Genome destabilization-associated phenotypes arising as a consequence of therapeutic treatment are suppressed by Olaparib

Mafuka Suzuki; Haruka Fujimori; Kakeru Wakatsuki; Yuya Manaka; Haruka Asai; Mai Hyodo; Yusuke Matsuno; Rika Kusumoto-Matsuo; Mitsunori Shiroishi; Ken-ichi Yoshioka

doi:10.1371/journal.pone.0281168

Peer Review History

Original SubmissionSeptember 15, 2022
28 Nov 2022 Decision Letter - Alvaro Galli, Editor PONE-D-22-25618Genome Destabilization-Associated Phenotypes Arising as a Consequence of Therapeutic Treatment are Suppressed by OlaparibPLOS ONE Dear Dr. Yoshioka, 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 Jan 12 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. 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Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. 6. 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 If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references.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 If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.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. 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: 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: In this manuscript the authors examine how the PARP inhibitor Olaparib suppresses cancer cells that developed resistance to camptothecin and radiation-induced double stranded breaks. While the data supports the observation that Olaparib activates the cGAS pathway, the evidence was only limited to the cytosolic cGAS observed in immunofluorescence microscopy. The conclusions about combining Olaparib and campthothecin for effective cancer cells killing are not unexpected since other groups have previously published that Olaparib disrupts ovarian and colorectal cancer cell lines (Ding et al Cell Rep, 2018; 25(11): 2972–2980.e5, Miura et al Radiat Oncol, 2012; 7, 62). The observations that Olaparib disrupts gamma H2AX and 53BP1 foci co-localisation suggest that PARP inhibition activates cGAS STING pathway that contributes to tumor cell death. It has recently been shown that the Fanconi Anemia DNA repair pathway bridges the HR and NHEJ pathway and would be useful to investigate further. Overall, I think the results will be useful to the field and may stimulate additional experiments by other investigators. However my enthusiasm is tempered since the major conclusions were already previewed in other published papers. If the authors could explain which DNA damage pathways (BRCA1 or 53BP1 mediated) that could be more exciting. Otherwise the observations may be more appropriate for another journal. Major comments: 1. Olaparib was previously shown to disrupt cGAS STING pathway and in BRCA1 deficient cells (Miura et al Radiat Oncol, 2012; Ding et al Cell Rep, 2018). The Miura et al, Radiat Oncol paper should be referenced in this manuscript. 2. PARP inhibitors are a classical example of a synthetic lethality relationship of diverging DNA repair mechanisms, and it is possible that Olaparib kills cancer cells after other HR pathways were compromised (eg. cells with stalled replication forks due to defective BRCA1 pathway). It was unclear which homologous repair pathway (eg BRCA1/Fanconi Anemia pathway) or non homologous end joining pathway (53BP1) that Olaparib affects. Cell imaging following Olaparib treatment with gH2AX and Rad51 staining should be repeated in 53BP1 and/or BRCA1/FANCD2 knock out cell lines. Alternatively, the authors could repeat the immunofluorescence imaging with BRCA1/FANCD2 to determine which pathway Olaparib affects. 3.Fig 5a – higher resolution of the figure containing the graph is needed. The number P=.. were ineligible. Fig 5c was missing two points for the gamma-ray irradiation (15 and 20 Gy). 4. Page 17 line 305 – there is already a paper that describes Olaparib association with BRCA or Fanconi Anemia pathway, please cite Ding et al Cell Rep. 2018 Dec 11; 25(11): 2972–2980.e5 as Olaparib has been shown to trigger cGas in BRCA1-deficient cells, which suggests it efficacy in killing cancer cells. 5. Please add the concentration of Olaparib as it was not described anywhere in the manuscript or methods. Minor comments: 1. page 18 line 308 is missing a reference after "..presumably by non-homologous end joining". 2. page 18 line 313 please add a sentence or two on how this work will bring impact to the cancer therapeutics or DNA damage fields. 3. Figure 4b remove the "With or without Olaparib" figure legend as the data was not shown in the figure. Alternatively, the authors could clarify that the graph on Fig 4b was without Olaparib. 