Novel Insights into the Role of Kras in Myeloid Differentiation: Engaging with Wnt/β-Catenin Signaling

Round 1

Reviewer 1 Report

The review entitled “Novel insights into the role of K-Ras in myeloid differentiation: 2 Crosstalk with Wnt/β-catenin signaling” focuses on a potential cross-talk between activated Ras proteins and Wnt/β-catenin signaling. The text of the review is divided into several chapters; with an abstract at the beginning and a conclusion at the end. The structure and organization of the review has a number of particularities:

1.     The abstract only treats Ras proteins and the Ras signaling pathway whereas the conclusions almost exclusively mentions the Wnt pathway. If a crosstalk exists this should be the topic of the conclusion.

2.     The Wnt-pathway diagram is not very well drawn and as such has been depicted in many reviews beforehand. It is unclear how this may help readers to understand the issue of the review.

3.     The cross talk between Ras and Wnt signaling is not really addressed. The canonical Wnt signaling should be put in perspective with this – non canonical – signaling. GSK3beta is certainly not only dependent on K-Ras signaling and the review should explain how the cells make a choice between the default pathway (FZD- Axin/DVL) and the K-Ras initiated pathway. The quality of the image in Figures 2 and 5 should be improved.

4.     It is unusual to include primary data such as diagrams and Western blots into a review. This should be avoided. The existing data that support the existence of a crosslink between K-Ras and Wnt signaling should be at the center of the review, which is not the case here.

In addition , the images in Figure (3 and 4) seem to be compressed. The diagrams appear somewhat out of context and appear too preliminary to be added into a manuscript – if it were an original article. Clearly, these Figures do not belong in a review.   

Author Response

Author's Reply to the Review Report (Reviewer 1)

The review entitled “Novel insights into the role of K-Ras in myeloid differentiation: Crosstalk with Wnt/β-catenin signaling” focuses on a potential cross-talk between activated Ras proteins

and Wnt/β-catenin signaling. The text of the review is divided into several chapters; with an abstract at the beginning and a conclusion at the end. The structure and organization of the review has a number of particularities:

1. The abstract only treats Ras proteins and the Ras signaling pathway whereas the conclusions almost exclusively mentions the Wnt pathway. If a crosstalk exists this should be the topic of the conclusion.

Thank you very much for your comments. Abstract lacks information of Wnt-signaling pathway. Therefore, we have incorporated background of Wnt/β-catenin signaling pathway (line 20-22). Moreover, we have revised and described Kras has distinct roles and more detail of how Kras linked to Wnt signaling pathway upon DMSO treatment (line 15-18 and 22-28).

• Line 15-18, Although the Ras proteins, a group of small GTP-binding proteins, are ubiquitously expressed and highly homologous, each has specific molecular functions. Kras was shown to be essential for normal mouse development, whereas Hras and Nras are not. Kras knock-out mice develop profound hematopoietic defects, indicating that Kras is required for hematopoiesis in adults.
• Line 20-22, The protein β-catenin is a key player in the Wnt/β-catenin signaling pathway. Many evidences show that the Wnt/β-catenin signaling pathway is deregulated in malignant tumors including hematological malignancies.
• Line, 22-28, Wild-type Kras acts as a tumor suppressor during DMSO-induced differentiation of HL-60 cells. Upon DMSO treatment, Kras translocates to the plasma membrane and its activity is enhanced. Inhibition of Kras attenuates CD11b expression. DMSO also elevates levels of GSK3β phosphorylation, resulting in the release of unphosphorylated β-catenin from β-catenin destruction complex and its accumulation in the cytoplasm. The accumulated β-catenin subsequently translocates into the nucleus. Inhibition of Kras attenuates Lef/Tcf-sensitive transcription activity.

We have amended the Conclusions (section 13, line 732-769) to contain therapy using inhibitors of Kras G12C (line 751-754). As described in section 11 (Application to treatment of patients with AML, line 586-669), therapies targeting Kras itself have not been successful in AML. Inhibitors of Kras G12C including AMG510 are effective in solid tumors. However, drugs that directly target Ras have not yet reached clinical trials in AML. We suggest that Kras G12C inhibitor may be a possible application in combination with other inhibitors. Therefore, in the Conclusion we did not elaborate.

Based on the comments of multiple reviewers and considering the concepts of this review, we feel that we have overestimated the concept of “crosstalk”. Therefore, we have substituted ‘engage’, ‘link’ or ‘sequentially activated’ instead of ‘crosstalk’. We have changed the title to“Novel insights into the role of Kras in myeloid differentiation: Engaging with Wnt/β-catenin signaling pathway”.  Thus, "crosstalk" does not apply in the revised review.

2. The Wnt-pathway diagram is not very well drawn and as such has been depicted in many reviews beforehand. It is unclear how this may help readers to understand the issue of the review.

