Dear Dr Vagner,

Thank you for submitting via Review Commons your manuscript entitled "An extra-glycolytic function for hexokinase 2 as an RNA-binding protein regulating SOX10 mRNA translation in melanoma" for consideration as a Research Article by PLOS Biology.

Your manuscript has now been evaluated by the PLOS Biology editorial staff as well as by an academic editor with relevant expertise and I am writing to let you know that we would like to invite you to submit a revision in due course.

However, before we can send you the decision with the details, we need you to complete your submission by providing the metadata that is required for full assessment. To this end, please login to Editorial Manager where you will find the paper in the 'Submissions Needing Revisions' folder on your homepage. Please click 'Revise Submission' from the Action Links and complete all additional questions in the submission questionnaire.

Once your full submission is complete, your paper will undergo a series of checks. After your manuscript has passed the checks I will send you the decision. To provide the metadata for your submission, please Login to Editorial Manager (https://www.editorialmanager.com/pbiology) within two working days, i.e. by Jan 30 2025 11:59PM.

Feel free to email us at plosbiology@plos.org if you have any queries relating to your submission.

Kind regards,

Ines

--

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

Dear Dr Vagner,

Thank you for providing the metadata for your manuscript entitled "An extra-glycolytic function for hexokinase 2 as an RNA-binding protein regulating SOX10 mRNA translation in melanoma" for consideration at PLOS Biology.

As I mentioned, I discussed the manuscript, the Review Commons reports and your revision plan with an Academic Editor expert in the field and we would like to invite you to submit a revision that thoroughly address the reviewers' reports. It will be important to add more information on the other identified targets to validate them and broaden the scope of the manuscript. Regarding the characterisation of SOX10, you should show whether HK2 specifically regulates SOX10 or has a broader role.

Given the extent of revision needed, we cannot make a decision about publication until we have seen the revised manuscript and your response to the reviewers' comments. Your revised manuscript is likely to be sent for further evaluation by all or a subset of the reviewers.

We expect to receive your revised manuscript within 3 months. Please email us (plosbiology@plos.org) if you have any questions or concerns, or would like to request an extension.

At this stage, your manuscript remains formally under active consideration at our journal; please notify us by email if you do not intend to submit a revision so that we may withdraw it.

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https://blogs.plos.org/plos/2019/05/plos-journals-now-open-for-published-peer-review/

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e) *Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosbiology/s/submission-guidelines#loc-materials-and-methods

Thank you again for your submission to our journal. We hope that our editorial process has been constructive thus far, and we welcome your feedback at any time. Please don't hesitate to contact us if you have any questions or comments.

Sincerely,

Ines

--

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

Dear Dr Vagner,

Thank you for your patience while we considered your revised manuscript entitled "An extra-glycolytic function for hexokinase 2 as an RNA-binding protein regulating mRNA translation" for publication as a Research Article at PLOS Biology. This revised version of your manuscript has been evaluated by the PLOS Biology editors, the Academic Editor and two of the original reviewers.

Based on the reviews, we are likely to accept this manuscript for publication, provided you satisfactorily address the remaining points raised by Reviewer 2. Please also make sure to address the data and other policy-related requests stated below my signature.

In addition, we would like you to consider a suggestion to improve the title:

"Hexokinase 2 is an RNA-binding protein that regulates mRNA translation and induces melanoma cell proliferation independently of glycolysis"

As you address these items, please take this last chance to review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the cover letter that accompanies your revised manuscript.

In addition to these revisions, you may need to complete some formatting changes, which you will receive in a follow up email. A member of our team will be in touch with a set of requests shortly. If you do not receive a separate email within a few days, please assume that checks have been completed, and no additional changes are required.

We expect to receive your revised manuscript within two weeks.

To submit your revision, please go to https://www.editorialmanager.com/pbiology/ and log in as an Author. Click the link labelled 'Submissions Needing Revision' to find your submission record. Your revised submission must include the following:

- a cover letter that should detail your responses to any editorial requests, if applicable, and whether changes have been made to the reference list

- a Response to Reviewers file that provides a detailed response to the reviewers' comments (if applicable, if not applicable please do not delete your existing 'Response to Reviewers' file.)

