­­A high-throughput screen identifies inhibitors of the interaction between the oncogenic transcription factor ERG and the cofactor EWS

Taylor R. Nicholas; Jingwei Meng; Benjamin M. Greulich; Teresa Stevie Morris; Peter C. Hollenhorst

doi:10.1371/journal.pone.0238999

Peer Review History

Original SubmissionJune 19, 2020
30 Jul 2020 Decision Letter - Irina U Agoulnik, Editor PONE-D-20-18931 A high-throughput screen identifies inhibitors of the interaction between the oncogenic transcription factor ERG and the cofactor EWS PLOS ONE Dear Dr. Hollenhorst, 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. Binding intrinsically disordered segments of transcription factors may lead to changes in either stability or transcriptional activity. Please determine whether treatment of RWPE1-ERG cells with hits B and D interfere with the stability or transcriptional activity of full length ERG using western blotting and expression of a couple of endogenous ERG target genes. Confirm this effect of the lead compound on ERG function in an independently derived cell line, such as VCaP. Add description of origin and culture conditions for the RWPE1-ERG cell line to materials and methods section. Correct typos throughout the manuscript, such as “both protein” and some others. Conduct statistical analysis wherever differences are claimed and add statistical analysis description to the materials and methods section. Please submit your revised manuscript by Sep 13 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, Irina U Agoulnik, Ph.D. Academic Editor PLOS ONE Journal Requirements: When submitting your revision, we need you to address these additional requirements. 1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf 2.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. 3. Please provide additional information about each of the RWPE1 cell line used in this work, including the culture conditions and any quality control testing procedures (authentication, characterisation, and mycoplasma testing). For more information, please see http://journals.plos.org/plosone/s/submission-guidelines#loc-cell-lines. 4. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses. For more information on PLOS ONE's expectations for statistical reporting, please see https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting. [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 ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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 ****** 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 ****** 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 manuscript by Nicholas et al. reports small molecules identified to inhibit the oncogenic ERG-EWS interaction. Authors previously identified ERG-EWS interaction as a driving factor of tumor growth in prostate cancer. Authors utilized AlphaScreen for highthrouput analysis and tested prostate cancer phenotypes in cultured cells to validate the molecules. One candidate lead inhibitor was identified from this scan. The manuscript is straight forward written and appropriate methods with adequate controls are used. There are minor issues that need to be addressed before it is ready for publication. This is not the first study aimed at targeting an ERG protein-protein interaction for the development of a small molecule-based prostate cancer therapy (PMID: 27223260). Authors may have to tone down such claims. Authors aim to demonstrate "Interestingly, an N-terminal degradation product arising from full-length ERG, and about the same size as EWS 1-355, also interacted with ERG better than full-length EWS. This suggests that the interaction with the N-terminus may be autoinhibited by the C-terminal region." however, the presented data does not fully support this conclusion. Authors acknowledge that wild type EWS interacting with rearranged ERG as an oncogenic mechanism. Would ERG fusion difference in cell lines used affect ERG/EWS interaction. What is the rational behind using PC3 nuclear extract for CoIP? How is AR dependence of cell lines affect ERG-EWS interaction? Authors need to acknowledge the limitations of the study such as engineered protein used in the assays. The reviewer was not able to judge the statistic rigor of the analyses. ****** 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:** Paul Basil [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.0238999.r001
Revision 1
26 Aug 2020 Author Response Binding intrinsically disordered segments of transcription factors may lead to changes in either stability or transcriptional activity. Please determine whether treatment of RWPE1-ERG cells with hits B and D interfere with the stability or transcriptional activity of full length ERG using western blotting and expression of a couple of endogenous ERG target genes. Confirm this effect of the lead compound on ERG function in an independently derived cell line, such as VCaP. The authors would like to very much thank the editor for these helpful suggestions, as they led to a result that nicely supports our hypothesis. As suggested, we used VCaP cells. In 2011, Mani et al. showed that the TMPRSS2/ERG fusion in VCaP cells encodes an N-terminally truncated version of ERG that can bind to the promoter of the other allele of full-length ERG and activate it. We treated VCaP cells