Funded model development efforts

Below are summaries of FCF-funded projects focused on developing improved disease models and diagnostic tools.

Project summaries: Model and diagnostic tools development

Development of a human-derived liver progenitor cell line with DNAJB1-PRKACA fusion gene and characterization of mitochondrial dynamics in fibrolamellar carcinomaBoston Children’s HospitalActive

Timeframe: 2022 - 2023

Goal: Development of a novel human-derived liver progenitor cell line model of fibrolamellar carcinoma

Principal Investigator: Khashayar Vakili, MD, Surgical Director of Liver, Kidney, Intestine, and Multivisceral Transplant Programs, Assistant Professor of Surgery, Harvard Medical School

Currently, very few cell-line models of FLC exist, so the development of additional cellular models is critical for the identification and testing of new therapies. Therefore, one of the aims of this proposal is to engineer normal liver cells in culture to express the DNAJB1-PRKACA gene fusion. This will be a novel model, which can provide insight into the mechanism of cancer development in addition to providing a platform for screening new therapeutic compounds. The second aim of this proposal is to assess the role of the mitochondria in FLC. Mitochondria are structures within cells that provide energy to the cells in addition to other vital functions. Mitochondria have been shown to develop abnormal characteristics in various types of cancers - leading to the hypothesis that they play a critical role in cancer survival and progression. Currently, there is very limited data available about the characteristics and role of mitochondria in FLC.

CRISPR-engineering of human liver organoids to study fibrolamellar carcinomaPrincess Maxima Center for Pediatric OncologyActive

Goal: Model development

Principal Investigator: Benedetta Artegiani, PhD, Artegiani Group, Princess Maxima Center

Research into FLC is complicated because of the limited experimental tools that can be used to study this rare cancer. While tests in laboratory animals such as mice are useful tools, they do not reflect the human disease due to inherent differences between mice and humans. To date, experimental models that can provide insights into human disease development and progression are highly sought after.

In this research project, the researchers will build new models using human cells to study FLC. The researchers will make use of lab-grown three-dimensional human mini-livers, so-called “organoids”. These organoids can be grown from the two most important cells in the liver: the ductal cells and the hepatocytes. The researchers will then precisely modify the DNA of the organoids using CRISPRCas9 technology, also called molecular scissors, to mimic the mutations (changes in the DNA) that have been found in FLC. The researchers will also try to identify from which particular liver cell type the cancer is initiated.

With these newly built human FLC models the researchers hope to increase our current understanding of FLC, which may lead in the future to the discovery and testing of novel therapeutic strategies.

Development and validation of an orthotopic, syngeneic, mouse model for fibrolamellar hepatocellular carcinomaJohns Hopkins UniversityActive

Timeframe: 2021 – 2022

Goal: Model development

Principal Investigator: Mark Yarchoan, MD, Associate Professor, Oncology/Division of GI Malignancies, Sidney Kimmel Comprehensive Cancer Center

Efforts to identify novel therapies for FLC have been confounded by a lack of preclinical models that accurately reflect the genetics and biology of the disease. This study aims to establish and validate an orthotopic, syngeneic, preclinical mouse model of FLC that faithfully recapitulate human FLC. Because the tumors in such models grow in the context of an intact immune system, they are therefore appropriate models to study agents that act on the host immune system to enhance tumor immunity such as checkpoint inhibitors. The resulting preclinical model of FLC will be openly shared with the larger research community. If successful, this model will offer unprecedented opportunities to investigate mechanisms underlying FLC pathogenesis, and will become a critical tool for investigating novel therapeutic strategies in FLC.

Creating a fibrolamellar cancer dependency mapBroad Institute of MIT and HarvardActive

Timeframe: 2020 – 2023

Goal: Create a comprehensive list of potential drug targets for FLC

Principal Investigator: Jesse Boehm, PhD, Institute Scientist, Director of the Broad Cancer Model Development Center

This project is part of the Broad Institute’s Rare Cancer Dependency Map Initiative. The project has three main goals to identify potential FLC therapeutics:

  1. Developing new cell models of FLC for the research community. Harnessing the Broad Institute’s Cancer Cell Line Factory laboratory and its combinatorial media screening technology will allow the team to systematically determine the conditions necessary to grow FLC samples as three-dimensional organoid models. They will also work in coordination with the FCF-sponsored Biobank to create a unified pipeline by which any patient can direct tissue to FLC researchers.
  2. Utilizing the developed cell culture models of FLC to create a comprehensive list of potential drug targets and to identify existing drugs that may have therapeutic potential against FLC.
  3. Empowering the entire FLC research community by sharing all developed genomically characterized and clinically annotated cell models and by making all the data and biologist-friendly analysis tools freely available online, pre-publication at

If fully successful, this effort will nominate high priority targets for drug discovery as well as new drug repurposing hypotheses.

