Funded disease mechanism research

Timeframe: 2022 - 2023

Goal: Identify functional role of mitochondrial alterations in FLC and FLC-Like BAP1 HCC

Principal Investigator: Jessica Zucman-Rossi, MD, PhD, Functional Genomics of Solid Tumors

Recently, important research studies have reported specific genomic abnormalities of tumor subtypes. One of these, representing the fusion between DNAJB1 and PRKACA genes, has been described to be typical of the classic fibrolamellar subgroup. Another subgroup of tumors harboring fibrolamellar-like features was recently identified by the Inserm lab and was characterized by inactivating mutations of BAP1 encoding the BRCA1 associated protein-1 (FLC-Like BAP1 HCC). Despite the fact that these two subtypes of tumors harbor particular tumor histology and specific major gene alterations, they also share common features including PKA activation and absence of liver disease. Potentially, other similarities could exist. Mitochondria, which are often referred to as the powerhouses of the cell, are critical for many cellular functions including energy production, metabolism or cell survival. They have their own DNA called mitochondrial DNA or mtDNA. It has been reported that mitochondria number abnomalities and mtDNA sequence and copy number alterations were found in some tumors including FLC. However, their biological and clinical significances in FLC malignancy is poorly understood.

In this study, the team aims to understand the mitochondria alterations in FLC and FLC-like BAP1 HCC at different levels. Thus, they will study both the nuclear and mitochondrial DNA alterations, the number and the distribution of mitochondria within the tumor, paying attention to the functional consequences of the abnormalities. The findings should provide new insights in mitochondria significance in FLC, prioritizing next steps toward the development of novel and effective therapeutics.

Timeframe: 2022 - 2023

Goal: Knock-out gene targets in a mouse model of FLC to identify potentially effective drug combinations for patients with advanced FLC

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

This study will apply an innovative gene-editing based technology (genome-wide CRISPR knockout screen) on a mouse cellular model to screen all possible cancer targets (i.e., all genes) coupled with promising drugs to precisely identify the most lethal drug combinations in FLC. This knowledge will enable discovery of new and effective combined drug treatments for FLC that can overcome the cancer cell adaptive mechanism, and work cooperatively to maximize the tumor-killing effect.

The rationale is threefold. First, combination drug therapy is requisite for treatment resistant cancers like FLC, yet identifying optimal therapeutic combinations has been exceedingly difficult with existing methods. Second, genome-wide CRISPR screens knockout each of the ~20,600 protein coding genes in the genome (one gene per cancer cell) to identify specific genes associated with cancer cell lethality. Remarkably, this unique approach can couple each possible target (i.e. entire genome) with concurrent promising drug treatment (i.e. a core ‘backbone’ drug) to precisely reveal clinically relevant multi-drug therapy for FLC. Third, this innovative approach could identify and exploit the essential pathways by which the DNAJB1-PRKACA driver gene mutation promotes FLC aggressiveness and treatment resistance.

In future studies, any novel drug treatments identified will be tested in pre-clinical animal models meant to closely mimic human FLC, followed by evaluation in patients with this deadly cancer.

Timeframe: 2020 - 2023

Goal: Investigate the impact on FLC growth and survival of inhibiting two specific oncogenes identified in previous works

Principal Investigator: Praveen Sethupathy Associate Professor Department of Biomedical Sciences, Cornell University, College of Veterinary Medicine

Based on previous epigenomic, metabolomic, and microRNA profiling, as well as initial drug studies, the study team has developed two new exciting hypotheses about therapeutic vulnerabilities in FLC. First, they hypothesize that inhibition of two candidate FLC oncogenes, CA12 or SLC16A14, independently and/or in conjunction with FDA-approved drugs, will dramatically reduce FLC cell viability, proliferation, and invasive capacity. Second, they hypothesize that the increase in LDHB promotes glycolysis and FLC tumor cell survival, which can be reversed by miR-375 mimics. In this project, the team proposes to test these hypotheses in several different disease models of FLC. The findings from the proposed studies could potentially lay the foundation for completely novel, effective strategies for molecular therapy.

Timeframe: 2021 - 2022

Goal: Elucidate the molecular mechanisms of FLC and identify new pharmacological agents that could lead to new, effective therapeutics

Principal Investigator: Jin Zhang, PhD, Vice Chair and Professor of Pharmacology, Department of Pharmacology

In recent studies, the investigation team discovered that the presence of the FLC oncogenic fusion protein disrupts a membraneless organelle. They showed that loss of this membraneless organelle in normal cells results in aberrant signaling as well as increased cell proliferation and transformation. Based on these data, they hypothesize that loss of this membraneless organelle induced by the oncogenic fusion leads to defects in cellular functions and drives tumor formation. The identification of pharmacological agents that recover this membraneless organelle in the presence of the oncogenic fusion protein, could provide useful leads for developing new therapeutics. In this proposed research, the team will test these hypotheses by combining a variety of novel approaches, including live-cell biochemistry. They will undertake mechanistic studies in the established model systems and perform drug screens, in conjunction with additional collaborators with complementary expertise. The study should provide new insights into the cause of FLC and enable new therapeutic strategies to tackle this lethal cancer.

