PONE-D-19-18074

Small Molecule Inhibition of Lysine-Specific Demethylase 1 (LSD1) and Histone Deactylase (HDAC) Alone and in Combination in Ewing Sarcoma Cell Lines

PLOS ONE

Dear Dr Reed,

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.

Although both the reviewers indicated an overall enthusiasm for the studies, it was however felt that further discussion and justifications/rationale for choosing certain compounds and their combination studies would be necessary. 

We would appreciate receiving your revised manuscript by Sep 12 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.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

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). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. 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,

Arun Rishi, 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

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. Please provide additional information about each of the cell lines used in this work, including history, 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.

3. Our staff editors have determined that your manuscript is likely within the scope of our Targeted Anticancer Therapies and Precision Medicine Call for Papers. This editorial initiative is headed by a team of Guest Editors for PLOS ONE: Andrew Cherniack, Anette Duensing, Steven Gray, Sunil Krishnan, Chandan Kumar-Sinha and Gayle Woloschak. The Collection will encompass a diverse range of research articles about the identification and classification of driver genes and somatic alterations, target and drug discovery, mechanisms of drug resistance, and early detection and screening.  Additional information can be found on our announcement page: https://collections.plos.org/s/targeted-anticancer-therapies.

If you would like your manuscript to be considered for this collection, please let us know in your cover letter and we will ensure that your paper is treated as if you were responding to this call. If you would prefer to remove your manuscript from collection consideration, please specify this in the cover letter.

4. Thank you for stating the following in the Financial Disclosure section: "This study was generously supported by the National Pediatric Cancer Foundation (www.nationalpcf.org). This work has been supported in part by the Translational Research Core at the H. Lee Moffitt Cancer Center & Research Institute, a NCI designated Comprehensive Cancer Center (P30-CA076292)."

We note that one or more of the authors are employed by a commercial company: 'Sunshine Lab LLC'.

a) Please provide an amended Funding Statement declaring this commercial affiliation, as well as a statement regarding the Role of Funders in your study. If the funding organization did not play a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials, please review your statements relating to the author contributions, and ensure you have specifically and accurately indicated the role(s) that these authors had in your study. You can update author roles in the Author Contributions section of the online submission form.

Please also include the following statement within your amended Funding Statement.

“The funder provided support in the form of salaries for authors [insert relevant initials], but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.”

If your commercial affiliation did play a role in your study, please state and explain this role within your updated Funding Statement.

b) Please also provide an updated Competing Interests Statement declaring this commercial affiliation along with any other relevant declarations relating to employment, consultancy, patents, products in development, or marketed products, etc.  

Within your Competing Interests Statement, please confirm that this commercial affiliation 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 this adherence statement is not accurate and  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 both an updated Funding Statement and Competing Interests Statement in 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: 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: The purpose of this study by Welch et al., was to define clinical agents that synergize with the LSD1 inhibitor SP2509 in Ewing sarcoma, an aggressive childhood cancer which has seen limited improvement in overall survival since the introduction of chemotherapy >50 years ago. The authors tested 7 clinically utilized chemotherapeutic agents in four different Ewing sarcoma cell lines with varying KDM1A expression levels and TP53/STAG2 mutational status. Order of addition of second line conventional combination therapy agents were also tested with the addition of SP2509.

The authors should be commended on several factors including their reproducible and high throughput method for exploring antitumor effects of combinations of therapies at clinically achievable concentrations. All concentrations used in experiments were well below observed maximal plasma concentrations. In addition, it is refreshing to see that the authors used active metabolites of cyclophosphamide, irinotecan, and temozolomide which is frequently overlooked. As all experimental considerations for combination therapy were developed and conducted with the end thought of rapid translation into current LSD1 trials, data presented in this manuscript has the potential to significantly improve the survival outcomes for Ewing sarcoma patients as well as demonstrating to the scientific community how synergy experiments should be conducted. The data is technically sound with multiple replicates and each conclusion is supported by the data presented.

Minor comments for consideration

Introduction

1) As synergism with SP2509 was the main premise for this study, the authors should explain why SP2509 was chosen over other LSD1 inhibitors (GSK2879552 and ORY-1001). It would be helpful to state in the introduction that SP2509 is currently in Phase I clinical testing for Ewing sarcoma patients (NCT03600649) and that Ewing sarcoma cell lines are resistant to reversible inhibitors (Romo‐Morales et al., Pediatric Blood & Cancer, 2019).

