Fibrolamellar received minimal research attention prior to the founding of FCF. Since 2010 FCF has invested nearly $7 million in research, across a wide range of industry-leading institutions, with the goal to accelerate the road to curative therapies. Research has been both in traditional and non-traditional approaches, led by respected M.D.’s and PhD’s who focus on clinical trials, translational, and basic research.
Below is a summary of the research grants awarded and committed by FCF from 2009 to 2019:
The following is a detailed list of FCF-sponsored research initiatives for each research institution:
FCF sponsored research initiatives
Boston Children’s Hospital (Harvard University)
- Development of a Human-Derived Liver Progenitor Cell Line with DNAJB1-PRKACA Fusion Gene
2019 – 2020
Principal Investigator: Khashayar Vakili, MD, Surgical Director of Liver, Kidney, Intestine, and Multivisceral Transplant Programs, Assistant Professor of Surgery, Harvard Medical School
Developing models to study the mechanism by which the DNAJB1-PRKACA fusion protein causes fibrolamellar hepatocellular carcinoma (FLC) is crucial for finding a cure for this cancer. In our lab, we have engineered a kidney cell line (HEK-DP) which contains the DNAJB1-PRKACA fusion gene. This cell line has demonstrated some interesting similarities to FLC tumors and has served as a platform for developing a better understanding of the actions of the fusion protein in the cellular environment.
In this project, we will apply the same strategy that we used to engineer the HEK-DP model to engineer normal human liver progenitor cells to express the DNAJB1-PRKACA fusion gene. This will provide us with a novel model to understand how the fusion protein reprograms the liver progenitor cells into becoming cancerous. Using cells derived from human livers, we have established three-dimensional “organoid” cultures which contain special precursor cells or stem cells. After introducing the fusion gene into these progenitor cells, we will examine changes induced in response to the presence of the fusion protein. Understanding the mechanisms involved in reprogramming of the cells by the fusion protein will give us insights into how to therapeutically target FLHCC.
- Creating a Fibrolamellar Cancer Dependency Map
2020 – 2022
Principal Investigator: Jesse Boehm, PhD, Institute Scientist, Director of the Broad Cancer Model Development Center
This resource project is part of the Broad Institute’s Rare Cancer Dependency Map Initiative. It aims to create cell culture research models of FLC and utilize them to determine both a comprehensive list of potential drug targets for FLC and to identify existing drugs that may have therapeutic potential against FLC. Over 3 years the project is designed to:
- In coordination with the FCF-sponsored BioBank, create a unified pipeline by which patients anywhere can direct living tissue to FLC researchers
- Systematically develop conditions to propagate multiple fibrolamellar carcinoma (FLC) samples as organoid models in the Broad Institute’s Cancer Cell Line Factory laboratory using its combinatorial media screening technology
- Create multiple publicly available, genomically characterized, and clinically annotated FLC cell models for the scientific community to support translational research
- Identify ways to enhance the proliferation rates of FLC models developed by the Bardeesy Lab and others, creating new opportunities for translational research
- Create a definitive list of the highest priority drug targets for FLC tumors together with predictive biomarkers
- Determine whether any existing drugs, originally developed for any disease, may have repurposing potential in FLC
- Empower the entire FLC research community by putting all data and biologist-friendly analysis tools freely available online, pre-publication at org.
- Establish proof of concept for further scaleup of the Fibrolamellar Dependency Map Initiative in Years 3 and beyond, including genome-wide CRISPR screening and large scale drug repurposing in an expanded number of models.
FCF has partnered with the prestigious Cancer Research Institute (“CRI”) to specifically focus on the role immunotherapy may have in creating curative therapies for fibrolamellar. CRI has been promoting immunotherapy research for 65 years, long before the establishment cancer treatment and research community recognized immunotherapy as a legitimate prospect for cancer patients.
For the past 5 years, FCF has worked with CRI to develop and fund prestigious three-year fibrolamellar (FLC) research fellowships. These young researchers are making great strides forward. FCF/CRI fellowships that FCF has funded include:
- Role of the innate immune system in Fibrolamellar Hepatocellular Carcinoma (FL-HCC) using zebrafish as a model system
2016 – 2019
Principal Investigator: Sofia de Oliveira, Ph.D., EMBO Postdoctoral Fellow, Huttenlocher Immunology Lab
About 80% of FHC cases are characterized by the presence of an activated form of a protein called Protein Kinase A (PKA). The mechanism of how active PKA leads to disease remains unclear. Normal PKA is involved in complex signaling pathways that among other processes control innate immune response. A better understanding of the mechanisms triggered by this active PKA and how it modulates innate immune response is crucial to develop more efficient drug therapies and increase the survival rate of fibrolamellar patients. Up to now, there are limited animal models to study FHC, which hampers our knowledge about the disease. This research proposes to take advantage of the outstanding characteristics of zebrafish and develop a FHC model. Zebrafish has proven to be an extremely useful tool to study innate immunity and cancer biology. Additionally, it has been used to study liver development, conventional hepatocellular carcinoma and several other liver disorders, displaying remarkable similarities and sharing genetic signatures with human diseases. More over zebrafish is the only vertebrate model with high homology with humans that allows visualization of cellular and molecular interactions in a whole organism context without the need to use invasive imaging methods since the larvae are transparent.
