ATM-deficient murine thymic T-cell lymphoblastic lymphomas are PTEN-deficient and require AKT signaling for survival

Joseph B. An; Karen S. Hathcock; Seth M. Steinberg; Hyoyoung M. Choo-Wosoba; Richard J. Hodes

doi:10.1371/journal.pone.0312864

Peer Review History

Original SubmissionMay 24, 2024
21 Jun 2024 Decision Letter - Alvaro Galli, Editor PONE-D-24-21090ATM-deficient murine thymic T-cell lymphoblastic lymphomas are PTEN-deficient and require AKT signaling for survivalPLOS ONE Dear Dr. Hodes, 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 Aug 05 2024 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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If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice. [Note: HTML markup is below. Please do not edit.] Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: Yes ******** 2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes Reviewer #3: 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 Reviewer #3: 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 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 study titled ATM-deficient murine thymic T-cell lymphoblastic lymphomas are PTEN-deficient and require AKT signaling for survival by An et al delves into the genetic and signaling aspects of T-cell lymphoblastic lymphoma cells from ATM-deficient mice, a model mimicking human T-ALL. The authors show that most ATM-deficient thymic T-LBLs exhibit genetic alterations in Pten, leading to the loss of PTEN expression and increased AKT activation. Furthermore, they show that all ATM-deficient T-LBLs are sensitive to AKT inhibition, suggesting the potential therapeutic potential of AKT inhibitors in treating human T-ALL. Although this study is conceptually interesting, the presented results are rather weak. Furthermore, several questions arise while reading the manuscript and the authors may address them during the revision process: - The first sentence of the abstract starts with the words “mice deficient in ATM…”. This is misleading as one immediately assumes that the experiments in this study are conducted in vivo. This is, however, not the case as the data in the manuscript are exclusively derived from cell culture-based experiments. In the Methods section, it is furthermore unclear, how the cells were extracted from mice to generate the analyzed cell lines. - The figures are pixelated and difficult to decipher - The figure captions are somehow embedded in the text and are easily missed. - In Figure 6A, define non-malignant versus malignant cells - In reference to Figure 6, would deleting Pten, e.g. using CRISPR-Cas9, in the PTEN-sufficient T-LBL cells mimic the phenotype observed in the PTEN-deficient cell lines? - What are the functional properties of the malignant T-LBL cells? Are they more proliferative? - Would these cells be resistant to standard treatment of, e.g., T-ALL? - Are the T-LBL cells responsive to Everolimus treatment? Reviewer #2: The authors reported the frequent pTEN deficiency either by complete deletion, partial deletion or mutations of PTEN, inducing pAKT on Ser 473 and sensitivity to inhibitors of AKT signaling among the 12 ATMKO mice with T-LBL in this series. Minor comments 1- The authors should explain how they select these 12 ATMKO T-LBL compared to their littermate of ATMKO mice and ATMKO with T LBL non selected for this study 2-the p values for figure 6B should be given Reviewer #3: In this manuscript by An et al., the authors confirm a previous discovery that ATMKO T-LBLs tend to exhibit Pten genetic alterations. The study characterized the mutations and found that most ATMKO T-LBLs have various genomic alterations in the Pten gene, resulting in the loss of functional PTEN protein. Consistently, all T-LBLs in the study demonstrated higher levels of phosphorylated AKT (pAKT), indicating the activation of AKT signaling. Importantly, this pAKT signaling appears to be crucial for the survival of the lymphomas, as indicated by the data on the AKT inhibitor MK-2206. The experiments were well-designed, and the results seem robust. This work thus nicely documents an interesting finding. I have no criticism except that in Figures 1, 3, 4, and 5, the authors only present data from representative ATMKO T-LBLs. Considering there are only 12 of them and not much mechanistic insight was demonstrated in this work, I think that all the data should be included in this manuscript, at least in the supplementary materials. ****** 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 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. 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.0312864.r001
Revision 1
8 Oct 2024 Author Response Reviewer #1: The study titled ATM-deficient murine thymic T-cell lymphoblastic lymphomas are PTEN-deficient and require AKT signaling for survival by An et al delves into the genetic and signaling aspects of T-cell lymphoblastic lymphoma cells from ATM-deficient mice, a model mimicking human T-ALL. The authors show that most ATM-deficient thymic T-LBLs exhibit genetic alterations in Pten, leading to the loss of PTEN expression and increased AKT activation. Furthermore, they show that all ATM-deficient T-LBLs are sensitive to AKT inhibition, suggesting the potential therapeutic potential of AKT inhibitors in treating human T-ALL. Although this study is conceptually interesting, the presented results are rather weak. Furthermore, several questions arise while reading the manuscript and the authors may address them during the revision process: - The first sentence of the abstract starts with the words “mice deficient in ATM…”. This is misleading as one immediately assumes that the experiments in this study are conducted in vivo. This is, however, not the case as the data in the manuscript are exclusively derived from cell culture-based experiments. In the Methods section, it is furthermore unclear, how the cells were extracted from mice to generate the analyzed cell lines. RESPONSE: Although the bulk of the data came from lymphoma cells acquired shortly after harvesting the cells from mice, because some of the data are from cells after they have been propagated multiple times in vitro, I have adjusted some of the words to reflect that this was an in vitro