Timeframe: 2019 - 2022

Goal: Investigate the potential of AURKA inhibitors for FLC treatment

Principal Investigator: John Gordan, MD, PhD, Assistant Professor, Department of Medicine, UCSF

Co-Investigator: Nabeel Bardeesy, Associate Professor, Massachusetts General Hospital, Harvard University

Even though the DNAJB1-PRKACA gene fusion (encoding a chimeric protein with a domain of heat shock protein 40, HSP40, fused to a majority of the enzymatically active subunit of protein kinase A, PKAc) is sufficient to trigger fibrolamellar liver cancer (FLC), no treatments directed at this target are clinically available. Most FLC patients receive chemotherapy and no PKA inhibitors are currently in clinical use.

Over the past two years, the study team has mapped the signaling cascade downstream of PRKACA in FLC and other tumors (see completed project below). 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 is 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. One of these new AURKA inhibitors does potently reduce the proliferation of FLC cells. The drug also reduces expression of MYC-family oncogenic transcription factors. The team hypothesizes 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, the team will studying them to understand if they are likely to be effective for FLC and whether they work well in combination with other available drugs. They 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.

Timeframe: 2019 - date

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, Edwin G. Krebs-Speights Professor of Cell Signaling and Cancer Biology, and Chair, Department of Pharmacology

Precision medicine approaches have identified the underlying genetic defect in FLC as a deletion in chromosome 19. Consequently, FLC patients produce a unique protein in which 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 expressed in FLC tumors where it hijacks normal cellular processes, leading to cancer.

It is believed 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 such key protein combinations represent a therapeutic opportunity. In a past study, the University of Washington team discovered drug pairs that halt the growth of genetically modified liver cells that mimic the human cancer. Their 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, they will use combinations of FDA-approved drugs and/or drugs already in clinical testing that neutralize proteins associated with this chimeric enzyme. In particular, they 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. They hope that such a pharmacological approach and the use of repurposed FDA-approved drugs will expedite a cure for FLC.

Renewal addition:

During the past funding cycle, the study team discovered drug combinations that target “signaling island” in FLC tumors that harbor DNAJ-PKAc. Eliminating these oncogenic focal points is a new frontier in FLC research. Accordingly, there are three main components of this funding renewal: 

1. Using innovative pharmacological approaches to determine which part of DNAJ-PKAc causes cancer. 
2. Advancing towards clinical trials any promising drug combinations that target DNAJ-PKAc signaling islands. 
3. Working in partnership with FCF to inform patients and families of the latest breakthroughs in translational research.

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 DepMap.org.

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

Timeframe: 2020 - 2021

Goal: Document mechanisms driving FLC and test if treatment with inhibitors of b-catenin may be a potential therapeutic approach for FLC

Principal Investigator:  Nikolai Timchenko, PhD, Head of Liver Tumor Biology, Liver Tumor Program; Professor, UC Department of Surgery

In the course of studies of pediatric liver cancer, we have identified chromosomal regions (Aggressive Liver Cancer Domains, ALCDs) in multiple liver cancer genes. Expression of the ALCD containing genes is increased in liver cancer. The project team identified several regions within ALCDs that might be activated by transcription factors and chromatin remodeling proteins. One of these regions contains an ideal binding site for b-catenin-TCF4 complexes. Given recent reports showing that the mutant kinase DNAJB1-PKAc phosphorylates b-catenin, they hypothesized that DNAJB1-PKAc activates ALCD-containing cancer genes leading to FLC pathology. Preliminary studies of FLC patient tumor samples show that the DNAJB1-PKAc phosphorylates b-catenin at Ser675 leading to the formation of 􀁅-catenin-TCF4 complexes and that these complexes bind to the ALCDs. They also identified the ALCD-containing cancer genes that are specifically upregulated in patients with FLC.

Therefore, the main hypothesis investigated in this effort is that DNAJB1-PKAc-b-catenin-ALCD axis plays a critical role in development of FLC. They:

• Examined if the DNAJB1-PKAc-b-catenin-TCF4 pathway activates known ALCD-dependent cancer genes in FLC patients
• Investigated new, FLC-specific ALCD-containing genes in FLC patients and determine mechanisms by which DNAJB1-PKAc-b-catenin pathway activates expression of these genes
• Examined if the inhibition of the DNAJB1-PKAc-b-catenin-TCF4 pathway by b-catenin inhibitor PRI-724 inhibits development of FLC in a mouse model of FLC.

