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Johns Hopkins University

2022

Multicenter consortium to define the single-cell activity landscape of fibrolamellar carcinoma

Goals: Advance immunotherapy for FLC by defining promising immunological targets that can be translated into effective cell-based immunotherapies

Principal Investigators: Praveen Sethupathy, PhD (Cornell University); Mark Yarchoan, MD (Johns Hopkins University); Paul G. Thomas, PhD (St. Jude Children’s Research Hospital)

Grant length: Two years

Study overview: Like tumors of other cancer types, the microenvironment of FLC tumors is highly complex, comprising many different cell types. It is now well-established from investigation of other cancer types (such as lung, breast, and pancreatic cancer) that cross-talk among these different cell types can promote tumor development, growth, and spread. A recent study in the Sethupathy lab identified critical regions of the genome that are uniquely activated in FLC. These regions offer clues about the genes that might be most critical for the development of FLC. However, an important limitation of this work is that it was performed on bulk FLC tissue, which does not resolve different cell types, and instead treats tumor tissue as one whole unit. This means that the specific cell types in which these FLC genes are active is not yet known. This represents a major knowledge gap. Identification of the specific cell types in which FLC genes are active would then allow more precise study of the functions of these genes in FLC, and facilitate the development of more effective targeted therapeutics.

To help bridge this knowledge gap, the Sethupathy (Cornell), Yarchoan (Johns Hopkins), and Thomas (St. Jude Children’s) labs will participate in a collaborative research consortium to develop an FLC tumor “atlas”. They will leverage state-of-the-art genome-scale technologies to provide unprecedented resolution of the cellular and molecular landscape of FLC. This consortium brings together three groups with longstanding interests and experience in FLC research, as well as specific expertise in genomics and gene regulation (Sethupathy), clinical oncology (Yarchoan), and immunology (Thomas and Yarchoan).

2022

Clinical trial protocol development

Goals: Accelerate development of potential clinical trial

Principal Investigator: Mark Yarchoan, MD

Grant length: Six months

Study overview: This grant to Dr. Yarchoan is meant to support the development of a protocol for a potential clinical trial. In 2020, the Johns Hopkins team began a phase I clinical trial of an experimental vaccine containing a peptide (small segment of a protein) that corresponds to the junction region between the two parts of FLC’s characteristic chimeric protein. Patients enrolled in that trial also simultaneously received two FDA-approved checkpoint inhibitors – Opdivo (nivolumab) and Yervoy (ipilimumab).

This new effort is focused on designing an appropriate follow-up clinical study as soon as possible, to build on the learnings of the peptide vaccine effort and better understand factors that can limit the immune system’s ability to fight FLC.

2019

A Pilot Study of a DNAJB1-PRKACA fusion kinase vaccine combined with nivolumab and ipilimumab for patients with fibrolamellar carcinoma (FLC)

Goal: Assess potential to induce immune response with a FLC therapeutic vaccine

Principal Investigator: Mark Yarchoan, MD

Grant length: Two years

Study overview: Many patients with FLC currently receive an immune checkpoint inhibitor off-label. These drugs “take the brakes off” the immune system, enhancing its ability to fight cancer. However, clinical experience suggests that they generally do not achieve strong anti-tumor responses from single checkpoint inhibitors. This study seeks to develop immunotherapy approaches to FLC that offer greater clinical benefit. In particular, it supports a clinical trial of an immune therapy for fibrolamellar carcinoma (FLC) at the Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins University in Baltimore, MD.

This clinical trial is 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 consistent across FLC tumors. Thus, 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. It also aims 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.

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.

Details of the trial and contact information can be found at: https://www.clinicaltrials.gov/ct2/show/NCT-04248569

2026

Systematic Profiling of Actionable Antibody–Drug Conjugate Targets in Fibrolamellar Carcinoma

Goal: Provide a biological rationale for biomarker-guided testing of antibody drug conjugates in FLC 

Principal Investigator: Mark Yarchoan, MD

Grant length: Six months

Study overview: Cancer cells often overexpress many proteins on their surface. Because of these differences, cancer cells can have unique surface “signatures” that can be used to specifically target them with therapies. These molecules can also be used as biomarkers. 