Innate immune cells, such as neutrophils and macrophages are important players in cancer development. Their presence in the tumor microenvironment might be beneficial or detrimental to cancer progression, depending on the type of cancer. This study will leverage the zebrafish models to study the role of different innate immune cells in FHC, and assess the potential role of neutrophils and macrophages as therapeutic targets for FHC.
- T cell immunotherapy in fibrolamellar cancer
2016 – 2019
Principal Investigator: Kevin M. Sullivan, M.D., General Surgery
Immunotherapy is a form of cancer treatment that harnesses the patient’s own immune system to fight the disease. The immune system can precisely target cancer cells, while also minimizing damage to the remainder of the body’s normal cells. In this project, we will investigate a well-established method of using the immune system, successful in treating other cancers such as melanoma, as a treatment for fibrolamellar. We will start by using a variety of techniques to look at which types of immune cells reside within the fibrolamellar tumors. One type of immune cell, called the T cell, is of particular interest because it can specifically recognize and destroy cancer cells. In preliminary work, our group has confirmed that T cells are found within a fibrolamellar tumor. In this project, we will gain a detailed understanding of the characteristics of the T cells that are active within fibrolamellar tumors. We then plan to grow and activate these T cells and test their ability to fight cancer cells in cell cultures and slices of fibrolamellar tumor grown in the laboratory. Ultimately, our project will provide the basis for making this type of T cell as a treatment against fibrolamellar a reality.
- Investigating immune checkpoint biomarkers in tissue and peripheral blood of patients with fibrolamellar hepatocellular carcinoma
2016 – 2019
Principal investigator: Amy K. Kim, M.D., Assistant Professor
The understanding of immune checkpoint molecules that suppress host immune response against tumor cells and the discovery of drugs that block these immune checkpoints have revolutionized current cancer treatment. Anti-PD1 (programmed cell death protein 1) immunotherapy has shown benefit in many cancer types, but certain cancers have also shown strong resistance to this immunotherapy. It is unclear how fibrolamellar cancer would respond to different immune checkpoint blockade, including anti-PD1 therapy. In addition, there is a need to investigate how circulating tumor cells (CTCs) in the blood that have disseminated from the primary tumor site induce anti-tumor immune response outside the tumor environment. This project will address these issues by pursuing the following specific aims: 1) to define the dominant immune checkpoint pathway in fibrolamellar cancer and its interaction with the patient’s immune response in the tumor, and; 2) to determine how immune checkpoint markers are associated with circulating tumor cells in the peripheral blood, in comparison to the primary tumor site. Understanding the immune checkpoint expressions in fibrolamellar cancers will guide in the future selection of immunotherapy and studying the role of circulating tumor cells and tumor-associated immune cells can reveal a novel way to predict cancer treatment response that does not require an invasive procedure for tumor tissue from the patients.
- Pre-clinical studies of the interactions of the immune system with FL-HCC
2016 – 2019
Principal Investigator: Kevin Barry, Ph.D., Postdoctoral Scholar
All sequenced human fibrolamellar hepatocellular carcinoma (FL-HCC) samples contain a mutation that leads to a fusion of the proteins DNAJB1 and PRKACA. The mutant DNAJB1-PRKACA protein is thought to drive the development of FL-HCC. There is a clear need to further understand the mechanism of FL-HCC tumorigenesis and to develop novel treatments.
Cancer immunotherapies harness the power of the immune system to kill tumors. Checkpoint blockade immunotherapies are an exciting class of cancer immunotherapies that remove the brakes from the immune system by targeting molecules that inhibit tumor-directed responses by immune cells called T cells. T cells are important for protecting patients from tumors as these cells directly kill tumor cells and modulate the global immune response towards tumors. Immunotherapies targeting T cells have been remarkably effective in treating cohorts of non FL-HCC cancer patients, leading to tumor regression and immune memory which offers long-term protection; effectively providing a cure to cancer in some patients. However, very little is known about how the immune system interacts with FL-HCC or if immunotherapy would be an effective treatment for FL-HCC. The study of the efficacy of immunotherapy in the treatment of FL-HCC is hampered by the fact that the current pre-clinical animal model of FL-HCC utilizes the transplantation of human tumor into immune compromised animals, making it impossible to study the interactions between the immune system and FL-HCC in a tractable system.