The project aimed to provide critical knowledge of the mechanisms of FLC and test if treatments with inhibitors of b-catenin might be considered a potential therapeutic approach for FLC.

Timeframe: 2019-2022

Goal: Investigate the potential of retinoic acid therapy

Principal Investigator: Andrew Yen, PhD, Professor, Department of Biomedical Sciences

Co-Principal Investigator: Praveen Sethupathy, PhD, Associate Professor, Department of Biomedical Sciences

FLC is driven by the DNAJ-PKAc fusion protein. A potential therapeutic strategy would be to induce loss of this key driver protein. One approach to substantially alter gene expression in cancer cells is differentiation induction therapy, which 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 proliferating malignant state resembling immature white blood cells to a non-transformed, arrested state resembling the corresponding normal, mature white blood cells. Preliminary observations in a model cell line engineered to stably express DNAJ-PKAc showed that RA causes loss of the fusion protein.

This suggests the possibility that retinoic acid could have therapeutic activity against FLC by causing loss of the transforming protein for this tumor, thereby relieving the hepatic cells of the tumor phenotype. The study exploited the observation in this experimental model and extended it to primary cultured FLC cells. The project goals were to:

1. Determine if retinoic acid causes loss of the fusion protein in FLC cells, and
2. Characterize the molecular signature and cellular attributes of the retinoic acid-induced FLC cell response.

If successful this effort will:

1. Demonstrate that retinoic acid, a drug already approved and used in leukemia therapy, has an off-label application for FLC, and
2. Identify candidates to target for more sophisticated combination therapy, an emerging therapeutic modality that is proving effective in retinoic acid based therapy against other tumors.

The basic rationale is that if a drug relieves the FLC cells of the tumor causing protein, then the tumor phenotype would be relieved.

Timeframe: 2017 - 2020

Goal: Investigate the role of microRNAs and long non-coding RNAs in fibrolamellar carcinoma and evaluate RNA-based therapeutics

Principal Investigator: Praveen Sethupathy Associate Professor Department of Biomedical Sciences, Cornell University, College of Veterinary Medicine

This study aimed to leverage genome-scale approaches to discover the molecular factors most critical to FLC tumor formation, metastasis, and drug resistance. MicroRNAs and long non-coding RNAs are RNA species that do not contain instructions for protein formation yet are vital to numerous biological processes including tumor formation. Specific study efforts included:

• Identifying microRNAs and long non-coding RNAs that facilitate FLC tumor formation/invasion
• Evaluating the potential of RNA-based therapeutics to inhibit their activity
• Integration of these results with multiple large-scale genomic and metabolomic datasets to identify critical druggable pathways.

For a description of some of the lab's published work, visit <https://www.cmghjournal.org/article/S2352-345X(19)30010-4/fulltext> and<http://news.cornell.edu/stories/2019/02/key-rare-aggressive-liver-cancer-found-rna-molecule>.

Timeframe: 2017 - 2019

Goal: Evaluate the role of Hedgehog and YAP signaling in fibrolamellar carcinoma

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 histological appearance consisting of large tumor cells surrounded by thick fibrous bands, the stroma. Molecular crosstalk between tumor and stroma is important in tumor maintenance and progression. Decoding the important signals in these interactions will reveal new potential therapeutic targets. This effort aimed to study the role of Hedgehog (Hh) signaling in tumor-stromal interactions. Hh signaling is important in normal liver development and regeneration as well as tumor-stromal interactions in other cancers. Active Hh signaling also activates a protein called Yap, which results in stroma accumulation and primitive stem cells, both of which are seen in FLC. The study examined the role of Hh and Yap signaling in tumor-stroma interactions and their effect on tumor growth and progression.

Timeframe: 2016 - 2018

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

Principal Investigator: Dr. Yi Guo, Ph.D, Associate Consultant – Asst Professor, Dept of Biochemistry and Molecular Biology

The DNAJB1-PRKACA fusion kinase is found in nearly 100% of FLC cases. While this novel fusion has 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. They has 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. They also exploited CRISPR/Cas9 genome-engineering technology in murine cultured hepatocytes to recreate the endogenous 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.

