TLR5 agonist entolimod reduces the adverse toxicity of TNF while preserving its antitumor effects

Gary J. Haderski; Bojidar M. Kandar; Craig M. Brackett; Ilia M. Toshkov; Christopher P. Johnson; Geraldine M. Paszkiewicz; Venkatesh Natarajan; Anatoli S. Gleiberman; Andrei V. Gudkov; Lyudmila G. Burdelya

doi:10.1371/journal.pone.0227940

Peer Review History

Original SubmissionSeptember 30, 2019
4 Nov 2019 Decision Letter - Partha Mukhopadhyay, Editor PONE-D-19-26443 Title - TLR5 agonist entolimod reduces the adverse toxicity of TNF while preserving its antitumor effects PLOS ONE Dear Dr. Burdelya, 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 received positive feedback on your manuscript from both experts. However, there are few technical issues requires your attention. We would appreciate receiving your revised manuscript by Dec 15 2019 11:59PM. When you are 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. 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This file should be uploaded as separate file and labeled 'Manuscript'. Please note while forming your response, if your article is accepted, you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. We look forward to receiving your revised manuscript. Kind regards, Partha Mukhopadhyay, Ph.D. Academic Editor PLOS ONE Journal Requirements: 1. When submitting your revision, we need you to address these additional requirements. 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2. At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines: (1) Please state the number of mice used in the study (2) Please provide details of animal welfare (e.g., shelter, food, water, environmental enrichment) (3) Please describe any steps taken to minimize animal suffering and distress, such as by administering anaesthesia, during intraperitoneal and subcutaneous injections (4) Please include the method of euthanasia (5) Please state specifically in your Methods section that no mice died before meeting the criteria for euthanasia Thank you for your attention to these requests. 3. At this time, we ask that you please report some additional details regarding the Entolimod used in this study. Specifically, we ask that you state the product number and any lot numbers provided when the compound was purchased from Cleveland BioLabs. 4. Thank you for stating the following in the Competing Interests section: I have read the journal's policy and the authors of this manuscript have the following competing interests: Andrei Gudkov is a consultant and shareholder of Cleveland BioLabs, Inc., the biotech company that provided EntolimodTM for this work. This did not alter authors’ adherence to PLOS ONE policies. Other authors declare no competing interests. We note that you received funding from a commercial source: Cleveland BioLabs, Inc. Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc. Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your amended Competing Interests Statement within your cover letter. We will change the online submission form on your behalf. Please know it is PLOS ONE policy for corresponding authors to declare, on behalf of all authors, all potential competing interests for the purposes of transparency. PLOS defines a competing interest as anything that interferes with, or could reasonably be perceived as interfering with, the full and objective presentation, peer review, editorial decision-making, or publication of research or non-research articles submitted to one of the journals. Competing interests can be financial or non-financial, professional, or personal. Competing interests can arise in relationship to an organization or another person. Please follow this link to our website for more details on competing interests: http://journals.plos.org/plosone/s/competing-interests [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: Yes Reviewer #2: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: No ****** 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: Significant work by Haderski et al suggesting the antitumor role of TNF. Below are the points which should be addressed before it's suitable for publication. 1.It's been shown by several groups that LPS activates NRF2 antioxidant pathways. Authors should discuss their perspective on the above mentioned topic on basis of this current study. 2. Authors must add a graphical abstract as a figure and discuss this to the text. It will help readers to get take-home message easily. 3.Fig 1 ang Fig 2 resolution is very low. Every lebel is very hazy and hard to read. Authors should work on that. Reviewer #2: In the study, 'TLR5 agonist entolimid reduces the adverse toxicity of TNF while preserving its antitumor effects,' the authors have successfully demonstrated that combining entolimid with TNF treatment significantly reduces related toxicity while maintaining its efficacy as an anti-cancer agent. This study was performed rigorously, and bears merit. Some minor revisions have been recommended as follows: 1) The authors use the term TNF, however, there are two members- TNF-alpha and TNF-beta. Please specify the correct member. 2) Abstract: Please describe actual results. Mention what was increased and decreased, by what treatment, and by how many fold/was it significant? 3) Introduction: Differentiate between a normal immune response triggered by bacteria which results in release of cytokines versus sepsis. Elaborate on 'cytokine storm'. 