This research, in collaboration with Dr. Julien Sage’s group and Stanford University, will generate a pre-clinical animal model of FL-HCC in mice with fully functional immune systems and will undertake the initial studies of how the immune system interacts with FL-HCC. These studies represent the first step in moving towards treating FL-HCC patients with immunotherapy in the clinic.
- Retinoic Acid-Induced Loss of DNAJB1-PRKACA Fusion Protein Expression
Principal Investigator: Andrew Yen, PhD, Professor, Department of Biomedical Sciences
Co-Principal Investigator: Praveen Sethupathy, PhD, Associate Professor, Department of Biomedical Sciences
Fibrolamellar carcinoma (FLC) is driven by the DNAJ-PKAc fusion protein. A potential therapeutic strategy would be to induce loss of this key driver protein of the cancer. One approach to substantially alter gene expression in cancer cells is differentiation induction therapy, in which a well-tolerated agent with low toxicity causes malignant cells to acquire more mature, specialized characteristics and to stop proliferating. The most successful differentiation therapy agent in current use is retinoic acid (RA), which has been the standard of care for acute promyelocytic leukemia (APL). RA, a metabolite of Vitamin A, induces APL cells to convert from a transformed/proliferating malignant state resembling certain immature white blood progenitor or stem cells to a non-transformed/cell cycle-arrested state resembling the corresponding normal, mature white blood cells.
Our preliminary observations in a model cell line engineered to stably express DNAJ-PKAc showed that RA causes loss of the fusion protein. We now will assess whether RA similarly decreases the level of DNAJ-PKAc in cultured human FLC cells and determine the optimal concentration and duration of RA treatment to effect a response that may suffice to mitigate the tumor phenotype and the virulence of FLC. We will then characterize the molecular signature of this response through high-throughput sequencing of messenger RNAs and small RNA species. Because they are known downstream effectors of RA action, we will look in particular for prominent responses in three classes of molecules, namely cyclin-related cell cycle regulators, MAPK pathway signaling regulators, and stemness/developmental regulators.
- Micro RNAs and long non-coding RNAs role in fibrolamellar and evaluation of RNA-based therapeutics
2017 – Current
Principal Investigator: Praveen Sethupathy Associate Professor Department of Biomedical Sciences, Cornell University, College of Veterinary Medicine
Fibrolamellar carcinoma (FLC) is a rare liver cancer that is characterized by multi-drug resistance, early onset, and high metastatic capacity. We are leveraging genome-scale approaches to discover the most critical molecular factors that promote and maintain these key features of FLC. Specifically, we are: (1) identifying microRNAs and long, non-coding RNAs (lncRNAs) that facilitate FLC tumor formation and/or invasion and evaluating the potential of RNA-based therapeutics; (2) mapping the chromatin activity patterns in FLC to identify master transcriptional regulators; (3) defining the super enhancer landscape in FLC to identify the regulatory elements and genes most critical for driving tumor behavior; and (4) integrating different types of large-scale datasets, including metabolomics data, to identify critical druggable pathways. We are actively engaged in collaborative ventures that bring together researchers with a shared commitment to tackle this devastating disease and bring relief and hope for patients. For a description of some of our recently published work, we encourage you to visit here <https://www.cmghjournal.org/article/S2352-345X(19)30010-4/fulltext> and here<http://news.cornell.edu/stories/2019/02/key-rare-aggressive-liver-cancer-found-rna-molecule>.
- Hedgehog and YAP signaling in fibrolamellar carcinoma: Tumor-stroma crosstalk and the cancer stem cell niche
2017 – 2019
Principal Investigators: Cynthia Guy, MD, Associate Professor of Pathology; Anna Mae Diehl, MD, Florence McAlister Professor of Medicine, Duke University School of Medicine
Fibrolamellar carcinoma has a unique appearance; it is made up of large tumor cells surrounded by thick fibrous bands (the stroma). We believe that the cancers growth may result from deregulated communications signals between the tumor cells and cells that produce the stroma.
