NRF2 co-opts the SWI/SNF complex to drive liver cell plasticity

  1. Athena Jessica S Ong
  2. Chapman CH Wong
  3. Anthony P Karamalakis
  4. Kimberley J Evason
  5. Kristin K Brown
  6. Andrew G Cox

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Abstract

Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays a role in the regulation of redox homeostasis and cellular metabolism. Activating mutations in the NRF2 pathway have been identified in approximately 15% of liver cancer patients. However, the mechanisms by which NRF2 promotes liver tumorigenesis are poorly understood. Employing a transgenic zebrafish model with hepatocyte-specific, inducible expression of a clinically relevant constitutively active NRF2 mutant (NRF2 T80K ), we show that constitutive activation of NRF2 drives hepatocyte to cholangiocyte transdifferentiation. Importantly, we demonstrate that NRF2 affects liver cell plasticity in a cell-autonomous, evolutionarily conserved, and reversible manner. Utilizing an epigenetic-focused chemical screen, the BRG1/BRM inhibitor FHD-286 was identified as a potent suppressor of NRF2-driven transdifferentiation. Overall, our study reveals a novel role for NRF2 in the regulation of liver cell plasticity during tumour initiation and identifies a therapeutic approach to overcome the oncogenic activity of NRF2.

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  1. Review Commons

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    Reply to the reviewers

    'The authors do not wish to provide a response at this time.'

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Liver cancer is a deadly disease that consists of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA). CCA can originate from hepatocytes. Interestingly, the NRF2 pathway is hyperactivated in 15% of HCC. To explore this, the authors developed a model of DOX-inducible hyperactivated NRF2T80K in hepatocytes of zebrafish. They suggest that hepatocyte to cholangiocyte transdifferentiation occurs, resulting in expansion of the cholangiocyte compartment. Importantly, they explore these phenotypes in larvae and adult zebrafish, clearly demonstrating that the impact of NRF2-driven cell plasticity is not limited to conditions of organogenesis. Using a pharmacologic inhibitor of BRG1/BRM, they also suggest that NRF2-driven cell plasticity is dependent on the SWI/SNF complex

    Major comments

    A major question regards the involvement of the SWI/SNF complex. The authors state, "NRF2 co-opts the SWI/SNF complex to drive liver cell plasticity". However, these conclusions are largely based on the phenotypes using the BRG1 inhibitor FHD-286. Currently, it is unclear the degree of on-target activity FHD-286 has on BRG1, and it would be important to potentially explore this. Further, did any other BRG1 inhibitors score from the screen, or could they explore BRG1 inhibition using distinct pharmacologic or genetic approaches? These experiments would strengthen the conclusions of the study.

    Significance

    This is a very interesting paper with broad implications. In future studies, it would be interesting to explore how NRF2 potentially impacts the SWI/SNF complex. It would also be interesting to explore which target genes downstream of NRF2 hyperactivation drive expansion of the cholangiocyte compartment. Finally, it would be interesting (in future studies) to explore metabolic changes caused by NRF2 hyperactivation in hepatocytes of zebrafish, and how these changes could cause transdifferentiation of hepatocytes into cholangiocytes

  3. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In the present manuscript, Dr. Ong and colleagues investigated the role of NRF2 activation in promoting liver cell plasticity. To support their conclusions, the authors use models of constitutive NRF2 activation, including KEAP1 KO and expression of the mutated NRF2T80K in HepG2 cells. In addition, they developed a transgenic zebrafish model with hepatocyte-specific inducible expression of mutated NRF2T80K to investigate the effect of NRF2 activation in liver tumour initiation. They found out that NRF2T80K expression promotes expansion of cholangiocyte compartment that resulted from hepatocyte transdifferentiation and provide evidence suggesting the potential role of SWI/SNF chromatin remodeling complex in this process.

    Major comments

    1. The major concern is to rule out the possibility that the observed phenotypes are influenced by off-target effects of doxycycline treatment in zebrafish. Although some control experiments are presented, they were performed at different experimental settings, or the methodology is not sufficiently clear. Therefore, control conditions should be presented more clearly. In particular, the authors should include analyses of liver plasticity markers and NRF2 activity markers in wild-type control animals treated with doxycycline. This would help demonstrate that the observed effects are due to NRF2T80K activation rather than doxycycline treatment itself. In addition, the experimental design is unclear in several panels (e.g. It is not clear if the zebrafish images were taken at 10 dpf and whether that also means the time of NRF2T80K induction).
    2. It would be important to determine whether systemic activation of NRF2WT on KEAP1 KO background produces similar effects.
    3. The data shown with the model keap1a/keap1b crispants needs more detailed explanation.
    4. The authors should better explain the criteria to select the hit from the compound screen. Does the selected compound also have effect on KEAP1 KO or NRF2 OE cells?
    5. Please double check the indications of treatment durations (time points/days) throughout the main text, figure legends, and experimental schemes to ensure consistency. Including a schematic overview that clearly distinguishes the larval and adult experimental settings would improve clarity and help readers follow the experimental design.
    6. The authors conclude the results section with "Together, these data highlight a role for SWI/SNF-dependent chromatin remodelling complex in regulating liver cell plasticity in the context of NRF2 pathway activation." However, the data presented only suggests a possible role of SWI/SNF. The conclusion is based solely on pharmacological treatment, without direct functional validation of the specific components (e.g. through genetic perturbations). Therefore, the current evidence supports an association rather than a definitive role in regulation of liver cell plasticity.
    7. Main conclusions derived from the imaging and gene expression analysis should be further validated at the protein level. Confirming the observed changes in the protein expression would provide stronger support for the proposed biological mechanism.