- a track-changes file indicating any changes that you have made to the manuscript.

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https://journals.plos.org/plosbiology/s/supporting-information

*Published Peer Review History*

Please note that you may have the opportunity to make the peer review history publicly available. The record will include editor decision letters (with reviews) and your responses to reviewer comments. If eligible, we will contact you to opt in or out. Please see here for more details:

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*Press*

Should you, your institution's press office or the journal office choose to press release your paper, please ensure you have opted out of Early Article Posting on the submission form. We ask that you notify us as soon as possible if you or your institution is planning to press release the article.

*Protocols deposition*

To enhance the reproducibility of your results, we recommend that if applicable you deposit your laboratory protocols in protocols.io, where a protocol can be assigned its own identifier (DOI) such that it can be cited independently in the future. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols

Please do not hesitate to contact me should you have any questions.

Sincerely,

Ines

--

Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org

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DATA POLICY: IMPORTANT - PLEASE READ

You may be aware of the PLOS Data Policy, which requires that all data be made available without restriction: http://journals.plos.org/plosbiology/s/data-availability. For more information, please also see this editorial: http://dx.doi.org/10.1371/journal.pbio.1001797

Note that we do not require all raw data. Rather, we ask that all individual quantitative observations that underlie the data summarized in the figures and results of your paper be made available in one of the following forms:

1) Supplementary files (e.g., excel). Please ensure that all data files are uploaded as 'Supporting Information' and are invariably referred to (in the manuscript, figure legends, and the Description field when uploading your files) using the following format verbatim: S1 Data, S2 Data, etc. Multiple panels of a single or even several figures can be included as multiple sheets in one excel file that is saved using exactly the following convention: S1_Data.xlsx (using an underscore).

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Fig. 1A, C, F; Fig. 2A-E, G, H; Fig. 3D; Fig. 4A-D, G, H, J, K; Fig. 5E, H; Fig. 6E, G, I, K; Fig. 7B, C, D; Fig. S2A-C; Fig. S3A-G; Fig. S4A-C; Fig. S6A-D; Fig. S7C; Fig. S8A, B; Fig. S9B, C; Fig. S10A and Fig. S11

NOTE: the numerical data provided should include all replicates AND the way in which the plotted mean and errors were derived (it should not present only the mean/average values).

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Please ensure that your Data Statement in the submission system accurately describes where your data can be found.

**In addition, please make publicly available the data you have deposited at the GEO database (ID: GSE274146).

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Please note that we cannot accept sole deposition of code in GitHub, as this could be changed after publication. However, you can archive this version of your publicly available GitHub code to Zenodo. Once you do this, it will generate a DOI number, which you will need to provide in the Data Accessibility Statement (you are welcome to also provide the GitHub access information). See the process for doing this here: https://docs.github.com/en/repositories/archiving-a-github-repository/referencing-and-citing-content

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BLOT AND GEL REPORTING REQUIREMENTS:

We require the original, uncropped and minimally adjusted images supporting all blot and gel results reported in an article's figures or Supporting Information files. We will require these files before a manuscript can be accepted so please prepare and upload them now. Please carefully read our guidelines for how to prepare and upload this data: https://journals.plos.org/plosbiology/s/figures#loc-blot-and-gel-reporting-requirements

We would need the original images for the gels shown in the following figures:

Fig. 1A, B; Fig. 1B-E; Fig. 3A-D; Fig. 5C, D, G; Fig. 6A, B; Fig. 7A, B, Fig. S1A, C; Fig. S2A; Fig. S3C; Fig. S5A-E and Fig. S7A, B, D, E

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Reviewers' comments

Rev. 2: Laura Broglia - note that this reviewer has signed the review

General Evaluation

This manuscript explores a novel, non-metabolic role of Hexokinase 2 (HK2) as an RNA-binding protein (RBP) that regulates mRNA translation in melanoma cells. While the metabolic functions of glycolytic enzymes in cancer are well established, their emerging roles in translational control are less well defined. The authors present compelling evidence that HK2 directly binds to the 5′ UTR of SOX10 mRNA and promotes its translation independently of its enzymatic activity and metabolic function. These findings highlight a previously unrecognized function of HK2 in tumor cell proliferation and offer valuable insights into the interplay between metabolism and gene expression regulation in cancer.