The major producers of stroma in the liver are called hepatic stellate cells (HSCs). Evidence from our laboratory has shown that in many different types of liver disease, HSCs promote repair of damaged livers by producing stroma and sending out signals that help surviving liver cells to regenerate. When repair is effective, stroma transiently accumulates and then regresses as healthy liver tissue is regenerated. However, when repair becomes dysregulated, excessive stroma (a.k.a., scar) accumulates and regeneration stalls before recovery of healthy liver tissue is accomplished. Our research revealed that HSCs regulate repair by controlling the activity of the Hedgehog (Hh) signaling pathway. We have demonstrated that while Hh signaling is helpful during normal liver development and repair, if it becomes deregulated, it can result in lead to pathologic processes. These processes include fibrosis (scarring) and the activation of a downstream signaling pathway that leads to the activation of Yap, a factor that can make liver cells become more primitive (and in many ways like cancer stem cells). Thus, dysregulation of Hh and Yap results in accumulation of stroma (scar) and primitive stem-like cells and as such, resembles key features of fibrolamellar carcinoma (FLC).
Given this background, we will evaluate evidence for and against the concept that Hh and Yap signaling between the tumor cells and the stroma is important for FLC growth, and for the perpetuation of cancer stem cells. This possibility is supported by a recent publication by a FCF- sponsored investigator, Lola Reid, PhD, who showed that malignant fibrolamellar cells produce Hh protein. Furthermore, there is growing evidence that Hh and Yap interact to control liver growth. However, to date, neither pathway has been formally studied in FLC, or considered as a possible diagnostic or therapeutic target in this cancer. Our project is the first to explore this possibility and offers the promise of novel interventions to prevent and treat this devastating disease. We will examine human fibrolamellar carcinoma samples to look for evidence of Hh and Yap signaling in the tumor cells and stroma. In addition, we will grow cells in culture with and without different types of HSCs to find the best cell-cell signals to target for anti-cancer therapy.
- Opening of FLC Peptide Vaccine Clinical Trial
A new clinical trial of an immune therapy for fibrolamellar carcinoma (FLC) is now recruiting subjects at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University in Baltimore, MD. The study asks if individuals can mount an effective immune response against FLC by specifically targeting the unique chimeric protein (resulting from a DNAJB1-PRKACA gene fusion) believed to drive the growth of almost all such tumors. Trial subjects will be given an experimental vaccine containing a peptide (small segment of a protein) that corresponds to the junction region linking the two parts of the chimeric protein. They will simultaneously receive two FDA-approved drugs, Opdivo (nivolumab) and Yervoy (ipilimumab), that may enhance the immune response against FLC by overcoming “checkpoint” systems that can limit the immune system’s ability to fight a cancer. The study’s principal investigator is Dr. Mark Yarchoan. Details and contact information can be found at: https://www.clinicaltrials.gov/ct2/show/NCT-04248569
- A Pilot Study of a DNAJB1-PRKACA Fusion Kinase Vaccine Combined with Nivolumab and Ipilimumab for Patients with Fibrolamellar Carcinoma (FLC)
Principal Investigator: Mark Yarchoan, MD, Assistant Professor, Oncology/Division of GI Malignancies, Sidney Kimmel Comprehensive Cancer Center
FLC is a rare and often lethal form of liver cancer for which there is no standard treatment option beyond surgery. Immune checkpoint inhibitors are a revolutionary new form of cancer therapy. These drugs “take the brakes off” the immune system, enhancing its ability to fight cancer. Examples of these new medicines include the PD1 inhibitor nivolumab (Opdivo), and the CTLA-4 inhibitor ipilimumab (Yervoy). Many patients with FLC currently receive an immune checkpoint inhibitor off-label. However, our clinical experience suggests that they generally do not achieve strong anti-tumor responses from single checkpoint inhibitors. We seek to develop immunotherapy approaches to FLC that offer greater clinical benefit.
This project entails the first test in patients of a combination of two checkpoint inhibitors (nivolumab and ipilimumab) plus a new vaccine designed to direct the immune response against FLC by targeting the DNAJ-PKAc fusion protein found in almost every case of this cancer. The fusion protein serves as a neoantigen – an abnormal protein found in the cancer but absent from normal cells. The selective component of the vaccine is a peptide (short segment of a protein) overlapping the junction between the DNAJ and PKAc segments of the fusion protein, which is precise and consistent among FLC tumors. Thus, potentially the vaccine could be harnessed by the immune system to recognize and eliminate cancer cells in any FLC patient with the characteristic gene fusion, in contrast to cancers for which a neoantigen vaccine must be personalized for each individual patient.
The primary goal of the study is to begin assessment of the safety and clinical activity of the FLC-vaccine in combination with nivolumab and ipilimumab in patients for whom complete surgical resection of the cancer is not possible. We also seek to determine if the combination of peptide vaccine and checkpoint inhibitors will promote induction and/or expansion of T cells that specifically recognize the DNAJ-PKAc fusion protein.
