PONE-D-20-00375

Synergistic apoptotic effects in cancer cells by the combination of CLK and Bcl-2 family inhibitors

PLOS ONE

Dear Dr. Araki,

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.

Additional experimental data are required to confirm the hypothesis. The specificity of T3 should be confirmed. More data are required to prove that the phenotypes observed upon treatment are CLK- or mRNA splicing-dependent. Non-cancerous cells should be tested.

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Irina V. Lebedeva, Ph.D.

Academic Editor

PLOS ONE

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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: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

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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: No

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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

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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 manuscript of Murai and co-authors examined the combined effects of a splicing inhibitor with an inhibitor of apoptosis for synergy in cell models of ovarian (A2780) and colorectal cancer (HCT116). In this study they present evidence that the putative CLK3 inhibitor T3 results in inhibition of splicing of anti-apoptotic proteins. Combined with an BCL-2 inhibitor (ABT-263), the authors provide evidence for synergistic increases in cell death as determined by caspase 3 assays. The authors provide further evidence that, in part, these effects are mediated through reduction of mTor signaling.

Critique

Overall these are carefully performed studies that provide initial evidence for a combinatorial approach to improve induction of cell death in cancer cells. However, there are a number of issues that need to be addressed before the manuscript is acceptable for publication.

1. Only one CLK inhibitor was examined and the specificity of T3 was not discussed. The authors need to add additional experiments with another CLK inhibitor to determine if this is a drug, or target specific response. Evidence of the selectivity of T3, with respect to other kinases, needs to be provided. These studies would be more convincing if synergy was observed with other CLK inhibitors as well.

2. The inhibition of mTOR signaling is suggested as a key part of the response although this is not well validated. Additional data demonstrating this (i.e. inhibition of S6 phos, EIF4EBP phos, or other) should be provided. At present the authors discuss the loss of S6- but no data is actually shown.

3. The explanation for the loss of MCL1 is not clear (Fig. 3C). In particular, the effects of MG132 are quite minor. Based on the PCR data shown, its possible that T3 is blocking total MCL1 expression. While the one splice (splice in) is shown, the total MCL1 message should also be presented. The potential effects of T3 on pTEF/transcriptional regulation of MCL1 have not been ruled out.

Reviewer #2: Authors showed that the small molecule splicing modulator T3, a CDC-like kinase (CLK) inhibitor, induced cell cycle changes and apoptosis in tumor cell lines A2780 and HCT116. T3 also showed synergistic pro-apoptotic effect with ABT-263. Authors also observed changes at protein levels for several apoptotic proteins following T3 treatment. This is an interesting study and has a potential in advancing cancer therapy. However, in the current version of this manuscript, there is a lack of direct evidence showing that the phenotypes are indeed CLK- or mRNA splicing-dependent. Limited studies on the changes in protein levels and the gene expression of only selected isoforms are not sufficient to reach the conclusion that the alternative splicing plays a critical role. Therefore, further studies are warranted before publication.

1. Authors need to directly show that the phenotypes induced by T3 are CLK- or mRNA splicing-dependent. For example, cells with mutant CLK (unable to bind to T3, or splicing function deficient), or CLK knockout, or through any other means to show that T3-induced cell cycle changes, apoptosis, and synergy with ABT-263 are CLK- or mRNA splicing-dependent. Comparing inactive T3 analog-induced vs T3–induced phenotypic changes would further help understanding the underlying mechanism.

2. Direct evidence at mRNA levels by such as RT-PCR showing alteration of splicing events after T3 treatment should be provided for each of the splicing variants, including cFLIP-L, cFLIP-S, Mcl-1L, and Mcl-1S. Whether the mRNA splicing of those apoptotic proteins is CLK dependent also needs to be investigated.

3. Authors claimed that some of the Bcl-2 family members such as Bcl-XL and Bcl-2 were resistant to T3-induced splicing modulation. Have authors investigated other isoforms of those proteins such as Bcl-xS, Bcl2L2, etc, to support the claim?

4. In Figure 3A, authors claimed “T3 altered splicing of the anti-apoptotic isoform MCL1L to the pro-apoptotic isoform MCL1S at 6 and 16 h in a dose-dependent manner”, however, no data for MCL-1S has been shown. PSI for both MCL-1L and MCL-1S need to be showed.

5. In Figure 3C, both western blot and RT-PCR for total MCl-1 and its isoforms Mcl-1L and Mcl-1S should be shown, in order to enable the comparison and delineate the effect of T3 on splicing alteration.

6. A section of statistical analysis should be included.

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Reviewer #1: No

Reviewer #2: 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 to be viewed.]

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Attachments

Attachment

Submitted filename: The manuscript of Murai and co.docx

PONE-D-20-00375R1

Synergistic apoptotic effects in cancer cells by the combination of CLK and Bcl-2 family inhibitors

PLOS ONE

Dear Dr. Araki,

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 the Reviewer 2 concerns

Please submit your revised manuscript by Aug 08 2020 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.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: http://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols

We look forward to receiving your revised manuscript.

Kind regards,

Irina V. Lebedeva, Ph.D.

Academic Editor

PLOS ONE

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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: (No Response)

Reviewer #2: (No Response)

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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: Partly

Reviewer #2: Partly

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

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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: No

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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

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: Please revise sentence on p.4 line 1-does not currently make sense.

The digitized blots in Figs 3 and 4 are unacceptable.

Reviewer #2: Authors did not directly and experimentally address many major concerns the reviewer has raised. One major point is the specificity aspect. Even though T3 has been reported to be potent, the off-target binding is still a possibility unless proven otherwise. Both the reviewers have specifically indicated that additional experiments with pharmacological and/or genetic approaches should be performed in order to show that the effect is CLK- or splicing-dependent. Unfortunately, authors did not attempt to address this point experimentally.

Figure 3A showed that the lower molecular weight BCL2L1 isoform Bcl-xS increased dose-dependently upon T3 treatment up to 1 uM, which is especially apparent after 16h treatment in both cell lines, and the amount of change is similar to MCL1 but with a different kinetics. However, authors described T3 “had no effect on splicing of the BCL2L1 gene”. This causes confusion.

There is also a considerable confusion regarding cFLIP. Authors stated that “T3 induced smaller size of cFLIPL and larger size of cFLIPL”, and then “T3 induced the smaller sized cFLIPL and cFLIPS”, and also stated “T3 alternatively induced multiple smaller sized transcripts of CFLAR-L and CFLAR-S”. In fact, the Fig 2 showed that T3 induced LARGER size of cFLIPs protein, but in Fig 4 there is NO LARGER size of cFLAR-S RNA but only SMALLER size cFLAR-S RNA. Would authors be able to consolidate?

Upon T3 treatment, Mcl-1S increased at RNA level (Fig 3A) but decreased at protein level (Fig 2). Reviewer’s Point 5 suggested to study the MCl-1 isoforms in Fig 3C (the original version), with using MG132 to investigate if the discrepancy in RNA and protein levels is at least due to proteasome degradation. Would the levels of Mcl-1S RNA become consistent with its protein levels if MG132 is present? Unfortunately, authors did not address this point experimentally.

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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

[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.

Synergistic apoptotic effects in cancer cells by the combination of CLK and Bcl-2 family inhibitors

PONE-D-20-00375R2

Dear Dr. Araki,

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,

Irina V. Lebedeva, 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

Reviewer #2: All comments have been addressed

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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

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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: No further changes necessary- although I am still not pleased with Figure 4. Otherwise the authors have done a good job responding to my critiques.

Reviewer #2: (No Response)

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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