From the journal RSC Chemical Biology Peer review history

02-May-2021

Dear Professor Zheng:

Manuscript ID: CB-REV-03-2021-000063
TITLE: Wnt signaling activation : targets and therapeutic opportunities for stem cell therapy and regenerative medicine

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I look forward to receiving your revised manuscript.

Yours sincerely,

Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

************

Reviewer 1

Major comments:
1) Brief summaries of Wnt/b-catenin signaling pathway, as well as stem cell therapy and regenerative medicine in the section of Introduction would be useful for readers not familiar to the field.
2) In the section of discussion, the applications were divided into 1) regeneration medicine and SC therapy, 2) SC expansion, 3) SC renewal/tissue homeostasis and repair/preventing ageing. These applications are quite overlapped, the concerns of Wnt activators in these applications were not clearly distinguished.
3) As a review article, the section of discussion could be replaced with “potential application of Wnt activators” instead.
4) It could be interesting to compare the advantages/disadvantages among the Wnt inhibitors with different targets regarding regeneration medicine and SC therapy.
5) Safety (side-effects) and selectivity (off-targets) are major concerns of Wnt activators, but not fully addressed.
6) The authors mentioned three different Wnt pathways: the canonical Wnt/β-catenin pathway, the non-canonical Wnt/planar cell polarity (PCP) pathway, and the non-canonical Wnt/Ca2+ pathway. What is the difference of the pathways in regeneration medicine and stem cells?
7) Some paragraphs were not fully referred.
8) Lithium chloride is not a natural product.
9) Full names for ROR and RYK should be added.

Reviewer 2

The topic is interesting and the manuscript is well-written. I believe researchers will benefit a lot from this review article in understanding Wnt signaling activators. My comments are listed below.
1. Page 8, e. Anti-NOTUM monoclonal antibodies. There is no NOTUM antibody discussed in this section. ABC99 is small molecule compound.
2. In Page 25, ABC99 should be deleted from the antibody panel in the figure.
3. Page 26, Table, ABC99 should be an inhibitor, not antagonist, of NOTUM.
4. The author should discuss another NOTUM inhibitor NOTUMib-1 (PMID: 31534655).

Point-by-Point Response to the Reviewers’ Comments

Referee: 1
1) Brief summaries of Wnt/b-catenin signaling pathway, as well as stem cell therapy and regenerative medicine in the section of Introduction would be useful for readers not familiar to the field.

Reply: We thank the reviewer for this comment. These definitions have been added in the Introduction, specifically in lines 51-54: “Wnt signaling include multiple functionally divergent pathways. Among them, the best characterized in the Wnt/β-catenin pathway, often referred to the canonical pathway. It regulates the β-catenin dependent genes expression that direct embryogenesis and SC fate.” Also, in lines 62-64, we added: “Theoretically, by activating Wnt, one could promote SC proliferation to restore tissue or organ impaired functions, the ultimate goal of SC therapy and regenerative medicine.”.

2) In the section of discussion, the applications were divided into 1) regeneration medicine and SC therapy, 2) SC expansion, 3) SC renewal/tissue homeostasis and repair/preventing ageing. These applications are quite overlapped, the concerns of Wnt activators in these applications were not clearly distinguished.

Reply: We thank the reviewer for this helpful comment. Accordingly, we have re-grouped the discussion into three potential applications: 1) a tool to dissect the regulatory roles of different Wnt signaling in SC, 2) potential reagents for regeneration medicine to promote SC renewal/tissue homeostasis in vivo, and 3) reagents for promoting ex-vivo SC expansion (for stem cell transplantation).

3) As a review article, the section of discussion could be replaced with “potential application of Wnt activators” instead.

Reply: The name of the section has been modified, in line 487: “Potential application of Wnt activators”.

4) It could be interesting to compare the advantages/disadvantages among the Wnt inhibitors with different targets regarding regeneration medicine and SC therapy.

Reply: We thank the reviewer for this comment. The advantages/disadvantages of Wnt inhibitors have been reviewed elsewhere, including a recent paper published by our team (reference 13). This review focuses on Wnt activators, a topic largely less described in the literature.