Thank you very much for your comment. We apologize that we did not present more clearly the diagram of Wnt signaling pathway. In the amended review, we have incorporated three diagrams of Wnt signaling pathways, Wnt/β-catenin signaling pathway and two non-canonical pathways (Wnt/PCP pathway and Wnt/Ca²⁺ pathway). Figure 1 (original version Figure 2) is Wnt/β-catenin signaling pathway in the presence or absence of Wnt. Please see section 3.1, line 160-204. In addition, Non-canonical Wnt signaling pathways (Wnt/PCP pathway and Wnt/Ca²⁺ pathway) are shown in Figure 2 (section 3. 2). Detailed explanations of non-canonical Wnt signaling pathways (Wnt/PCP pathway and Wnt/Ca²⁺ pathway) are described in section 3. 2 (line 205-246). Furthermore, we have incorporated two more sections (section 4, line 247-290 and section 5, 291-313) and described the involvement of two pathways in normal hematopoiesis and AML. We hope these changes better facilitate readers’ understanding of Wnt signaling pathways.

To avoid confusions, our findings are separately presented in the section 12 “Perspective” (line 670-731, Figure 5). In this model, we show that WT-Kras engage with Wnt/β-catenin signaling to promote DMSO-induced differentiation in HL-60 cells. We also described the difference between the Wnt/β-catenin signaling pathway and DMSO-induced differentiation pathway in section 12 ”Perspective” line 671-677.

3. The cross talk between Ras and Wnt signaling is not really addressed. The canonical Wnt signaling should be put in perspective with this – non canonical – signaling. GSK3beta is certainly not only dependent on K-Ras signaling and the review should explain how the cells

make a choice between the default pathway (FZDAxin/DVL) and the K-Ras initiated pathway. The quality of the image in Figures 2 and 5 should be improved.

Following to multiple comments from reviewers and we have overestimated the concept of “crosstalk”, we have substituted ‘engage’, ‘link’ or ‘sequentially activated’ instead of ‘crosstalk’. Thus, "crosstalk" does not apply in the revised review.

Regarding of function of GSK3β, we agree with you. GSK3β involved in multiple signaling pathways and regulates many cellular events. As suggested, we have amended section 6 “The role of GSK3β in hematopoietic stem cells” (line 314-359). Please see lines 316-321, 325-330, and 331-359 in the revised version of review. We describe that GSK3β activity is regulated by phosphorylation/dephosphorylation and GSK3β functioning as either tumor suppressor or tumor enhancer (line 316-321 and 325-330). We also describe possible applications of GSK3β inhibitor in combination therapy (line 331-343). Finally, we mention roles of GSK3β in immunotherapy against cancer (line 344-359).

Canonical Wnt signaling pathway is triggered by the binding of Wnt to LRP5/6 and FZ receptors. Wnt binding triggers the recruitment and polymerization of Dishevelled. Dishevelled polymerization triggers binding of Axin together with GSK3β to form Wnt/signalosome. Eventually, accumulated β-catenin translocate to nuclei, and stimulate expression of target genes. In contrast, WT-Kras engages with Wnt/β-catenin signaling pathway to differentiate HL-60 cells by DMSO treatment. In this case, DMSO is an inducer and treatment with DMSO triggered activation of downstream signaling. Treatment with DMSO affects the fluidity of plasma membranes and activates several biological functions of cells by changing characters of lipid bilayers. However, the detailed mechanism of DMSO has not yet been elucidated. Both signaling pathways initiate accumulation of unphosphorylated β-catenin in cytoplasm. However, the mechanism of inhibition of GSK3β activity is known. In the Kras signaling pathway, GSK3β inhibition is achieved by its phosphorylation by AKT. In contrast, there are two possible models of how GSK3β activity is inhibited in the Wnt/β-catenin signaling pathway. GSK3β and CK1 phosphorylate PPPSPxS motifs in the cytoplasmic tail of LRP5/6, and this motif binds to the catalytic pocket of GSK3β, thereby blocking GSK3β activity towards β-catenin. Thus, direct catalytic inhibition of Axin-mediated GSK3β is caused by the phosphorylated PPPSPxS motif in LRP5/6. The other is an indirect cell biological model based on LRP5/6 and Axin-mediated GSK3 sequestration by multivesicular bodies (MVBs). Thus, inhibition of GSK3β　(phosphorylation of GSK3β) is the point, which these pathway may linked each other. We also described the difference between the Wnt/β-catenin signaling pathway and DMSO-induced differentiation pathway in section 12 ”Perspective” line 671-677.

Knockdown of Kras by siRNA attenuates Lef/Tcf transcription activity (indicator of activation of Wnt/β-catenin signaling pathway) in the DMSO-induced differentiation of HL-60 cells (shown in Figure 4), supporting an idea Kras signaling pathway is linked to Wnt/β-catenin signaling. We agree with your comment.