4. Figures 2a and 3c was unclear. Higher resolution images are required to see the foci. Reviewer #2: Suzuki et al. presented a concise study on the suppression of radio- and CPT surviving cells by Olaparib co-treatment. Although the enhanced anti-tumour activity of Olaparib and CPT or gamma-rays was presented several years ago (Miura et al 2012 doi: 10.1186/1748-717X-7-62), the authors are addressing important aspect of simultaneous treatment that enhance elimination of resistant cancer cells, using MCF7 and to some extent HeLa and SW480 models. Overall, the text is well-written and data presented on figures are complete. Thus, I recommend the manuscript for publication, however, for increasing accessibility of the manuscript content I suggest some minor modifications of text, listed below. 1) The quality of figures (at least in my version, even after download) was low and should be enhanced in final version – the authors should clearly export the files at least in 300 DPI. 2) Fig 4 b and c – the authors claim that ‘no cells irradiated three times were obtained in the presence of Olaparib, because growth failed to recover in the presence of Olaparib’. This description is unclear to me and should be extended / commented. It is a bit disturbing that absolutely no cells were found in the presence of Olaparib. I convince the authors to rewrite the description of this results to explain the observed strong effects. 3) Lines 272-273 require edition as it reads strange in current version. 4) Lines 302-304 The authors should state here that it is simultaneous treatment of CPT or gamma-rays with Olaparib. 5) The final sentence of Discussion (lines 311-313) should be followed by a major conclusion on the findings described in this work to leave the reader with a clear message. 6) Methods – line 319 – used or prepared? ****** 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: Winnie Tan 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.0281168.r001
Revision 1
10 Jan 2023 Author Response Reviewer #1: In this manuscript the authors examine how the PARP inhibitor Olaparib suppresses cancer cells that developed resistance to camptothecin and radiation-induced double stranded breaks. While the data supports the observation that Olaparib activates the cGAS pathway, the evidence was only limited to the cytosolic cGAS observed in immunofluorescence microscopy. The conclusions about combining Olaparib and campthothecin for effective cancer cells killing are not unexpected since other groups have previously published that Olaparib disrupts ovarian and colorectal cancer cell lines (Ding et al Cell Rep, 2018; 25(11): 2972–2980.e5, Miura et al Radiat Oncol, 2012; 7, 62). The observations that Olaparib disrupts gamma H2AX and 53BP1 foci co-localisation suggest that PARP inhibition activates cGAS STING pathway that contributes to tumor cell death. It has recently been shown that the Fanconi Anemia DNA repair pathway bridges the HR and NHEJ pathway and would be useful to investigate further. Overall, I think the results will be useful to the field and may stimulate additional experiments by other investigators. However my enthusiasm is tempered since the major conclusions were already previewed in other published papers. If the authors could explain which DNA damage pathways (BRCA1 or 53BP1 mediated) that could be more exciting. Otherwise the observations may be more appropriate for another journal. Response: We thank the reviewer for these valuable comments and have revised the manuscript according to the reviewer’s suggestions. We have addressed the issues raised by the reviewer by adding references to the suggested studies. Although those studies are certainly important, they differ from our study is several respects. We would like to explain the differences as follows. First, Ding et al. studied STING pathway activation in BRCA1-mutated tumor cells that are sensitive to the PARP inhibitor Olaparib (Ding et al. Cell Rep, 2018; 25(11): 2972–2980.e5). By contrast, we studied the effects of Olaparib on BRCA1-wt cells that are not sensitive to Olaparib. Since cells treated with CPT and/or irradiated with gamma-rays are often subject to genomic instability that increases the risks of cell migration and treatment resistance, we believed it was important to suppress these risks. Our results revealed that the increase risk of resistance associated with genomic instability is suppressed by Olaparib, which to the best of our knowledge has not been reported in previous studies. In addition, Miura et al. studied the effects of Olaparib as a sensitizer of CPT and gamma-ray irradiation by monitoring cell