Pilot study evaluating everolimus molecular mechanisms in tuberous sclerosis complex and focal cortical dysplasia

Dominique F. Leitner; Evgeny Kanshin; Manor Askenazi; Yik Siu; Daniel Friedman; Sasha Devore; Drew Jones; Beatrix Ueberheide; Thomas Wisniewski; Orrin Devinsky

doi:10.1371/journal.pone.0268597

Peer Review History

Original SubmissionJanuary 21, 2022
7 Mar 2022 Decision Letter - Emilio Russo, Editor Transfer Alert This paper was transferred from another journal. As a result, its full editorial history (including decision letters, peer reviews and author responses) may not be present. PONE-D-22-02043Pilot Study Evaluating Everolimus Molecular Mechanisms in Tuberous Sclerosis Complex and Focal Cortical DysplasiaPLOS ONE Dear Dr. Devinsky, 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 address all Reviewers' comments and make clear in the manuscript the limitations of the study Please submit your revised manuscript by Apr 21 2022 11:59PM. 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Thank you for stating the following in the Competing Interests section: [I have read the journal's policy and the authors of this manuscript have the following competing interests: All the authors report no conflicts of interest. Below are the disclosures of the three authors: Daniel Friedman receives salary support for consulting and clinical trial related activities performed on behalf of The Epilepsy Study Consortium, a non-profit organization. Dr. Friedman receives no personal income for these activities. NYU receives a fixed amount from the Epilepsy Study Consortium towards Dr. Friedman’s salary. Within the past two years, The Epilepsy Study Consortium received payments for research services performed by Dr. Friedman from: Alterity, Baergic, Biogen, BioXcell, Cerevel, Cerebral, Jannsen, Lundbeck, Neurocrine, SK Life Science, and Xenon. He has also served as a paid consultant for Neurelis Pharmaceuticals and Receptor Life Sciences. He has received research support from NINDS, CDC, Epitel, and Neuropace unrelated to this study. He holds equity interests in Neuroview Technology. He received royalty income from Oxford University Press. Sasha Devore receives salary support from the National Institutes of Health, Department of Defense, and the Templeton World Charity Foundation unrelated to this study. Orrin Devinsky receives grant support from NINDS, NIMH, MURI, CDC and NSF. He has equity and/or compensation from the following companies: Tilray, Receptor Life Sciences, Qstate Biosciences, Tevard, Empatica, Engage, Egg Rock/Papa & Barkley, Rettco, SilverSpike, and California Cannabis Enterprises (CCE). He has received consulting fees from Zogenix, Ultragenyx, BridgeBio, and Marinus. He holds patents for the use of cannabidiol in treating neurological disorders but these are owned by GW Pharmaceuticals and he has waived any financial interests. 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Please amend either the abstract on the online submission form (via Edit Submission) or the abstract in the manuscript so that they are identical. 6. Please ensure that you refer to Figure 5 in your text as, if accepted, production will need this reference to link the reader to the figure. 7. Please include a copy of Table 2 which you refer to in your text on page 23. [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 Reviewer #3: Partly ******** 2. 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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 Reviewer #3: 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 authors aimed at evaluating safety of the mTOR inhibitor everolimus in treatment resistant (failure of > 2 anti-seizure medications) TSC and FCD patients undergoing surgical resection and to assess changes in mTOR signaling and molecular pathways. I have some queries a) the authors should provide the range age of active patients and controls since they do not appear to be age-matched. Active patients’ mean age is > 18 years old while controls’ mean age is < 18 years old. b)How did the authors deal with the variable levels of total S6 in their analysis, particularly among the Active group? c) A better delineation of histopathology is needed d) The most relevant result was that no safety concerns were found in everolimus treated patients, with no reported adverse events before, during, or after surgical resection. However, owing to the low number of recruited patients and the short treatment time the authors should soften their conclusions e) To increase the interest of the study, the authors should discuss the correlation of their results with seizure outcome after surgery in their sample Reviewer #2: The authors report findings from their study evaluating safety and molecular mechanisms of short-term everolimus (7 days) in TSC and FCD patients. The study has conducted well. I would like to give a few suggestions to improve the manuscript. 