Clinical implication and potential function of ARHGEF6 in acute myeloid leukemia: An in vitro study

Kang Li; Haiquan Wang; Chaofan Yang; Chaojun Li; Bin Xue; Jiankui Zhou

doi:10.1371/journal.pone.0283934

Peer Review History

Original SubmissionApril 29, 2022
12 Jan 2023 Decision Letter - Senthilnathan Palaniyandi, Editor PONE-D-22-12620Clinical implication and potential function of ARHGEF6 in acute myeloid leukemiaPLOS ONE Dear Dr. Zhou, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. We have received the opinions of expert reviewer and we invite you to submit a revised version of the manuscript. Please consider and address each of the comments raised by the reviewers. Please submit your revised manuscript by Feb 09 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Senthilnathan Palaniyandi, Ph.D Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. Thank you for stating in your Funding Statement: “This work was supported by Guangdong Basic and Applied Basic Research Foundation (2020A1515110581 to J. Zhou), start-up funds from Guangzhou University to J. Zhou, Chinese National Science Foundation (32071145 and 31771572 to B. Xue), the Nature Science Foundation of Jiangsu Province (BK20191356 to B. Xue).” Please provide an amended statement that declares all the funding or sources of support (whether external or internal to your organization) received during this study, as detailed online in our guide for authors at http://journals.plos.org/plosone/s/submit-now. Please also include the statement “There was no additional external funding received for this study.” in your updated Funding Statement. Please include your amended Funding Statement within your cover letter. We will change the online submission form on your behalf. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Partly Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 3. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This article is bioinformatic and it just collected published data and then analyzed them. Most of data derived from cell lines, the experiment result of cell line are artificial data not is not real data. we discuses on AML it meaning patients life, by statically analysis we can reach to use one drug is good or not. Any Patient is unique. Reviewer #2: Thank you for the opportunity to review the manuscript titled ‘Clinical implication and potential function of ARHGEF6 in acute myeloid leukemia’. The authors have highlighted an important finding of high ARHGEF6 expression in patients with AML being associated with a favorable prognosis and low expression being associated with a poor prognosis. The authors have also highlighted the improvement of outcomes by using hematopoietic stem cell transplantation in patients with ARHGEF6 low expression. Additionally, the role of the HOX gene family and TRH has been highlighted which is an important prognostic finding. The paper is well written, informative and I believe will be valuable to readers given its scientific merit. The only point I would like to make is that FAB classification of AML is now obsolete and it would be interesting to see the trends of ARHGEF6 expression with the new classification. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No Reviewer #2: Yes: Supriya Gupta, MD ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0283934.r001
Revision 1
10 Feb 2023 Author Response Dear Dr. Senthilnathan Palaniyandi, Thank you for your letter dated January 12. We were pleased to know that our work was rated as potentially acceptable for publication in PLOS ONE, subject to adequate revision. We thank the reviewers for the time and effort that they have put into reviewing the previous version of the manuscript. Their suggestions have enabled us to improve our work. Based on the instructions provided in your letter, we uploaded the file of the revised manuscript. Accordingly, we have uploaded a copy of the original manuscript with all the changes highlighted by using the track changes mode in MS Word. Appended to this letter is our point-by-point response to the comments raised by the reviewers. The comments are reproduced and our responses are given directly afterward. We would like also to thank you for allowing us to resubmit a revised copy of the manuscript. We hope that the revised manuscript is accepted for publication in PLOS ONE. Sincerely, Jiankui Zhou Encl. Responses to the comments from Reviewers #1 and #2. Reply to Reviewer #1 Dear Reviewer, Thank you very much for your time involved in reviewing the manuscript; your responsible attitude toward patients is admirable. Comments: “This article is bioinformatic and it just collected published data and then analyzed them. Most of data derived from cell lines, the experiment result of cell line are artificial data not real data. we discuss on AML it meaning patients life, by statically analysis we can reach to use one drug is good or not. Any Patient is unique.” We also appreciate your clear and detailed feedback and hope that the explanation has fully addressed all of your concerns. In this work, we focused on the expression characteristics, clinical prognosis, and potential function of the Rho guanine nucleotide exchange factor6 (ARHGEF6) in acute myeloid leukemia (AML). Through analysis of multiple databases (2 datasets on cell line; 4 datasets on RNA levels of patients; 2 datasets on patients’ clinical records), we found that: (1) As a member of Rho guanine nucleotide exchange factors (GEFs), ARHGEF6 expression in AML has not been reported. By analyzing several databases, we found that ARHGEF6 is highly expressed in tissues and cell lines of AML. Pan-cancer analysis of ARHGEF6 showed its highest expression in AML. In the analysis of several common AML recurrence-associated karyotypes, we found that ARHGEF6 expression was significantly higher in t(8;21) compared to other karyotypes. (2) Previous studies have generally concluded that Rho GEFs play a role in cancer promotion because they activate Rho GTPases. But a large part of Rho GEFs has not been sufficiently studied in various cancer types. Our analysis of the TCGA database and LinkedOmics database revealed that overall survival was significantly higher in patients with high ARHGEF6 expression, suggesting that overexpression of ARHGEF6 is associated with a good prognosis in AML. Besides, low ARHGEF6 expression patients can benefit from auto/allo-HSCT. These results can guide clinical treatment. (3) ARHGEF6 downregulates the expression level of HOX family genes which inhibit hematopoietic stem cell differentiation, meanwhile increasing the expression of TRH, a favorable prognostic factor in AML. These results somewhat corroborate our previous findings and reveal the role of ARHGEF6 in AML. Although we used data from cell lines, this part of the data only presented in FigS1, and all data presented in the text are from AML patients. Providing treatment plans to patients need to be personalized, and we should be responsible