4) Introduction: Give a bit more detail about the mechanism of action of TNF. How does TLR4 and NFKB fit in (provide more details)? 5) Introduction: Page 5 Line 88: The authors say TLR5 has narrow tissue distribution. So, if TLR4 is more widespread, how then, does TLR5 neutralize the toxic effects of TLR4 effectively? Is the effect tissue specific and restricted, or is it systemic which would make more sense? 6) Introduction: Page 5, Line 93: How does entolimid preferentially protect only normal tissues? What is the mechanism? Anything previously published? 7) Introduction: Page 6, Line 110: Elaborate on how D-galactosamine administration sensitizes cells to TNF? 8) Materials and methods: Mention how the gene expression profiling was done exactly here. 9) Materials and methods: Please clarify how many mice were in each treatment group exactly? 10) Statistical analysis: Student's t test was performed in some animal studies. If distribution was skewed, a non parametric test should be applied, not t test. 11) Statistical analysis: Was power analysis performed to determine sample size? 12) Figures: Images need to be of higher quality, as some of the labels are blurred. 13) Figure 2F: There should be an Entolimid/TNF/D-GalN only control. 14) Please separate the results section and figure legends sections completely. ****** 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: Yes: Suman Mukhopadhyay 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 to be viewed.] 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 us at figures@plos.org. Please note that Supporting Information files do not need this step. https://doi.org/10.1371/journal.pone.0227940.r001
Revision 1
15 Dec 2019 Author Response Editorial Comments: 1. When submitting your revision, we need you to address these additional requirements. 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 http://www.journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and http://www.journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf We have checked the revised manuscript for full compliance with the style requirements of PLOS ONE. 2. At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines: (1) Please state the number of mice used in the study We have added information on the number of mice used for each experiment to the figure legends. (2) Please provide details of animal welfare (e.g., shelter, food, water, environmental enrichment) Details regarding animal maintenance and welfare have been added to the Materials and Methods section. (3) Please describe any steps taken to minimize animal suffering and distress, such as by administering anesthesia, during intraperitoneal and subcutaneous injections Steps taken to minimize animal suffering and distress are included in the Ethics Statement (Materials and Methods). These included close monitoring of mice after treatment with LPS and TNF for signs of discomfort, pain and distress (i.e., hourly for 8 hours after treatment, then twice a day for 2 days, then daily until the end of the experiment). Mice were euthanized as soon as signs of morbidity developed. In accordance with the IACUC-approved protocol, no anesthesia was used during intraperitoneal and subcutaneous injections of drugs or tumor cells. (4) Please include the method of euthanasia The method of euthanasia used in this study was CO2 asphyxiation followed by cervical dislocation, per IACUC guidelines. This information has been added to the revised manuscript in the Ethics Statement (Materials and Methods). (5) Please state specifically in your Methods section that no mice died before meeting the criteria for euthanasia As described in the Ethics Statement (Materials and Methods), we aimed to avoid treatment- or tumor-related pain or death as an endpoint by closely monitoring animals for signs of discomfort, pain and distress hourly for 8 hours after treatment, then twice a day for 2 days, then daily until end of experiments. This allowed us to euthanize animals, as required by IACUC guidelines, when indicators of severe morbidity such as weight loss, large or necrotized tumors, ruffled fur developed. 3. At this time, we ask that you please report some additional details regarding the Entolimod used in this study. Specifically, we ask that you state the product number and any lot numbers provided when the compound was purchased from Cleveland BioLabs. Requested details regarding the entolimod (CBLB502) lot used in the study have been added to the Methods section. 