AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent in vitro pancreatic cancer growth inhibitor

Komala Ingle; Joseph F. LaComb; Lee M. Graves; Antonio T. Baines; Agnieszka B. Bialkowska

doi:10.1371/journal.pone.0294065

Peer Review History

Original SubmissionMay 4, 2023
18 Jun 2023 Decision Letter - Wagdy Mohamed Eldehna, Editor PONE-D-23-13615AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent pancreatic cancer growth inhibitorPLOS ONE Dear Dr. Bialkowska, 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 02 2023 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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[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: Partly 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: Bialkowska et al compare the effect in viablity, proliferation, cell cycle and biomarker modulation of a PIM-i with a triple inhibitor PIm/PI3K/mTOR in five pancreatic cell lines. Moreover, they compare their activity in one cell line resistant to gemcitabine. When they study the effect on biomarkers they treat the cells for 72h, a time point in which AUM-302 usually has a strong effect in viability or proliferation, so this data are not valid. Major revision: The authors should also include a PI3K/mTOR inhibitor to compare with and really demonstrate that the triple inhibitor is more potent Minor revisions 1. Correct table 1. When a cell line is resistant to a drug de GI50 should be higher than the highest concentration tested 2. In order to be able to take any conclusions regarding the effect in biomarker modulation, on one hand the authors should provide a quantification of the bands and in the case of phosphorylation take into account the total levels of each corresponding protein. Moreover, 72h is a very long time treatment to see effect in phosphorylation as compensatory mechanisms could take place. 3. In the case of resistant cell line, the highest concentration tested of AUM-302 for the evaluation of biomarkers has a very strong effect in proliferation at 72h then this results are not reliable. Shorter treatments should be included. Reviewer #2: Running manuscript discuss a potential therapy of pancreatic cancer, UM302, as a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent pancreatic cancer. Although the point is very interesting, authors have to made major revision of their manuscript as recommended in the following points: Methodology: Why authors did not determine PIM1/2 inhibition to ensure the correlation with the demonstrated results and the correlation of inhibited molecular components of PIM/PI3K/m TOR It is advisable for author to make a semiquantitative analysis of western plot bands to facilitate the interpretation of inhibited proteins Results part: Table one should include standard deviation of demonstrated IC50s, its title should be also more elucidated to be self-explanatory. Quality of Figures 2-8 should be improved in their quality Discussion: Authors should reconstruct the discussion part to be more focused on discussing the demonstrated results and the correlation of inhibited molecular components of PIM/PI3K/m TOR. Reviewer #3: Dr. Bialkowska and co-authors report that “AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent pancreatic cancer growth inhibitor.” The authors should add the words in vitro to this title. Overall, this is a well-executed and well-written study demonstrating that AUM302 inhibits the proliferation and viability of different human pancreatic cancer cell lines in culture and is capable of overcoming gemcitabine resistance in a gemcitabine-resistant MIA PaCa2 cell line. Since the authors did not carry out in vivo studies with either intra-pancreatic or autochthonous models of PDAC, the authors should add a statement or two to the discussion section to indicate that such studies are necessary to determine whether AUM302 can be effective in vivo, and whether AUM302 is capable of altering in a beneficial manner the immune-excluding tumor microenvironment in this immune-cold cancer. ****** 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: Carmen Blanco-Aparicio 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.0294065.r001
Revision 1
19 Sep 2023 Author Response September 12th, 2023 Editor and Reviewers PLOS ONE Re: PONE-D-23-13615 AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent pancreatic cancer growth inhibitor Dear Editor and Reviewers, We want to thank the Reviewers for their invaluable comments. We also acknowledge the concerns raised by the Reviewers and now provide a revised copy of the manuscript, considering all of the Reviewers' concerns. This revised manuscript addressed all the comments and suggestions the Editor and Reviewers provided. Please see our responses to your comments below. Reviewer 1. Comment 1. The authors should also include a PI3K/mTOR inhibitor to compare with and really demonstrate that the triple inhibitor is more potent. Response 1. We agree with the Reviewer comment. We performed cell viability studies in BxPC-3, Capan-2, MIA PaCa-2, PANC-1, and Hs766T using two compounds, GDC-0941 and BEZ235, both PI3K and mTOR inhibitors. We included the results in Figure 1 and Table 1 and added an appropriate statement in the Results section. Comment 2. Correct table 1. When a cell line is resistant to a drug de GI50 should be higher than the highest concentration tested. Response 2. We provided IC50 values in nanomolar concentrations. We performed all experiments using low concentrations of AUM-302 of 10nM and 100nM as we have observed its significant effects using these concentrations on cell proliferation and cell cycle of tested PDAC cell lines. Comment 3. In order to be able to take any conclusions regarding the effect in biomarker modulation, on one hand the authors should provide a quantification of the bands and in the case of phosphorylation take into account the total levels of each corresponding protein. Moreover, 72h is a very long time treatment to see effect in phosphorylation as compensatory mechanisms could