2) To prevent confusion, it should be noted in the text that SP2509 was formally known as HCI2509.

Results section

1) Minor concern for this study was the decision to use Romidepsin which is only clinically approved for T-cell lymphoma. Considering the Phase 2 clinical trial of Romidepsin (NCT00112463) failed in 40 patients with metastatic or unresectable sarcoma, what is the likely hood of this HDAC inhibitor being chosen for further study in solid tumors?

2) Reference is required for the following statement “HDAC1 and HDAC2 inhibitor romidepsin and the reversible LSD1 inhibitor SP2509, were selected based on their respective targets in the NuRD complex” Lines 171-172.

3) Figure 1: Viability time point should be listed in the figure legend ie 24 or 72hr treatment. Image quality is also quite poor and should be corrected.

4) The authors state on Line 200 that “SP2509 showed synergy with all drugs apart from vincristine”. Can the authors postulate why? Is anything known for the cell lines in terms of previous chemotherapy treatment and does this explain sensitivity/resistance to the agents tested?

5) On lines 285-286, the authors elude to sensitivity of drugs may “simply be due to differences in doubling times”. This is quite possible considering the doubling times for the majority of cell lines ranges from 21-25hrs except for RDES (60hrs) (May et al., PLOS ONE, 2013). The authors should include doubling rates for each cell line in Table 1.

6) Is there any correlation between sensitivity to SP2509 and the other chemotherapeutic agents tested?

7) With all the different dosing schedules and agents, it was quite difficult throughout the manuscript to track which agents were synergistic over multiple cell lines. It would be nice to present a final table/figure summarizing which agents where synergistic across all four Ewing sarcoma cell lines and dosing schedules.

8) Minor notes, gene names should be in italics eg Line 275 TP53 and STAG2.

Discussion

1) Do the authors know whether SP2509 and SP2577 have similar IC50 values? This will have implications as to whether their synergistic findings can be replicated with SP2577 in clinical settings.

2) Although not used for clinical settings in Ewing sarcoma, SP2509 has been shown to synergise with docetaxel in prostate cancer (Gupta et al., 2016). This study should be referenced.

3) The dosing model presented in Supplementary Figure 2 is quite interesting and should be moved to the main text.

Reviewer #2: This study describes in vitro 2D analyses of cell viability for drug combination indices of an HDAC and an LSD1 inhibitor combined with standard chemotherapies used for treatment of ewing sarcomas. Overall the experiments appear well executed and described.

Although this is clearly not a mechanistic paper, the authors should better consider and discuss

i. the reasons supporting their hypothesis “that agents that interact with components of the NuRD complex would work synergistically with agents already utilized in the treatment of ES”.

ii. the rationale behind the choice of HDAC and LSD1 inhibitors including recent comments about how SP2509 may work (PMID: 29205263 PMID:31207107)

iii. possible mechanistic explanations for the results of the most and least synergistic combinations in different cell lines. For example it has been published that cells with low EWS-FLI1 expression are less proliferative PMID:28135250. Does this impact on the sensitivity of the agents used? The mechanism of action of some chemotherapeutic agents used depends on rapid cell cycling, e.g. SN-38. Can this be considered in light of A673’s relative insensitivity to topoisomerases? comments/considerations around reversal of insensitivity of

iv. the importance of scheduling. They only do one sequential experiment in which the order of drugs is changed.

v. the importance around time of treatment. Activity is reported at 72hours, however drugs targeting epigenetic modifying enzymes have been shown to benefit from prolonged exposure (EZH2 inhibitors in lymphoma). Longer-term assays could be useful to achieve maximal drug efficacy and therefore give a broader overview of their activity in combination with chemotherapeutic agents.

vi. Given the previous effects of LSD1 on migration (PMID:18381423) and the potential clinical significance of this phenotype, why was this not assessed or discussed?

Minor comments:

Page 3 line 44 sentence unclear – most commonly t(11;22)(q24;12) between the amino … – “12” should be q12 is missing and that fuses would be better than “between”

**********

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 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.

[EXSCINDED]

Small Molecule Inhibition of Lysine-Specific Demethylase 1 (LSD1) and Histone Deactylase (HDAC) Alone and in Combination in Ewing Sarcoma Cell Lines

PONE-D-19-18074R1

Dear Dr. Reed,

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,

Arun Rishi, Ph.D.

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: N/A

**********

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: (No Response)

**********

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

Reviewer #2: No