with DMSO, hit B, or hit D and measured protein levels of ERG and EWS by immunoblot. We found that expression of EWS was unchanged while levels of full-length, but not TMRPSS2/ERG encoded ERG protein were decreased by Hit B, but not Hit D. This would be consistent with what we would expect if Hit B can disrupt the transcriptional activation activity of ERG. We then looked at expression of two known ERG target genes in VCaP cells after treatment. Both HSPA8 and ERG expression at the RNA level were significantly decreased upon treatment with hit B. Treatment with hit D had no impact. These new data are in the revised Figure 5. Add description of origin and culture conditions for the RWPE1-ERG cell line to materials and methods section. Correct typos throughout the manuscript, such as “both protein” and some others. Conduct statistical analysis wherever differences are claimed and add statistical analysis description to the materials and methods section. A description of origin and culture conditions for the RWPE1-ERG cell line was added to the materials and methods section under “Cell Culture and Antibodies”. Typos have been corrected. Statistical analyses were added when appropriate and a “Statistical Analysis” paragraph was added to the materials and methods section. 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 Our manuscript now meets the PLOS ONE style requirements. 2.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. We have provided the original uncropped and unadjusted images in the supplemental figure file S1_File.pdf 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. 3. Please provide additional information about each of the RWPE1 cell line used in this work, including the culture conditions and any quality control testing procedures (authentication, characterisation, and mycoplasma testing). For more information, please see http://journals.plos.org/plosone/s/submission-guidelines#loc-cell-lines. Cell culture conditions have been updated in the Materials and methods section. We have also detailed how cell lines have been authenticated and tested for mycoplasma. 4. To comply with PLOS ONE submission guidelines, in your Methods section, please provide additional information regarding your statistical analyses. For more information on PLOS ONE's expectations for statistical reporting, please see https://journals.plos.org/plosone/s/submission-guidelines.#loc-statistical-reporting. The authors have added a “Statistical analysis” section to the material and methods. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 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 manuscript by Nicholas et al. reports small molecules identified to inhibit the oncogenic ERG-EWS interaction. Authors previously identified ERG-EWS interaction as a driving factor of tumor growth in prostate cancer. Authors utilized AlphaScreen for highthrouput analysis and tested prostate cancer phenotypes in cultured cells to validate the molecules. One candidate lead inhibitor was identified from this scan. The manuscript is straight forward written and appropriate methods with adequate controls are used. There are minor issues that need to be addressed before it is ready for publication. This is not the first study aimed at targeting an ERG protein-protein interaction for the development of a small molecule-based prostate cancer therapy (PMID: 27223260). Authors may have to tone down such claims. Authors aim to demonstrate "Interestingly, an N-terminal degradation product arising from full-length ERG, and about the same size as EWS 1-355, also interacted with ERG better than full-length EWS. This suggests that the interaction with the N-terminus may be autoinhibited by the C-terminal region." however, the presented data does not fully support this conclusion. Authors acknowledge that wild type EWS interacting with rearranged ERG as an oncogenic mechanism. Would ERG fusion difference in cell lines used affect ERG/EWS interaction. What is the rational behind using PC3 nuclear extract for CoIP? How is AR dependence of cell lines affect ERG-EWS interaction? Authors need to acknowledge the limitations of the study such as engineered protein used in the assays. The reviewer was not able to judge the statistic rigor of the analyses. The authors would like to thank this reviewer for a thoughtful review. We will address each comment with our response below: This is not the first study aimed at targeting an ERG protein-protein interaction for the development of a small molecule-based prostate cancer therapy (PMID: 27223260). Authors may have to tone down such claims. Thanks to the reviewer for pointing out that the BRD4 inhibitors JQ1 and iBET762 are reported to inhibit the protein-protein interaction between BRD4 and ERG. We have toned down our claim. Authors aim to demonstrate "Interestingly, an N-terminal degradation product arising from full-length ERG, and about the same size as EWS 1-355, also interacted with ERG better than full-length EWS. This suggests that the interaction with the N-terminus may be autoinhibited by the C-terminal region." however, the presented data does not fully support this conclusion. We agree that the data inferred such conclusion but did not directly support the claim. We therefore toned down our claim. Authors acknowledge that wild