Timeframe: 2017 - 2019

Goal: Characterize inhibition of the DnaJ-PKAc chimeric protein in fibrolamellar carcinoma

Principal Investigator: Hibba tul Rehman, M.D., University of Vermont

The DNAJB1-PRKACA fusion gene produces the DnaJ-PKAc fusion kinase protein, which is present in nearly all FLC tumors and promotes liver tumor formation in mice. Kinases, including fusion kinases, have been successful drug targets in numerous cancer types. Inhibition of DnaJ-PKAc may provide the first targeted therapy for FLC. This study proposed a two pronged approach towards identifying therapeutic inhibitors of the fusion:

1. Screening a previously developed peptide library to identify peptides that preferentially bind chimeric DnaJ-PKAc over normal, wide-type protein in vitro.
2. Developing a library of inhibitory peptides that would preferentially inhibit DnaJ-PKAc.

These aim of these efforts was to develop an understanding that will support the development of inhibitors that regulate the function of the chimeric kinase without affecting the wild-type kinase, thus selectively targeting cancer cells without affecting healthy tissue.

Timeframe: 2016 - 2019

Goal: Understand growth mechanisms and identify potential therapeutic targets

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 nearly all cases of fibrolamellar liver cancer (FLC) creates hope that targeted therapy against PKA will have potent effects for FLC patients. However, progress has been limited due to 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. The study team hypothesized 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.

This study applied cutting-edge proteomic methods to comprehensively map biochemical processes controlled by GPCRs and PKA across a number of cancer cell lines. These efforts were complemented with genetic approaches to identify other genes essential for PKA-driven cancer growth. Finally, newly developed FLC models were used to test key targets identified with our screening techniques. By identifying and rigorously testing the importance of the mediators of PKA signaling in FLC, the study hoped to lay groundwork for the repurposing of existing drugs to accelerate progress in the treatment of patients with FLC.

Timeframe: 2017 - 2019

Goal: Understand growth mechanisms and identify potential therapeutic targets

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 affects nearly 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, this study aimed to advance our understanding of the mechanisms that drive FLC development by:

• Identifying protein kinase pathways that are activated downstream of the mutation 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.

Timeframe: 2016 - 2017

Goal: Develop new therapeutics for FLC

Principal Investigator: Sandy Simon, Ph.D., Professor
Investigator Collaboration: Barbara A. Lyons, Ph.D., Professor, New Mexico State University

The goal of this work was to develop therapeutics for FLC. The team identified three independent strategies based on their laboratory’s work showing that a single common DNA alteration is found in all fibrolamellar tumors leading to the DNAJB1-PRKACA fusion and that this chimera is sufficient to cause fibrolamellar. The three projects covered included:

1. A high-throughput screen for molecules that directly block the chimera (covering millions of compounds with no prior assumptions about which compounds might work).
2. A screen to identify molecules that are directly phosphorylated by the chimera.
3. An analysis of the structural dynamics of the chimera using a molecular dynamics simulation to identify sites on the chimera that would be appropriate for targeting therapeutics.

Timeframe: 2014 - 2015

Goal: Identify genetic mutations in FLC

Principal Investigator: Sandy Simon, Ph.D., Professor, Rockefeller University

The goal of this project was to identify genetic mutations present in FLC. The team used whole genome sequencing together with RNA sequencing to identify mutations in FLC tumors compared to their normal liver counterparts. Identification of mutations common in FLC tumors is critical to enhancing the understanding of the underlying biology of this tumor and allow for development of novel therapeutics.

This research ultimately resulted in game-changing discovery of a unique genetic mutation, a chimeric gene, common to all fibrolamellar tissues studied. This research was conducted at the Tucker Davis Research Facility at Rockefeller University, led by Dr. Sandy Simon. 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.

Timeframe: 2014 - 2015

Goal: Investigate the potential of radioactive iodine as a therapeutic in FLC

Principle Investigator: Nancy Carrasco, MD, Professor, Cellular and Molecular Physiology, Yale University (Currently at Vanderbilt University)

FLC cells express high levels of the iodine transporter NIS, raising the possibility that radioactive iodine may be a potential therapeutic for FLC. Radioactive iodine has a long and effective history as a treatment for thyroid cancer, which effectively takes in iodine. This study investigated whether FLC cells readily uptake iodine and if radioactive iodine may serve as an effective therapeutic for FLC patients.

[NOTE:  Use of radioactive iodine on several patients did not confirm this theory and the project was terminated]

Timeframe: 2011

Goal: Profile microRNA in metastatic Fibrolamellar Carcinoma

Principal Investigator: Yuzhou Wang, PhD, University of British Columbia

MicroRNAs are small RNA species that regulate the expression of other genes and are important in many biological processes, including tumor development, growth, and metastasis. MicroRNAs have become increasingly useful as diagnostic markers, prognostic indicators, and therapeutic targets. This study examined the microRNA profile of FLC tumors which metastasize and matched tumors that do not metastasize. By identifying microRNAs that predict or lead to tumor metastasis, the study results may help to create new diagnostic and therapeutic tools and strategies.