- Blood markers for fibrolamellar
While at Johns Hopkins (he is now at Mayo Clinic in Rochester, MN), Dr. Michael Torbenson wrote the first paper on blood markers for fibrolamellar which was published in the journal, Modern Pathology, in late 2010. The article recognized FCF for their financial support. Dr. Torbenson was studying the microRNA of fibrolamellar cells and his laboratory was working on a genetic sequencing study of fibrolamellar to determine if there are genetic mutations unique to fibrolamellar cells.
Massachusetts General Hospital (Harvard University)
- Fibrolamellar Carcinoma Model Development and Analysis
2019 – 2021
Principal Investigator: Nabeel Bardeesy, PhD, Associate Professor, Massachusetts General Hospital, Harvard University
This is a resource development project for the fibrolamellar cancer research community. The primary goal is to derive a new series of transplantable human FLC tumors grown in immune-deficient mice [patient-derived xenograft (PDX) models]. The starting biospecimens will be obtained through the Fibrolamellar Cancer BioBank established by the FCF at Massachusetts General Hospital. The project also will contribute to the generation of three-dimensional cell culture models (3D tumor organoids) and established cell lines from human FLC tumors, in coordination with the FCF-funded project at the Broad Institute.
Analyses of organoid and cell line models will entail close interactions with the Broad Institute investigators and others in the FCF’s research network. Key shared goals include the identification of genetic dependencies and drug sensitivities of FLC tumor models, and “single cell landscape” analysis of gene expression. Studies of this type define the specific properties of individual cells among the various types found in a tumor: cancer cells; non-cancerous cells that may help to support the cancer such as the “stromal” cells that make up the characteristic fibrous bands for which fibrolamellar carcinoma was named; blood vessel cells; and cells of the immune system that may have potential to attack the cancer cells and stop tumor growth.
- Kinase fusion function investigation
2016 – 2018
Principal Investigator: Dr. Yi Guo, Ph.D, Associate Consultant – Asst Professor, Dept of Biochemistry and Molecular Biology
A recurring chromosomal deletion on human chromosome 19 was detected in at least 80% of FL-HCC cases, resulting a novel kinase fusion of DNAJB1-PRKACA (Cornella et al., 2015; Honeyman et al., 2014; Xu et al., 2015). While this novel fusion is identified as a potential oncogenic factor, the establishment of the causal and mechanistic relationship between the DNAJB1-PRKACA fusion and FL-HCC is critical for developing targeted cancer therapy.
This study aims to investigate the function of DNAJB1-PRKACA fusion in FL-HCC oncogenesis using both Drosophila and mice models. We established a DNAJB1-PRKACA transgenic Drosophila model and discovered abnormal phenotype affecting both proliferation and differentiation of Drosophila eyes. We also exploited CRISPR/Cas9 genome-engineering technology in murine cultured hepatocytes to recreate the endogenous chromosomal deletion as found in FL-HCC patients. For this Research Grant application, we proposed to 1) characterize the oncogenic and fibrogenic activities of genetic engineered murine hepatocytes in vitro and in vivo; and 2) screen potent therapeutics using the human DNABJ1-PRKACA over-expression model in Drosophila menalogaster. This study will provide essential resources and knowledge for fighting this aggressive hepatocellular carcinoma.
- Clinical trial
2012 – 2014
FCF funded the first clinical trial of drugs aimed specifically at fibrolamellar liver cancer. This trial was coordinated by Dr. Ghassan Abou-Alfa at Memorial Sloan Kettering Cancer Center (MSKCC). The trial was also conducted at other consortium members, including the University of California San Francisco, Johns Hopkins, and Dana Farber. Two major pharmaceutical companies donated the drugs.
MSKCC is sequencing the exome of the fibrolamellar genome.
MSKCC is the coordinator of the Fibrolamellar Consortium.
- Developing Therapeutics for Fibrolamellar Hepatocellular Carcinoma
2016 – 2017
Principal Investigator: Sandy Simon, Ph.D., Professor
Investigator Collaboration: Barbara A. Lyons, Ph.D., Professor, New Mexico State University
The goal of this work is to develop a therapeutic for fibrolamellar hepatocellular carcinoma (FLHCC). These are three different two-year projects, with independent synergistic strategies, to identify small molecules to treat fibrolamellar. The strategy is based on this laboratory’s published work that there is a single alteration in the DNA that is found in all fibrolamellar tumors: a deletion of 400kB that results in a fusion gene, a chimera of the heat shock protein DNAJB1 and the catalytic subunit of protein kinase A, PRKACA and on this laboratory’s unpublished work that the chimera with an active kinase is sufficient to cause fibrolamellar.
