Disulfiram and copper combination therapy targets NPL4, cancer stem cells and extends survival in a medulloblastoma model

Riccardo Serra; Tianna Zhao; Sakibul Huq; Noah Leviton Gorelick; Joshua Casaos; Arba Cecia; Antonella Mangraviti; Charles Eberhart; Renyuan Bai; Alessandro Olivi; Henry Brem; Eric M. Jackson; Betty Tyler

doi:10.1371/journal.pone.0251957

Peer Review History

Original SubmissionDecember 23, 2020
8 Feb 2021 Decision Letter - Ilya Ulasov, Editor PONE-D-20-38226 Disulfiram and Copper Combination Therapy Targets NPL4, Cancer Stem Cells and Extends Survival in a Medulloblastoma Model PLOS ONE Dear Dr. Serra, 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 Mar 25 2021 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file. 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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: Chelation with copper is essential for the anticancer effects of the old alcohol reversion drug disulfiram. As such, administration of DSF along with copper gluconate has emerged as a drug repurposing strategy with clinical trials underway in multiple cancers. This strategy is of particular interest for brain cancers, because, DSF can cross the blood-brain barrier. There have been several preclinical studies published on DSF-Cu therapy in glioblastoma and other brain cancer models. The authors here have performed extensive cell culture and orthotopic xenograft studies using standard cytotoxicity, flow cytometry, western blot, and immunochemical analyses in a panel of human medulloblastoma cell lines. The results are along the expected lines of strong cytotoxicity, antitumor effects, expression of apoptotic regulatory proteins, etc by DSF-Cu. Apart from confirming the anticancer effects of Cu-DSF, this study lacks novelty and does not shed light on the probable mechanisms of action of the copper chelated disulfiram. However, it is acceptable for publication in PLOSone given the journal policy for acceptance. Nevertheless, the following points require attention before acceptance. 1) The strategy of copper gluconate supplementation with DSF has been tried in many trials, however, the results have not been encouraging. The authors should discuss the possible reasons and future directions in Cu-DSF treatments. 2) Sure Cu-DSF may cause aggregation of NPL4, however, this is not likely the only mechanism. Can NPL4 clustering be a response seen in dying cells? Were any major targets affected by NPL4 aggregation? Currently, there is no information on the role of NPL4 in therapy-targeted ubiquitin destruction of proteins. How were the cell extracts prepared for NPL4 Western blots? Soluble or insoluble fractions used for SDS-PAGE? If aggregated, much of the protein is likely to remain with cell debris after lysis. Did authors find NPL4 aggregation in xenografted tumor specimens after Cu-DSF administration? 3) How do the DSF and Cu doses used in animal experiments compare with those used in clinical trials? ****** 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 [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
26 Apr 2021 Author Response Response to Reviewers We thank the Reviewer for her/his time and helpful comments on our manuscript. In response to her/his critiques, we have introduced several changes to our manuscript. Furthermore, in the present Letter we tried to explain our rationale and perspective on the points brought up by the Reviewer. 1) The strategy of copper gluconate supplementation with DSF has been tried in many trials, however, the results have not been encouraging. The authors should discuss the possible reasons and future directions in Cu-DSF treatments. We thank the reviewer for this comment. Although some of the results of large randomized clinical trials in brain cancers with the combination of Disulfiram and Copper are not encouraging, a recent Phase I/II study of disulfiram and copper with concurrent radiation therapy and temozolomide for newly diagnosed glioblastoma showed promising preliminary efficacy for a subset of glioblastoma with IDH1, BRAF, and NF1 mutations. Among these patient glioblastomas with IDH1 (n = 6), BRAF (n = 2), or NF1 (n = 1) mutations had significantly better PFS and OS than those without the mutations (1-year PFS: 100% vs 22%, respectively, p = 0.001; 1-year OS: 100% vs 42%, respectively, p = 0.006) (2). These data might therefore support the use of this combination in specific subsets of patients, or at least the possibility of expanding the current trials to include larger samples of newly diagnosed glioblastoma. Furthermore, a major advantage of Disulfiram-Copper is their relatively benign and well-known safety profile, especially when compared to novel anti-cancer strategies. Finally, their limited cost will allow for a larger use and more widespread testability in resource-limited settings. As suggested by the Reviewer, we included these considerations in our Discussion. 