University of Washington

Goal: Investigate the potential of heat shock protein 70 (Hsp70) and kinase inhibitors as potential therapeutic options in pre-clinical models

Principal Investigator: John Scott, PhD

Grant length: Two years

Study overview: In the previously funded work, the investigators discovered that DNAJ-PKAc forms a complex with heat shock protein 70 (Hsp70) and mitogenic kinases (kinase enzymes that function in the cell proliferation pathway) by acting as a scaffold. This feature creates a subcellular focal point for the transmission of aberrant chemical signals throughout FLC tumors and may be recruiting a host of other protein beyond what has been already reported. Pharmacological targeting of these oncogenic focal points was the next logical step in the continuum of this research. The goals of this study were:

1. Determine if FLC is driven by the DNAJ-PKAc kinase activity or by association with oncogenic binding partners.
2. Test efficacy of Hsp70/kinase inhibitor combinations in human FLC cells, organotypic tumor slices, hepatic organoids expressing the chimeric protein and PDX models.

Results: The investigators used a proximity assay to characterize the range of binding partners for DNAJ-PKAc ‘scaffold’ as compared to the native protein.  Results showed that the fusion protein is promiscuous (binds to various partners) as compared to the native protein. Within these wide range of binding partners, they discovered increased association of Bcl2-associated athanogene 2 (Bag 2), with the fusion protein. Bag2 is a co-chaperone protein linked to Bcl-2, an anti-apoptotic factor (an inhibitor of programmed cell death which can contribute to the development of cancer), which was also observed at elevated levels in FLC patient samples. Treatment of hepatocyte cell lines overexpressing the DNAJ-PKAc fusion protein with drug combinations targeting this DNAJ-PKAc/Bag2 axis confirmed a chemoresistant function of Bag2, which could explain the increased levels of this protein seen in FLC samples. This observation opened up potential therapeutic targets.

Implications: This study provided a strong candidate for therapeutic target explorations. Bag2 interacts with Bcl-2, which is an anti-apoptotic protein. Several targeted therapies against Bcl-2 are used for treatment of a wide range of cancers. However, targeting Bcl-2 in FLC has not been successful, so possible combinations of therapies targeting Bcl2 and associated factors, such as Bag2 could be a good strategy for FLC.

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

Grant length: Two years

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.

Goal: Understand growth mechanisms and identify potential therapeutic targets

Principal Investigator: Raymond Yeung, MD, Professor of Surgery

Grant length: Two years

Study overview: Fibrolamellar cancer (FLC) is one of the most lethal form of liver cancer in adolescents and young adults. Currently, there is no effective therapy for these patients besides surgery. Armed with a novel set of immortalized cell lines developed at the University of Washington that bear the characteristic FLC mutation (the DNAJB1-PRKACA gene fusion), this study sought to advance the understanding of the mechanisms that drive FLC development by:

• Identifying protein kinase pathways that are activated downstream of the DNAJB1-PRKACA gene fusion using a combination of global and targeted phosphoproteomic analyses.
• Functionally validating these results using FLC cell lines as well as human FLC samples.
• Investigating the role of HSPs in FLC, including their pro-survival function in keeping cells alive during stress, based on preliminary observations that the mutant protein associates with heat shock protein (HSP) 70.

Together, these studies aimed to unveil mechanistic insights and new therapeutic targets that will accelerate a cure for this deadly disease.

Key findings: The study team identified approximately10 kinases that affected the proliferation of FLC-expressing cells. In this study, researchers also found that DNAJ-PKAc acts like a scaffold, bringing together other proteins that contribute to the development of the cancer. They established that the fusion protein, DNAJ-PKAc is stabilized by its association with A-kinase anchoring proteins and the chaperon protein Hsp70. It also interacts with a kinase module known as RAF-MEK-ERK. This leads to the activation ERK signaling pathways, which in turn activates other downstream kinases which culminate in increased cell proliferation.

Drug combinations that inhibit Hsp70 and MEK (from the RAF-MEK-ERK module) led to the reduced proliferation of cells expression the DNAJ-PKAc protein. As a next step, these results were being tested in patient-derived cell lines and mouse models of FLC.

In May 2019, the key results of this study were published in the journal eLife. The full text of the published article, “An acquired scaffolding function of the DNAJ-PKAc fusion contributes to oncogenic signaling in fibrolamellar carcinoma“, can be read here.

Implications: These results established the importance of interaction between mutant fusion kinase and scaffolding proteins and chaperones.  Through such functional cross-talk, DNAJ-PKAc most likely affects many signaling pathways in specific cellular subdomains to promote oncogenesis. This study provided a functional model of the fusion protein and effect on kinases.

The study also suggested that a combination of Hsp70 and MEK inhibition may offer a potential combination therapy for FLC. However, that utility is not clear and should be tested in other model systems in addition to the engineered AML-12 system used in this study.