R-Spondin Mimetic, SZN-043, Induced Proliferation and Expression of Wnt Target Genes, Two Impaired Features in Human Alcohol-Associated Liver Disease
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eLife Assessment
This important study highlights the novel role of RSPO mimetic SZN-043 in the activation of hepatic WNT signaling and promoting hepatocyte regeneration. The authors provide convincing evidence of SZN-043 increasing hepatocytes proliferation in various mouse models, including a humanized mouse liver model, ALD model and CCL4 fibrosis model. This study will be of interest to researchers in liver regeneration and repair mechanisms.
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Abstract
Abstract
Liver regeneration is impaired in patients suffering from alcohol-associated liver (ALD) diseases. Wnt ligands and their FZD receptors are dysregulated in diseased livers. R-spondin and their receptors are known to regulate Wnt activity via the stabilization of FZD receptors. Here, we investigated the components of the Wnt and R-Spondin-signaling pathways and their activity in patients with ALD. We found that while hepatocytes retained high levels of differentiation markers such as ASGR1 and ASGR2, the expression of two R-spondin co-receptors, LGR4 and LGR5, and of Wnt target genes, CYP1A2 and others, were strongly reduced.
SZN-043, a hepatocyte-targeted R-Spondin mimetic, is a new investigational drug that stimulates the physiological Wnt repair pathway and proliferation of hepatocytes. Here, we show that SZN-043 induced hepatocyte proliferation in all models tested, including humanized mouse livers, a chronic-binge alcohol-induced liver injury, and a CCl4-induced fibrosis mouse model. Altogether, SZN-043 could be beneficial for the treatment of ALD.
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eLife Assessment
This important study highlights the novel role of RSPO mimetic SZN-043 in the activation of hepatic WNT signaling and promoting hepatocyte regeneration. The authors provide convincing evidence of SZN-043 increasing hepatocytes proliferation in various mouse models, including a humanized mouse liver model, ALD model and CCL4 fibrosis model. This study will be of interest to researchers in liver regeneration and repair mechanisms.
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Reviewer #1 (Public review):
Summary:
The work by Fisher et al describes the role of novel RSPO mimetics in the activation of WNT signaling and hepatocyte regeneration. However, the results of the experiments and weaknesses of the methods used do not support the conclusions of the authors that the new therapy can promote liver regeneration in alcohol-induced liver cirrhosis.
Strengths:
Similarly to its precursor, aASGR1-RSPO2-RA-IgG, SZN-043 can upregulate Wnt target genes and promote hepatocyte proliferation in the liver.
Comments on revisions:
The authors responded to all my comments and concerns.
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Reviewer #2 (Public review):
Summary:
The study by Fisher et al investigates therpauetic role for SZN-043, a hepatocyte-targeted R-spondin mimetic, for its potential role in restoring Wnt signaling and promoting liver-regeneration in alcohol-associated liver disease (ALD). Using multiple preclinical models, the compound was shown to promote hepatocyte proliferation and reduce fibrosis. This study highlights the efficacy in promoting liver regeneration while maintaining controlled signaling. Limitations include a need for further exploration of off-target effects and fibrosis mechanisms. The findings support SZN-043 as a promising candidate for ALD therapy, warranting further clinical evaluation. This is a well deigned study with thorough investigation using multiple disease models.
Strengths:
(1) Well-written manuscript with clear …
Reviewer #2 (Public review):
Summary:
The study by Fisher et al investigates therpauetic role for SZN-043, a hepatocyte-targeted R-spondin mimetic, for its potential role in restoring Wnt signaling and promoting liver-regeneration in alcohol-associated liver disease (ALD). Using multiple preclinical models, the compound was shown to promote hepatocyte proliferation and reduce fibrosis. This study highlights the efficacy in promoting liver regeneration while maintaining controlled signaling. Limitations include a need for further exploration of off-target effects and fibrosis mechanisms. The findings support SZN-043 as a promising candidate for ALD therapy, warranting further clinical evaluation. This is a well deigned study with thorough investigation using multiple disease models.
Strengths:
(1) Well-written manuscript with clear design, robust methods, and discussion.
(2) Using multiple models strengthens the findings and expands beyond ALD.
(3) Identification of SZN-043 as a novel potent drug for liver regeneration.
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Author response:
Response to Comments from reviewer #1
Many thanks for appreciating that SZN-043 can promote hepatocyte proliferation via the Wnt-signaling pathway.