Antibody–drug conjugates (ADCs) are an important class of targeted therapies. ADCs use antibodies that recognize specific proteins on cancer cells to deliver toxic drugs directly to the tumor, reducing damage to normal cells. This strategy has already shown major success in certain cancers. For example, in breast cancer patients with high levels of the HER2 surface protein, HER2-targeted ADCs have significantly improved outcomes. FDA has approved the HER2 ADC for all solid cancers that overexpress a certain level of HER2 molecule. Interestingly, some FLC patients also overexpress HER2, making them potential candidates for treatment with the FDA-approved ADC, ENHERTU. Currently, more than 100 ADCs are being developed that target different surface proteins common to various cancers. 

This grant focuses on identifying cell surface proteins that are expressed in FLC that are being targeted by ADCs under development. The team will analyze patient tumor samples from the Johns Hopkins and the FCF Biobank to profile ADC-relevant surface antigens on the tumor cells. Such a systematic characterization of highly expressed surface molecules in FLC could open new treatment options for patients, potentially by repurposing currently available or soon-to-be-approved ADCs. 

2023

Exploiting a Critical Vulnerability to Glutamine Antimetabolite Therapy in Fibrolamellar Hepatocellular Carcinoma (FLC)

Goals: Assess the potential of an inhibitor of glutamine metabolism (DRP-104; sirpiglenastat) in combination with an immune checkpoint inhibitor (durvalumab) as a treatment option for unresectable disease

Principal Investigators: Marina Baretti, MD and Mark Yarchoan, MD

Grant length: Five years

Study overview: This phase IB/2 clinical study, led by Dr. Marina Baretti and Dr. Mark Yarchoan of Johns Hopkins University, will test a new cancer treatment that capitalizes on FLC’s unique metabolism. Recent work from several research teams indicates that the DNAJB1-PRKACA fusion causes a metabolic rewiring of FLC tumor cells that makes them dependent on breaking down large amounts of the amino acid glutamine. This “glutamine addiction” leads to a nutrient-depleted tumor environment that is also enriched in immunosuppressive metabolites including ammonia. This could impair a patient’s ability to launch an effective immune response to the cancer.

The study plans to test the effectiveness of treating FLC patients with an inhibitor of glutamine metabolism (DRP-104; sirpiglenastat) in combination with an immune checkpoint inhibitor (durvalumab). The team’s hypothesis is that this treatment could deliver a “one-two punch” against FLC by:

  • Directly attacking an important metabolic pathway of FLC tumors
  • Restoring an immune supportive tumor environment, and therefore enabling a patient’s immune cells to attack the cancer.

The glutamine antagonist drug DRP-104 is being developed by Dracen Pharmaceuticals. Initial testing of DRP-104 in patients with solid tumors has already been completed. Dracen will provide DRP-104 for this FLC trial, as well as the necessary regulatory and operational support for the effort.

The phase 1b/2 clinical trial at Johns Hopkins will enroll patients diagnosed with unresectable or metastatic FLC whose disease progressed on prior immune therapy. The study will have two key aims:

  1. Testing the safety and clinical activity of the DRP-104/durvalumab drug combination in children and adults with advanced FLC.
  2. Determining whether the treatment suppresses glutamine-dependent processes and increases the number of activated FLC-specific T cells around FLC tumors.

Patient enrollment is anticipated to begin in late 4Q 2023. More information will be released once the trial receives all the necessary institutional and regulatory approvals.