for the patient's life. Basic research is a long process from bench to bedside. Research starts from the cellular level, then move up to the animal, and finally is validated with precious clinical patient samples. For humans, the number of genes to be studied is massive, and screen for some genes with key roles in a limited time is crucial. Bioinformatic analysis can accelerate this process and guide subsequent laboratory and clinical studies. We would like to take this opportunity to thank you for all your time involved and this great opportunity for us to improve the manuscript. We hope you will find this revised version satisfactory. Sincerely, Jiankui Zhou -----End of Reply to Reviewer #1------ Reply to Reviewer #2 Dear Dr. Supriya Gupta, Thank you very much for your time in reviewing the manuscript and your encouraging comments on the merits. Comments: “The authors have highlighted an important finding of high ARHGEF6 expression in patients with AML being associated with a favorable prognosis and low expression being associated with a poor prognosis. The authors have also highlighted the improvement of outcomes by using hematopoietic stem cell transplantation in patients with ARHGEF6 low expression. Additionally, the role of the HOX gene family and TRH has been highlighted which is an important prognostic finding. The paper is well written, informative and I believe will be valuable to readers given its scientific merit.” We also appreciate your clear and detailed feedback and hope that the explanation has fully addressed all of your concerns. In the remainder of this letter, we discuss each of your comments individually along with our corresponding responses. Comment1: “The only point I would like to make is that FAB classification of AML is now obsolete and it would be interesting to see the trends of ARHGEF6 expression with the new classification.” Response1: Thanks for your great suggestion on improving the accessibility of our manuscript. We have re-analyzed the data using the WHO classification published in 2016. The relevant contents are provided below as a screen dump for your quick reference. The WHO classification and risk stratification were significantly different in patients with different ARHGEF6 expressions. In the WHO classification distribution, high ARHGEF6 expression was significantly associated with genetic abnormalities. We would like to take this opportunity to thank you for all your time involved and this great opportunity for us to improve the manuscript. We hope you will find this revised version satisfactory. Sincerely, Jiankui Zhou -----End of Reply to Reviewer #2------ Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0283934.r002
8 Mar 2023 Decision Letter - Senthilnathan Palaniyandi, Editor PONE-D-22-12620R1Clinical implication and potential function of ARHGEF6 in acute myeloid leukemiaPLOS ONE Dear Dr. Zhou, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. We have now received comments from the referee of your manuscript, we invite you to submit a revised version of the manuscript. Would you be able to consider adding in vitro in the title? Please submit your revised manuscript by Apr 22 2023 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. Please include the following items when submitting your revised manuscript: A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'. A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'. An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'. If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter. If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols. We look forward to receiving your revised manuscript. Kind regards, Senthilnathan Palaniyandi, Ph.D Academic Editor PLOS ONE Journal Requirements: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't Know ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: I read the answers of correspond auteur, it described very well. as I suggest in last review. Same experiment is needed on primary AML cells derived from patients in two groups: M3 and non M3 groups. Or change the title : added in vitro results ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No ******** [NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.] While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0283934.r003
Revision 2
20 Mar 2023 Author Response Dear Dr. Senthilnathan Palaniyandi, Thank you for your letter dated March 9. We are pleased to know that our work was rated as potentially acceptable for publication in PLOS ONE, subject to adequate revision. We thank the reviewer for the time and effort in reviewing the previous version of the manuscript. The suggestions have enabled us to improve our work. Based on the instructions provided in your letter, we revised the article title and uploaded the file of the revised manuscript. Accordingly, we have uploaded a copy of the original manuscript with all the changes highlighted by using the track changes mode in MS Word. Appended to this letter is our response to the comment raised by the reviewer. We would like also to thank you for allowing us to resubmit a revised copy of the manuscript. We hope that the revised manuscript is accepted for publication in PLOS ONE. Sincerely, Zhou Jiankui Encl. Responses to the comments from Reviewer #1. Reply to Reviewer #1 Dear Reviewer, Thank you very much for your time involved in reviewing the manuscript. Your suggestions are of great help to us. Comments: “I read the answers of correspond auteur, it described very well. as I suggest in last review. Same experiment is needed on primary AML cells derived from patients in two groups: M3 and non M3 groups. Or change the title : added in vitro results” We also appreciate your clear and detailed feedback. In this revised version we have changed the title to “Clinical implication and potential function of ARHGEF6 in acute myeloid leukemia: an in vitro study”. We would like to take this opportunity to thank you for all your time involved and this great opportunity for us to improve the manuscript. We hope you will find this revised version satisfactory. Sincerely, Zhou Jiankui -----End of Reply to Reviewer #1------ Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0283934.r004
22 Mar 2023 Decision Letter - Senthilnathan Palaniyandi, Editor Clinical implication and potential function of ARHGEF6 in acute myeloid leukemia：an in vitro study PONE-D-22-12620R2 Dear Dr. Zhou, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Senthilnathan Palaniyandi, Ph.D Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0283934.r005
Formally Accepted
29 Mar 2023 Acceptance Letter - Senthilnathan Palaniyandi, Editor PONE-D-22-12620R2 Clinical implication and potential function of ARHGEF6 in acute myeloid leukemia: an in vitro study Dear Dr. Zhou: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Senthilnathan Palaniyandi Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0283934.r006

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