The first project is a high-throughput screen for molecules that directly block the chimera. This is an agnostic screen, which presumes no advanced knowledge about the chimera, with the goal of blocking its kinase activity. The screen will cover millions of compounds with no prior assumptions about what might work.
The second project is a screen to identify the molecules that are directly phosphorylated by the chimera (in contrast to identifying downstream elements that change as a consequence of the activity of the chimera).
The third project is an analysis of the structural dynamics of the chimera using a molecular dynamics simulation. This project is based on to-date-unpublished x-ray and NMR data on the structure of the chimera, together with molecular dynamics simulations, to identify sites on the chimera that would be appropriate for targeting therapeutics. Unlike the first project, which is an agnostic screen, this project is based on the hope that domains can be identified that are critical to the chimera’s.
- Discovery of the chimera genetic mutation
2014 – 2015
FCF funding to Rockefeller University resulted in a potentially game-changing discovery of a unique genetic mutation common to all fibrolamellar tissues studied, a chimera. This research was conducted at the Tucker Davis Research Facility at Rockefeller University. Dr. Sandy Simon is head of that facility and his daughter Elana, who is a fibrolamellar patient, was a lead researcher. The results were published in the preeminent medical journal, Science and reported in The Wall Street Journal, US News and World Report, AP, The Today Show, NBC Nightly News, and presented to President Obama.
The Foundation granted Dr. Sandy Simon funds to study immunotherapy and fibrolamellar. Rockefeller University has put their full support behind Dr. Simon and charges no administrative fees for this research. Rockefeller University has provided Dr. Simon with a dedicated space exclusively for fibrolamellar research, The Tucker Davis Fibrolamellar Research Facility. FCF provided a freezer for fibrolamellar tissue samples. Dr. Simon’s goal is finding a cure – total eradication from the body. He feels this path is through the immune system using the patients’ own antibodies to track and kill the cancer cells. His research also includes melanoma and breast cancer cells.
Dr. Simon has already discovered a way to extract antibodies from a patient, mark them, and re-introduce them into the body. The marked antibodies can attach to the smallest of cancer cells which will help surgeons and pathologists determine, during surgery, whether all the cancer has been removed.
- Developing pre-clinical models for fibrolamellar FL-HCC: Therapeutic target identification and testing
2016 – 2018
Principal Investigator: Dr. Julien Sage, Ph.D., Associate Professor, Department of Pediatrics and Genetics
Fibrolamellar hepatocellular carcinoma (FL-HCC) is a rare but lethal form of liver cancer for which few therapeutic options are available. Major barriers hampering the development of better therapies for FL-HCC patients include the rarity of the disease and the fact that many of these patients are children, limiting the implementation of clinical trials. One solution to this problem can come from the development of accurate pre-clinical models of FLHCC; such models can be used both to investigate the basic mechanisms of FL-HCC development, which may help identify new therapeutic targets, and to test novel therapeutic strategies. Here we propose to generate a mouse model for FL-HCC. We and others have analyzed the sequence of FL-HCC patients and identified a distinct genetic alteration resulting in the fusion of two proteins (a small piece of “DnaJ” and a larger piece of “PKA” are fused together). This DnaJ-PKA fusion is present in all the FL-HCC tumors sequenced so far and we showed it has pro-tumorigenic effects in cells.
Based on the pattern of development of FL-HCC, mostly in children and young adults, we hypothesize that the expression of the DnaJ-PKA fusion initiates cancer in specific liver stem/progenito cell populations during liver development. We have introduced the DnaJ-PKA DNA fusion into the genome of mice but the fusion protein can only be expressed upon activation of an activating enzyme (named “Cre”). To test our hypothesis, we will introduce the Cre enzyme at specific stages of liver development using genetic tools in mice.
If tumors develop, this will conclusively demonstrate that expression of the DnaJ-PKA fusion is an essential step in FL-HCC development, providing novel insights into the mechanisms of FL-HCC development. In addition, the generation of mice developing FL-HCC tumors resembling human tumors would provide a pre-clinical platform to test new therapeutic strategies.
For additional details see the article published by Stanford on FCF’s grant and collaboration.
