FCF Funded Projects

Molecular therapies for fibrolamellar carcinoma (FLC)

Status: Completed

Timeframe: 2019 – 2021

Goal: Investigate the potential of heat shock protein 70 (Hsp70) and mitogen-activated protein kinases (MAPKs) as therapeutic options for FLC

Principal Investigator: John Scott, PhD

Study overview: A main goal of this project was to determine how the abnormal form of protein kinase A (PKA) present in fibrolamellar carcinoma (FLC) cells differs in its biochemical function from normal PKA, and to determine if the differences can be exploited to find drugs to treat FLC.

The driver of fibrolamellar carcinoma (FLC), specified by the DNAJB1-PKACA fusion gene, is a fusion protein (“chimera”) in which most of the biochemically active catalytic subunit of PKA (designated PKAc, or simply C), is linked to a domain of a “heat shock protein 40” (HSP40) coded for by a segment of the gene DNAJB1. The chimera can be designated the DNAJB1-PKACA protein, or more simply, DNAJ-PKAc.

Protein kinases are enzymes that attach phosphate groups to specific sets of protein substrates in the cell. In turn, this phosphorylation by a kinase influences the substrates by turning on or off their activity and/or by changing their location within the cell.

This project aimed to determine how attaching a piece of the DNAJB1-encoded HSP40 to PKAc contributes to the ability of the DNAJ-PKAc fusion protein to serve as the driver of fibrolamellar carcinoma (FLC). One possibility was that the fusion protein would be fully active as a kinase even in the absence of cAMP. However, John Scott’s lab previously had found that DNAJ-PKAc is regulated similarly to normal PKAc by the R subunit, and still requires cAMP to be activated.

Given the underlying biochemical similarities between the PKAc and DNAJ-PKAc kinases, the Scott lab explored other ways in which they differ that could explain the cancer-driving property of the chimera, and might also suggest new therapeutic strategies. They asked broadly whether the normal and oncogenic forms of the protein differ substantially in their interaction with other proteins in the cell, their localization within the cell, and/or the sets of proteins they phosphorylate.

The HSP40 (DNAJ) domain of the FLC-associated chimera is well-known to bind and “recruit” to protein complexes another heat-shock protein, HSP70. The HSPs play important roles in biology by assisting in the folding of proteins into their correct three-dimensional shapes, preventing or correcting misfolding of proteins, and helping to eliminate badly misfolded or aggregated proteins that can damage cells. Cells in stressed environments, or which are challenged to grow in a deregulated manner as in cancer, often require elevated levels of certain HSPs, including HSP70. Indeed, high expression of HSP70 is a frequent feature of cancer cells, and the protein contributes to tumor survival and growth in multiple ways.

The goals of the study were:

  1. Establish if the unique combination of DNAJ-PKAc-interacting proteins stabilize the signaling islands and lead to the FLC pathology.
  2. Test drug combinations that target the DNAJ-PKAc interacting proteins.

Results: In the funded project, the Scott lab confirmed their hypothesis that DNAJ-PKAc serves as a scaffold for a protein complex that includes HSP70. They utilized biochemical, cell biology, and visual imaging methods to document the presence of DNAJ-PKAc/HSP70 complexes in a model cell system (cultured mouse cells, similar to normal liver cells, in which the DNAJB1-PRKACA fusion was introduced by genetic engineering) and found that these complexes also are prevalent in FLC cells in human tumor biopsies. In the model system they further demonstrated that inducing an artificial dimer of normal HSP70 and PKAc inside cells mimicked DNAJ-PKAc in activating a proliferation-associated protein kinase (named ERK) that is known to be up-regulated in FLC tumor cells.

Additionally, the team found that AKAP-Lbc, in particular, is up-regulated in FLC tumors and that it sequesters DNAJ-PKAc/HSP70 sub-complexes and most likely forms a focal point for signaling. They suggest that this brings the oncogenic PKA and HSP70 into association with several protein kinases that are associated with active cell division.

Finally, the team demonstrated that the proliferation in culture of their model mouse liver cell line containing DNAJ-PKAc could be blocked by a combination of two drugs, Ver155008, an inhibitor of HSP70, and Cobimetinib, an inhibitor of MEK, one of the “mitogenic” protein kinases in the AKAP-Lb-associated complex. However, the efficacy of the same drug combination against human FLC cells grown in mice was not determined. (Such experiments were not feasible at the investigators’ institution during the COVID pandemic.)

Implications: This study provided proof of concept that proteins binding to DNAJ-PKAc or activated by the fusion protein can be targeted instead of the fusion protein itself. The fusion protein is difficult to target by existing protein kinase inhibitors due to structural similarity with the native PKAc. Therefore, these results offer an alternative method of targeting the pathways activated by DNAJ-PKAc.

Elements of the team’s work was published and acknowledged in an article appearing in Cell Reports in April 2020. The full text of that publication can be read here.

The preceding efforts of the team defining the scaffolding function of the PNAJ-PKAc fusion were published in the journal eLife. Click here to access that article.