Inhibition of casein kinase 2 induces cell death in tyrosine kinase inhibitor resistant chronic myelogenous leukemia cells

Ondřej Mitrovský; Denisa Myslivcová; Tereza Macháčková-Lopotová; Adam Obr; Kamila Čermáková; Šárka Ransdorfová; Jana Březinová; Hana Klamová; Markéta Žáčková

doi:10.1371/journal.pone.0284876

Peer Review History

Original SubmissionOctober 1, 2022
24 Jan 2023 Decision Letter - Rama Krishna Kancha, Editor PONE-D-22-27194Inhibition of Casein Kinase 2 induces cell death in chronic myelogenous leukemia cells resistant to tyrosine kinase inhibitorsPLOS ONE Dear Dr. Obr, 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 10 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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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 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. 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(Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: The manuscript entitled “Inhibition of Casein Kinase 2 induces cell death in chronic myelogenous leukemia cells resistant to tyrosine kinase inhibitors” by Adam Obr is interesting however the manuscript has many flaws which needed to be answered before its publication. Throughout the manuscript the authors used sentences like “somewhat affected” and “slight growth slowing”. These sentences need to be avoided and “percentage of growth inhibition” would be recommended. Instead of denoting K562 cells which have never been exposed to inhibitors as control, representation of them as näive K562 cells would be more appropriate. Results part needs to be written very precisely and the sub heading should have a title which explains the outcome of the results otherwise it is highly confusing what authors want to claim. When there is no explanation for CK2 levels and chromosomal abnormality devoting one big paragraph for cytogenetics is unnecessary and this part is highly confusing and should move to supplementary information. Most importantly, authors should check the mutational status of BCR-ABL in the established TKI resistant cell lines. Without this information, concluding the results part is highly indecisive. Best example is Figure 1, TKI resistant JURL-MK1 and MOLM-7 cells showed persistent p-CRKL, whereas K562 could not show suggesting that in K562 cells TKI resistance might be due to BCR-ABL independent mechanism. When data is not significant, showing them in the main figures is also confusing. Keeping them in the supplementary information and giving detailed explanation would be advisable. Example is Figure 2 CDC37 levels. It is also highly recommended to authors to show the dose dependent growth curves in order to claim the specificity of the CX-4945 in the mentioned cell lines. Apoptosis assays such as annexin stating needed to perform with different doses of CX-4945 showing the PARP cleavage is not enough. However, PARP cleavage was also noticeable in OCI-AML 3 cells with dasatinib and CX-4945 treatment. Do they get affected by TKIs treatment? Authors claimed that OCI-AML 3 cell line is BCR-ABL negative and in Figure 3 C it is mentioned as imatinib resistant (IR) and Dasatinib resistant (DR) OCI-AML3, this is confusing, did they develop OCI-AML resistant clones against the imatinib and dasatinib? Authors should clearly check this information. As said earlier, concluding the Figure 4 and relationship between the BCR-ABL and CK2 is indecisive without knowing the mutational status of BCR-ABL and mechanism of resistance in these clones. Based on these points, the manuscript required major revision for publication. Reviewer #2: This is a very nice study that can be accepted for publication with minor modifications and suggestions. The authors used proper analyses techniques and evaluate the results properly. 1. Page 7, Line 122 : T315I Mutation in BCR-ABL.. It is written as’’ In both JURL-MK1 IR and DR cells, there was a T315I mutation in the kinase domain. In resistant cells 123 derived from MOLM-7 and K562, respectively, no mutation in the kinase domain of BCR-ABL was 124 detected.’’in the results section of the manuscript. However, no DNA sequence data related to this mutation were included in this article. I think this result should be added to the article. 2. Please: consider adding a paper I wrote below to your references. • ’’The casein kinase 2 inhibitor, CX-4945, as an anti-cancer drug in treatment of human hematological malignancies.’’ ****** 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. 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Revision 1
5 Apr 2023 Author Response We thank both reviewers for their valuable input to our work. Their specific points were addressed as follows. Reviewer #1: The manuscript entitled “Inhibition of Casein Kinase 2 induces cell death in chronic myelogenous leukemia cells resistant to tyrosine kinase inhibitors” by Adam Obr is interesting however the manuscript has many flaws which needed to be answered before its publication. Throughout the manuscript the authors used sentences like “somewhat affected” and “slight growth slowing”. These sentences need to be avoided and “percentage of growth inhibition” would be recommended. - We changed the language used in the manuscript in order to be more precise (see the version with tracked changes). The results & discussion sections were largely rewritten for clarity and precision. Instead of denoting K562 cells which have never been exposed to inhibitors as control, representation of them as näive K562 cells would be more appropriate. - The part is edited now (line 167 in the revised version). Results part needs to be written very precisely and the sub heading should have a title which explains the outcome of the results otherwise it is highly confusing what authors want to claim. - The headings in the results section were updated. When there is no explanation for CK2 levels and chromosomal abnormality devoting one big paragraph for cytogenetics is unnecessary and this part is highly confusing and should move to supplementary information. - The part served mainly as a verification of the novel cell line origin and as a method for seeking BCR-ABL amplifications, which could be potentially relevant. To avoid confusion with detailed description, we