(1) The reviewer is concerned with using only CYP1A2 expression as an endpoint to make a conclusion about the effect of SZN-043 on Wnt activity in human ALD samples. The reviewer raises a good point as the more commonly used Wnt target gene, AXIN2, is not consistantly changed in both cohorts. We were at first also surprised by this finding. However, upon closer analysis we found that the expression of hepatocyte-specific target genes such as CYP1A2 (Figure 2), CYP2E1, OAT, LGR5, GLUL (Table 1) and ZNRF3 were mostly expressed in hepatocytes and ductal cells were all down-regulated in ALD samples. Others Wnt target genes expressed in epithelial and mesenchymal liver cell …
Author response:
Response to Comments from reviewer #1
Many thanks for appreciating that SZN-043 can promote hepatocyte proliferation via the Wnt-signaling pathway.
(1) The reviewer is concerned with using only CYP1A2 expression as an endpoint to make a conclusion about the effect of SZN-043 on Wnt activity in human ALD samples. The reviewer raises a good point as the more commonly used Wnt target gene, AXIN2, is not consistantly changed in both cohorts. We were at first also surprised by this finding. However, upon closer analysis we found that the expression of hepatocyte-specific target genes such as CYP1A2 (Figure 2), CYP2E1, OAT, LGR5, GLUL (Table 1) and ZNRF3 were mostly expressed in hepatocytes and ductal cells were all down-regulated in ALD samples. Others Wnt target genes expressed in epithelial and mesenchymal liver cell populations, such as AXIN2, CCND1 and NOTUM are indeed not consistently and significantly changed. Given that SZN-043 is not active on mesenchymal cells, this discrepancy could be best explained by the large increase in mesenchymal cells in ALD tissue samples, thereby confounding the results. We have now clarified this in the discussion. Another method to assess Wnt activity is to measure b-catenin phosphorylation and nuclear transfer. In our hands, this method was found to be better suited for tissue culture than histological sections from in vivo studies. We have also amended the manuscript title to refer to expression of Wnt target genes, rather than Wnt activity.
(2) We have now added a supplemental figure to show the lack of Ki-67+ human hepatocytes in the cirrhotic tissue samples to confirm the absence of hepatocyte proliferation (Figure S1).
(3) The differences in amino acid sequence between SZN-043 and its precursor, αASGR1-RSPO2-RAIgG, can be found in the material and method section. These changes in amino acid sequences improved the biophysical properties of the final clinical candidate, such as oxidation and nonspecific binding. The biochemical analysis of those differences exceeds the scope of the current manuscript. We present here the pharmacokinetic properties of SZN-043 only, as this was the only molecule advanced to clinical trial and used in the studies presented here.
(4) The reviewer suggests to assess the effect of SZN-043 in Ctnnb1-KO mice to confirm that SZN043 acts via a canonical Wnt pathway. Indeed, there were several reports on the ability of Rspondin to act on other pathways besides the Wnt signaling pathway (for recent review, Niehrs et al, 2024, Bioessays). However, while an interesting suggestion, this line of investigation belongs to MOA studies and exceeds the scope of the current manuscript. An additional manuscript presenting MOA studies for SZN-043 was recently submitted elsewhere. Still, we have added this possibility in the discussion section.
(5) The reviewer is asking how SZN-043 is affecting liver functions in general. Indeed, we have observed a consistent reduction in the international normalized ratio of prothrombin time using the thioacetamide (TAA)-induced fibrosis model and previously published those findings (Zhang, 2020). In our hands, the TAA is the only liver injury model that significantly increases INR. This increase is modest compared to that observed in clinical patients. Therefore, we do not report INR findings for other models. We have not seen any effects of SZN-043 on hepatocyte differentiation markers such as HNF4A (data not shown) and the hepatocyte specific ASGR1/2 as shown in Figure 5. Rather we focused on proliferation as the main potentially beneficial endpoint, to restore the parenchymal mass in injured livers. Finally, consistent with what was reported in the literature, we have observed a transient and reciprocal effect on albumin and alfa-fetoprotein expression during the proliferative phase of liver regeneration. These results are detailed in an additional manuscript presenting MOA studies for SZN-043, which was recently submitted elsewhere.
(6) We have used females only in the ethanol-induced injury models because there are numerous reports in the literature stating that males are not as susceptible to those injuries.
(7) The reviewer questions the relevance of the ethanol-induced injury model used to evaluate SZN043 efficacy. Indeed, none of the disease model developed to date reproduce the severity and complexity of alcohol-associated liver diseases, although some, such as the ethanol supplemented Lieber DeCarli diet, are more commonly used than others – which is the reason why this model was selected.
(8) The reviewer questions the relevance of the fibrosis model used to evaluate SZN-043 efficacy. Indeed, none of the fibrosis models developed to date reproduce the severity and complexity of cirrhosis in human livers. While combining ethanol with CCl4 would lead to more severe fibrotic livers, CCl4 itself is not involved in ALD in humans. Both models are likely to result in similar pericentral fibrosis with central-to-central bridging. In this study, we were mostly interested in addressing the effects of SZN-043 in a tissue affected by fibrotic scars.