We have amended Figure1 (original version, Figure 2) and Figure 5. Figure 1 presents Wnt/β-catenin signaling pathway with or without Wnt. Figure 5 shows that WT-Kras is linked to Wnt/β-catenin signaling to promote DMSO-induced differentiation in HL-60 cells. Thus, these Figures have been revised to show differences of two signaling pathways.

4. It is unusual to include primary data such as diagrams and Western blots into a review. This should be avoided. The existing data that support the existence of a crosslink between K-Ras and Wnt signaling should be at the center of the review, which is not the case here.

In addition, the images in Figure (3 and 4) seem to becompressed. The diagrams appear somewhat out of context and appear too preliminary to be added into a manuscript – if it were an original article. Clearly, these Figures do not belong in a review.

Thank you for the valuable comments. Regarding diagrams, we also have described in the comment 2. We apologize that we did not present more clearly the diagrams of Wnt signaling pathway. In the amended review, we have incorporated three diagrams of Wnt signaling pathways, Wnt/β-catenin signaling pathway and two non-canonical pathways (Wnt/PCP pathway and Wnt/Ca²⁺ pathway). Figure 1 (original version Figure 2) is Wnt/β-catenin signaling pathway and the other is non-canonical Wnt signaling pathway (Figure 2). Amended Figure 1 (original version Figure 2) presents Wnt /β-catenin signaling pathway with or without Wnt. Please see section 3.1, line 160-204. In addition, Non-canonical Wnt signaling pathways (Wnt/PCP pathway and Wnt/Ca²⁺ pathway) are shown in Figure 2 (section 3. 2). Detailed explanations of non-canonical Wnt signaling pathways (Wnt/PCP pathway and Wnt/Ca²⁺ pathway) are included in section 3. 2 (line 205-246). To avoid confusions, our findings are separately presented in the section 12 “Perspective” (line 670-731, Figure 5). According to other reviewer comments, Figure 5 has been modified. In this model, we show that WT-Kras engage with Wnt/β-catenin signaling to promote DMSO-induced differentiation in HL-60 cells. We also described the difference between the Wnt/β-catenin signaling pathway and DMSO-induced differentiation pathway in section 12 ”Perspective” line 671-677.

Previously, we have published the fact that WT-Kras regulates the differentiation of HL-60 via engaging with Wnt/β-catenin signaling pathway (Yokoyama, N. et. al. FASEB Bioadv, 2019. 1,7, 435-449). Hematopoietic differentiation is a very complicated process. MPP (multipotent progenitor) divides to CMP (common myeloid progenitor), CDP (common DC precursor) and CLP (common lymphoid progenitor). Monocyte and granulocyte are derived from GMP (granulocyte-macrophage progenitor), in turn derived from CMP. Many modulator proteins are involved in the differentiation of hematopoietic cells. During the maturation process, distinct transcription factors are upregulated, and cooperate to enhance the maturation process. Therefore, we feel it is important to show which and when transcriptions are involved in the process. The data in Figure 3 show DMSO induced granulocytic differentiation in a time-dependent manner. Figure 4 shows that knockdown of Kras by siRNA attenuates Lef/Tcf transcription activity. These data show that Wnt/β-catenin signaling pathway is downstream of Kras. In this review, we describe the pathway, in which WT-Kras engages with Wnt/β-catenin signaling pathway to enhance the differentiation of HL-60 cells. However, many signaling pathways regulate the differentiation of hematopoietic cells. The pathway we have presented in the review is just one of the mechanisms and this pathway does not control all phenomenon. We have inserted the sentences in “perspective” (section 12, line 726-731). The phenomenon of DMSO-induced differentiation of HL-60 cells was reported in 1978. Differentiation of HL-60 cells by DMSO is used as a common model for differentiation in human leukemia, although the molecular mechanism of differentiation in this cell line has not been elucidated. Therefore, we wished report how DMSO induced the differentiation of HL-60 cells. We would like to report that this pathway as one of them and prefer to keep these figures in the review.

As we have explained in the response of Comment 3, and also based on the comments of multiple reviewers and considering the concepts of this review, we have substituted ‘engage’, ‘link’ or ‘sequentially activated’ instead of ‘crosstalk’. Thus, "crosstalk" does not apply in the revised review. We have changed the title to “Novel insights into the role of Kras in myeloid differentiation: Engaging with Wnt/β-catenin signaling pathway”.  

We have amended the compressed Figures 3 and 4（Original version, numbers are same).

Author Response File: Author Response.pdf

Reviewer 2 Report

This paper provided an introduction to the differentiation of the myeloid leukemia cell line HL- 60 cells and the novel functions of WT-Kras in the DMSO-induced differentiation of HL-60 cells. However, your review lacked novelty and features. It lacked emphasis and lost the depth correlation with further clinical application. Besides, there exist several typographical, and grammatical errors. Due to the competitive nature of the articles submitted to this journal, the paper does not meet the criteria for publication. 

