PONE-D-21-38368Non-canonical driver mutations of urethane carcinogenesisPLOS ONE

Dear Dr. Counter,

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Comments to the Author

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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: Yes

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

Reviewer #1: Yes

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Reviewer #1: Yes

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Reviewer #1: 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: Thank you for the opportunity to review the manuscript entitled "Non-canonical driver mutations of urethane carcinogenesis" by Drs. Li and Counter for PLOS ONE. This is an important manuscript, describing mutations observed in pulmonary tumors that arise in mice as a result of urethane exposure when the typical mutation observed in this scenario (Kras Q61L/R) is selected against. This is an important study in terms of describing fully the mechanism of urethane carcinogenesis as well as defining pathways that can functionally substitute for Kras Q61L. Overall the manuscript is well written and the experiments are well done. I have several suggestions for further analyses that could strengthen the conclusions of the paper. These suggestions and some additional comments are outlined below

1) Please be careful about the use of the term "driver" without functional validation. The functional validation, such as the possibility of creating transgenic mouse models expressing Rab3gap2 Y467C and KrasG12D, is so exciting, but clearly outside the scope of the current paper. Perhaps the term "genomic driver" would be more accurate.

2) The authors could perform copy number analysis (CNVkit) using the whole exome sequencing data generated for this manuscript. This analysis could provide possible genomic drivers for the tumors that don't have an obvious SNV genomic driver.

3) Have the authors considered performing subclonal analysis and/or attempting to predict the timing of the genetic events contributing to carcinogenesis in these tumors? This analysis could add depth to the description of urethane carcinogenesis. Please see PLoS Genet

. 2015 Mar 13;11(3):e1005075. doi: 10.1371/journal.pgen.1005075. eCollection 2015 Mar. for an example of this analysis. WES of the germline in these mice would be required to conduct the analysis

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Non-canonical genomic driver mutations of urethane carcinogenesis

PONE-D-21-38368R1

Dear Dr. Counter,

Congratulations on an interesting manuscript that hypothesizes an interaction between carcinogen-induced and spontaneous mutations in the context of tumor development.  I am 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,

Shawn Ahmed

Academic Editor

PLOS ONE

ps. How about them Tar Heels? This might be a fitting ending to a storied career with many titles to reflect on.

Additional Editor Comments (optional):

Your manuscript looks pretty clean, but I offer a few minor comments:

Line 30: ‘for specific genomic driver mutation’. Mutations?

Line 30: ‘Small Nucleotide Variations (SNVs)’. I am not familiar with this term. There is a related term ‘Single Nucleotide Variations (SNVs) that is more prominent in the literature. If you choose to use Small Nucleotide Variations, perhaps provide a reference that helps to clarify how this differs from Single Nucleotide Variations.

Line 33: ‘oncogenic mutation in the proto-oncogene BRAF conforming to the mutation signature’. Do you mean ‘oncogenic mutation in the proto-oncogene BRAF that conforms to the mutation signature’?

Line 162: ‘The previously published study has shown’. Perhaps ‘A previously published study has shown’

Line 164: ‘MDS has high mutation frequency’. Do you mean ‘MDS has a high mutation frequency’?

Line 191: ‘convert the mutation’ might be ‘converting the mutation’

Line 191: ‘multiply the calculated’ might be ‘multiplying the calculated’

Figure 2. What is the significance of the black squares of the nonsense mutations that matches the color of the urethane-induced mutations? Do the nonsense mutations also possess a urethane signature?

Figure 2. Could some of the purple mutations represent an unknown mutational signature of urethane? This might be apparent if there is a specific pattern of base change that is common to ⅓ or more of these mutations.

Line 422: ‘function as tumor suppressor in krasG12D-driven mouse model’. ‘function as tumor suppressors in a krasG12D-driven mouse model’

Line 440: ‘Interestingly, there was no mutagenic signature specific for just one KRAS mutation.’ Perhaps clarify what you mean by just one KRAS mutation? Does this refer to a single base change in KRAS? Does this mean that although urethane-induced mutations were apparent in KRAS, that there were also other mutations whose origin is unclear?

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

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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

**********

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

Reviewer #1: 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

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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

**********

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: Thank you so much for your careful responses to my comments and I look forward to reading follow up papers to this important work.

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Reviewer #1: No