Massachusetts General Hospital Archives - Fibrolamellar Cancer Foundation

2025

Dissecting and Harnessing SIK Tumor Suppressor Function in FLC

Goal: Identify mechanisms driving FLC and identify potential treatment targets

Principal Investigator: Nabeel Bardeesy, PhD

Grant length: Two years

Study overview: Typically, cancers requires activation of a “driver” of uncontrolled growth, such as the DNAJB1-PRKACA (DP) fusion in FLC, coupled with the loss of a “tumor suppressor” function. Tumor suppressors act as “brakes” on cancers, by causing cells that have gone out of control to undergo programmed suicide.

Surprisingly, studies of FLC have not revealed consistent losses or inactivation of known tumor suppressor genes. However, in previous work funded by FCF, Dr. Nabeel Bardeesy discovered why the production of DP apparently suffices to cause FLC. His lab recently showed that the DP protein causes sustained inhibition of a family of “salt inducible protein kinases” (SIKs)*. The SIK proteins are known tumor suppressors. The Bardeesy team, therefore, concluded that, rather than depending on independent genetic events to inactivate a tumor suppressor, the DP protein itself simply turns OFF the activity of the SIK protein(s). This finding reveals a “core mechanism” by which DP causes FLC.

In that work, Dr. Bardeesy provided a mechanistic model for how inactivation of SIK function by DP contributes to FLC formation. Like all protein kinases, the SIKs work by attaching a small chemical “tag” (a phosphate group) onto specific sites on various target proteins to regulate their activity. The addition or subtraction of the tag often toggles a protein between ON (active) and OFF (inactive) states. The presence or absence of the tag also may change the stability of the protein and/or alter its location in the cell. When the SIKs are ON, they suppress (turn OFF) the expression of key gene programs that drive cells to proliferate and to produce essential metabolites for growth. Conversely, when DP switches the SIKs OFF, an important consequence is the switching ON of the genetic programs which enable cells to proliferate.

The proposal seeks to leverage the knowledge that DP works directly through inactivating SIKs to devise new treatments for FLC. The immediate proximity of SIK inactivation to the fundamental driver of FLC could make it especially attractive for targeted cancer therapy. Through this study, Dr. Bardeesy proposes to explore the feasibility of two classes of therapies:

Drugs that inhibit proteins which are responsible for cancer-specific gene expression, and which are made in FLC cells as a direct result of switching OFF SIK. Two important examples of such proteins are named P300 and CRTC. These are “transcription coactivators” which cooperate to promote the expression of genes essential for the survival and malignant growth of FLC cancers.
Drugs that activate SIK kinase(s) in a manner that overcomes the block caused by DP. This should restore SIK’s tumor suppressor activity, thereby preventing the proliferation of FLC cells, and potentially triggering “programmed cell death,” a crucial feature of many successful cancer therapies.

If successful, this project could:

Confirm the working model that an important element of cancer causation in FLC is the inactivation of the SIK family kinases by DP, which turns OFF their tumor suppressor function.
Determine whether drugs being developed as activators of SIK kinases can kill FLC cells and whether they deserve further attention as potential FLC therapeutics.
Provide mechanistic understanding of how inhibition of SIK by DP reprograms gene expression in FLC cancer cells.

* Gritti, et al., Cancer Discovery, 2025

2025

Fibrolamellar Carcinoma Cell Line Derivation and Characterization

Goal: Model development

Principal Investigator: Nabeel Bardeesy, PhD

Grant length: One year

Study overview: Cell line models and patient-derived xenographs (PDXs) are important tools that researchers can use to study disease. For FLC research to move forward, stable, reliable, patient-derived cell lines that truly represent the human disease are needed. While multiple FLC models have recently been developed, additional model development remains a priority for the FLC community.

Previously, the Bardeesy lab developed a FLC cell line model (FLX1) that shows molecular and biological
faithfulness to the human disease. However, the model is slow to grow and requires a high level of care by the investigator. In this effort, the Bardeesy lab will use a systematic approach to identify among a set of FLX1 derivatives (including very early passage models) the most robust FLX1 derivative and to expand it under standardized protocols, providing a model with uniformity, integrity, and reproducibility.

They also plan to work with two additional, new PDXs and associated in vitro cultures that showed initial promise but were confounded by the presence of competing murine cells. Early passage materials from these cultures will be carefully recovered, purified, expanded, and tested, potentially providing additional models.

If successful, this effort will:

provide robust and well characterized stocks of FLX1 cells that can be distributed to interested FLC researchers, and
develop new cell lines for studying the disease.