1. The manuscript can be structured better and sections can be organized better for the flow of the article. Meanwhile, the author did not propose new perspectives and breakthroughs in treatment and other aspects of the Kras.

2. A review should be a critical and personal view of a subject supported by data from the literature. This review lack innovation, it pays much attention to listing the existing research results and needs to add a summary and review description. E.g. “ Crosstalk of WT-Kras and Wnt signaling cascade in the differentiation of HL-60 cells”, “Wnt/β-catenin signaling pathway is located downstream of WT-Kras”.

3. It is unreasonable to show pictures of other people's research results, it is recommended to delete related pictures.

4. This review is about the differentiation of HL-60 cells, what is the clinical significance, future clinical application, or prospect? It is suggested to add this description to the article.

5. Figures can be improved. It would be even better if Figure 2 could be more informative and Figure 5 could be more beautified.

6. The various mechanisms mentioned in the text, how do they relate to clinical drugs. It is suggested to add the table to new therapeutics and ongoing clinical trials.

7. Additionally, please check the sentence structure to make it easier to be understood and embellish figures.

Author Response

Author's Reply to the Review Report (Reviewer 2)

Comments And　Suggestion　for Authors

This paper provided an introduction to the differentiation of the myeloid leukemia cell line HL- 60 cells and the novel functions of WT-Kras in the DMSO-induced differentiation of HL-60 cells.

However, your review lacked novelty and features. It lacked emphasis and lost the depth correlation with further clinical application. Besides, there exist several typographical, and

grammatical errors. Due to the competitive nature of the articles submitted to this journal, the paper does not meet the criteria for publication.

We thank the reviewer for the thorough reading of our original review. As you suggested, we have refined, redacted, and focused the flow of the review in the revised version. We appreciate the comments. Your comments made this review more valuable to the leaders of Cells. We have arranged the order of sections and incorporated new sections “Non-canonical Wnt signaling pathways” (section 3. 2, line 205-247) including Planar cell polarity (Wnt/PCP) pathway (line 224-236) and Wnt calcium (Wnt/Ca²⁺) pathway (line 237-247). “Wnt-β-catenin signaling in normal hematopoiesis and AML” (section 4, line 248-291) and “Non-canonical Wnt signaling pathways in AML” (section 5, line 292-314) are incorporated following these “Application to treatment of patients with AML” (section 11, line 586-669, including Tables 1 and 2) has also been incorporated. We have introduced application therapies targeting Kras (line 587-634) and Wnt/β-catenin signaling pathway (line 635-669). As suggested, we have deleted Figure 1. We have incorporated new Figure (Figure 2) describing non-canonical Wnt signaling pathway. The order of some sections has been changed to improve the flow of the review.

According to multiple reviewer’ comments and considering the concept of this review, in the revised review, we have substituted ‘engage’, ‘link’ or ‘sequentially activated’ instead of ‘crosstalk’. We have changed the title to“Novel insights into the role of Kras in myeloid differentiation: Engaging with Wnt/β-catenin signaling pathway”.  

1. The manuscript can be structured better and chapters can be organized better for the flow of the article. Meanwhile, the author did not propose new perspectives and breakthroughs in treatment and other aspects of the Kras.

We very much appreciate your comments and agree with them. Following your suggestion, we have arranged the order of sections and incorporated new ones in the new version review. New section, “Application to treatment of patients with AML” (section 11, line 586-669, including Tables 1 and 2), has been incorporated. We have introduced application therapies targeting Kras (line 587-634) and Wnt/β-catenin signaling pathway (line 635-669). We also propose a possible terminal differentiation therapy for AML.

2. A review should be a critical and personal view of a subject supported by data from the literature. This review lack innovation, it pays much attention to listing the existing research results and needs to add a summary and review description. E.g. “ Crosstalk of WT-Kras and Wnt signaling cascade in the differentiation of HL-60 cells”, “Wnt/β-catenin signaling pathway is located downstream of WT-Kras”.

According to multiple reviewer’ comments and considering the concept of this review, in the revised version, we have used ‘engage’, ‘link’ or ‘sequentially activated’ instead of ‘crosstalk’.

The original versions of section 7. DMSO-induced differentiation of HL-60 cells and section 8. Crosstalk of WT-Kras and Wnt signaling cascade in the differentiation of HL-60 cells have been combined as section 9. “WT-Kras engages with Wnt/β-catenin signaling pathway in the DMSO-induced differentiation of HL-60 cells” in the revised version. The previous section 9. WT-Kras positively regulates the DMSO-induced differentiation of HL-60 cells and section 10. Wnt/β-catenin signaling pathway is located down stream of WT-Kras have been combined as section 10. “Level of β-catenin regulates DMSO-induced differentiation in HL-60 cells” in the revised review.