5) Safety (side-effects) and selectivity (off-targets) are major concerns of Wnt activators, but not fully addressed.

Reply: As mentioned above, the potential applications of Wnt activators section has been revised to emphasize the current limitations of Wnt activators as therapeutics, in lines 516-524: “The ability of Wnt activators to promote SC proliferation, the purpose of SC therapy, poses safety concerns, as dysregulation in the mechanisms that keep SC in a quiescent, non-proliferating state can lead to cancer197. Overactivation of Wnt has been reported in numerous cancer types198. Wnt is a powerful and complex morphogen, temporal, dosage, and tissue specific, that exhibits numerous cross-talks with other crucial signaling pathways. Targeting such major developmental pathway utilized by both physiologic SC and by cancer SC can have dramatic teratogenic effect and poses safety concerns given the duality of such therapeutics 139.”

6) The authors mentioned three different Wnt pathways: the canonical Wnt/β-catenin pathway, the non-canonical Wnt/planar cell polarity (PCP) pathway, and the non-canonical Wnt/Ca2+ pathway. What is the difference of the pathways in regeneration medicine and stem cells?

Reply: We do not have a good answer for this question, and we believe this question is one of the active research frontiers in the field. However, the differences between the pathways depends on the targets of one Wnt activator, and its position in the pathway. As mentioned in the potential application of Wnt activators section, we modified the text in lines 508-510: “Nevertheless, the abilities of activating different Wnt signaling pathways make these reagents as perfect tools to dissect regulating roles of different Wnt pathways in SC.”

7) Some paragraphs were not fully referred.

Reply: We thank the reviewer for this comment; the manuscript has been carefully reviewed to complete references, notably the NOTUM section. Newly added references are:

90. J. E. Tarver, Jr., P. K. Pabba, J. Barbosa, Q. Han, M. W. Gardyan, R. Brommage, A. Y. Thompson, J. M. Schmidt, A. G. E. Wilson, W. He, V. K. Lombardo and K. G. Carson, Bioorg Med Chem Lett, 2016, 26, 1525-1528.,
91. R. Brommage, J. Liu, P. Vogel, F. Mseeh, A. Y. Thompson, D. G. Potter, M. K. Shadoan, G. M. Hansen, S. Jeter-Jones, J. Cui, D. Bright, J. P. Bardenhagen, D. D. Doree, S. Movérare-Skrtic, K. H. Nilsson, P. Henning, U. H. Lerner, C. Ohlsson, A. T. Sands, J. E. Tarver, D. R. Powell, B. Zambrowicz and Q. Liu, Bone Res, 2019, 7, 2.
93. B. N. Atkinson, D. Steadman, Y. Zhao, J. Sipthorp, L. Vecchia, R. R. Ruza, F. Jeganathan, G. Lines, S. Frew, A. Monaghan, S. Kjær, M. Bictash, E. Y. Jones and P. V. Fish, Medchemcomm, 2019, 10, 1361-1369.
188. T. Zhan, N. Rindtorff and M. Boutros, Oncogene, 2017, 36, 1461-1473.”

8) Lithium chloride is not a natural product.

Reply: We thank the reviewer for this comment. The title of the section has been revised accordingly. Specifically, we added in line 327: “Lithium Chloride” and line 328: “Lithium chloride (LiCl) is a chemical compound that has been used for decades as a mood stabilizer for bipolar disorder, schizophrenia, depression, and other mental illnesses, with little toxicity92.”

9) Full names for ROR and RYK should be added.

Reply: Full names for ROR, receptor tyrosine kinase-like orphan receptor, and RYK, receptor like tyrosine kinase, have been added, lines 94-95.

Referee: 2
1. Page 8, e. Anti-NOTUM monoclonal antibodies. There is no NOTUM antibody discussed in this section. ABC99 is small molecule compound.
Reply: We thank the reviewer for this comment. The title has been revised in line 288: “Anti-NOTUM small molecules”.

2. In Page 25, ABC99 should be deleted from the antibody panel in the figure

Reply: We thank the reviewer for this comment, the figure has been revised accordingly, and the compound has been removed.