4. Thank you for stating the following in the Competing Interests section: I have read the journal's policy and the authors of this manuscript have the following competing interests: Andrei Gudkov is a consultant and shareholder of Cleveland BioLabs, Inc., the biotech company that provided EntolimodTM for this work. This did not alter authors’ adherence to PLOS ONE policies. Other authors declare no competing interests. We note that you received funding from a commercial source: Cleveland BioLabs, Inc. Please provide an amended Competing Interests Statement that explicitly states this commercial funder, along with any other relevant declarations relating to employment, consultancy, patents, products in development, marketed products, etc. Within this Competing Interests Statement, please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your amended Competing Interests Statement within your cover letter. We will change the online submission form on your behalf. The following amended Competing Interests Statement has been included in the cover letter: “My relationship with Cleveland BioLabs, Inc. (CBLI), the company that developed and holds the rights to entolimod, was limited to receiving entolimod for this study. I, Lyudmila Burdelya, did not receive any monetary funding for this study from CBLI or any other commercial source, nor was I employed or paid by CBLI during the time period when the study was conducted. The fact that I am a co-inventor of entolimod as listed on several filed patent applications does not alter my adherence to PLOS ONE policies on sharing data and materials. Andrei Gudkov is also a co-inventor of entolimod and is a consultant and shareholder of CBLI; this does not alter the author’s adherence to PLOS ONE policies on sharing data and materials. Other authors declare no competing interests.” Reviewer #1: Significant work by Haderski et al suggesting the antitumor role of TNF. Below are the points which should be addressed before it's suitable for publication. 1. It's been shown by several groups that LPS activates NRF2 antioxidant pathways. Authors should discuss their perspective on the above-mentioned topic on basis of this current study. In the Introduction of the revised manuscript, we have added more information on LPS-induced inflammatory and pro-survival pathways, including the Nrf2 pathway (lines 100-102). However, because the goal of our study was to assess the ability of entolimod to mitigate tissue damaging effects of LPS and TNF, our work and the manuscript text is focused on the deleterious aspects of LPS responsible for excessive inflammation and septic shock syndrome. 2. Authors must add a graphical abstract as a figure and discuss this to the text. It will help readers to get take-home message easily. We appreciate this suggestion and have provided a schematic illustration of the mechanisms involved in entolimod-mediated protection of the liver from LPS and TNF toxicity as Figure 5. This figure is referred to in the Discussion section (lines 561-564). 3. Fig. 1 ang Fig. 2 resolution is very low. Every label is very hazy and hard to read. Authors should work on that. The revised manuscript includes higher quality figures. Reviewer #2: In the study, 'TLR5 agonist entolimod reduces the adverse toxicity of TNF while preserving its antitumor effects,' the authors have successfully demonstrated that combining entolimod with TNF treatment significantly reduces related toxicity while maintaining its efficacy as an anti-cancer agent. This study was performed rigorously and bears merit. Some minor revisions have been recommended as follows: 1) The authors use the term TNF, however, there are two members- TNF-alpha and TNF-beta. Please specify the correct member. We used only TNF-alpha in this study, which is commonly abbreviated as TNF (https://en.wikipedia.org/wiki/Tumor_necrosis_factor_alpha). The fact that we used TNF-alpha abbreviated as TNF is described now in the Abstract, Introduction, and Materials and Methods sections of the revised manuscript (lines 16, 55, 170). 2) Abstract: Please describe actual results. Mention what was increased and decreased, by what treatment, and by how many fold/was it significant? The abstract has been revised to more clearly describe specific results of the study in addition to the overall conclusions. 3) Introduction: Differentiate between a normal immune response triggered by bacteria which results in release of cytokines versus sepsis. Elaborate on 'cytokine storm'. We have revised the Introduction section to more clearly describe and define the conditions of normal immune responses to bacteria, cytokine storm and septic shock (line 55). 4) Introduction: Give a bit more detail about the mechanism of action of TNF. How does TLR4 and NFKB fit in (provide more details)? We have revised the Introduction section of the manuscript to include more detail regarding what is known about the mechanisms of action responsible for the toxicities of LPS and TNF. 5) Introduction: Page 5 Line 88: The authors say TLR5 has narrow tissue distribution. So, if TLR4 is more widespread, how then, does TLR5 neutralize the toxic effects of TLR4 effectively? Is the effect tissue specific and restricted, or is it systemic which would make more sense? We have modified the text (line 96) to more clearly present our statement (different from TLR4 rather than narrow). We also explain that entolimod (i) has direct local protective effects in the liver due to TLR5-NF�B signaling in hepatocytes, and (ii) protects distant organs (including TLR5 non-expressing organs) through secretion of cytoprotective factors. 6) Introduction: Page 5, Line 93: How does entolimod preferentially protect only normal tissues? What is the mechanism? Anything previously published? Our previous studies have shown that entolimod does not protect tumors from various stress-induced therapies (Burdelya et al 2008, 2012, 2013, 2016, Kojouharov et al 2014). Our current explanation is that the entolimod’s protective effects towards normal cells are mediated via NF-�B-mediated activation of pro-survival factors. Since tumors commonly have constitutively active NF-�B, they cannot benefit, in terms of survival under conditions of stress, from NF-�B-activating mechanisms such as TLR5 activation. This is discussed in the text of the manuscript (Introduction, lines 94-104 and 111-117. 