(9) The sex of CCl4-treated mice is male. We added this information in the methods section.
(10) A summary of histology and fibrosis assessment data for alcohol-fed mice was added in supplemental Table S3. In our hands, the use of aging mice did not induce the presence of fibrosis, in contrast to published results.
(11) The rationale for using 13.5-month-old mice in the alcohol studies and scid mice in the CCl4 studies has been clarified in the results and discussion sections.
a. Briefly, aging mice were reported to be more susceptible to ethanol-induced injury than young mice and to include induction of fibrosis. However, we were unable to reproduce the presence of fibrosis reported in the literature.
b. Scid mice were used in the CCl4 studies to test whether a stronger response could be observed in the absence of a potential anti-drug antibodies response. While a modest reduction in fibrosis was observed in both B6 and scid mice following the SZN-043 treatment, the effect size did not seem affected by the mouse strain.
Response to Comments from reviewer #2
Many thanks for appreciating that the use of multiple disease models to identify SZN-043 as a potential novel drug for liver regeneration.
(1) The importance of restoring liver regeneration capacity to reduce the need for liver transplantation had been emphasized in the introduction.
(2) There is continuous damage to the mouse hepatocytes in the FRG mice, due to the Fah mutation. They undergo repair mechanisms favoring the proliferation of human hepatocytes during the production period. Injury models that affect the human hepatocytes population have been developed in these mice. However, the primary goal of this study was to confirm that SZN043 was efficacious in inducing human hepatocytes proliferation, a feature difficult to reproduce in primary hepatocyte cultures. Given the artefactual nature of the chimeric liver in FRG mice and the high cost of these mice, further studies were not judged to be necessary.
(3) Corrected
(4) A figure including DAPI staining has now been included in supplemental Figure S2.
(5) Clarification that the 8 weeks alcohol feeding used in our study design is a modification of the NIAAA model. While some ASGR1 has been reported on the surface of macrophages, additional data from MOA studies strongly suggest that the effect of SZN-043 is mediated via a hepatocytespecific mechanism (submitted manuscript).
(6) The reviewer inquired about the potential role of macrophages in promoting an antiinflammatory state in response to SZN-043. While a direct effect is unlikely, a potential effect of macrophages in response to SZN-043 is plausible. Wnt activation is known to induce the secretion of hepatokines, such as LECT2, which in turn can influence macrophage activity. This possibility is discussed in the discussion section.
(7) The potential off-target effects of SZN-043 such as stellate cell activation is discussed in the discussion section.
(8) The discussion of the limitations of current models has been included in the discussion section of the manuscript.
(9) We have now included a discussion of prior RSPO-based therapies, such as OMP-131R10. We explain why the hepatocyte-targeting of RSPO activity minimizes undesired effects.
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eLife Assessment
This important study highlights the novel role of RSPO mimetic SZN-043 in the activation of hepatic WNT signaling and promoting hepatocyte regeneration. The authors provided solid evidence of SZN-043 increasing hepatocyte proliferation in various mouse models, including a humanized mouse liver model, ALD model, and CCL4 fibrosis model, although mechanistic characterization of liver pathology remains incomplete. This study will be of interest to researchers in liver regeneration and repair mechanisms.
-
Reviewer #1 (Public review):
Summary:
The work by Fisher et al describes the role of novel RSPO mimetics in the activation of WNT signaling and hepatocyte regeneration. However, the results of the experiments and weaknesses of the methods used do not support the conclusions of the authors that the new therapy can promote liver regeneration in alcohol-induced liver cirrhosis.
Strengths:
Similarly to its precursor, aASGR1-RSPO2-RA-IgG, SZN-043 can upregulate Wnt target genes and promote hepatocyte proliferation in the liver.
Weaknesses:
(1) The authors rely on the expression of a single gene, CYP1A1, as a readout of Wnt/ß-catenin target gene expression. A more systemic evaluation of Wnt/ß-catenin activity should be performed.
(2) The lack of the mRNA upregulation of cell cycle genes is not sufficient to draw a conclusion of the impaired …
Reviewer #1 (Public review):
Summary:
The work by Fisher et al describes the role of novel RSPO mimetics in the activation of WNT signaling and hepatocyte regeneration. However, the results of the experiments and weaknesses of the methods used do not support the conclusions of the authors that the new therapy can promote liver regeneration in alcohol-induced liver cirrhosis.
Strengths:
Similarly to its precursor, aASGR1-RSPO2-RA-IgG, SZN-043 can upregulate Wnt target genes and promote hepatocyte proliferation in the liver.
Weaknesses:
(1) The authors rely on the expression of a single gene, CYP1A1, as a readout of Wnt/ß-catenin target gene expression. A more systemic evaluation of Wnt/ß-catenin activity should be performed.