take place. Response 3. We agree with the Reviewer's comment. In this revised manuscript, we included the results of the 24-hour treatment of five tested PDAC cell lines with 10nM and 100nM concentrations of TP-3654 and AUM-302 in Figure 6 and the quantification of the western blot results in Figure 7. Comment 4. In the case of resistant cell line, the highest concentration tested of AUM-302 for the evaluation of biomarkers has a very strong effect in proliferation at 72h then this results are not reliable. Shorter treatments should be included. Response 4. We provided western blot results from 24-, 48- and 72-hour treatment in Figure 9 and appropriate quantifications of the western blot results in Supplementary Figures 1 through 3. Reviewer 2. Comment 1. Why authors did not determine PIM1/2 inhibition to ensure the correlation with the demonstrated results and the correlation of inhibited molecular components of PIM/PI3K/m TOR? Response 1. The AUM-302 compound does not impact the levels of PIM1/2 on RNA or protein levels. The compound has been designed to affect their activity. Thus, the levels of PIM1/2 are unchanged, but their activities are decreased upon AUM-302 treatment. Comment 2. It is advisable for author to make a semiquantitative analysis of western plot bands to facilitate the interpretation of inhibited proteins. Response 2. We agree with the Reviewer's suggestions. We performed appropriate quantifications and included them in Figure 7 and Supplementary Figures 1 through 3. Comment 3. Table one should include standard deviation of demonstrated IC50s, its title should be also more elucidated to be self-explanatory. Response 3. Thank you for your suggestion. We modified the title of Table 1 and provided IC50 values in nanomolar concentrations with standard deviations. Comment 4. Quality of Figures 2-8 should be improved in their quality. Response 4. All figures were prepared in Adobe Photoshop according to the PLOS ONE requirements with at least 300-600 dpi. All figures are submitted as .tif files. In addition, we utilized Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool recommended by PLOS ONE that tests the quality of prepared images and their compliance with the journal requirements. It could be that the journal provides low-quality figures for the primary review. We hope that the quality of the revised figures is adequate. Comment 5. Authors should reconstruct the discussion part to be more focused on discussing the demonstrated results and the correlation of inhibited molecular components of PIM/PI3K/mTOR. Response 5. We want to thank the Reviewer for this suggestion. We revised the Discussion section and added more information relevant to inhibiting PIM/PI3K/mTOR pathway components. Reviewer 3. Comment 1. The authors should add the words in vitro to this title. Response 1. We modified the title of the manuscript to reflect its in vitro studies. Comment 2. Since the authors did not carry out in vivo studies with either intra-pancreatic or autochthonous models of PDAC, the authors should add a statement or two to the discussion section to indicate that such studies are necessary to determine whether AUM302 can be effective in vivo, and whether AUM302 is capable of altering in a beneficial manner the immune-excluding tumor microenvironment in this immune-cold cancer. Response 2. Thank you for this suggestion. We modified the Discussion section and added a statement emphasizing the necessity for in vivo studies to assess AUM-302 full impact on the PDAC microenvironment. We confirm that neither the manuscript nor any parts of its content are currently under consideration or published in another journal. We changed the revised manuscript to ascertain compliance with the PLOS ONE journal's requirements. We hope the incorporated changes will satisfy the Reviewers and Editor and render the revised manuscript suitable for publication. Thank you for being so considerate. All authors have approved the manuscript and agree with its submission to PLOS ONE. Please feel free to contact me if I can be of further assistance, Sincerely Yours, Agnieszka B. Bialkowska, PhD Associate Professor Renaissance School of Medicine at Stony Brook University Department of Medicine GI Translational Research Lab HSC-T17 Room 090 Stony Brook, NY 11794-8176 Phone: (631) 638 2161 Email: Agnieszka.Bialkowska@stonybrookmedicine.edu Attachments Attachment Submitted filename: Response to the Reviewers comments.pdf https://doi.org/10.1371/journal.pone.0294065.r002
25 Oct 2023 Decision Letter - Wagdy Mohamed Eldehna, Editor AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent in vitro pancreatic cancer growth inhibitor PONE-D-23-13615R1 Dear Dr. Bialkowska, 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 #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: Yes Reviewer #3: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: 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: 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: 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: Authors have addressed all my comments properly. They just only need to rewrite lines 244 and 245 regarding the time treatment, as for DMSO they refer 72h and for treatment inhibitors 24h. all time points should be 24h. Reviewer #3: Dr. Bialkowska and co-authors have addressed my concerns and the paper in my opinion presents important new information in a valid manner and is acceptable for publication. ****** 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: Yes: Carmen Blanco-Aparicio Reviewer #3: No ******** https://doi.org/10.1371/journal.pone.0294065.r003
Formally Accepted
31 Oct 2023 Acceptance Letter - Wagdy Mohamed Eldehna, Editor PONE-D-23-13615R1 AUM302, a novel triple kinase PIM/PI3K/mTOR inhibitor, is a potent in vitro pancreatic cancer growth inhibitor Dear Dr. Bialkowska: 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. Wagdy Mohamed Eldehna Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0294065.r004

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