Selective killing of homologous recombination-deficient cancer cell lines by inhibitors of the RPA:RAD52 protein-protein interaction

Mona Al-Mugotir; Jeffrey J. Lovelace; Joseph George; Mika Bessho; Dhananjaya Pal; Lucas Struble; Carol Kolar; Sandeep Rana; Amarnath Natarajan; Tadayoshi Bessho; Gloria E. O. Borgstahl

doi:10.1371/journal.pone.0248941

Peer Review History

Original SubmissionMay 28, 2020
8 Jul 2020 Decision Letter - Alvaro Galli, Editor PONE-D-20-16153 Selective killing of homologous recombination-deficient cancer cell lines by inhibitors of the RPA:RAD52 protein-protein interaction PLOS ONE Dear Dr. Borgstahl, 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. Please submit your revised manuscript by Aug 22 2020 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Alvaro Galli 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. Please ensure you have thoroughly discussed any potential limitations of this study within the Discussion section. [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: Partly ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: No 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: This is a very well written manuscript with novel and exciting data on using FDA approved drugs for targeted therapy of BRCA-1/2 cancers by specifically target the RPA:RAD52 protein interaction. Comments: 1. Figures need to be of higher quality/resolution/contrast 2. Identification of the axes in some figure need to be more clear for example figure 1 y axes "% inhibition of " what?) while it is in the text and the legend it will be more clear if it was on the axes it self 3. Most of the supplemental data should be actually in the main manuscript that S2-6 figures as the some claims and conclusions in the manuscript can not stand without the data in these figures. The data in the manuscript should stand by it self even without the supporting material. 4. Indicate significance in each figure where applied ( use * or similar marking) 5. Unpaired student t test is not an adequate statistical tool for some the data that have more than three variables authors need to do ANOVA with honesty testing. 6 Minor spelling and grammar issues that can be easily fixed Overall all the manuscript have excellent experimental design, adequate controls and pass the scientific rigor and it would be outstanding with these minor suggestion an comments. Reviewer #2: In current manuscript, authors identified three FDA-approved drugs Mitoxantrone, doxorubicin and quinacrine as the inhibitors of the RPA:RAD PPI by high throughput screening from chemical libraries. They demonstrated that these drugs could induce synthetic lethality in BRCA deficient cancer cell lines in vitro. Overall this is a preliminary study. Some points need to be addressed. 1. To address that RAD52 PPI inhibitors induce synthetic lethality in HR-deficient cancer cell lines, in Fig2-4, it should show the RAD52 expression in the tested cell lines before and after treatment. RAD52 is DNA Repair Protein. It also would be better to have the evidence that these drugs induced DNA damage in the tested cell lines, such as markers γH2AX. 2. Fig3A, X axis missed concentration label “uM”. 3. Fig 5C, is it possible to show western blot original images and FACS image? 4. FigS3A, B, it was not clear which cell line was treated. Can authors label them? 5. Fig S4, Western blot didn’t show loading control although authors claimed there were GAPDH controls in the experiments. (Not sure it was the image loading problem, it seemed Fig S4 missed left panel?) 6. Labels in figure make people confuse. Fig S5, what does mean “1,2,3,4,5,6”? Was drug dose? FigS4, S5, was “c” control? Scel expressed in Fig S4”c” but not in Fig S5 “c”. What was the difference of the “C”? ******** 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/. 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Revision 1
29 Jan 2021 Author Response The reviewers’ comments are summarized below and followed by our response in purple italics. Places in the manuscript that were modified were highlighted in gray. Reviewer #1: This is a very well written manuscript with novel and exciting data on using FDA approved drugs for targeted therapy of BRCA-1/2 cancers by specifically target the RPA:RAD52 protein interaction. Comments: 1. Figures need to be of higher quality/resolution/contrast The resolution of all figures has been improved. 2. Identification of the axes in some figure need to be more clear for example figure 1 y axes "% inhibition of " what?) While it is in the text and the legend it will be more clear if it was on the axes itself The y-axis for Figure 1B was revised to indicate what is being inhibited (RPA:RAD52 complex). 3. Most of the supplemental data should be actually in the main manuscript that S2-6 figures as the some claims and conclusions in the manuscript cannot stand without the data in these figures. The data in the manuscript should stand by itself even without the supporting material. Although the data in the supplement support the manuscript conclusions, we consider these data as preliminary and this is an active area of research in the lab. Therefore, we believe the supplementary section is the appropriate place so they do not detract from the focus of the manuscript. 4. Indicate significance in each figure where applied (use * or similar marking) The data in Figures 2, 3, and 4 were re-evaluated using ANOVA honesty testing as described in the edited method section of the manuscript and an asterisk sign was indicated where significance was found. 5. Unpaired student t test is not an adequate statistical tool for some the data that have more than three variables authors need to do ANOVA with honesty testing. ANOVA with honesty testing was used to re-evaluate the statistical significance of the data as described in the revised method section. 6. Minor spelling and grammar issues that can be easily fixed The manuscript was edited for any spelling or grammar issues Overall all the manuscript have excellent experimental design, adequate controls and pass the scientific rigor and it would be outstanding with these minor suggestion an comments. Reviewer #2: In current manuscript, authors identified three FDA-approved drugs mitoxantrone, doxorubicin and quinacrine as the inhibitors of the RPA:RAD PPI by high throughput screening from chemical libraries. They demonstrated that these drugs could induce synthetic lethality in BRCA deficient cancer cell lines in vitro. Overall this is a preliminary study. Some points need to be addressed. 