PONE-D-21-29989Ataxia telangiectasia and Rad3-related inhibition by AZD6738 enhances gemcitabine-induced cytotoxic effects in bladder cancer cellsPLOS ONE

Dear Dr. Isono,

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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: Yes

Reviewer #3: Partly

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

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: I Don't Know

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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: Yes

Reviewer #3: 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

Reviewer #3: 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: This article has a straightforward take home message that AZD6738 can increase the sensitivity of bladder cancer cells to gemcitabine.

The data seem strong. However, the quality of the images is very poor and most of them are not readable at all.

One other issue is a complete lack of statical analyses on all the figures although some error bars are present. Appropriate statics should be included for significance of the data.

More specifically:

Fig1: authors mention that the number of morpholigally abnormal cells increase. Yet there is no quantification provided, only images. Either this statement has to be removed or quantifications have to be performed and provided with appropriate statiscal analyses.

Sup Fig 3: the legend such as “lower left” etc...does not seem appropriate. Authors may consider naming them viable, early, late directly.

Finally, can the authors comment on why CHk1 phosphorylation on S345 is still happening while ATR is inhibited. It seems counter-intuitive and should be further discussed.

Reviewer #2: Overall: Isono et al investigated the synergistic use of gemcitabine and ATR inhibitor AZD6738 to treat bladder cancer. In vitro studies using 3 bladder cancer cell lines demonstrated clear promise for this combination, as results indicated induction of apoptosis, as well as inhibition of cell viability, colony formation, and cell cycle progression to a greater extent than was seen with either gemcitabine or AZD6738 alone. Moreover, Western blot analyses of checkpoint factors provided evidence that AZD6738 works by inhibiting the repair of gemcitabine-induced double stranded breaks. The study is well conduced, and the results are well described and support the conclusions. While this study is an important and vital step towards understanding the potential for this drug combination in bladder cancer, enthusiasm is a bit diminished due to previous works demonstrating the success of this combination, and its gemcitabine sensitizing mechanism, in other cancer types.

Minor:

The last sentence on the first page of the introduction loses clarity starting with “… or resected DBSs recruits and activates…”

Reviewer #3: In this study, the authors report that inhibition of ATR kinase by AZD6738 can sensitize bladder cancer cells to gemcitabine therapy. The design of the study is straightforward, including analyses of the combined effects of gemcitabine and AZD6738 on cell viability, clonogenicity, cell cycle, apoptosis, DNA damage, DNA repair and DNA damage checkpoint signaling. Three bladder cancer cells are included in the experiments. While the similar synergistic effects have been observed for many human cancer cell lines, this study add bladder cancer cell lines to the list. Overall, this is an incremental study to extend our understanding on the significance of ATR inhibition in sensitizing cancer cells to gemcitabine.

1. Since the quality of all figure images is extremely poor and the images are at very low resolution, the data cannot be read by this reviewer to make proper judgements on the quality of data and how the experiments were designed and performed. Thus, no comments can be made.

2. AZD6738 inhibits both ATR and ATM. There are more specific ATR inhibitors such as VX970 or BAY 1895344, which should be used.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Ataxia telangiectasia and Rad3-related inhibition by AZD6738 enhances gemcitabine-induced cytotoxic effects in bladder cancer cells

PONE-D-21-29989R1

Dear Dr. Isono,

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.

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Kind regards,

Robert W Sobol, PhD

Academic Editor

PLOS ONE

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 #2: All comments have been addressed

Reviewer #3: (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 #2: Yes

Reviewer #3: (No Response)

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

Reviewer #2: I Don't Know

Reviewer #3: (No Response)

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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 #2: Yes

Reviewer #3: (No Response)

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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 #2: Yes

Reviewer #3: (No Response)

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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 #2: (No Response)

Reviewer #3: I was disappointed that the authors have made no improvement on the display quality of the figures. All the figures are basically the same as in the original manuscript and are unreadable as all data labels look fuzzy. There is no way reviewers can read and review this manuscript. I wonder if the authors submitted a wrong version of their revised manuscript.

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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 #2: No

Reviewer #3: No