(2) The lack of the mRNA upregulation of cell cycle genes is not sufficient to draw a conclusion of the impaired regeneration in cirrhotic livers.
(3) The authors present single-dose pharmacokinetic (PK) profile of SZN-04. It is not clear how that compares to its precursor, to justify better pharmacokinetic properties.
(4) The specificity of Wnt/ß-catenin activation should be evaluated in ß-catenin KO mice to show no target gene induction in the absence of ß-catenin.
(5) The authors demonstrated that the drug promoted hepatocyte proliferation. How it affects liver functional parameters in alcohol-fed mice, hepatocyte differentiation markers, albumin production, and coagulation factor synthesis is not clear.
(6) Female mice only were used for alcohol studies; the effect on the male mice needs to be evaluated as well.
(7) Alcohol feeding did not reduce Wnt/ß-catenin target gene expression in mice suggesting that it is a bad model to study the efficacy of the SZN-043 in alcohol-induced liver cirrhosis.
(8) The authors used CCl4-induced fibrosis as a model of ALD fibrosis. However, this is not a suitable fibrosis model for ALD studies. Adding alcohol to CCl4 treatment could potentially address this issue. Alternatively, the authors should use an ALD model that produces significant fibrosis.
(9) Sex for the CCl4-treated mice is not indicated.
(10) Histology and fibrosis assessment data for alcohol-fed mice should be presented.
(11) The rationale for using 13.5-month-old aging mice for alcohol studies and immunodeficient mice only for CCl4 studies is not clear.
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Reviewer #2 (Public review):
Summary:
The study by Fisher et al investigates a therapeutic role for SZN-043, a hepatocyte-targeted R-spondin mimetic, for its potential role in restoring Wnt signaling and promoting liver regeneration in alcohol-associated liver disease (ALD). Using multiple preclinical models, the compound was shown to promote hepatocyte proliferation and reduce fibrosis. This study highlights the efficacy of promoting liver regeneration while maintaining controlled signaling. Limitations include a need for further exploration of off-target effects and fibrosis mechanisms. The findings support SZN-043 as a promising candidate for ALD therapy, warranting further clinical evaluation. This is a well-designed study with thorough investigation using multiple disease models.
Strengths:
(1) Well-written manuscript with clear …
Reviewer #2 (Public review):
Summary:
The study by Fisher et al investigates a therapeutic role for SZN-043, a hepatocyte-targeted R-spondin mimetic, for its potential role in restoring Wnt signaling and promoting liver regeneration in alcohol-associated liver disease (ALD). Using multiple preclinical models, the compound was shown to promote hepatocyte proliferation and reduce fibrosis. This study highlights the efficacy of promoting liver regeneration while maintaining controlled signaling. Limitations include a need for further exploration of off-target effects and fibrosis mechanisms. The findings support SZN-043 as a promising candidate for ALD therapy, warranting further clinical evaluation. This is a well-designed study with thorough investigation using multiple disease models.
Strengths:
(1) Well-written manuscript with clear design, robust methods, and discussion.
(2) Using multiple models strengthens the findings and expands beyond ALD.
(3) Identification of SZN-043 as a novel potent drug for liver regeneration.
Weaknesses:
(1) The introduction needs to be re-structured with an emphasis on liver regeneration. It seems that the entire manuscript is focused on liver regeneration, however, only the last two sentences or so describe liver regeneration. The frequency of liver transplants owing to a reduced ability for liver regeneration in AH patients needs to be highlighted.
(2) In Figure 4, it appears that the humanized mice liver was injected with the SZN-043. Is it possible that using a partial hepatectomy model will be beneficial for assessing the effects of SZN-043 rather than using them in mice without any hepatocyte damage?
(3) Figure 4B. Panel 3 has 10mpk merged inside the figure. Please correct this.
(4) Figure 4B. DAPI staining will be vital to show the Ki67 staining specific to hepatocytes (at least visually we can do co-localization with a double nucleus in each cell). The current image shows some cells show Ki67 staining which shows some cells which are not binuclear.
(5) The alcohol feeding was performed for 8 weeks and is described as NIAAA model in the methods section. NIAAA model is 11 days of alcohol+ one binge. Please correct this or clarify it in the methods section, as this is not reflected. ASGR1 may be also expressed by macrophages so it's important to show the specificity.
(6) Is it possible that the SZN-043 also has effect on macrophages promoting an anti-inflammatory state? This should be discussed.
(7) Potential off-target effects of SZN-043, particularly in stellate cell activation in the context of fibrosis should be discussed.
(8) Discuss the limitations of current models and how they might influence the interpretation of the results.
(9) Clearly explain how SZN-043 overcomes limitations of prior RSPO-based therapies.
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