survival rates and gammaH2AX/Rad51 foci (Miura et al Radiat Oncol, 2012; 7, 62). Although the results of their study are important, we still think that our study is informative, because we studied the effects of Olaparib on genomic instability and on the increase in cell migration and resistance associated with genome instability. In addition, we also found that Olaparib suppressed all those genomic instability-associated effects. Regarding cGAS/STING pathway activation, the reviewer remarked that “the evidence was only limited to the cytosolic cGAS observed in immunofluorescence microscopy”. However, this criticism is not merited, because we also provided supporting data showing the expression of downstream factors Ifnβ, Ifit1, and Ifit3. The reviewer’s remark on repair pathways is related to major comment 2. Please see our response there. We carefully rewrote the manuscript to address the issues raised by the reviewer, which we think has improved the manuscript. Major comments: 1. Olaparib was previously shown to disrupt cGAS STING pathway and in BRCA1 deficient cells (Miura et al Radiat Oncol, 2012; Ding et al Cell Rep, 2018). The Miura et al, Radiat Oncol paper should be referenced in this manuscript. Response: Thank you for this suggestion. We have added references to those manuscripts and related background information. 2. PARP inhibitors are a classical example of a synthetic lethality relationship of diverging DNA repair mechanisms, and it is possible that Olaparib kills cancer cells after other HR pathways were compromised (eg. cells with stalled replication forks due to defective BRCA1 pathway). It was unclear which homologous repair pathway (eg BRCA1/Fanconi Anemia pathway) or non homologous end joining pathway (53BP1) that Olaparib affects. Cell imaging following Olaparib treatment with gH2AX and Rad51 staining should be repeated in 53BP1 and/or BRCA1/FANCD2 knock out cell lines. Alternatively, the authors could repeat the immunofluorescence imaging with BRCA1/FANCD2 to determine which pathway Olaparib affects. Response: As pointed out, a PARP inhibitor is a drug that causes a synthetic lethal effect in HR deficient cells. However, in our manuscript, we did not use a PARP inhibitor for this purpose. We studied the effects of Olaparib on HR proficient cancer cells. In our experiments, the major type of DNA damage caused by CPT and ionizing radiation is replication stress-associated DSBs (Matsuno et al., iScience 2021; Atsumi et al., JBC 2012). Although DSBs can also directly result from irradiation, those are usually repaired within a few hours. Persistent DSBs that increase the risk of genomic instability occur during the S phase that follows irradiation and are associated with replication stress (Matsuno et al., iScience 2021), similar to the cells treated with CPT. The resulting genomic instability further increases the risk of cell migration (Matsuno et al., iScience 2021) and the clonal evolution of cells with increased resistance (Matsuno et al., Nature Com 2019); therefore, it is important to suppress the effects of genomic instability. Replication stress-associated DSBs are the prime target of homologous recombination (HR). Genomic instability is usually caused by erroneous DNA repair. Therefore, we simply tested the effects of PARP inhibitor, because PARP inhibitor effectively blocks the induction of HR (Hochegger et al., EMBO J. 2006; Sugimura et al., JCB 2008). The results were basically as expected, in which the killing effect was elevated. In our experiments, we monitored Rad51/gammaH2AX foci to see if HR-associated damage and 53BP1/gammaH2AX foci determine the resulting DSBs. We did not examine the involvement of BRCA1/FANCD2, because our study was not designed to address mechanism questions relating to the Fanconi pathway. In fact, our basic aim was to study cellular responses to replication stress-associated DSBs, not to ICL type DNA damage, which involves the Fanconi pathway. We carefully rewrote the manuscript to address these issues, which we believe has made the manuscript clearer. 3.Fig 5a – higher resolution of the figure containing the graph is needed. The number P=.. were ineligible. Fig 5c was missing two points for the gamma-ray irradiation (15 and 20 Gy). Response: Thank you for this comment. We have improved the resolution of the images. The missing two points were because there were no surviving cells under this condition. The issue has been addressed in the legend. 