type EWS interacting with rearranged ERG as an oncogenic mechanism. Would ERG fusion difference in cell lines used affect ERG/EWS interaction. In our previous publication (PMID: 27783944) we found that the first 275 amino acids of ERG are not necessary for the interaction with EWS, and identified a region in the C-terminus of ERG that is necessary for the interaction. All known ERG fusion gene products have this region and would be predicted to bind EWS equally. What is the rational behind using PC3 nuclear extract for CoIP? PC3 nuclear extract is purchased from Santa Cruz Biotechnology and is an economical source of native EWS protein. How is AR dependence of cell lines affect ERG-EWS interaction? AR dependence of cell lines does not appear to impact the ERG-EWS interaction. The protein-protein interaction is maintained in VCaP cells (androgen sensitive) and RWPE1-ERG cells (androgen insensitive). The interaction is also maintained in primary patient tumors. These data are provided in our previous publication (PMID: 27783944). Authors need to acknowledge the limitations of the study such as engineered protein used in the assays. We have now acknowledged this limitation in the discussion. The reviewer was not able to judge the statistic rigor of the analyses. The authors have added statistical analyses to appropriate figures and added a “Statistical analysis” section to the material and methods. The authors would like to thank this reviewer for a thoughtful review. We will address each comment with our response below: This is not the first study aimed at targeting an ERG protein-protein interaction for the development of a small molecule-based prostate cancer therapy (PMID: 27223260). Authors may have to tone down such claims. Thanks to the reviewer for pointing out that the BRD4 inhibitors JQ1 and iBET762 are reported to inhibit the protein-protein interaction between BRD4 and ERG. We have toned down our claim. Authors aim to demonstrate "Interestingly, an N-terminal degradation product arising from full-length ERG, and about the same size as EWS 1-355, also interacted with ERG better than full-length EWS. This suggests that the interaction with the N-terminus may be autoinhibited by the C-terminal region." however, the presented data does not fully support this conclusion. We agree that the data inferred such conclusion but did not directly support the claim. We therefore toned down our claim. Authors acknowledge that wild type EWS interacting with rearranged ERG as an oncogenic mechanism. Would ERG fusion difference in cell lines used affect ERG/EWS interaction. In our previous publication (PMID: 27783944) we found that the first 275 amino acids of ERG are not necessary for the interaction with EWS, and identified a region in the C-terminus of ERG that is necessary for the interaction. All known ERG fusion gene products have this region and would be predicted to bind EWS equally. What is the rational behind using PC3 nuclear extract for CoIP? PC3 nuclear extract is purchased from Santa Cruz Biotechnology and is an economical source of native EWS protein. How is AR dependence of cell lines affect ERG-EWS interaction? AR dependence of cell lines does not appear to impact the ERG-EWS interaction. The protein-protein interaction is maintained in VCaP cells (androgen sensitive) and RWPE1-ERG cells (androgen insensitive). The interaction is also maintained in primary patient tumors. These data are provided in our previous publication (PMID: 27783944). Authors need to acknowledge the limitations of the study such as engineered protein used in the assays. We have now acknowledged this limitation in the discussion. The reviewer was not able to judge the statistic rigor of the analyses. The authors have added statistical analyses to appropriate figures and added a “Statistical analysis” section to the material and methods. Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0238999.r002
28 Aug 2020 Decision Letter - Irina U Agoulnik, Editor A high-throughput screen identifies inhibitors of the interaction between the oncogenic transcription factor ERG and the cofactor EWS PONE-D-20-18931R1 Dear Dr. Hollenhorst, 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, Irina U Agoulnik, Ph.D. Section Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0238999.r003
Formally Accepted
3 Sep 2020 Acceptance Letter - Irina U Agoulnik, Editor PONE-D-20-18931R1 A high-throughput screen identifies inhibitors of the interaction between the oncogenic transcription factor ERG and the cofactor EWS Dear Dr. Hollenhorst: 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. Irina U Agoulnik Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0238999.r004

Open letter on the publication of peer review reports

PLOS recognizes the benefits of transparency in the peer review process. Therefore, we enable the publication of all of the content of peer review and author responses alongside final, published articles. Reviewers remain anonymous, unless they choose to reveal their names.

We encourage other journals to join us in this initiative. We hope that our action inspires the community, including researchers, research funders, and research institutions, to recognize the benefits of published peer review reports for all parts of the research system.

Learn more at ASAPbio .