study. I have also modified the methods section to reflect how the cells were obtained. - The figures are pixelated and difficult to decipher RESPONSE: We have worked on getting this fixed - The figure captions are somehow embedded in the text and are easily missed. RESPONSE: The addition of the figure captions into the main text body of the manuscript was done according to the guidelines of the PLOS journal. - In Figure 6A, define non-malignant versus malignant cells RESPONSE: Like in Petinot et al. 2000, we determined tumors by their significantly increased weight and size of the thymus compared to those of littermates without ATM deficiency. Necropsy was performed on all mice upon detection of rapid/shallow breathing or spontaneous death. Tumors frequently had an abnormal immunophenotype by expressing CD25/CD44 despite expressing CD4 and/or CD8. In addition, in contrast to T-LBLs, non-malignant thymocytes cannot self-propagate in our standard culture media. - In reference to Figure 6, would deleting Pten, e.g. using CRISPR-Cas9, in the PTEN-sufficient T-LBL cells mimic the phenotype observed in the PTEN-deficient cell lines? RESPONSE: Are you referring to PTEN deletion in PTEN+ non-malignant ATMKO thymocytes or in PTEN+ ATMKO T-LBL cells? In PTEN+ non-malignant thymocytes, selective deletion of Pten using either Lck-cre or CD4-Cre models (Hagenbeek and Spits, 2008; Liu et al., 2010) have been described and do indeed promote T cell lymphomagenesis. Our ATMKO lymphoma model, contrary to the one aforementioned which involves genetically knocking out Pten, demonstrates that these lymphomas undergo a selective pressure to lose PTEN activity. In addition, most of the lymphomas generated from the CD4/Lck-Cre model are more developmentally mature (after pre-TCRB expression/the beta checkpoint), whereas some of our tumors possess a more developmentally early immunophenotype that resembles human pre-T cell ALL. In regards to PTEN+ T-LBLs, because these cells are still sensitive to the pan-AKT inhibitor, I don't expect a change in consequence if targeting Pten by CRISPR, at least in cell viability. - What are the functional properties of the malignant T-LBL cells? Are they more proliferative? RESPONSE: I can answer these questions in comparison to the non-malignant thymocytes. Whereas normal thymocytes are unable to proliferate in standard culture media, our T-LBL cells can propagate virtually indefinitely in vitro. In vivo, these T-LBLs proliferate to the point of causing mass effect in the mediastinum, ultimately leading to premature death of these mice. - Would these cells be resistant to standard treatment of, e.g., T-ALL? RESPONSE: The standard of care treatment for T-ALL is a multi-chemotherapy regimen such as HyperCVAD, which has a very high initial complete response rate. Thus, I would expect a very good anti-lymphoma response with these chemotherapeutics in vitro. The issue is that disease recurrence in vivo is common, and that response to salvage therapy is very poor. - Are the T-LBL cells responsive to Everolimus treatment? RESPONSE: We have some data indicating high sensitivity of our T-LBL cells to rapamycin but not everolimus. We did not include the data due to insufficient replicated data Reviewer #2: The authors reported the frequent pTEN deficiency either by complete deletion, partial deletion or mutations of PTEN, inducing pAKT on Ser 473 and sensitivity to inhibitors of AKT signaling among the 12 ATMKO mice with T-LBL in this series. Minor comments 1- The authors should explain how they select these 12 ATMKO T-LBL compared to their littermate of ATMKO mice and ATMKO with T LBL non selected for this study RESPONSE: We included for analysis all of the 12 lines successfully adapted into in vitro culture 2-the p values for figure 6B should be given RESPONSE: The p-values for Fig 6B were listed in the figure legend instead of in the manuscript itself, which have been fixed Reviewer #3: In this manuscript by An et al., the authors confirm a previous discovery that ATMKO T-LBLs tend to exhibit Pten genetic alterations. The study characterized the mutations and found that most ATMKO T-LBLs have various genomic alterations in the Pten gene, resulting in the loss of functional PTEN protein. Consistently, all T-LBLs in the study demonstrated higher levels of phosphorylated AKT (pAKT), indicating the activation of AKT signaling. Importantly, this pAKT signaling appears to be crucial for the survival of the lymphomas, as indicated by the data on the AKT inhibitor MK-2206. The experiments were well-designed, and the results seem robust. This work thus nicely documents an interesting finding. I have no criticism except that in Figures 1, 3, 4, and 5, the authors only present data from representative ATMKO T-LBLs. Considering there are only 12 of them and not much mechanistic insight was demonstrated in this work, I think that all the data should be included in this manuscript, at least in the supplementary materials. RESPONSE: We have adjusted the figures to include all of the lines analyzed Attachments Attachment Submitted filename: Response to the reviewers.docx https://doi.org/10.1371/journal.pone.0312864.r002
15 Oct 2024 Decision Letter - Alvaro Galli, Editor ATM-deficient murine thymic T-cell lymphoblastic lymphomas are PTEN-deficient and require AKT signaling for survival PONE-D-24-21090R1 Dear Dr. Hodes, 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 will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. 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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 ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes Reviewer #2: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes Reviewer #2: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Dear authors, thank you for addressing my remarks. Please pay attention to the figure quality, they still appear extremely blurry in the new version Reviewer #2: the authors responded adequately to all the comments made and modified the manuscript as well as figures accordingly ****** 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 ******** https://doi.org/10.1371/journal.pone.0312864.r003
Formally Accepted
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