2022

Elucidation of nuclear-mitochondrial signaling by DNAJ-PKAc to define targeted vulnerabilities in FLC

Goals: Identifying how the DNAJ-PKAc/SIK/p300 program controls mitochondrial function and define how these abnormal mitochondria affect the metabolism of FLC cells

Principal Investigator: Nabeel M. Bardeesy, PhD

Grant length: Two years

Study overview: Signals that control the growth and metabolism of cells often are relayed through a series of reactions in which proteins are sequentially modified with a chemical tag, a phosphate group. Each of the reactions in a cascade of phosphate-transfers is carried out by a specific “protein kinase,” the major class of signaling enzymes. In fibrolamellar carcinoma (FLC), an abnormal protein kinase (labeled DNAJ-PKAc) that results from FLC’s DNAJB1-PKACA gene fusion drives the development and growth of tumor cells. The functions of that kinase remain incompletely understood. Another characteristic of FLC is the presence of exceptionally large numbers of abnormal mitochondria, organelles that play a key role in cellular energy generation and metabolism.

Past studies by Dr. Bardeesy revealed that DNAJ-PKAc inactivates three related protein kinases, the Salt-Inducible Kinases (SIKs). In turn, inactivation of the SIKs leads to the mitochondrial abnormalities in FLC. Additional data indicate that an important element of this signaling cascade is the activation of a protein called P300 histone acetyltransferase (abbreviated simply as P300). P300 exerts broad effects in controlling gene expression by modifying proteins in chromatin, the complex structure in which DNA is packaged and organized in living cells.

In this study, Bardeesy proposes to “explore precisely how the DNAJ-PKAc/SIK/P300 program controls mitochondrial function” and to define how the mitochondrial abnormalities associated with P300 activation contribute to cancerous growth in FLC. Most importantly, his preliminary data suggests that the mitochondrial abnormalities could potentially be exploited in a novel approach to therapy for this cancer. He proposes to further characterize the functional impact of aberrant mitochondria in tumor cells, and to test whether FLC is sensitive to drugs against P300 and drugs that target the altered mitochondria.

Successful completion of the study aims should have three key benefits:

Improving our understanding of a key signaling pathway central to the metabolism, growth, and survival of FLC cancer cells
Validating P300 as a therapeutic target for FLC and selection of a best-in-class inhibitor with potential to treat patients
Defining the metabolic changes in FLC mitochondria that are likely to contribute to the cancer’s growth, survival, and resistance to therapy.

2019

Targeting DNAJB1-PRKACA driven signaling dependencies in FLC

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.

2020

Fibrolamellar carcinoma model development and analysis

Goal: Model development

Principal Investigator: Nabeel Bardeesy, PhD

Grant length: Two years

Study overview: While multiple FLC models have recently been developed, additional model development remains a priority for the FLC community. This effort aimed to develop multiple different FLC models as resources for the FLC research fibrolamellar cancer research community, collaborating with the Fibrolamellar Cancer Biobank established at Massachusetts General Hospital. Specimens from the biobank were used to attempt to create a 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 hoped to harness these newly developed models to identify genetic dependencies of the disease, better understand the molecular mechanisms underlying FLC formation and growth, and ultimately will set the stage for the development of new therapeutics.

Key Findings: Through this effort, the investigators successfully developed a new cell line model (FLX1) derived from a previously-established PDX model. The investigators reported high level of fusion protein expression in that FLX1 cell line and were able to successfully propagate it in lab as a functional cell line.

During the effort, organoids and patient derived 3D cultures proved more difficult to establish. In all, 28 patient tissue samples were received by the study team from the FCF biobank and other sources to drive PDX, cell line and organoid model development attempts. While 3 PDX models engrafted to a size allowing re-implantation and expansion, all were eventually lost to contamination, murine lymphoma or lack of fusion detection. A second established cell line (FLX2) had a histopathology more like HCC instead of FLC. For 3-dimensional organoids, growth in many attempts was good initially, but senesce (age deterioration) occurred after a limited number of passages in all cases, preventing the establishment of a useful model.

The successfully established FLX1 model has already been used by the study team to make significant advances in understanding the networks mediated by fusion signaling. Studies by Dr. Bardeesy using FLX1 have revealed that that DNAJ-PKAc inactivates three related protein kinases, the Salt-Inducible Kinases (SIKs), which in turn leads to the mitochondrial abnormalities observed in FLC. Currently, Dr. Bardeesy is exploring specifically how that DNAJ-PKAc/SIK pathway controls mitochondrial function and how the mitochondrial abnormalities contribute to cancerous growth in FLC.

Efforts are also underway to make this new FLX1 cell line broadly accessible to the research community as a research tool.

2016

Flipping the switch on PKA: synthetic lethal approaches to block PKA-driven tumor growth in fibrolamellar liver cancer

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.