    Minor comments

    1. Immunoblot of KEAP1 is missing to confirm KO in Fig1B and Immunoblot of NRF2 is missing to confirm OE in Fig1D.
    2. Review whether the data normalization appropriate matches the statistical test applied (In FigS1A, are the reference/control values random set to 1 or the references values used were the average of the controls?).
    3. Please provide a more detailed explanation of the data processing and curation used to generate the graphs on Fig4B.
    4. The legend indication of the panels on FigS4 is wrong. Please revise all figure legends and main text.

    Significance

    The conclusions are relevant for the field of basic research on cancer and cell biology. However, would be essential to clarify the points raised. The major limitation is that conclusions were made based on one model.

  4. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    While the study is technically ambitious, challenging in vivo zebrafish, scRNA-seq, and chemical library screen. On the other hand, the NRF2-SWI/SNF causal link is not well supported yet to fully back the claims. This reviewer's background is in redox signaling, so does not comment on the zebrafish technical methodology.

    Major comments

    1. No wild-type NRF2 control is studied. Everything is done only with NRF2T80K, but no WT NRF2 overexpression comparison. Therefore, this reviewer cannot tell if the phenotype is mutant-specific or just a general consequence of NRF2 pathway activation.
    2. FHD-286 does not establish an NRF2-SWI/SNF link. As BRG1/BRM are broadly acting, but not NRF2-specific, identification of FHD-286 does not prove SWI/SNF's action downstream of NRF2. In fact, Figs. S4C & D show that FHD-286 does not fully suppress NRF2 target genes even while blocking trans-differentiation. This disconnect suggests FHD-286 may act on a parallel or downstream pathway, not addressed in this version.
    3. Several other hits in the Fig. 4A screen show similar suppression to FHD-286. The authors need to explain the rationale for picking FHD-286 as the lead compound.
    4. There are no functional cancer assays. Only histology/fluorescence for trans-differentiation were conducted, but no clonogenic assay, anchorage-independent growth, organoids, nor transplant tumor-initiation studies. The link to actual tumorigenesis remains speculative.
    5. The Introduction says NRF2-driven tumorigenesis mechanisms are "poorly understood", but there are substantial existing literatures (e.g., PMID: 38308097, 37364049, 31961719).
    6. No mention about where NRF2T80K mutation was first identified, its prevalence across liver cancer subtypes, nor known functional consequences beyond disrupting KEAP1 binding. This point should be stated more in detail.
    7. In Fig 2A, text describes CCA-like glandular structures after DOX treatment, but they are not marked with arrows or outlines in the figure.

    Minor comments

    1. GSEA results would benefit from heatmaps showing the leading-edge genes driving the enrichment scores.
    2. NQO1 and HMOX1 are the standard markers as NRF2 target genes. Incorporating them would strengthen the validation of NRF2 pathway engagement in this model.
    3. The figure legend for Figure 1G states that it shows magnifications from Figure 1G. This should refer to Figure 1E

    Significance

    This manuscript uses a zebrafish model with inducible NRF2T80K expression, plus HepG2 cells, to show that constitutive NRF2 activation drives hepatocyte-to-cholangiocyte trans-differentiation. This phenotype is cell-autonomous, conserved between zebrafish and human cells, and reversible. The authors conducted a small-molecule screen to identify the BRG1/BRM inhibitor FHD-286 as a suppressor of this trans-differentiation. The authors propose this screening result links NRF2 to the SWI/SNF complex. The phenotypic work is clear and interesting, but the mechanistic experimental design has fundamental gaps that remain unaddressed.

    If the mechanism holds up, this is a meaningful finding for the liver cancer field: it points to hepatocytes as a possible cell-of-origin for NRF2-driven cholangiocarcinoma and suggests SWI/SNF as a druggable vulnerability. The study is technically ambitious, challenging in vivo zebrafish, scRNA-seq, and chemical library screen.

  5. Version published to 10.64898/2026.02.09.704724 on bioRxiv