We have revised these sections to be simple and clear to understand.

3. It is unreasonable to show pictures of other people's research results, it is recommended to delete related pictures.

As suggested, we have deleted Figure 1.

4. This review is about the differentiation of HL-60 cells, what is the clinical significance, future clinical application, or prospect? It is suggested to add this description to the article.

We have incorporated a new section described “Application to treatment of patients with AML” (section 11, line 586-669, including Tables, 1 and 2). We have introduced application therapies targeting Kras (line 587-634) and Wnt/β-catenin signaling pathway (line 635-669). Unfortunately, clinical drugs for AML are still under development. AML is a remarkably heterogeneous disease. For this reason, combination therapy is suitable for AML. In Conclusion (section 13, line 732-768), we describe that future directions should focus on identification of additional mutations in signaling molecules and determination of the connection of different genotypes and specific signaling pathways. Thus, we feel that basic research regarding fundamental phenomenon such as differentiation or apoptosis etc. is important for the development of novel therapy. We believe that the addition of this section makes this review more valuable, since therapies for AML are still under development. We appreciate your comments.

5. Figures can be improved. It would be even better if Figure 2 could be more informative and Figure 5 could be more beautified.

We have amended Figures 2 and 5 (in the revised version, Figure 1, and Figure 5). Figure 1 shows Wnt/β-catenin signaling pathway with and without Wnt. We first presented canonical Wnt/β-catenin signaling pathway and our findings are separately presented in the section 12 “perspective” (line 670-731). As suggested, Figure 5 has been modified. We have incorporated new Figure (Figure 2) describing non-canonical Wnt signaling pathway.

6. The various mechanisms mentioned in the text, how do they relate to clinical drugs. It is suggested to add the table to new therapeutics and ongoing clinical trials.

We have incorporated “Application to treatment of patients with AML” (section 11, line 586-669, including Tables, 1 and 2). We have introduced application therapies targeting Kras (line 587-634) and Wnt/β-catenin signaling pathway (line 635-669). Direct inhibition of Ras is desirable for treatment of mutant Ras-induced tumors. AMG510, inhibitor of Kras G12C, is the first molecular treatment in ongoing clinical trials of inhibitors targeting Kras. AMG510 is effective in solid tumors and was approved as a first line treatment for nonsmall-cell lung cancer (NSCLC) by the U.S. Food and Drug Administration. Many clinical trials are in progress, however there is yet no effective therapy targeting Kras mutations in AML. We propose a possible application in which terminal differentiation therapy has great possibility. We describe palmitoylation as a possible target for AML. For therapy targeting Wnt/β-catenin signaling pathway, there are many clinical trials in progress (please see Table 2). Currently, there is no FDA-approved Wnt-targeting agent. We appreciate the valuable comments and criticisms of the original review.             

7. Additionally, please check the sentence structure to make it easier to be understood and embellish figures.

As suggested, we have amended the sentence structure to make clear to understand through manuscript.

The paper has been checked by http://www.english-for-scientists.com/ and a certificate of proofreading is available.

Author Response File: Author Response.pdf

Reviewer 3 Report

The authors have given an account of the role of the Kras gene in myeloid differentiation ad crosstalk with Wnt/β-catenin signaling. The review written by the authors Yokoyama et al. is worth appreciation. There are some suggestions that need to be considered to enhance the quality of the review.  

1. Abstract is slightly less informative. It should contain the background information in the starting part of the abstract.

2. In the first and second paragraphs of the introduction, there is a lack of connection. Incorporate one line for the connectivity between the molecular mechanism responsible for the myeloid differentiation 32 of HL-60 cells and Ras proteins.

3. Give some more data related to the Wnt/β-catenin signaling path- 57 way is deregulated in hematological malignancies.

4. Authors have mentioned the role of GSK3 in hematopoietic stem cells. The information given is very less as this is the major part. Please explain in detail

5. Authors have not used the uniform format. Maintain uniformity throughout the manuscript.

6. Give citations for figure 2. Explain the proteosomal degradation of beta-catenin.

7. Conclusion should also address little info about the background of the study. Please reframe the conclusion also.

8. Kindly improve the quality of English and avoid grammatical errors.

9. Please explain in the introduction, why authors have reviewed the most discussed topic in the literature. What is the gap in the review?

Author Response

Author's Reply to the Review Report (Reviewer 3)

Comments and Suggestions for Authors

The authors have given an account of the role of the Kras gene in myeloid differentiation ad crosstalk with Wnt/β-catenin signaling. The review written by the authors Yokoyama et al. is worth appreciation. There are some suggestions that need to be considered to enhance the quality of the review.