Fibrolamellar carcinoma model development and analysisMassachusetts General HospitalActive

Timeframe: 2019 – 2022

Goal: Model development

Principal Investigator: Nabeel Bardeesy, PhD, Associate Professor, Massachusetts General Hospital, Harvard University

Multiple FLC models have recently been developed, which have advanced our understanding of this disease. However, additional model development remains a priority for the FLC community. This effort aims to develop multiple different FLC models as resources for the FLC research fibrolamellar cancer research community, and will collaborate with the Fibrolamellar Cancer Biobank established at Massachusetts General Hospital to obtain biological specimens. These specimens will be used to create series of transplant human FLC tumors grown in immune-deficient mice (patient-derived xenograft [PDX] models), three-dimensional cell culture models (3D tumor organoids), and cell lines in partnership with the Broad Institute.

Together with collaborators at the Broad Institute and throughout the FCF research network, the study team will harness these newly developed models to identify genetic dependencies and drug sensitivities of the disease. (see also the Broad Institute initiative). Additional studies will be aimed at further understanding this cancer by examining gene expression at the single cell level and characterizing non-cancerous cells that support or interact with tumor cells such as immune cells and stromal cells that make up the characteristic fibrous bands for which fibrolamellar carcinoma was named. These studies aim to better understand the molecular mechanisms underlying FLC formation and growth and ultimately will set the stage for the development of new therapeutics.

Liquid Biopsy for Active Surveillance of Fibrolamellar Hepatocellular CarcinomaUniversity of VermontActive

Timeframe: 2021 – 2022

Goal: Develop a blood-based screening method to detect relapse in FLC patients

Principal Investigator: Gary S. Stein, PhD, Department of Biochemistry, University of Vermont Cancer Center

This study is focused on developing a blood-based screening method to detect relapse in FLC patients that have been diagnosed and treated by surgery. This screening protocol is intended to be non-invasive, to overcome limitations of current imaging strategies, and to be accessible and affordable for patients and their families. Currently, treatment of patients with FLC is limited by the understanding of how this cancer responds to therapies. Cancer physicians currently have limited ability to test for, and monitor patients’ response to therapy, and to detect disease progression. It is therefore important to have effective monitoring strategies for physicians to determine if patients are successfully remaining in remission, or if they are progressing towards the earliest stages of relapse. Catching relapsed tumors early, prior to spreads throughout the body, is critically important to increase FLC patient survival.

The study is evaluating the use of DnaJ-PKAc transcripts in circulating nucleated cells as a proxy for detecting the presence of circulating tumor cells (CTCs). A major challenge to detecting and quantitating CTCs is their extremely low abundance – roughly 1 to >100 per 7.5 mL of whole blood, compared to the billions of other cells typically found in blood. The objective of this initiative is to test whether the DnaJ-PKAc transcript can be detected in blood samples and to establish the threshold of transcript detection. We will utilize an engineered model of FLC that possesses the DnaJ-PKAc fusion gene as a substitute for actual CTCs. These pseudo-CTCs will be spiked into blood samples from normal donors to simulate whole blood from a metastatic or progressive disease scenario.

Development of a human-derived liver progenitor cell line with DNAJB1-PRKACA fusion geneBoston Children’s HospitalCompleted

Timeframe: 2019 – 2021

Goal: Development of a novel human-derived liver progenitor cell line model of fibrolamellar carcinoma

Principal Investigator: Khashayar Vakili, MD, Surgical Director of Liver, Kidney, Intestine, and Multivisceral Transplant Programs, Assistant Professor of Surgery, Harvard Medical School

The study team's lab previously engineered a kidney cell line (HEK-DP) which contains the DNAJB1-PRKACA fusion gene found in FLC tumors. This cell line demonstrates interesting similarities to FLC tumors and serves as a proof of concept for the development of additional cell lines. This effort applied apply the same strategy used to engineer the HEK-DP model to engineer normal human liver progenitor cells to express the DNAJB1-PRKACA fusion gene. These liver progenitor cells were to be grown as three-dimensional “organoid” cultures to better replicate in vivo conditions.