2025

A therapeutic cancer vaccine plus ipilimumab and nivolumab for fibrolamellar hepatocellular carcinoma (FLC)

Goal: Assess potential to induce immune response with a FLC therapeutic vaccine

Principal Investigator: Mark Yarchoan, MD and Marina Baretti, MD

Grant length: Two years

Study overview: This grant allows Dr. Mark Yarchoan and Dr. Marina Baretti of Johns Hopkins University to keep JHU’s successful “peptide vaccine” clinical trial open to enrollment by fibrolamellar patients. 

In the study, patients receive a vaccine created to train the immune system to recognize and attack cells carrying the abnormal fusion protein that causes FLC. Because nearly all FLC tumors share this same fusion protein, the same “off-the-shelf” vaccine made from a small peptide representing the junction of the fused proteins can be used for all patients. During the clinical trial, the vaccine is administered alongside two immune checkpoint inhibitors (ICIs) already approved for use in other cancers (nivolumab and ipilimumab). 

Research published in Nature Medicine in November 2025 summarized the results of this clinical trial for the first 12 evaluable participants:

  • The vaccine appears safe.
  • 75% of participating patients mounted the desired immune response; all of these responders achieved some disease control (at least several months without tumor growth).
  • One-third of responders (3 patients) experienced dramatic, long-lasting tumor shrinkage—and remain cancer-free.

An ultimate objective of the peptide vaccine study is to obtain FDA approval of a new, effective treatment regimen for FLC. The additional data provided by this expanded Phase 1 study will be crucial to determine the size (number of patients) and precise design of the Phase 2/3 clinical trial aimed at obtaining regulatory approval. It also will shed light on whether specific subgroups of patients are more likely to respond to the therapy.

Details of the trial and contact information can be found at: https://www.clinicaltrials.gov/ct2/show/NCT-04248569.

2015

Generation of T cells with specificity to FLC’s chimeric fusion

Goal: Determine if T cells can be generated that react to FLC’s characteristic fusion protein

Principal Investigator: Ephraim Fuchs, MD

Grant length: One year

Study overview: In FLC, the characteristic DNAJB1-PRKACA fusion creates a novel protein with a unique amino acid sequence occurring at the junction of these two proteins.  This unique amino acid sequence could function as a “neoantigen” that can be recognized as foreign by the immune system of the patient or even the immune system of a healthy donor such as a sibling or parent.  The goal of this project was to identify the novel amino acid sequence that occurs in patients with FLC and to generate a response to this sequence from T cells of the immune system.

If T cells that react to the chimeric fusion protein of FLC could be generated and expanded in tissue culture, such T cells may be capable of killing cells that express this protein.  One possibility is to raise T cells against the fusion protein from a healthy donor and infuse these T cells after partial liver transplantation and bone marrow transplantation from this donor.  Such an infusion would be expected to reduce the risk of tumor relapse.

Key elements of the effort included:

  1. Obtaining tumor tissue from a biopsy and determining the sequence of chimeric fusion protein.
  2. Create peptides that spanned the junction between the DNAJB1 and PRKACA proteins.
  3. Collecting blood from the patient and tissue-matched relatives and culturing those cells in presence of the peptide, interleukin-2 (IL-2) and IL-15
  4. Testing the T cells for specificity to the peptide.

Results: A biopsy specimen from an FLC patient was sent for whole genome sequencing and targeted mutation analysis, which identified the fusion gene and predicted amino acid sequence.  The team synthesized 14 overlapping peptides, each containing the fusion amino acids and overlapping 13 amino acids from its neighboring peptide.  These fusion peptides were used to stimulate peripheral blood cells of the recipient and tumor-infiltrating lymphocytes (TILs) of the recipient.  In parallel, relatives of the patient donated blood and their lymphocytes were stimulated with overlapping peptides encompassing the fusion region of the patients DNAJB1-PRKACA fusion protein. 

The study identified that it was possible to grow T cells specific for the fusion protein in both cases. Both the patient and the donors had T cells that recognized and proliferated in response to the unique protein presented by the patient’s FLC tumor. 