1. In abstract, in conclusion sub-section, authors should avoid using "These and other findings, such as changes in coagulation system activation, should be assessed in...". I would rather stop at saying that "Our findings should be confirmed in larger studies." Talking off coagulation system proteins and others is not worthwhile here. 2. The discussion should be reduced by at least a third. Also, authors should discuss the role of phospho-S6 in development of dysplastic cortex and abnormal synatptogenesis. Reviewer #3: Leitner et al present an interesting approach to target molecular mechanisms of everolimus treatment in patients who underwent epilepsy surgery. Whilst I find merrit in the study design the presented results are very limited unless the note that it is a 'pilot study'. - the conclusions that can be drawn are limited - it would be an improvement if the authors show validation of their proteomics findings besides pS6 stainings; was there any difference in glutamate receptor expression throughout the individuals or myelination? - how was the mitochondrial status? - these data would be helpful to point out the importance of changes in metabolites ****** 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: Yes:** Vishal Sondhi Reviewer #3: 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. 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Revision 1
25 Mar 2022 Author Response We thank the editor and reviewers for their comments and the opportunity to revise our manuscript. Below we have provided responses to reviewer and editor comments in red. We thank the reviewers for these suggestions, as we believe these revisions have improved our manuscript. 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. We have confirmed the manuscript meets the style formatting requirements. 2. We note that the grant information you provided in the ‘Funding Information’ and ‘Financial Disclosure’ sections do not match. When you resubmit, please ensure that you provide the correct grant numbers for the awards you received for your study in the ‘Funding Information’ section. We confirm that these sections are updated and matching on the submission site. 3. Thank you for stating the following in the Competing Interests section: [I have read the journal's policy and the authors of this manuscript have the following competing interests: All the authors report no conflicts of interest. Below are the disclosures of the three authors: Daniel Friedman receives salary support for consulting and clinical trial related activities performed on behalf of The Epilepsy Study Consortium, a non-profit organization. Dr. Friedman receives no personal income for these activities. NYU receives a fixed amount from the Epilepsy Study Consortium towards Dr. Friedman’s salary. Within the past two years, The Epilepsy Study Consortium received payments for research services performed by Dr. Friedman from: Alterity, Baergic, Biogen, BioXcell, Cerevel, Cerebral, Jannsen, Lundbeck, Neurocrine, SK Life Science, and Xenon. He has also served as a paid consultant for Neurelis Pharmaceuticals and Receptor Life Sciences. He has received research support from NINDS, CDC, Epitel, and Neuropace unrelated to this study. He holds equity interests in Neuroview Technology. He received royalty income from Oxford University Press. Sasha Devore receives salary support from the National Institutes of Health, Department of Defense, and the Templeton World Charity Foundation unrelated to this study. Orrin Devinsky receives grant support from NINDS, NIMH, MURI, CDC and NSF. He has equity and/or compensation from the following companies: Tilray, Receptor Life Sciences, Qstate Biosciences, Tevard, Empatica, Engage, Egg Rock/Papa & Barkley, Rettco, SilverSpike, and California Cannabis Enterprises (CCE). He has received consulting fees from Zogenix, Ultragenyx, BridgeBio, and Marinus. He holds patents for the use of cannabidiol in treating neurological disorders but these are owned by GW Pharmaceuticals and he has waived any financial interests. He holds other patents in molecular biology.] Please confirm that this does not alter your adherence to all PLOS ONE policies on sharing data and materials, by including the following statement: "This does not alter our adherence to PLOS ONE policies on sharing data and materials.” (as detailed online in our guide for authors http://journals.plos.org/plosone/s/competing-interests). If there are restrictions on sharing of data and/or materials, please state these. Please note that we cannot proceed with consideration of your article until this information has been declared. Please include your updated Competing Interests statement in your cover letter; we will change the online submission form on your behalf. We confirm that the potential competing interests do not alter adherence to PLOS ONE policies on sharing data and materials. 4. 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. We are providing the uncropped and unadjusted original blot data in the supporting information file S1_raw_images. 