Goal: Investigate ways to therapeutically target DNAJ-PKAc
Principal Investigators: John Gordan, MD, PhD
Grant length: Two years
Study overview: Testing drugs in cells and living organisms is a critical step in developing new treatments. For a drug to work, it must move through complex cell structures and, in animals and eventually humans, overcome challenges like absorption, distribution, metabolism, and excretion (often called ADME). Studying how these drugs behave in cells and in animal models not only shows whether they work but also helps us understand which cancer pathways they affect. This knowledge can guide combination therapies and predict which patients might respond—or develop resistance.
This is especially important for Fibrolamellar Carcinoma (FLC), where the cancer-driving kinase DNAJ-PKAc operates as part of a larger protein complex. Its partners may influence how drugs bind and work, so these interactions need to be studied carefully.
This effort is led by Dr. John Gordan, a physician-scientist with deep experience in cancer drug development. His team will create specialized cell models that can tell the difference between the DNAJ-PKAc fusion protein and its normal counterpart. This is essential for designing drugs that target only the cancer-driving fusion. They will also test promising compounds in animal models to move candidate drugs closer to treatments for patients.
Goal: Testing the drug zotatifin (eFT226) against human FLC tumors grown in mice
Principal Investigator: John Gordan, MD, PhD
Grant length: One year
Study overview: This “proof of concept” study focuses on testing the drug zotatifin (eFT226) for activity against human FLC tumors grown in mice. This drug is being developed by eFFECTOR Therapeutics (Solana Beach, CA). The drug is currently in Phase I clinical studies as a potential therapy for a variety of cancers.
The application follows from a completed FCF-funded project led by Dr. Gordan as Principal Investigator, together with Nabeel Bardeesy, PhD (Massachusetts General Hospital). They found that the primary driver of FLC, the DNAJ-PKAc fusion protein kinase (DP), increases the expression of a cancer-promoting protein called c-MYC. This occurs because DP activates an enzyme, eIF-4A, that substantially increases the amount c-MYC made from each copy of the corresponding messenger RNA – such regulation of protein synthesis is known as “translational control.” Furthermore, they showed that c-MYC is required for robust proliferation of FLC cells in culture. Zotatifin was developed as an inhibitor of eIF-4A. Dr. Gordan has postulated that this drug should prevent the DP-dependent overproduction of MYC in FLC cells, and thereby slow tumor growth and potentially cause the cancer cells to die.
Previously, the Gordan / Bardeesy team found that zotatifin indeed inhibits the growth and may cause the death of human FLC cancer cells grown in laboratory culture. The main goal of the new project will be to determine whether zotatifin is similarly active against FLC cells growing in living animals, using immune-deficient mice that can serve as hosts for human tumors (PDX models). Dr. Gordan also will assess combinations of zotatifin with other potential anti-FLC therapeutics.
If successful, the experiments will provide proof of concept for the use of translation inhibition as a potential treatment strategy for FLC patients.
Goals: Investigate the potential of AURKA inhibitors for FLC treatment
Principal Investigators: John Gordan, MD, PhD (UCSF) and Nabeel Bardeesy, PhD (MGH)
Grant length: Two years
Study overview: Even though the DNAJB1-PRKACA gene fusion 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.
In previous work, the study team mapped the signaling cascade downstream of PRKACA in FLC and other tumors. This analysis highlighted 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 drugs in clinical trials. However, a new class of AURKA inhibitors have emerged that are designed to disrupt its interaction with the Myc family of oncoproteins, which are critical drivers of many cancers. The team believed that that AURKA-mediated stabilization of MYC is necessary to maintain growth of FLC cancer cells, and that one of these new AURKA inhibitors could potentially reduce the proliferation of FLC cells.
Although these new AURKA inhibitors are not yet ready for human use, this study aimed to understand if they are likely to be effective for FLC and whether they work well in combination with other available drugs. Their effort planned to assess the activity and mechanism of action of these 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.
Key Findings: The authors reported that the inhibitor of AURKA (alisertib) did not show any effect on Myc protein levels in FLX1, a patient-derived cell line of FLC. They also reported that the combination of alisertib with an inhibitor of Pim had a mild effect on reducing MYC levels (the Pim kinase acts along with Myc during the formation of a tumor). However, the combination had no effect on cell viability.
The authors then focused on identifying other mechanisms of regulation of MYC family of oncogenic factors to identify strategies to selectively target it. It was observed that PKA stimulation increases phosphorylation of a translation factor, eIF4A, thereby increasing its activity and causing rapid cell proliferation. This suggested that PKA effects on the initiation of transcription (via eIF4A) might be responsible for its induction of c-MYC expression and increased cell proliferation. Consistent with this, the inhibition of eIF4A with the natural product rocaglamide, or its clinically used derivative zotatifin (now being investigated in clinical trials for other cancers), significantly reduced c-MYC protein levels and potently inhibited proliferation of a fibrolamellar cell line (FLX1).
This work was completed in collaboration with another FCF grantee, John Scott of the University of Washington. An article summarizing the work was published by eLife in January 2023. The full text of the publication can be read here: https://prod–journal.elifesciences.org/articles/69521.
Implications: This study established a clearly defined mechanism of MYC regulation by PKA. It also identified compounds currently in clinical trials that can selectively disrupt this mechanism of MYC activation. In the next steps for this investigation, these compounds will be tested in patient-derived organoid models.