1. To address that RAD52 PPI inhibitors induce synthetic lethality in HR-deficient cancer cell lines, in Fig2-4, it should show the RAD52 expression in the tested cell lines before and after treatment. RAD52 is DNA Repair Protein. It also would be better to have the evidence that these drugs induced DNA damage in the tested cell lines, such as markers γH2AX. Thank you, this was an excellent idea and the resulting data proved to be very informative. All cell lines were grown and cells were harvested without treatment and treated with doses of mitoxantrone near their EC50 values. The level of DNA damage induced (�-H2AX) and RAD52 expression was measured by western analysis as detailed in the method section. The western data were measured in triplicate, normalized with actin and graphed (average and standard deviation) with significance calculated using ANOVA: Single factor in Excel. These graphs were included as a new figure (Fig. 5) and so the figures after that were renumbered. Individual data measurements were included in the supplementary section using the lane representation (S2 Fig.). Western data were measured using a Protein Simple Peggy Sue instrument and analyzed using the associated Compass for Simple Western software. 2. Fig3A, X axis missed concentration label “uM. Corrected. 3. Fig 5 (now Fig 6), is it possible to show western blot original images (parts and FACS image? Western images are in Fig S6. The detailed FACS analysis from our core facility was added to Fig S6. 4. FigS3A, B, it was not clear which cell line was treated. Can authors label them? Corrected. 5. Fig S4, Western blot didn’t show loading control although authors claimed there were GAPDH controls in the experiments. (Not sure it was the image loading problem, it seemed Fig S4 missed left panel?) We did not describe that we used GAPDH as a loading control. We used p70 subunit of RPA to normalize the expression of SceI. All information is given in the legend. 6. Labels in figure make people confuse. Fig S5, what does mean “1,2,3,4,5,6”? Was drug dose? FigS4, S5, was “c” control? Scel expressed in Fig S4”c” but not in Fig S5 “c”. What was the difference of the “C”? We have modified the labeling of Fig. S4 so the meaning is clearer. Now, in both figures, C stands for no SceI control. The 1,2,3,4,5,6 in Fig. S5 are simply lane numbers as described in the figure legend. We have revised the figure legends for both Figs. S4 and S5 for clarity. Attachments Attachment Submitted filename: Response to Review_1_29_2021.docx https://doi.org/10.1371/journal.pone.0248941.r002
11 Feb 2021 Decision Letter - Alvaro Galli, Editor PONE-D-20-16153R1 Selective killing of homologous recombination-deficient cancer cell lines by inhibitors of the RPA:RAD52 protein-protein interaction PLOS ONE Dear Dr. Borgstahl, 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. The reviewer is asking why some results are included in the supplementary materials and not in the manuscript figure; these results are discussed in the text and should be shown in the main text. I believe that that fugure should be shown in the manuscript properly. Please submit your revised manuscript by Mar 28 2021 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: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols We look forward to receiving your revised manuscript. Kind regards, Alvaro Galli Academic Editor PLOS ONE [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: Partly ****** 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 addressed all my comment. I still stand by my comment that supplementary data presented was used in the claims and conclusions of the study and the manuscript cannot stand without the data in these figures. I am surprised that the authors characterized these data as preliminary and not complete and still under investigation. The authors have the option to remove the claims and the conclusions related to these supplementary (preliminary) data from the main manuscript. The figure are still have poor resolution, but I think that not the fault of the authors just technology not working appropriately! ****** 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.0248941.r003
Revision 2
5 Mar 2021 Author Response We have implemented the suggestions from the reviewer by moving three figures from the supplement into the manuscript for the data that was directly cited in the text. There is a total of 9 figures in the main manuscript. The figures that remain in the supplement are only cited in the figure legends as they contain the original data that is summarized in graphs in the main figure. Some methods were moved from figure legends (previously in the supplement) to the methods section. These changes are highlighted in grey in the “Revised manuscript with Track Changes” document. Concerning the resolution of the figures, we have uploaded high resolution figures. We are not sure why the figures the reviewer sees appear to be low resolution. Attachments Attachment Submitted filename: PLOS_One_response to review 2.docx https://doi.org/10.1371/journal.pone.0248941.r004
9 Mar 2021 Decision Letter - Alvaro Galli, Editor Selective killing of homologous recombination-deficient cancer cell lines by inhibitors of the RPA:RAD52 protein-protein interaction PONE-D-20-16153R2 Dear Dr. Borgstahl, 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, Alvaro Galli 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: (No Response) ****** 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 addressed all of my comments. I think the manuscript in this revised version is highly enhanced and will attract a lot of attention and citations. ****** 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.0248941.r005
Formally Accepted
18 Mar 2021 Acceptance Letter - Alvaro Galli, Editor PONE-D-20-16153R2 Selective killing of homologous recombination-deficient cancer cell lines by inhibitors of the RPA:RAD52 protein-protein interaction Dear Dr. Borgstahl: 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. Alvaro Galli Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0248941.r006

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