If successful, this novel model will facilitate understanding how the fusion protein reprograms normal liver progenitor cells to become cancerous. Understanding the precise mechanisms underlying the formation of FLC will provide insights to future therapeutic strategies.

A gene editing approach to develop new and effective models for FLCUniversity of WisconsinCompleted

Timeframe: 2021

Goal: Generate a mouse model of FLC

Principal Investigator: Sean Ronnekleiv-Kelly, MD, Surgical Oncology

The purpose of this study was to generate a robust pre-clinical murine model of FLC that can provide a basis for understanding factors contributing to FLC formation, and for therapeutic development. The study proposed to develop a mouse model of FLC using a gene editing approach in a susceptible population, using hydrodynamic delivery of gene editing material to the liver via retro-orbital injection at age 7-8 weeks in mice with varying susceptibility to liver tumor formation. This included C57BL/6 mice, FVB mice, C3H mice, Balb/c mice and DBA/2 mice, which have a range of 2-3 fold lower vulnerability to 3-7 fold higher susceptibility to liver cancer development. In this manner, they wanted to identify if different susceptibilities to liver tumor formation causes earlier onset / more aggressive cancer, which could then ultimately improve the understanding of FLC development. In a second subset of the same mouse strains, they performed retro-orbital injection of the mice at age 2 weeks, a timeframe selected because the fusion gene mutation is an early somatic event in humans (i.e. peak age of FLC diagnosis is 21 years). Most existing studies targeted the DNAJB1-PRKACA mutation to the liver at age 7-8 weeks. Targeting the gene editing material to generate the fusion gene at 2 weeks (when liver cells are still actively dividing) may create a different tumor phenotype compared to existing models.

Role of the innate immune system in fibrolamellar hepatocellular carcinoma (FL-HCC) using zebrafish as a model systemUniversity of WisconsinCompleted

Timeframe: 2016 – 2019

Goal: Use zebrafish as a model system for fibrolamellar carcinoma to study the immune system

Principal Investigator: Sofia de Oliveira, Ph.D., EMBO Postdoctoral Fellow, Huttenlocher Immunology Lab

Few FLC animal models currently exist limiting our ability to study FLC in the context of a complete organism. While cell-based models are extremely useful, animal models allow scientists to study biological processes involving multiple organs and cell types, such as tumor immunology and metastasis. Zebrafish are a valuable tool to study many diseases including cancer and have been used as a model system by the genetics community for decades. They display remarkable similarities and share many genetic signatures with humans and have been used to study liver development, hepatocellular carcinoma, and several other liver disorders. This effort planned on developing a zebrafish model of FLC and harnessing this model to study how the immune system interacts with FLC.

Developing pre-clinical models for fibrolamellar FL-HCC: therapeutic target identification and testingStanford UniversityCompleted

Timeframe: 2016 – 2018

Goal: Develop a pre-clinical mouse model for fibrolamellar carcinoma

Principal Investigator: Dr. Julien Sage, Ph.D., Associate Professor, Department of Pediatrics and Genetics

There are few effective therapies for FLC patients and development of improved therapeutics are hampered by the rarity of the disease and the challenge of including pediatric patients in many clinical trials. One solution to this problem is the development of accurate models of FLC. This study proposed to generate a mouse model for FLC. Because FLC occurs primarily in children and young adults, they hypothesized that the expression of the DnaJ-PKA fusion found in FLC tumors cells initiates cancer in specific liver stem/progenitor cell populations during development. Using genetic engineering techniques, they attempted to introduce the DnaJ-PKA fusion into the mouse genome in an inducible manner. Therefore, they hoped to be able to switch on expression of DnaJ-PKA at specific stages of liver development to examine exactly how this fusion contributes for FLC formation. Such mouse models would provide a novel preclinical platform to test future therapeutic strategies.

For additional details see the article published by Stanford on FCF’s grant and collaboration.

Blood markers for fibrolamellarJohns Hopkins UniversityCompleted

Timeframe: 2010

Goal: Identify biomarkers

Principal Investigator: Michael Torbenson, MD, Department of Pathology, Johns Hopkins University (Currently at Mayo Clinic)

Early identification of tumors is essential for aggressive treatment. The majority of fibrolamellar carcinoma (FLC) patients have metastatic disease at the time of diagnosis. Therefore, identification of biomarkers for FLC is essential. Using a broad collection of FLC pathology specimens, the study planned to identify diagnostic biomarkers for FLC. In addition, they investigated whether these biomarkers are linked to any genetic mutations or microRNA expression profiles unique to FLC.