We have incorporated new sections “Non-canonical Wnt signaling pathways” (section 3.2 line 205-246), “Wnt/β-catenin signaling pathway in normal hematopoiesis and AML” (section 4, line 247-290), “Non-canonical pathway in AML” (section 5, line 291-313) and “Applications to treatment of patients with AML” (section 11, line 586-669, including Tables 1 and 2) in the revised review. We have introduced application therapies targeting Kras (line 586-634) and Wnt/β-catenin signaling pathway (line 635-669). We have deleted Figure 1 as suggested by a reviewer comment, and therefore original Figure 2 is Figure 1 in the revised version of review. We have incorporated new Figure (Figure 2) describing non-canonical Wnt signaling pathway.

The order of some sections has been changed to improve the flow of the review.

According to multiple reviewer’ comments and considering the concept of this review, in the revised review, we have substituted ‘engage’, ‘link’ or ‘sequentially activated’ instead of ‘crosstalk’. We have changed the title to “Novel insights into the role of Kras in myeloid differentiation: Engaging with Wnt/β-catenin signaling pathway”.

1. Abstract is slightly less informative. It should contain the background information in the starting part of the abstract.

We have amended the Abstract, described Kras background and more detail of how Kras linked to Wnt signaling pathway upon DMSO treatment (line 15-18 and 22-28). We also described background of Wnt/β-catenin signaling pathway (line 20-22).

• Line 15-18, Although the Ras proteins, a group of small GTP-binding proteins, are ubiquitously expressed and highly homologous, each has specific molecular functions. Kras was shown to be essential for normal mouse development, whereas Hras and Nras are not. Kras knock-out mice develop profound hematopoietic defects, indicating that Kras is required for hematopoiesis in adults.
• Line 20-22, The protein β-catenin is a key player in the Wnt/β-catenin signaling pathway. Many evidences show that the Wnt/β-catenin signaling pathway is deregulated in malignant tumors including hematological malignancies.
• Line, 22-28, Wild-type Kras acts as a tumor suppressor during DMSO-induced differentiation of HL-60 cells. Upon DMSO treatment, Kras translocates to the plasma membrane and its activity is enhanced. Inhibition of Kras attenuates CD11b expression. DMSO also elevates levels of GSK3β phosphorylation, resulting in the release of unphosphorylated β-catenin from β-catenin destruction complex and its accumulation in the cytoplasm. The accumulated β-catenin subsequently translocates into the nucleus. Inhibition of Kras attenuates Lef/Tcf-sensitive transcription activity.

The addition of these sentences makes Abstract more informative. Thank you for your comment.

2. In the first and second paragraphs of the introduction, there is a lack of connection. Incorporate one line for the connectivity between the molecular mechanism responsible for the myeloid differentiation 32 of HL-60 cells and Ras proteins.

We have amended the Introduction. In order to make a connection to these two paragraphs, we have inserted several sentences, line 43-44, line 48-49 and line 54-56 in the text.

• Line 43-44, Elucidation of this differentiation mechanism in acute myeloid leukemia (AML) cells is indispensable in the development of effective treatments.
• Line 48-49, Two members of the Ras gene family, Nras and Kras are frequently mutated in AML and related myelodysplastic syndromes [5].
• Line 54-56, In contrast, knock-out of Ras genes has shown that wild type Kras (WT-Kras) is involved in normal mouse development, whereas Hras and Nras are redundant [10]

3. Give some more data related to the Wnt/β-catenin signaling path- 57 way is deregulated in hematological malignancies.

As suggested, we have described the deregulation of Wnt signaling pathway from line 69-73. And we also provided the references.

• Line 69-73, Wnt signaling in AML is deregulated at various levels, involving extracellular factors, cell surface receptors, cytoplasmic components, and activators of transcriptions [26-28]. Extracellular factors include ligands, agonists, and antagonists; and cytoplasmic components include adaptors and components of destruction complexes involved in β-catenin stability and nuclear translocation.

4. Authors have mentioned the role of GSK3 in hematopoietic stem cells. The information given is very less as this is the major part. Please explain in detail

Thank you very much for the comment. We have agreed that information is not enough to address the importance roles of GSK3. We have amended this section 6. Please see lines 316-321, 325-330, and 331-359 in the revised version. We describe that GSK3β activity is regulated by phosphorylation/dephosphorylation and GSK3β functioning as either tumor suppressor or tumor enhancer (line 316-322 and line 325-330, respectively). We also describe possible applications of GSK3β inhibitor in combination therapy (line 331-343). Finally, we mention roles of GSK3β in immunotherapy against cancer (line 344-359).

5. Authors have not used the uniform format. Maintain uniformity throughout the manuscript.

 As suggested, we have carefully amended the style throughout the manuscript.

6. Give citations for figure 2. Explain the proteosomal degradation of beta-catenin.

Original version Figure 2 is the revised version Figure 1.

