Combination of triciribine and p38 MAPK inhibitor PD169316 enhances the differentiation effect on myeloid leukemia cells

Yuri Sato-Nagaoka; Susumu Suzuki; Souma Suzuki; Shinichiro Takahashi

doi:10.1371/journal.pone.0312406

Peer Review History

Original SubmissionJune 26, 2024
21 Aug 2024 Decision Letter - Jinsong Zhang, Editor PONE-D-24-25935Combination of triciribine and p38 MAPK inhibitor PD169316 enhances the differentiation effect on myeloid leukemia cellsPLOS ONE Dear Dr. Takahashi, 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. Your manuscripts have been reviewed by two experts in the field of cancer and gene transcription. While they found your results promising, they also identified some important issues, including (but are not limited to) cell line differences, some poor-quality data, and questions about the clinical relevance of the conditions used in your study. All these issues must be addressed by substantially revising the current manuscript. 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We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed: https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0303428 https://karger.com/aha/article-abstract/145/2/113/821321/Kinase-Inhibitors-and-Interferons-as-Other-Myeloid?redirectedFrom=fulltext https://linkinghub.elsevier.com/retrieve/pii/S0006291X24000767 In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed. 4. Thank you for stating the following financial disclosure: Grant-in-Aid for Scientific Research (21K07346) from the Ministry of Education, Science and Culture, Japan, to ST. Kyowa-Kirin Research Support (Kyowa-Kirin Co. Ltd.), Japan, to ST. Daiichi-Sankyo Research Support (Daiichi-Sankyo Inc.), Japan, to ST. Please state what role the funders took in the study. 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When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. [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: No Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: I Don't Know ****** 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: In this manuscript Combination of triciribine and p38 MAPK inhibitor PD169316 enhances the differentiation effect on myeloid leukemia cells, Sato-Nagaoka et al used two cell lines NB4 and HL-60 to study the effects of TCN and PD169316 on leukemic cell differentiation. They showed that the combination of two agents had an enhanced effect on cell differentiation. They further investigated the mechanism underlying the observed effect and found that multiple pathways and genes were involved. Overall, this is an interesting study with intriguing observations. However, there are some concerns which need to be addressed. 1.Both TCN and PD169316 were used at 10uM. How did the authors choose this dosage? Do lower doses have an effect on cell differentiation? Is this dose clinically relevant if a clinical trial is to be conducted. Authors need to provide more information on their choice of dosage. 2.Figure 2. CD13 expression was increased after TCN+PD169316 in HL-60 cells, but CD13 was decreased after TCN+PD169316 treatment based on the heatmap shown. Please explain this result in the text. 3.Figure 4. p-ERK1/2 Western blot in NB4 cells was overexposed and saturated. The difference between bands was not obvious. Please repeat the experiemnts and have high quality publishable band images available. The b-Actin loading control bands were not of the publication quality for NB4 cells in Figure 4. Reviewer #2: This study evaluates the effects of the p38 MAPK inhibitor PD169316 in enhancing the differentiation effects of the Akt inhibitor triciribine triciribine (TCN) on myeloid leukemia cell differentiation. In a previous study the authors showed that PD169316, SB203580, SB202190 (p38 MAPK inhibitors), and TCN potently increased the expression of CD11b in the NB4 acute promyelocytic leukemia cell line (PLoS One. 2024 May 14;19(5):e0303428). While that study focuses on TCN, this study focuses on the combined effects of PD169316 and TCN. The methods used are very similar. The results show enhancement of combination of TCN and PD169316 potently induces myeloid differentiation markers in NB4 and HL-60 cells, changes the morphology and decreases the nuclear-to-cytoplasmic ratio, induced phosphorylation of ERK, and induces a panel of chemokines as well as cytokines and their receptors. The data is of high quality. However, the effects are shown in M3 NB4 cells which contains two t(15;17) chromosomes and other cytogenetic abnormalities, and the M2 HL-60 cells, both of which can be differentiated into granulocytic and mononuclear lineage. The effects were absent in M5 acute monoblastic and monocytic leukemia cell lines (THP-1 and U937). In the future, if the authors could demonstrate the key effects (e.g. myeloid differentiation markers and nuclear-to-cytoplasmic ratio) in a couple more AML cell lines, it could further validate the effects of TCN and PD169316. ****** 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: 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.0312406.r001
Revision 1
18 Sep 2024 Author Response Reviewer #1: In this manuscript Combination of triciribine and p38 MAPK inhibitor PD169316 enhances the differentiation effect on myeloid leukemia cells, Sato-Nagaoka et al used two cell lines NB4 and HL-60 to study the effects of TCN and PD169316 on leukemic cell differentiation. They showed that the combination of two agents had an enhanced effect on cell differentiation. They further investigated the mechanism underlying the observed effect and found that multiple pathways and genes were involved. Overall, this is an interesting study with intriguing observations. However, there are some concerns which need to be addressed. 1.Both TCN and PD169316 were used at 10uM. How did the authors choose this dosage? Do lower doses have an effect on cell differentiation? Is this dose clinically relevant if a clinical trial is to be conducted. Authors need to provide more information on their choice of dosage. To address this issue, we examined the expression levels of differentiation marker CD11b, in lower doses of TCN and PD169316. As a result, we observed that above 5 µM of TCN and PD169316 had sufficient induction activity both in NB4 and HL-60 cells (Fig S1, S2). These are added as follows (page 9, line 167). We further examined the lower (0, 0.1, 0.5, 1.0 and 5.0 µM) dosages of these two combinations. As a result, below 1.0 µM of TCN, there were almost no effect for the combination, in both NB4 (Fig. S1) and HL-60 cells (Fig. S2). However, we found that 5.0 µM of TCN had sufficient, and 10 µM of TCN had potent combination effect. These concentrations are clinically achievable, since specimens from primary AML blasts accumulated a median peak concentration of 4 µM (range of 2.1-7.5µM) [14]. To see the maximal effect, we choose 10 µM concentration for TCN and PD169316, for further study. 