Goal: Develop updated estimate for US national incidence of FLC
Investigators: John Gordan, MD, PhD (UCSF); Travis Zack, MD (UCSF); and Kurt Losert (FCF)
Grant length: One year
Study overview: Rare diseases like fibrolamellar are often poorly understood because of the difficulty in assembling accurate patient cohorts and the lack of ICD billing codes specific to the disease. The most cited study of national incidence of FLC, using data from the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) Program, reports an incidence of approximately 0.02 per 100,000. However, this rate is significantly lower than might be expected based on observations from clinical practice which indicate that the incidence of FLC in the United States equals approximately 1% that of HCC. The main goal of this study was to analyze and combine data from nationally aggregated billing record datasets and major healthcare organization’s electronic medical records to re-estimate the national incidence of FLC.
Key Findings: This study used a unique approach that combined “narrow but detailed” EMR data with “broad but less specific” national payer billing information to define FLC incidence. The analysis suggests that FLC’s incidence rate is likely 5-8 times higher than the 0.02 per 100,000 rate reported in the SEER Program statistics. While this approach might be artificially inflated by long-distance referrals of FLC patients to major medical centers, even the lowest estimate made was still five times higher than current SEER data after adjusting the analysis for referral distances.
The study also included an analysis of clinical trial data which identified a higher level of hyperammonemia in FLC patients than is currently recognized in clinical practice, suggesting that hyperammonemia is an underappreciated source of comorbidity in FLC patients. By employing unsupervised machine learning on clinical laboratory data from patients with hyperammonemia, it also found that FLC-associated hyperammonemia mirrors metabolic dysregulation in urea cycle disorders.
The results of the effort were published in NPJ Precision Oncology in March 2023. Click here to read or download the complete article.
Goal: Understand growth mechanisms and identify potential therapeutic targets
Principal Investigators: John Gordan, MD, PhD (UCSF) and Nabeel Bardeesy, PhD (Mass General Hospital)
Grant length: Two years
Study overview: 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 would 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.
Results: To understand PKA’s oncogenic mechanism and identify its downstream targets, the investigators generated genetic cell models with doxycycline-inducible PRKACA or its dominant negative counterpart, a mutant form of the PRKAR1A regulatory subunit. These cell models were then subjected to mass spectrometry for kinome profiling in order to detect kinases with significant altered activity following PKA modulation. This was integrated with small molecule inhibition and siRNA knockdown to identify PKA-regulated kinases that modify cell proliferation. This analysis revealed activation of the aurora-family kinase AURKA, with preferential sensitivity to the confirmation-disrupting AURKA inhibitor (CD-AURKAi) CD532 compared to other AURKA inhibitors.
Further experiments showed that the level of both c-MYC and n-MYC, known to be stabilized by AURKA, were both inhibited by CD532 in the FLC cell line. However, the reduction in proliferation in FLX1 by either c-MYC or n-MYC siRNAs treatment was not as much as the DNAJB1 siRNA treatment, suggesting that both c-MYC and n-MYC must be targeted together. Or more likely, there are other factors contributing to cell proliferation still to be uncovered.
Implications: The results from this study revealed a key molecular target, Aurora kinase A, that could be inhibited by pharmaceutical agents in combination with other therapeutic agents.
Research efforts continued under a follow-on grant from FCF.
Goal: Study the interactions between the immune system and FLC in a mouse model
Principal Investigator: Kevin Barry, PhD
Grant length: Multiple years; part of CRI fellowship
Study overview: 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 and allow effective killing of tumor cells. These therapies function 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 non-FLC cancer patients, leading to tumor regression and offering long-term protection, effectively providing a cure to cancer in some patients. However, at the time of this study,very little was known about how the immune system interacts with FLC or if immunotherapy would be an effective treatment for FLC.
The study of the efficacy of immunotherapy in the treatment of FLC is hampered by the fact that the current patient-derived xenograft animal models of FLC utilize the transplantation of human tumor into immune compromised animals. The lack of an effective immune system allows the host mice to grow tumors, but also makes it impossible to study the interactions between the immune system and FLC in this model system. This study harnessed a preclinical mouse model of FLC developed by Dr. Julien Sage and Stanford University with a fully functional immune system to understand how the immune system interacts with FLC. These studies represented the first steps towards treating FLC patients with immunotherapy in the clinic.
Results: The investigator analyzed a mouse model that conditionally expressed the DNAJB-PRKACA fusion protein. While the hepatocytes expressed the fusion protein, there was no tumor development once the mice were aged. This suggested that the expression of fusion protein in hepatocytes may not be sufficient to drive tumorigenesis in FLC.
Additionally, to explore the immune cells present in FLC, the researchers obtained fresh patient samples and dissociated the tumor into a single cell suspension. Those suspensions were sorted into a non-immune fraction, an immune fraction consisting of all immune cells except T cells, and a T cell fraction. The cells were then analyzed to identify the transcriptional makeup of the immune cells in FLC at the single cell level.
The study founds that the T cell population was made up of a large segment of exhausted T cells, which is indicative of a suppressive immune microenvironment and usually reduces the efficacy of immunotherapy approaches. Follow up analysis of the dataset will help to identify unique changes in the genes expressed in the immune cells within FLC and understand the characteristics of how the immune system responds to FLC.
Implications: These studies will help to fully characterize the distinct transcription programs in immune cells in FLC. These studies are critically important to understand the interaction of the immune system with FLC and to design effective immunotherapy approaches.