- Characterizing enzyme inhibition of the DnaJPKAc chimeric protein derived from fibrolamellar hepatocellular carcinomas
2017 – 2019
Principal Investigator: Hibba tul Rehman, M.D., University of Vermont
Scientists have identified a unique fusion protein, DnaJ-PKAc, which causes this cancer. When overexpressed, this protein causes havoc in the downstream signaling pathways that are involved in cell growth and metabolism and is thought to be the key driver for cancer growth. Based on the 100% presence of the chimeric DnaJ-PKAc protein in FL-HCC patients, we are proposing a two pronged approach towards the regulation of the cancer cells. First, we will screen a previously developed peptide library to test the substrates against purified wild-type vs. the chimeric DnaJ-PKAc in vitro. Second, we propose to develop a library of inhibitory peptides that would preferentially inhibit DnaJ-PKAc. The studies suggested here should allow us to develop inhibitors that regulate the function of the chimeric enzyme without affecting the wild- type enzyme, thus selectively targeting cancer cells without affecting healthy tissue.
- MicroRNA research support
Dr. Y.Z. Wang published his findings on the microRNA research he conducted on cancer. He has thanked FCF for supporting his effort. His findings will help other microRNA researchers who are studying other cancers, including Dr. Torbenson at Johns Hopkins University who is studying the microRNA of fibrolamellar. Dr. Wang’s paper sets an important precedent in cancer research in that it reports discrete molecular (microRNA) differences between tumors which metastasize and perfectly matched tumors that do not. These findings may have diagnostic, prognostic and most important of all, therapeutic implications.
- Targeting DNAJB1-PRKACA Driven Signaling Dependencies in Fibrolamellar Liver Cancer
2019 – 2021
Principal Investigator: John Gordan, MD, PhD, Assistant Professor, Department of Medicine, UCSF
Co-Investigator: Nabeel Bardeesy, Associate Professor, Massachusetts General Hospital, Harvard University
Even with the identification of a near universal DNAJB1-PRKACA gene fusion (encoding a chimeric protein with a domain of heat shock protein 40, HSP40, fused to most of the enzymatically active subunit of protein kinase A, PKAc) as sufficient to trigger fibrolamellar liver cancer (FLC), no treatments directed at this target are clinically available, and most patients with FLC receive cytotoxic chemotherapy. In particular, no PKA inhibitors ready for human studies are yet in hand.
Over the past two years, we have mapped the signaling cascade downstream of PRKACA in FLC and other tumors. This analysis highlights Aurora Kinase A (AURKA) as a key mediator of oncogenic growth. AURKA is best known for regulating the cell cycle, but also promotes cell survival and the expression of oncogenic genes (i.e., those that contribute to cancerous growth). Most conventional AURKA inhibitors fail to strongly inhibit the growth of human FLC cells. This finding seems consistent with limited activity observed with such a drug in clinical trials. However, colleagues at UCSF described a novel class of AURKA inhibitors designed to disrupt its interaction with members of the MYC family of oncoproteins, which are critical drivers of many cancers. We find that one of these new AURKA inhibitors does potently reduce proliferation of FLC cells. The drug also reduces expression of MYC-family oncogenic transcription factors. We hypothesize that AURKA-mediated stabilization of MYC is necessary to maintain growth of FLC cancer cells. Although these new AURKA inhibitors are not yet ready for human use, by studying them now we can understand if they are likely to be effective for FLC and whether they work well in combination with other available drugs. We plan to assess the activity and mechanism of conformation disrupting AURKA inhibitors in FLC laboratory models, including human tumors grown in mice, with the goal of identifying a drug in this class that could be advanced to clinical testing in FLC patients.
[Note that the original grant to U Washington with Ray Yeung as PI does not seem to have been listed on the web site. I suggest using the original lay Abstract, with minor edits. MEF]
- Flipping the switch on PKA: synthetic lethal approaches to block PKA-driven tumor growth in fibrolamellar liver cancer
2016 – 2019
Principal Investigators: John Gordan. M.D., Ph.D, Clinical Instructor, University of California San Francisco; Nabeel Bardeesy, Ph.D, Associate Professor, Harvard University
The discovery of a genetic change in the protein kinase A (PKA) gene in most cases of fibrolamellar liver cancer (FLC) creates hope that targeted therapy against PKA will have potent effects for FLC patients. However, progress is impeded by the relative scarcity of established model systems and the current lack of an effective anti-PKA drug. PKA is a component of the G protein-coupled receptor (GPCR) pathway, which is thought to play a role in many other cancer types. However, little is known about how this pathway makes tumors grow, and if it creates any specific liabilities in tumor cells that can be effectively targeted even when PKA is still active. We hypothesize that common mechanisms support the growth of different cancers where PKA is abnormally activated and that deciphering these mechanisms will lead to new treatment strategies for FLC.
In this project, we will apply cutting-edge proteomic methods to comprehensively map biochemical processes controlled by GPCRs and PKA across a number of cancer cell lines. We will complement these efforts with genetic approaches to identify other genes that are essential for PKA-driven cancer growth. Finally, we will use newly developed FLC models to test key targets identified with our screening techniques.