The citation is given in line 184-185.

• Line 184-185, The model presented is a modified version, and part of previous model (FASEB Bioadvances, 2019).

We have described β-catenin degradation by proteasome in line 174-176. More detailed description is line 190-192 in the text.

• Line 174-176, Ubiquitylated and phosphorylated β-catenin is degraded by the proteasome. PP2A dephosphorylates β-catenin and inhibits its degradation.
• Line 190-192, These sequential phosphorylations of β-catenin generate a recognition site for the E3-ubiquitin ligase β-TrCP, with ubiquitinated β-catenin subsequently degraded by proteasomes (Fig.1, left side) [57, 58].

7. Conclusion should also address little info about the background of the study. Please reframe the conclusion also.

Thank you very much for the comment. We have amended the Conclusion part (section 13, line 732-768). We have described differences in gene mutations among solid tumors and AML (line 736-740). Next, we have addressed character of AML and an importance of deregulation of signaling pathway in AML (line 741-747). A future direction is mentioned in line 748-750. Our findings suggest the possibility that activation of WT-Kras can modulate the differentiation of HSC and AML cells, and combination therapy may be effective for AML. We have described a possible contribution of our finding in therapy for AML, although more detailed analysis is required (line 757-768). 

8. Kindly improve the quality of English and avoid grammatical errors.

The paper has been checked by http://www.english-for-scientists.com/ and a certificate of proofreading is available.

9. Please explain in the introduction, why authors have reviewed the most discussed topic in the literature. What is the gap in the review?

The phenomenon of DMSO-induced differentiation of HL-60 cells was reported in 1978. Differentiation of HL-60 cells by DMSO is a common model for differentiation in human leukemia, although the molecular mechanism of differentiation in this cell line has not been elucidated. Therefore, we wished know how DMSO induced the differentiation of HL-60 cells. We found that both WT-Kras and Wnt/β-catenin signaling pathways regulate these processes, we feel this is worth reporting. Therefore, the introduction of DMSO-induced differentiation, background of Kras, and Wnt signaling pathways are important topics described in the Introduction. In Introduction text, we have already described that both Kras and Wnt signaling pathways play as oncogenic factors and enhance formation of tumors (line 49-54 and line 67-69). These facts are the most discussed topics in Ras/Wnt signaling pathways, and well accepted. In contrast, roles of WT-Kras as tumor suppressor have not been fully elucidated, because a few cases are reported. Ras proteins have been dismissed as undruggable target for many decades. Even great discovery of Kras G12C inhibitor brings light to many patients with solid tumors, but much less benefits to patients with AML. How WT- Kras functions as a tumor suppressor has attracted national attention. Molecules targeting other than Kras itself may involves in regulation of differentiation of process in AML cell lines. Wnt/β-catenin signaling pathway regulates both differentiation and proliferation. Switching mechanism of Wnt/β-catenin signaling between the differentiation and proliferation processes are not fully elucidated, because a complication is that Wnt/β-catenin signaling pathway also plays as either a tumor suppressor or a tumor enhancer. In the hematopoietic cells, the concept that fold changes of β-catenin, not absolute levels of β-catenin, govern downstream signaling events such as differentiation and proliferation, is accepted at present so far. Our finding suggests that switching between differentiation and proliferation is depending on the links of two signaling pathways. Therefore, we would like to introduce this novel link of Kras and Wnt/β-catenin signaling pathway to the readers of Cells. In the revised review, we have proposed that more attentions need to be paid the functions as tumor suppressors of not only WT-Kras but also Wnt/β-catenin signaling pathways, and must be focusing on how these pathways govern the downstream signaling pathway in the hematopoietic cells. we hope this basic research may contribute to development of effective therapies. We have amended and incorporated the description in lines 80-89. 

Line 80-89, The functions of both pathways are very delicately and finely regulated to control many cellular events. In this review, we are paying attention to the roles of tumor suppressor rather than tumor enhancer, although the functions of WT-Kras have not fully elucidated. Ras proteins have been dismissed as undruggable target for many decades. Great discovery of Kras G12C inhibitor [31] brings light to many patients with solid tumors, but much less benefits to patients with AM. Here, we have shown that WT-Kras linked to Wnt/β-catenin signaling pathway and regulated DMSO-induced differentiation of HL-60 cells. Engagement of WT-Kras and Wnt/β-catenin signaling may govern downstream signaling pathway. We have described and discussed here the functions of tumor suppressor in these two signaling pathways including our finding.　

Author Response File: Author Response.pdf

Reviewer 4 Report

1. In figure 3C the blot of Catenin is not clear at all.
2. In line 164, the clarity of the presentation needs to be improved.
3. The author stated the results in line 503. However, more details are needed so that the reader can understand the impact of this information. 

