From the journal RSC Chemical Biology Peer review history

03-Sep-2020

Dear Dr Monchaud:

Manuscript ID: CB-REV-08-2020-000151
TITLE: The threat from within: how DNA folds threaten genetic stability and for which chemotherapeutic payoffs

Thank you for your submission to RSC Chemical Biology, published by the Royal Society of Chemistry. I sent your manuscript to reviewers and I have now received their reports which are copied below.

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************

Reviewer 1

The review article by Zell et al. is comprehensive and details nicely the history and current state of targeting DNA as a therapy in cancer. The review is timely and gives a nice balance between non-specialist and specialists information. There is something for everyone in this review and it was a very interesting read. This review will make a great addition to the literature describing this area of research. Below are a list of issues/comments that hopefully will be useful to the authors in finalizing this manuscript for publication.

1. Title: It is a little unclear what “and for which chemotherapeutic payoffs” means. Should consider editing title for clarity.

2. abstract: either a new perspective or delete a to read new perspectives.

3. Introduction – DNA modifications is a confusing way to talk about DNA lesions or damage as DNA modifications often refer to methylation. Would suggest changing modification here.

4. There are many types of DNA structures. Figure 7 nicely shows this but the authors may want to consider other repair intermediates including single strand and DSB DNA and holiday junctions, where protein trapped molecules have been reported (reviewed in PMID: 30962000).

5. recombination (HR and NHEJ) – NHEJ is not a recombination mechanism so this wording should be changed. (pg 3).

6. Figure 1 – It is known that several of the DNA-protein crosslinking agents, especially topoisomerase poisons, induce DSBs as a consequence of their formation. This figure lacks this important concept so the authors should consider modifying to include this concept.

7. pg 6, may want to include meiotic recombination and SPO11 site-specific breaks in the section with RAG and AID.

8. Figure 3. There is growing evidence that PARP1 is involved in DSB repair. The authors should considering discussing this and showing it in Figure 3.

9. ATM is also activated by oxidative stress which could be mentioned.

10. A major pathway involved in PARPi resistance is through replication fork stabilization, independent from HR, in BRCA-deficient cells.

11. pg 18 DNA base lesion repairs – should be repaired

12. pg 19. It is worth making it clear that quadruplex-forming sequences have the potential to fork G4 but don’t necessarily do in cells and at all times. There are likely many processes that regulate the actual forming of G4 in cells at these sequences. This is an important concept to make clear, especially to the non-specialist.

13. pg. 20 – earliest of evidence – earliest evidence

14. pg 28 – PDS has also been shown to down-regulate SRC and block cancer cell migration PMID: 22306580. This is unlikely to be due to DNA damage per se as DNA damage does not impede cancer cell migration in this same cell line PMID: 26030852. This provides an additional example of a G4 ligands targeting of a cancer-relevant pathway.

15. A very recent Cell paper from Oliviera et al (Durocher) lab identified PDS as a TOP2 inhibitor. This paper would be worth citing and discussing.
16. pg 29. The fact that BRCA2-depleted cells are sensitive to G4 compound may also suggest an effect on replication, in addition to repair.

17. Conclusion: Given that the genome is organized by chromatin, which will impact DNA structures, it would be interesting to discuss this in the conclusions as chromatin modulators are therapeutic targets and may represent combinatorial treatments that could be considered with DNA structure targeting drugs (ex. see PMID: 25311424).

Reviewer 2

This is an interesting and timely review that is well-written and easily understandable for a general readership. However, I think that a few minor revisions could improve the manuscript, as outlined below:

1. The majority of the manuscript is very well written for readers from a variety of scientific fields. However, the initial introduction section seemed like it was written for a lay audience, and this could be improved by revisions to make it more suitable for a scientific audience.

2. While the focus of the review was on G4-DNA structures, the authors did mention other non-B DNA structures (e.g. hairpins, cruciforms, H-DNA, Z-DNA, slipped DNA, etc.) but perhaps did not reference these appropriately. It seems that these other structures were simply mentioned without literature citations to support their roles in genetic instability and disease, for which there are many.