The authors have addressed all my major concerns raised in the first round of revision. The manuscript is now much improved, with the inclusion of key experiments that convincingly support the role of HK2 as a regulator of SOX10 mRNA translation.

Comments

- While I understand the rationale for focusing on the selected targets, the purpose of the translatome and RNA-seq analyses remains somewhat unclear and not connected with the rest of the paper. Since the six targets of interest were ultimately identified by another strategy, it would be helpful to specify whether they were absent from the RNA-seq data due to a lack of statistical significance. If so, please indicate in page 12, line 9 the p-values for those genes to show how close they were to the significance threshold (e.g., p < 0.05).

- Figure 1F: For the qPCR results, please avoid setting the control arbitrarily to 1. Use proper normalization, as done for the Western blot quantification.

- Figure 2A: Please include statistical analysis for the different fractions.

- Figure 6E: Statistical information is missing in the plot. Please include it for completeness.

- Regarding the rescue experiment: I originally suggested including ectopic expression of SOX10 ORF with both the WT 5′ UTR and the 5′ UTR harboring deletion #4 (i.e., the deletion that showed reduced luciferase activity and lower enrichment in IP).

The authors respond that this experiment is not feasible due to inefficient translation of the transduced ORF with the deleted 5′ UTR. My original point was that such a control would be critical to demonstrate that the deleted region is indeed necessary for regulation (i.e. no rescue should be observed when using that construct). That said, I acknowledge that this conclusion is already well supported by the other complementary approaches.

Minor Comments

- Abstract, lines 25–26: The sentence "by promoting the release of HK2..." is unclear. Please clarify—do high glucose conditions promote HK2 release from the outer mitochondrial membrane (OMM)? Rephrasing is needed for clarity.

- Introduction, page 7, line 31: Please define or briefly explain SNAIL-mediated EMT for clarity.

- Introduction, page 8, line 6: Spell out and define "OXPHOS" at first mention.

- Page 14, lines 33–35: Rephrase this sentence. It is currently unclear and appears to misuse “either…or.”

- Page 28, lines 16–18: Add a reference to the relevant figure.

Rev. 3:

Accept.

Attachments

Attachment

Submitted filename: 250708_LB_revision.docx

Dear Dr Vagner,

Thank you for the submission of your revised Research Article entitled "Hexokinase 2 is an RNA-binding protein that regulates mRNA translation independently of glycolysis and induces melanoma cell proliferation" for publication in PLOS Biology. On behalf of my colleagues and the Academic Editor, Elena Rainero, I am delighted to let you know that we can in principle accept your manuscript for publication, provided you address any remaining formatting and reporting issues. These will be detailed in an email you should receive within 2-3 business days from our colleagues in the journal operations team; no action is required from you until then. Please note that we will not be able to formally accept your manuscript and schedule it for publication until you have completed any requested changes.

Please take a minute to log into Editorial Manager at http://www.editorialmanager.com/pbiology/, click the "Update My Information" link at the top of the page, and update your user information to ensure an efficient production process.

PRESS

We frequently collaborate with press offices. If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximise its impact. If the press office is planning to promote your findings, we would be grateful if they could coordinate with biologypress@plos.org. If you have previously opted in to the early version process, we ask that you notify us immediately of any press plans so that we may opt out on your behalf.

We also ask that you take this opportunity to read our Embargo Policy regarding the discussion, promotion and media coverage of work that is yet to be published by PLOS. As your manuscript is not yet published, it is bound by the conditions of our Embargo Policy. Please be aware that this policy is in place both to ensure that any press coverage of your article is fully substantiated and to provide a direct link between such coverage and the published work. For full details of our Embargo Policy, please visit http://www.plos.org/about/media-inquiries/embargo-policy/.

Many congratulations and thanks again for choosing PLOS Biology for publication and supporting Open Access publishing. We look forward to publishing your study. 

Sincerely, 

Ines

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Ines Alvarez-Garcia, PhD

Senior Editor

PLOS Biology

ialvarez-garcia@plos.org