5. Please amend either the abstract on the online submission form (via Edit Submission) or the abstract in the manuscript so that they are identical. We will confirm this formatting at submission, including confirmation that the abstract is 300 words or less. 6. Please ensure that you refer to Figure 5 in your text as, if accepted, production will need this reference to link the reader to the figure. We have updated the manuscript to reflect the text and corresponding figure references. 7. Please include a copy of Table 2 which you refer to in your text on page 23. We have updated the reference to “Table 2” to “S1 Table.” Reviewer #1: The authors aimed at evaluating safety of the mTOR inhibitor everolimus in treatment resistant (failure of > 2 anti-seizure medications) TSC and FCD patients undergoing surgical resection and to assess changes in mTOR signaling and molecular pathways. I have some queries a) the authors should provide the range age of active patients and controls since they do not appear to be age-matched. Active patients’ mean age is > 18 years old while controls’ mean age is < 18 years old. The age range was included in the abstract and we note in the methods section that there was no difference in the age between the Active and Control participants (p = 0.48). We have added age range for both groups in the methods section as well. Although there was no significant difference in age between the two groups, we noted in the discussion that were limitations to the study (including the broad age range), “There were several limitations to this study. There was a low number of participants due to recruitment difficulties as a result of multiple factors (few eligible candidates, few parents considered participation, many traveling long distances), several heterogeneous variables (age, brain region, neuropathology), no baseline pre-drug blood or brain tissue analyses, short-term treatment duration of 7 days, inclusion of patients with FCD that may not be mTORopathies, and lack of genetic risk factor evaluation.” b)How did the authors deal with the variable levels of total S6 in their analysis, particularly among the Active group? Total S6 levels have previously been reported as being variable in glioblastoma patients but similarities are seen when comparing western blot and histology. Further, we noted in the discussion section, “Similarities in brain phospho-S6 levels between western blot and histology was reported after rapamycin in glioblastoma, with substantial variation in intra-tumoral phospho-S6 relative to baseline surgery levels but with agreement between methods and differing expression magnitudes with more dramatic differences by western blot [16]. It has also been suggested that total S6 levels may be influenced by mTOR dependent translation, in which total S6 was reduced in 2/3 patients after rapamycin [16].” We report total S6 levels as measured by western blot, histology, and proteomics (also noted below). However, one of the main outcomes was to evaluate percent of phospho-S6 relative to total S6 levels, as has been reported in human and animal model studies to decrease in response to mTOR inhibitors rapamycin and everolimus. c) A better delineation of histopathology is needed Additional details have been added to the immunohistochemistry section in the methods. d) The most relevant result was that no safety concerns were found in everolimus treated patients, with no reported adverse events before, during, or after surgical resection. However, owing to the low number of recruited patients and the short treatment time the authors should soften their conclusions We acknowledge that this is a pilot study with some limitations. e) To increase the interest of the study, the authors should discuss the correlation of their results with seizure outcome after surgery in their sample Correlation of our results to any changes in seizure frequency are certainly of interest, thus we noted in the methods section, “Changes to seizure frequency were not evaluated, given the brief study duration and small participant numbers with high intra-individual variability in seizure frequency.” Reviewer #2: The authors report findings from their study evaluating safety and molecular mechanisms of short-term everolimus (7 days) in TSC and FCD patients. The study has conducted well. I would like to give a few suggestions to improve the manuscript. 1. In abstract, in conclusion sub-section, authors should avoid using "These and other findings, such as changes in coagulation system activation, should be assessed in...". I would rather stop at saying that "Our findings should be confirmed in larger studies." Talking off coagulation system proteins and others is not worthwhile here. We note the evaluation of coagulation system activation in particular, as coagulation should be evaluated in the context of safety in a long-term study with a larger group, thus we think it is worth mentioning as safety was one of the primary outcomes in this pilot study. 