largely redacted this part and removed is as separate section. The reference in the main text is now included at the beginning of the results section (lines 100-102 in the revised version, and referenced in other places) and the original, detailed commentary is moved to the supplementary material. Most importantly, authors should check the mutational status of BCR-ABL in the established TKI resistant cell lines. Without this information, concluding the results part is highly indecisive. Best example is Figure 1, TKI resistant JURL-MK1 and MOLM-7 cells showed persistent p-CRKL, whereas K562 could not show suggesting that in K562 cells TKI resistance might be due to BCR-ABL independent mechanism. - We did check the mutation status in the cell lines. The sequences are now included as the Supplementary Table 1. The commentary was present in the original manuscript, and is also present in the revised version (lines 124-126). When data is not significant, showing them in the main figures is also confusing. Keeping them in the supplementary information and giving detailed explanation would be advisable. Example is Figure 2 CDC37 levels. - CDC37 levels in figure 2, though statistically insignificant, show a clear trend. We feel that albeit not significant in this particular figure, without the results the manuscript would be more difficult to follow through, because we use CDC37 as a surrogate marker of CK2 activity. We did our best to reduce the main results to absolute minimum we feel is necessary to comprehend the main point (for example, we eliminated the PARP cleavage in OCI-AML3 from Fig 3C, see below). It is also highly recommended to authors to show the dose dependent growth curves in order to claim the specificity of the CX-4945 in the mentioned cell lines. - The concentration of CX-4945 was chosen based on several publications and our own preliminary data. Since CX-4945 is a clinically tested inhibitor and therefore proven highly specific at given concentrations, addition of more results would not benefit the comprehensibility of the manuscript. Apoptosis assays such as annexin stating needed to perform with different doses of CX-4945 showing the PARP cleavage is not enough. However, PARP cleavage was also noticeable in OCI-AML 3 cells with dasatinib and CX-4945 treatment. Do they get affected by TKIs treatment? - This is actually a very interesting phenomena. In our lab experience, OCI-AML 3 cells show weak PARP cleavage even when they are sustained in a culture, untreated, with good proliferation and viability markers. You can see this in Fig 3C of the original manuscript in the control OCI-AML3. We currently have no explanation of this phenomena. For the sake of this manuscript, we would argue that the changes in PARP cleavage in OCI-AML3 cells are not significant (see quantification in Supplementary Fig 6). However, seeing that it might cause some confusion, we removed OCI-AML3 cells from the main Fig 3C, and kept the data only as a quantification in Supplementary Fig 6). Authors claimed that OCI-AML 3 cell line is BCR-ABL negative and in Figure 3 C it is mentioned as imatinib resistant (IR) and Dasatinib resistant (DR) OCI-AML3, this is confusing, did they develop OCI-AML resistant clones against the imatinib and dasatinib? Authors should clearly check this information. - BCR-ABL negative cells are technically not TKI resistant (TKIs have no significant effect on them, since there is no BCR-ABL proliferation and cell survival dependence). Despite this, we did cultivate these cells in the same conditions as the new BCR-ABL positive cells (that means, in 2 µM imatinib or 2 nM dasatinib), and we labeled cells cultivated in those conditions as „TKI resistant“ for the sake of simplicity. We now clarified this information in the results section (lines 97-99 in the revised version). As said earlier, concluding the Figure 4 and relationship between the BCR-ABL and CK2 is indecisive without knowing the mutational status of BCR-ABL and mechanism of resistance in these clones. Based on these points, the manuscript required major revision for publication. - Hopefully, we addressed your points well enough. Thank you for the comments. Reviewer #2: This is a very nice study that can be accepted for publication with minor modifications and suggestions. The authors used proper analyses techniques and evaluate the results properly. 1. Page 7, Line 122 : T315I Mutation in BCR-ABL.. It is written as’’ In both JURL-MK1 IR and DR cells, there was a T315I mutation in the kinase domain. In resistant cells 123 derived from MOLM-7 and K562, respectively, no mutation in the kinase domain of BCR-ABL was 124 detected.’’in the results section of the manuscript. However, no DNA sequence data related to this mutation were included in this article. I think this result should be added to the article. - The sequences related to line 122 in the original manuscript are now included as Supplementary Table 1. The commentary is also present in the revised version, lines 124-126. 2. Please: consider adding a paper I wrote below to your references. • ’’The casein kinase 2 inhibitor, CX-4945, as an anti-cancer drug in treatment of human hematological malignancies.’’ - The reference was added, thank you for the suggestion. Thank you for your kind comments, hopoefully we addressed them well enough. Attachments Attachment Submitted filename: Response to reviewers.docx https://doi.org/10.1371/journal.pone.0284876.r002
11 Apr 2023 Decision Letter - Rama Krishna Kancha, Editor Inhibition of Casein Kinase 2 induces cell death in chronic myelogenous leukemia cells resistant to tyrosine kinase inhibitors PONE-D-22-27194R1 Dear Dr. Obr, 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, Rama Krishna Kancha Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: https://doi.org/10.1371/journal.pone.0284876.r003
Formally Accepted
18 Apr 2023 Acceptance Letter - Rama Krishna Kancha, Editor PONE-D-22-27194R1 Inhibition of Casein Kinase 2 induces cell death in tyrosine kinase inhibitor resistant chronic myelogenous leukemia cells Dear Dr. Obr: 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. Rama Krishna Kancha Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0284876.r004

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