation and differentiation. Genetic inhibition by simply feeding the flies hPKI (a human inhibitory peptide for PKA) reversed the hDNAJB1-PRKACA over-expression eye phenotype. However, testing with a commercially available inhibitor caused toxic side effects and that molecule had to be discontinued as a potential therapeutic candidate.

Implications: These studies show the utility of fly eye morphology change as an easy, fast, and functional relevant in vivo readout for hDNAJB1-PRKACA-dependent developmental perturbations. This model is being subsequently used for the screening of several FDA-approved small molecule kinase inhibitors.