By identifying and rigorously testing the importance of the mediators of PKA signaling in FLC, we will be positioned to repurpose existing drugs to accelerate progress in the treatment of patients with FLC.
- Molecular Therapies for Fibrolamellar Carcinoma (FLC)
2019 – 2021
Principal Investigator: John Scott, PhD, Edwin G. Krebs-Speights Professor of Cell Signaling and Cancer Biology, and Chair, Department of Pharmacology
Successes in precision medicine have identified the underlying genetic defect in FLC as a deletion in chromosome 19. Consequently, FLC patients produce a unique protein where an important part of heat shock protein 40 (DNAj) is fused to a key cellular enzyme called protein kinase A (PKAc). This chimeric protein, DNAJ-PKAc, is only expressed in FLC tumors where it hijacks normal cellular processes, leading to cancer.
We believe that DNAJ-PKAc brings together unique combinations of cellular enzymes that cause FLC. These protein complexes activate the biochemical pathway downstream from the fusion protein, deregulating cell proliferation. Thus, drugs that target the key protein combinations represent a therapeutic opportunity. We have discovered drug pairs that halt the growth of genetically modified liver cells that mimic the human cancer. Our experimental plan is to test a new concept in drug treatments for FLC. Rather than blocking the action of the DNAJ-PKAc protein kinase enzyme itself, we will use combinations of FDA-approved drugs and/or drugs already in clinical testing that neutralize proteins associated with this chimeric enzyme. In particular we believe that combinations of drugs targeting proteins that bind tightly to DNAJ-PKAc, such as heat shock protein 70 (Hsp70) and mitogen-activated protein kinases (MAPKs), will offer a viable therapeutic option. The motivation behind this pharmacological approach comes from our conviction that repurposing these drugs will expedite a cure for FLC.
- Modulating stromal-immune cell interactions to activate anti-tumor immunity to fibrolamellar carcinoma
Principal Investigator: Venu Pillarisetty, MD, Associate Professor, Division of General Surgery [Extension after CRI Fellowship grant to Kevin Sullivan in Dr. Pillarisetty’s lab, 2016-2019]
Immunotherapy, harnessing the patient’s immune system to precisely target cancer cells, has emerged as a promising approach to treat many cancers. We and others have found that fibrolamellar carcinoma (FLC) tumors contain a class of immune cells called T cells that potentially could recognize and destroy the cancer cells. However, the therapeutic ability of T can be limited by suppressive factors made by both cancer cells and other cells in the tumor such as the abundant fibroblastic stromal cells that give FLC its name. Suppressive molecules in the tumor microenvironment that may limit anti-cancer immune responses include “immune checkpoints” and signaling molecules known as cytokines. The latter include chemokines, small protein chemical messengers that influence cell migration. We will determine whether blockade of checkpoints and/or signaling by a specific chemokine (CXCL12) can enhance T cell mediated immunity against fibrolamellar cancer cells, using slice cultures of human FLC tumors.
- Therapeutic Innovations in Fibrolamellar Cancer
2017 – 2019
Principal Investigator: Raymond Yeung, MD, Professor of Surgery
Fibrolamellar cancer (FLC) is the most lethal form of liver cancer in adolescents and young adults. Currently, there is no effective therapy for these patients besides surgery. With the identification of a unique genetic defect that likely affects all FLCs (the DNAJB1-PRKACA gene fusion), the opportunity to find a cure for this disease is now within reach. Armed with a novel set of immortalized cell lines developed at the University of Washington that bear the FLC mutation, we will advance our understanding of the mechanisms that drive FLC development. Firstly, we will identify protein kinase pathways that are activated downstream of the mutation using a combination of global and targeted phosphoproteomic analyses. These results will be functionally validated using our FLC cell lines as well as human FLC samples. Secondly, based on our preliminary observations that the mutant protein associates with heat shock protein (HSP) 70, we will investigate the role of HSPs in FLC, including their pro-survival function in keeping cells alive during stress. Together, our proposed studies will unveil mechanistic insights and new therapeutic targets that will bring us closer towards a cure for this deadly disease.
- Iodine Transporter Research
2014 – 2015
FCF funded Dr. Nancy Carrasco at Yale School of Medicine to analyze FLC cells to discover whether they could be effectively targeted by radioactive iodine. Radioactive iodine has a long history as a safe and effective treatment for thyroid cancer. Research had indicated that fibrolamellar cells might have pathways similar to those of thyroid cancer cells. Use of radioactive iodine on several patients did not confirm this theory and the project was terminated.