2.Figure 2. CD13 expression was increased after TCN+PD169316 in HL-60 cells, but CD13 was decreased after TCN+PD169316 treatment based on the heatmap shown. Please explain this result in the text. To follow this comment, we added following sentence in the text (line 192). CD13 expression was induced by TCN, but this was suppressed by PD169316, and the combination of these two agents decreased its expression, in contrast to the expression in HL-60 cells (Fig. 2B). 3.Figure 4. p-ERK1/2 Western blot in NB4 cells was overexposed and saturated. The difference between bands was not obvious. Please repeat the experiemnts and have high quality publishable band images available. The b-Actin loading control bands were not of the publication quality for NB4 cells in Figure 4. To follow this comment, we repeated experiments for NB4 cells and obtained better quality, not saturated, blot for revised Figure 4A. Following these comments, we feel the improvement of our paper. We appreciate very much for these comments. Reviewer #2: This study evaluates the effects of the p38 MAPK inhibitor PD169316 in enhancing the differentiation effects of the Akt inhibitor triciribine triciribine (TCN) on myeloid leukemia cell differentiation. In a previous study the authors showed that PD169316, SB203580, SB202190 (p38 MAPK inhibitors), and TCN potently increased the expression of CD11b in the NB4 acute promyelocytic leukemia cell line (PLoS One. 2024 May 14;19(5):e0303428). While that study focuses on TCN, this study focuses on the combined effects of PD169316 and TCN. The methods used are very similar. The results show enhancement of combination of TCN and PD169316 potently induces myeloid differentiation markers in NB4 and HL-60 cells, changes the morphology and decreases the nuclear-to-cytoplasmic ratio, induced phosphorylation of ERK, and induces a panel of chemokines as well as cytokines and their receptors. The data is of high quality. However, the effects are shown in M3 NB4 cells which contains two t(15;17) chromosomes and other cytogenetic abnormalities, and the M2 HL-60 cells, both of which can be differentiated into granulocytic and mononuclear lineage. The effects were absent in M5 acute monoblastic and monocytic leukemia cell lines (THP-1 and U937). In the future, if the authors could demonstrate the key effects (e.g. myeloid differentiation markers and nuclear-to-cytoplasmic ratio) in a couple more AML cell lines, it could further validate the effects of TCN and PD169316. We agree with this suggestion. Indeed, we are now investigating this combination in many AML cells, because we also think it is important and to increase the relevance of this series of research. However, it would be grateful if you allow as to move forward at this time, and clarify this as a next project. Thank you so much for this constructive comment. 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 We confirmed. Thank you. 2. In your Methods section, please report the source of cell lines used for your study. We cited the source of cell lines used for this study. 3. We noticed you have some minor occurrence of overlapping text with the following previous publication(s), which needs to be addressed: https://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0303428 https://karger.com/aha/article-abstract/145/2/113/821321/Kinase-Inhibitors-and-Interferons-as-Other-Myeloid?redirectedFrom=fulltext https://linkinghub.elsevier.com/retrieve/pii/S0006291X24000767 In your revision ensure you cite all your sources (including your own works), and quote or rephrase any duplicated text outside the methods section. Further consideration is dependent on these concerns being addressed. We ensured to cite all of our sources, and all of these are from this corresponding author’s paper, as ref [6], [11], [13]. Furthermore, we rephrased related sentences. 4. Thank you for stating the following financial disclosure: Grant-in-Aid for Scientific Research (21K07346) from the Ministry of Education, Science and Culture, Japan, to ST. Kyowa-Kirin Research Support (Kyowa-Kirin Co. Ltd.), Japan, to ST. Daiichi-Sankyo Research Support (Daiichi-Sankyo Inc.), Japan, to ST. Please state what role the funders took in the study. If the funders had no role, please state: ""The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."" If this statement is not correct you must amend it as needed. Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf. We added, Role of Funder statement: The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript, in the cover letter. 5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions. We submitted our blot/gel image data as supporting information and this was noted in our cover letter. Thank you. Attachments Attachment Submitted filename: Pointbypointreply.docx https://doi.org/10.1371/journal.pone.0312406.r002
7 Oct 2024 Decision Letter - Jinsong Zhang, Editor Combination of triciribine and p38 MAPK inhibitor PD169316 enhances the differentiation effect on myeloid leukemia cells PONE-D-24-25935R1 Dear Dr. Takahashi, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, 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, Jinsong Zhang Academic Editor PLOS ONE Additional Editor Comments (optional): 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: Yes ****** 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: The authors have answered my questions and addressed my concerns. The manuscript could be considered for publication after editorial review. ****** 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 ******** https://doi.org/10.1371/journal.pone.0312406.r003
Formally Accepted
10 Oct 2024 Acceptance Letter - Jinsong Zhang, Editor PONE-D-24-25935R1 PLOS ONE Dear Dr. Takahashi, I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team. At this stage, our production department will prepare your paper for publication. This includes ensuring the following: * All references, tables, and figures are properly cited * All relevant supporting information is included in the manuscript submission, * There are no issues that prevent the paper from being properly typeset If revisions are needed, the production department will contact you directly to resolve them. If no revisions are needed, you will receive an email when the publication date has been set. At this time, we do not offer pre-publication proofs to authors during production of the accepted work. Please keep in mind that we are working through a large volume of accepted articles, so please give us a few weeks to review your paper and let you know the next and final steps. Lastly, 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 customercare@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. Jinsong Zhang Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0312406.r004

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