Implications: This preliminary data supports the possibility of generating and expanding T cells that recognize proteins that are uniquely expressed by FLC. This ability to grow T cells that recognize the DNAJB1-PRKACA from patients and healthy, related donors, PD-L1 expression on FLC cells, and the presence of tumor-infiltrating lymphocytes in FLC, indicate potential promise for new immunotherapy approaches to treating FLC. 

2014

Sequential related donor partial liver followed by bone marrow transplantation for treatment of extensive, liver-confined fibrolamellar hepatocellular carcinoma (FL-HCC)

Goals: Develop and implement a clinical trial for combined liver and bone marrow transplantation as a treatment for liver-confined FLC

Principal Investigator: Ephraim Fuchs, MD

Grant length: Three years

Study overview: This grant supported the development of a phase II, single center study to assess the feasibility and effectiveness of combining bone marrow transplantation and partial liver transplant from a matched living donor in patients with FLC.

The primary objective of this trial was to characterize recurrence-free survival at 1 year following bone marrow transplantation among recipients of prior partial liver transplantation from the same donor.

Under the proposed protocol, patients would have received a combination of:

  • A living related donor partial liver transplantation
  • Total body irradiation, and
  • Bone marrow transplant from same donor.

The treatment was meant to apply to patients whose cancer remained confined to the liver but was too widespread to be removed by surgery or treated by a liver transplant from a deceased donor. These patients have a poor prognosis, with a median survival of 20 months. This protocol was designed to prolong the survival of these patients or perhaps even cure their disease. The purpose of the combined treatment was to reduce the risk of the cancer coming back after the liver transplant. The study team believed that bone marrow transplantation could reduce the risk of cancer relapse in two ways:

  • Patients who have combined bone marrow and solid organ transplants may minimize the use of anti-rejection drugs, which inhibit the immune system from destroying cancer cells.
  • The donor’s bone marrow contains cells of their immune system, which can attack any cancer cells that remain after the liver transplant.

Details of the proposed trial approach can be found at: https://clinicaltrials.gov/study/NCT02702960

Results: The clinical study was withdrawn in 2018 before any patient enrollment. Most surgeons were concerned about management of the risks to the living donor as a consequence of such a high risk transplant approach.

2021

Development and validation of an orthotopic, syngeneic, mouse model for fibrolamellar hepatocellular carcinoma

Goal: Model development

Principal Investigator: Mark Yarchoan, MD

Grant length: One year

Study overview: 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 aimed to establish and validate the first orthotopic, syngeneic, preclinical mouse model of FLC that reproduces the key biological behavior and tumor microenvironment (TME) of 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 and immunotherapy agents. Once successfully established,the resulting preclinical model of FLC will be openly shared with the larger research community. This model could offer unprecedented opportunities to investigate mechanisms underlying FLC pathogenesis, and become a critical tool for investigating novel therapeutic strategies in FLC.

Key findings: The study team has successfully established a new pre-clinical model of FLC. They created a FLC-like murine cell line by inducing hepatoblast cells (TIB-75) to express the DNAJB1-PRKACA fusion and implanted the tumor line orthotopically into the livers of syngeneic BALB/c mice. In mice, these FLC-like tumors have growth kinetics, high levels of infiltrating lymphocytes, and histological features, such as fibrosis, that are consistent with the clinical phenotypes of FLC observed in humans.

The study team is committed to further describing and validating this new model. Additional planned analyses include characterizing the metabolomics of the FLC-like and parenteral cell lines. They intend to publish their findings, and are open to widely distributing the model within the FLC community. The hope is that this model can serve as the basis for in-depth investigation of the DNAJB1-PRKACA chimeric transcript-dependent pathway in the FLC tumor microenvironment, as well as efforts to drug the fusion and/or its downstream factors. Already, the model has proved instrumental in performing pre-clinical assessments of the impact of glutamine antagonist treatment in combination with checkpoint inhibition – work critical to the development of the upcoming DRP-104 clinical trial at Johns Hopkins.