7) Introduction: Page 6, Line 110: Elaborate on how D-galactosamine administration sensitizes cells to TNF? We have added a description of (and references for) how D-galactosamine affects hepatocytes and sensitizes them to LPS- and TNF-induced toxicity in the Results section of the revised manuscript (lines 329-332). 8) Materials and methods: Mention how the gene expression profiling was done exactly here. The methodological details of our gene expression profiling experiment are provided in the Results section of the manuscript (line 433 and below). 9) Materials and methods: Please clarify how many mice were in each treatment group exactly? We have added sample size numbers for both in vivo and in vitro experiments (see Materials and Methods and figure legends). 10) Statistical analysis: Student's t test was performed in some animal studies. If distribution was skewed, a non-parametric test should be applied, not t test. Data were evaluated using the multiple distribution tests available in GraphPad Prism software to determine whether the data followed the normal distribution. In each instance, the data were found to fit the normal distribution, thus allowing use of the parametric t-test for data analysis. 11) Statistical analysis: Was power analysis performed to determine sample size? Minimum sample sizes for all experiments were determined using a significance level alpha (probability of a Type I error) value of 0.05 and a beta (probability of a Type II error) value of 0.20. The study power (probability of detecting a true positive) was then 80%. The calculations used are customary based on normal distributions. Ref: Rosner B. Fundamentals of Biostatistics. 8th ed. Cengage Learning, Inc. 2015. Animal protocols were reviewed by RPCCC’s biostatistician as part of IACUC protocol review and approval process. 12) Figures: Images need to be of higher quality, as some of the labels are blurred. We have included higher quality figures in the revised manuscript. 13) Figure 2F: There should be an Entolimod/TNF/D-GalN only control. The relevant controls without antibody treatment to deplete neutrophils are presented in Figures 2A (for entolimod/LPS/D-GalN) and 2D (for entolimod/TNF/D-GalN) of the revised manuscript. 14) Please separate the results section and figure legends sections completely. The figure legends are inserted into the Results section and separated from the text according to PLOS One instructions. Additional changes: In Figure 3, panels B, C, D have been changed to bar graphs (instead of line graphs as included in our original manuscript submission) since the data were obtained from individual treatment groups which are more accurately represented by independent bars than connected lines. Attachments Attachment Submitted filename: Response to Reviewers.pdf https://doi.org/10.1371/journal.pone.0227940.r002
6 Jan 2020 Decision Letter - Partha Mukhopadhyay, Editor TLR5 agonist entolimod reduces the adverse toxicity of TNF while preserving its antitumor effects PONE-D-19-26443R1 Dear Dr. Burdelya, We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements. Within one week, you will receive an e-mail containing information on the amendments required prior to publication. When all required modifications have been addressed, you will receive a formal acceptance letter and your manuscript will proceed to our production department and be scheduled for publication. Shortly after the formal acceptance letter is sent, an invoice for payment will follow. To ensure an efficient production and billing process, please log into Editorial Manager at https://www.editorialmanager.com/pone/, click the "Update My Information" link at the top of the page, and update your user information. 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 enable them to help maximize its impact. If they will be preparing press materials for this manuscript, you must inform our press team as soon as possible and 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. With kind regards, Partha Mukhopadhyay, Ph.D. Section 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: Significant work. All concerns have been addressed and modified the manuscript by incorporating the changes. Manuscript is ready for acceptance in plosone. ****** 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: Yes: Suman Mukhopadhyay https://doi.org/10.1371/journal.pone.0227940.r003
Formally Accepted
22 Jan 2020 Acceptance Letter - Partha Mukhopadhyay, Editor PONE-D-19-26443R1 TLR5 agonist entolimod reduces the adverse toxicity of TNF while preserving its antitumor effects Dear Dr. Burdelya: I am 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 notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, 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. For any other questions or concerns, please email plosone@plos.org. Thank you for submitting your work to PLOS ONE. With kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Partha Mukhopadhyay Section Editor PLOS ONE https://doi.org/10.1371/journal.pone.0227940.r004

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