2) Cu-DSF may cause aggregation of NPL4, however, this is not likely the only mechanism. Can NPL4 clustering be a response seen in dying cells? We thank the Reviewer for commenting on this important and still debated point of DSF cytotoxicity. Skrott et al. showed that NPL-4 is usually not a physiologic response seen in cell death, and that it is triggered by the combination of DSF-Cu++ and their metabolite Cuet. Specifically, NPL-4 aggregation is the mechanism involved in p97 blockage, leading to the downstream activation of the Heat Shock Response that causes cell death(1). However, we agree with the reviewer that NPL4 aggregation is likely not the only cytotoxic mechanism mediated by DSF-Cu++ administration. DNA damage and stem-cell targeting seem in fact to be involved in the anti-cancer effects of the combination, as demonstrated by several studies in CNS and non-CNS malignancies (1, 3, 4). In our study these mechanisms were confirmed by different tests, and we believe that they still partially contribute to the final cytotoxic and anticancer effect of this combination. Were any major targets affected by NPL4 aggregation? Currently, there is no information on the role of NPL4 in therapy-targeted ubiquitin destruction of proteins. We thank the reviewer for this comment. As shown by Skrott et al. the major downstream effector of NPL4 aggregation resides in the activation of Heat-Shock Response proteins, with consequent triggering of cell apoptosis (1). As a mediator of the p97 cascade, NPL4 plays an indirect role in ubiquitin-mediated destruction of proteins. How were the cell extracts prepared for NPL4 Western blots? Soluble or insoluble fractions used for SDS-PAGE? We thank the reviewer for pointing this out. Insoluble fractions were used for Western Blotting, and a more detailed description of the methods used for sample preparation is now included in the Supplementary files. If aggregated, much of the protein is likely to remain with cell debris after lysis. Did authors find NPL4 aggregation in xenografted tumor specimens after Cu-DSF administration? We thank the reviewer for this comment. WB showed a significant increase in NPL-4 signal after in vivo treatment with DSF-Cu++. A larger cohort, better timing of sample collection and the possible transient expression of NPL4 aggregation may explain this finding. Furthermore, while other groups demonstrated in vitro clustering of this mediator, in vivo evidence has been so far very limited and not definitive, somehow reinforcing the idea of the ephemeral action of NPL-4(1). 3) How do the DSF and Cu doses used in animal experiments compare with those used in clinical trials? We thank reviewer for this comment. The DSF and Cu++ dosages used in mouse are significantly lower than the maximum tolerated dose in humans. Recent trials used doses of 125 mg/2 mg, twice daily, compared to our dosage of 150 mg/kg/day and 2 mg/kg/day (2). A case report in a Glioblastoma patient also reported a daily dosage of DSF and Cu++ of 250 and 6 mg, respectively (5). However, given the faster metabolic rate typical of rodents, preliminary studies in Glioblastoma and AT/RT used higher dosages than those administered to human patients. Specifically, 100mg/kg/day was used in Glioblastoma (4) and AT/RT (3). For the dosage used in our study, we did not determine any behavioral changes or body weight loss during our treatment. FUNDING The Funding Statement and Funding section in the Manuscript do not coincide because I was unable to include the Ms. Kimberly Spiro, the Donald W. Spiro Foundation, and Donald R. Spiro donations as a source of funding. No commercial interests are connected with this donation. I would like to include Ms. Kimberly Spiro, the Donald W. Spiro Foundation, and Donald R. Spiro donations in the final manuscript. Thank you COMMERCIAL INTERESTS The Commercial Interests statement in the manuscript is up to date. 1. Skrott Z, Mistrik M, Andersen KK, Friis S, Majera D, Gursky J, et al. Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4. Nature. 2017;552(7684):194. 2. Huang J, DeWees T, Campian JL, Chheda MG, Ansstas G, Tsien C, et al. A TITE-CRM phase I/II study of disulfiram and copper with concurrent radiation therapy and temozolomide for newly diagnosed glioblastoma. American Society of Clinical Oncology; 2019. 3. Choi SA, Choi JW, Wang K-C, Phi JH, Lee JY, Park KD, et al. Disulfiram modulates stemness and metabolism of brain tumor initiating cells in atypical teratoid/rhabdoid tumors. Neuro-oncology. 2015;17(6):810-21. 4. Lun X, Wells JC, Grinshtein N, King JC, Hao X, Dang N-H, et al. Disulfiram when combined with copper enhances the therapeutic effects of temozolomide for the treatment of glioblastoma. Clinical Cancer Research. 2016;22(15):3860-75. 5. Karamanakos PN, Trafalis DT, Papachristou DJ, Panteli ES, Papavasilopoulou M, Karatzas A, et al. Evidence for the efficacy of disulfiram and copper combina-tion in glioblastoma multiforme-A propos of a case. Journal of BU ON: official journal of the Balkan Union of Oncology. 2017;22(5):1227-32. https://doi.org/10.1371/journal.pone.0251957.r002
7 May 2021 Decision Letter - Ilya Ulasov, Editor Disulfiram and Copper Combination Therapy Targets NPL4, Cancer Stem Cells and Extends Survival in a Medulloblastoma Model PONE-D-20-38226R1 Dear Dr. Serra, 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, Ilya Ulasov, 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 ******** 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 ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This is an emerging therapeutic area, particularly for brain cancers. It will further evoke interest in the concerned scientific community. ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review?** For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: No https://doi.org/10.1371/journal.pone.0251957.r003
Formally Accepted
26 Aug 2021 Acceptance Letter - Ilya Ulasov, Editor PONE-D-20-38226R1 Disulfiram and Copper Combination Therapy Targets NPL4, Cancer Stem Cells and Extends Survival in a Medulloblastoma Model Dear Dr. Serra: 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. Ilya Ulasov Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0251957.r004

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