Author Response

Author's Reply to the Review Report (Reviewer 4)

We have incorporated new sections “Non-canonical Wnt signaling pathways” (section 3.2 line 205-246), “Wnt/β-catenin signaling pathway in normal hematopoiesis and AML” (section 4, line 247-290), “Non-canonical Wnt signaling pathways in AML” (section 5, line 291-313) and “Application to treatment of patients with AML “(section 11, line 586-669, including Tables 1 and 2). We have introduced application therapies targeting Kras (line 587-634) and Wnt/β-catenin signaling pathway (line 635-669). We have deleted Figure 1 suggested by a reviewer comment. We have incorporated new Figure (Figure 2) describing non-canonical Wnt signaling pathway. The order of some sections has been changed to improve the flow of the review.

According to several reviewers’ comments and considering the concept of this review, in the revised review, we have substituted ‘engage’ or ‘link’ or ‘sequentially activated’ instead of ‘crosstalk’. We have changed the title to “Novel insights into the role of Kras in myeloid differentiation: Engaging with Wnt/β-catenin signaling pathway”.

1. In figure 3C the blot of Catenin is not clear at all.

A previous report showed that β-catenin level is low in HL-60 cells (Chung, E.J. et al, Blood, 2002. 100,3, 982-990). Similarly, we detected low levels of basal of β-catenin as shown in Figure 2. C (Original version, Figure. 3 C). Thus, without DMSO (basal level) the band is very faint and almost detectable. Upon DMSO stimulation, β-catenin levels are enhanced in a time-dependent manner. These results were replicated and a limitation of this study, and therefore we have not replaced the blot. As described in the manuscript, fold of β-catenin rather than amount of β-catenin plays important roles for the differentiation of myeloide cells (Goentoro, L. and M.W. Kirschner. Mol Cell, 2009. 36,5, 872-884., Luis, T.C. et.al,. Leukemia, 2012. 26,3, 414-421., Luis, T. C. et.al, Cell Stem Cell, 2011. 9,4, 345-356). Shown in Figure 2. C, the fold change of β-catenin is clearly detectable. This is another reason that why we kept the blot in the amended review.

2. In line 164, the clarity of the presentation needs to be improved.

For clarity, we have replaced the Figure 1 (original version, Figure 2). We have described more detail about Wnt/β-catenin signaling pathway with or without Wnt in Figure 1. First, we explained the β-catenin degradation via β-catenin destruction complex in the absence of Wnt (shown in left panel). We have described β-catenin degradation by proteasome in line 174-176. More detailed description is given in line 190-192 in the text.

The signaling pathway with Wnt is presented in the right panel of Figure 1. We have explained the canonical signaling pathway, by which Wnt ligand transduces the signal via Wnt signalosome (line 193-204, original version line 164). We apologize that we did not present clearly the diagram of Wnt/β-catenin signaling pathway in the original version of review and hope this has now been achieved.

3. The author stated the results in line 503. However, more details are needed so that the reader can understand the impact of this information.

Thank you very much for the comment. We have amended Conclusion part (section 13, line 732-768). We have described the differences in gene mutations among solid tumors and AML (line 736-740). Next, we have addressed character of AML and an importance of deregulation of signaling pathway in AML (line 741-747). A future direction is mentioned in line 748-750. Our findings suggest the possibility that activation of WT-Kras can modulate the differentiation of AML cells and combination therapy may be effective for AML. We have described a possible contribution of our finding in therapy for AML, although more detailed analysis is required (line 757-768). 

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The authors present a revised of their manuscript which takes many comments of all reviewers into account. the revised version is clearly an improvement over the original submission. However, it remains unclear why the authors include data such as western blots and diagrams into this manuscript, which is a review. The two Figures, which contain data should be deleted from the review. If these data or similar data represent findings that are generally accepted knowledge and that have been published, the authors could be represented the finding as a diagram or a schema - but not in form of western blots or diagrams. 

Author Response

Thank you very much for your thoughtful comments. According to your suggestions, we have deleted two figures (Figures 3 and 4). We have incorporated the new Table 1, which presents the movement of various signaling molecules during DMSO-induced differentiation of HL-60 cells. Therefore, original Table 1 and 2 are Table 2 and 3, respectively in the revised version of Review. Thank you very much for your advice.

Author Response File: Author Response.docx

Reviewer 2 Report

This paper provided an introduction to the differentiation of the myeloid leukemia cell line HL- 60 cells and the novel functions of WT-Kras in the DMSO-induced differentiation of HL-60 cells. The authors have carefully revised the article and enriched the discussion content according to the previous opinions. The article provides certain clinical guiding value. Except for a few typographical and format errors, this letter can be accepted for publication.

Author Response

Thank you very much for the comments.

We have amended typographical and format errors.

Author Response File: Author Response.docx