2. The discussion should be reduced by at least a third. Also, authors should discuss the role of phospho-S6 in development of dysplastic cortex and abnormal synatptogenesis. We note in the introduction section studies related to phospho-S6 and dysplasia, “FCD may result from somatic mosaicism with pathogenic gene variants that impact the mTOR signaling pathway, particularly FCD type II with dysmorphic neurons (6, 7). Dysmorphic and balloon neurons are found histologically in TSC and FCD cases, with high ribosomal protein S6 phosphorylation (phospho-S6) levels due to increased mTOR pathway activation (6, 8).” Regarding synaptogenesis, it is reported that mTOR activity is associated with dendrite formation, including the increased activity that occurs in long-term potentiation and is altered in epilepsy models. We have emphasized this in the discussion. Interestingly, we reported that increasing phospho-S6 levels correlated with decreased synaptic transmission via proteins involved in transport and localization (Figure 3), thus synaptic transmission proteins had higher expression levels in those cases with lower phospho-S6 (seen among several of the Active participants). We have also shortened the discussion section by about 200 words. Reviewer #3: Leitner et al present an interesting approach to target molecular mechanisms of everolimus treatment in patients who underwent epilepsy surgery. Whilst I find merrit in the study design the presented results are very limited unless the note that it is a 'pilot study'. - the conclusions that can be drawn are limited The results we describe in the manuscript are indeed a smaller pilot study. We agree that follow up studies are needed in larger groups and with long-term everolimus treatment to evaluate brain molecular changes and any correlations to seizure frequency. - it would be an improvement if the authors show validation of their proteomics findings besides pS6 stainings; was there any difference in glutamate receptor expression throughout the individuals or myelination? We noted in the results section, “The histology quantification of total S6 (RPS6, p = 0.90, Figure 2) provides validation of the proteomics results (p = 0.94, Supplemental Table 1) with some variability in the Active group that is also seen by western blot (p = 0.97, Figure 1B).” Below is a graphical representation of the validation for RPS6. From proteomics, we did not identify differentially expressed glutamate receptors or myelin proteins (including detected MBP, CNP, PLP1, PLP2, MAG, MOG, MOBP), only those associated with coagulation and acute phase response activation (S1 Figure). The glutamate receptor signaling pathway (p = 1.70 x 10-5, z = 0.71; S2 Table) did include detected glutamate receptors (GRIA2, GRIA3, GRIA4, GRIN1, GRIN2B, GRM2, GRM3), however this pathway was not significantly activated. There may be increased activity at the inhibitory GRM3 receptor in the Active participants related to the increased NAAG we identified by metabolomics. - how was the mitochondrial status? We reported that mitochondrial cellular respiration negatively correlated to higher phospho-S6 levels in Figure 3 (the most significantly correlated module to P235/236), thus cellular respiration proteins had higher expression levels in those cases with lower phospho-S6 (seen among several of the Active participants). - these data would be helpful to point out the importance of changes in metabolites The altered metabolites that we identified, particularly NAAG, is not necessarily directly related to mitochondrial activity. NAAG is synthesized from NAA in the cytosol by NAAG synthetase (PMID 35163193). Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0268597.r002
3 May 2022 Decision Letter - Emilio Russo, Editor Pilot Study Evaluating Everolimus Molecular Mechanisms in Tuberous Sclerosis Complex and Focal Cortical Dysplasia PONE-D-22-02043R1 Dear Dr. Devinsky, 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. 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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 Reviewer #3: 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) Reviewer #2: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: (No Response) Reviewer #2: Yes Reviewer #3: 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: (No Response) Reviewer #2: Yes Reviewer #3: 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: (No Response) Reviewer #2: Yes Reviewer #3: 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: All my queries have been addressed. The manuscript is now acceptable in current format from my side. Reviewer #3: (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: Yes:** Vishal Sondhi Reviewer #3: No https://doi.org/10.1371/journal.pone.0268597.r003
Formally Accepted
9 May 2022 Acceptance Letter - Emilio Russo, Editor PONE-D-22-02043R1 Pilot study evaluating everolimus molecular mechanisms in tuberous sclerosis complex and focal cortical dysplasia Dear Dr. Devinsky: 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 Prof Emilio Russo Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0268597.r004

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