4. Page 17 line 305 – there is already a paper that describes Olaparib association with BRCA or Fanconi Anemia pathway, please cite Ding et al Cell Rep. 2018 Dec 11; 25(11): 2972–2980.e5 as Olaparib has been shown to trigger cGas in BRCA1-deficient cells, which suggests it efficacy in killing cancer cells. Response: We have added a reference to this paper. 5. Please add the concentration of Olaparib as it was not described anywhere in the manuscript or methods. Response: This information has been added to the Methods section. Minor comments:1. page 18 line 308 is missing a reference after "..presumably by non-homologous end joining". 2. page 18 line 313 please add a sentence or two on how this work will bring impact to the cancer therapeutics or DNA damage fields. 3. Figure 4b remove the "With or without Olaparib" figure legend as the data was not shown in the figure. Alternatively, the authors could clarify that the graph on Fig 4b was without Olaparib. 4. Figures 2a and 3c was unclear. Higher resolution images are required to see the foci. Responses to minor comments 1 to 4: We have addressed these issues in the revised manuscript. Reviewer #2: Suzuki et al. presented a concise study on the suppression of radio- and CPT surviving cells by Olaparib co-treatment. Although the enhanced anti-tumour activity of Olaparib and CPT or gamma-rays was presented several years ago (Miura et al 2012 doi: 10.1186/1748-717X-7-62), the authors are addressing important aspect of simultaneous treatment that enhance elimination of resistant cancer cells, using MCF7 and to some extent HeLa and SW480 models. Overall, the text is well-written and data presented on figures are complete. Thus, I recommend the manuscript for publication, however, for increasing accessibility of the manuscript content I suggest some minor modifications of text, listed below. Response: We very much appreciated for the reviewer’s encouraging comments. We also appreciated the other comments, which we have addressed on a one-to-one basis below. 1) The quality of figures (at least in my version, even after download) was low and should be enhanced in final version – the authors should clearly export the files at least in 300 DPI. Response: We have improved the quality of the figures. 2) Fig 4 b and c – the authors claim that ‘no cells irradiated three times were obtained in the presence of Olaparib, because growth failed to recover in the presence of Olaparib’. This description is unclear to me and should be extended / commented. It is a bit disturbing that absolutely no cells were found in the presence of Olaparib. I convince the authors to rewrite the description of this results to explain the observed strong effects. Response: We have carefully revised the text to address this comment. 3) Lines 272-273 require edition as it reads strange in current version. Response: We have revised the text. 4) Lines 302-304 The authors should state here that it is simultaneous treatment of CPT or gamma-rays with Olaparib. Response: We now include this statement. 5) The final sentence of Discussion (lines 311-313) should be followed by a major conclusion on the findings described in this work to leave the reader with a clear message. Response: We have added conclusion statements to the final paragraph. 6) Methods – line 319 – used or prepared? Response: We have corrected this. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0281168.r002
17 Jan 2023 Decision Letter - Alvaro Galli, Editor Genome Destabilization-Associated Phenotypes Arising as a Consequence of Therapeutic Treatment are Suppressed by Olaparib PONE-D-22-25618R1 Dear Dr. Yoshioka, 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 Reviewer #2: 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 Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: 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 Reviewer #2: 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 Reviewer #2: 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: (No Response) Reviewer #2: In current version, the authors have addressed all the points raised in the first round of review. Present text reads well, Line 151 requires correction: stability should be replaced by instability. Overall, the manuscript is now ready for publication. ****** 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: Winnie Tan Reviewer #2: No ******** https://doi.org/10.1371/journal.pone.0281168.r003
Formally Accepted
19 Jan 2023 Acceptance Letter - Alvaro Galli, Editor PONE-D-22-25618R1 Genome Destabilization-Associated Phenotypes Arising as a Consequence of Therapeutic Treatment are Suppressed by Olaparib Dear Dr. Yoshioka: 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.0281168.r004

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