3. Page 26, Table, ABC99 should be an inhibitor, not antagonist, of NOTUM.

Reply: We thank the reviewer for this comment, the table has been revised accordingly. The term antagonist has been removed, the compounds LP-922056 and 2-phenoxyacetamides have been added.

4. The author should discuss another NOTUM inhibitor NOTUMib-1 (PMID: 31534655).

Reply: We thank the reviewer for this comment. The NOTUM section has been entirely revised accordingly. Specifically, new sentences have been added in lines 289-304: “Wnts ligands are post-transcriptionally modified by palmitoleoylation of a conserved serine by Porcupine, an event that is required for Wnt trafficking and binding to Frizzled receptors. NOTUM is a serine hydrolase, involved in the deacylation of the palmitoylated serine residue, leading to Wnt/β-catenin inactivation89. Only a few reports describe the use of NOTUM inhibitors, such as LP-922056, to activate Wnt signaling, promote bone formation and treat osteoporosis in animal models90, 91. However, these compounds could present off-targets effects, as they can penetrate the blood-brain barrier. Suciu et al., using an activity-based protein profiling method, engineered a more specific inhibitor, N-hydroxyhydantoin carbamate inhibitor (ABC99) that potently and selectively inhibits NOTUM, while the activation of the Wnt/β-catenin pathway was confirmed in vitro using a super TOPflash assay92. However, it tends to form covalent adduct with NOTUM, limiting its use in vivo. Atkinson et al. recently developed 2-phenoxyacetamides compounds as specific NOTUM inhibitors that restore Wnt/β-catenin signaling in vitro93. They are interesting compounds to study in vitro models of Alzheimer’s disease but to date, these compounds are not metabolically stable.”

Also, three new references have been added in the References sections:

90. J. E. Tarver, Jr., P. K. Pabba, J. Barbosa, Q. Han, M. W. Gardyan, R. Brommage, A. Y. Thompson, J. M. Schmidt, A. G. E. Wilson, W. He, V. K. Lombardo and K. G. Carson, Bioorg Med Chem Lett, 2016, 26, 1525-1528.
91. R. Brommage, J. Liu, P. Vogel, F. Mseeh, A. Y. Thompson, D. G. Potter, M. K. Shadoan, G. M. Hansen, S. Jeter-Jones, J. Cui, D. Bright, J. P. Bardenhagen, D. D. Doree, S. Movérare-Skrtic, K. H. Nilsson, P. Henning, U. H. Lerner, C. Ohlsson, A. T. Sands, J. E. Tarver, D. R. Powell, B. Zambrowicz and Q. Liu, Bone Res, 2019, 7, 2.
93. B. N. Atkinson, D. Steadman, Y. Zhao, J. Sipthorp, L. Vecchia, R. R. Ruza, F. Jeganathan, G. Lines, S. Frew, A. Monaghan, S. Kjær, M. Bictash, E. Y. Jones and P. V. Fish, Medchemcomm, 2019, 10, 1361-1369.

01-Jun-2021

Dear Professor Zheng:

Manuscript ID: CB-REV-03-2021-000063.R1
TITLE: Wnt signaling activation : targets and therapeutic opportunities for stem cell therapy and regenerative medicine

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

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With best wishes,

Cai-Guang Yang, Ph.D.
Associate Editor/RSC Chemical Biology
Professor/Shanghai Institute of Materia Medica, CAS
Phone: +86-021-50806029
Email: yangcg@simm.ac.cn

Reviewer 1

There are still too many mistakes in grammar, below are examples in the sections of the Abstract and ITntroduction:
line 35: "activation of Wnt/β-catenin signaling is a target of interest..." should be " Wnt/β-catenin signaling is a target to be targeted.."
Line 36： high throughput should be high-throughput
Line 39：““mechanisms of actions” should be “mechanisms of action”
Line 51: "include" should be "initiates"
Line 52: "in" should be "is"
Line 54: “beta-catenin dependent” should be "beta-catenin-dependent", "direct" should be "directs"
Line 62: Wnt should be Wnt signaling pathway
...