3. In the introduction on NER, the authors mention ERCC1-XPF as the structure-specific nuclease that cleaves the DNA at site of damage, but I did not see where they mentioned XPG, which cleaves on the 3' side of the lesion. The authors should revise this section to include both nucleases that are required for NER.

Reviewer 3

This is an extremely well written and original review, which presents in a very accessible way the basics of DNA-damage lesions and DDR machineries available in the cells to repair them, putting those into context of their function within standard chemotherapy and projecting a vision towards the targeting of alternative DNA-structural features, particularly G4s, that could be used to develop novel targeted therapies or synthetic lethality strategies. The narrative of the document is flowing and I was particularly impressed by the accuracy in the chronology of the key discoveries illustrated in the manuscript. I feel that this review will be of particular interest and use to students who can use it as document where all the key information about DNA-targeting and response are located, but also established researchers novel to this area. Overall, I am very supportive for publication of this review in RSC Chemical Biology.

I have only few very minor comments for the authors to address prior publication:

1) In section 3a (Prevalence of G4s in the human genome), it would be helpful to have a figure/scheme of a G4 to guide non expert reader through the section and follow the description of what a G-tetrad is an how an assembled G4-structure looks like

2) In the Pyridostatin section when discussing about reference 88 and the ability of PDS to induce a DNA damage response in the form of gH2AX, it would be worth mentioning briefly also that this study was used to create the first genomic map of G4 in a chromatin context looking at gH2AX sites generated by the ligand. This is important to show that not only the DDR eliciting properties of the molecules where characterised but also used to map (for the first time at the time) the distribution of G4s in chromatin, which will important to underline as a chemical-biology approach and also for its relevance in the context of G4 investigation.

3) Page 17 first line "clinics" should be clinic. This is the only typo I spotted but is worth giving a second round of checking given is a long document and some more typos might be present.

To Prof. H. Suga
Chair of the Editorial Board of RSC Chemical Biology

To Dr. G. Bernardes
Associate Editor of RSC Chemical Biology

Dear Prof. Suga, Dear Dr. Bernardes,

I would like to sincerely thank you for giving us the opportunity to submit a revised version of our manuscript CB-REV-08-2020-000151 initially entitled “The threat from within: how DNA folds threaten genetic stability and for which chemotherapeutic payoffs”, now entitled “DNA folds threaten genetic stability and can be leveraged for chemotherapy”. We were delighted to see that the three reviewers judged our review article very positively, worthy of interest and suited to publication in RSC Chem. Biol., after fixing the issues listed below.

It is our pleasure to send you today a revised version of our manuscript, which has been modified according to the reviewers’ comments. Our point-by-point responses to the reviewers’ comments is provided below. All modifications are highlighted in the track-change version of this manuscript, which is provided along with a clean version in which all corrections were accepted for reading fluidity. Of note, bibliographies have been added at the end of the manuscript, along with the authours’ biographies, a graphical abstract and a short sentence for the Table of Contents.

We hope that this review will be judged as valuable for RSC Chemical Biology.

With our best wishes,

David Monchaud, Ph.D.
On behalf of all authors


---- Referee: 1
The review article by Zell et al. is comprehensive and details nicely the history and current state of targeting DNA as a therapy in cancer. The review is timely and gives a nice balance between non-specialist and specialists information. There is something for everyone in this review and it was a very interesting read. This review will make a great addition to the literature describing this area of research. Below are a list of issues/comments that hopefully will be useful to the authors in finalizing this manuscript for publication.
Answer: we sincerely thank Referee 1 for her/his very kind words and helpful comments.

1. Title: It is a little unclear what “and for which chemotherapeutic payoffs” means. Should consider editing title for clarity.

Answer: we agree. The title has now been changed to “DNA folds threaten genetic stability and can be leveraged for chemotherapy”.

2. abstract: either a new perspective or delete a to read new perspectives.

Answer: done.

3. Introduction – DNA modifications is a confusing way to talk about DNA lesions or damage as DNA modifications often refer to methylation. Would suggest changing modification here.

Answer: agreed; we selected “DNA lesions” as it is indeed more accurate.

4. There are many types of DNA structures. Figure 7 nicely shows this but the authors may want to consider other repair intermediates including single strand and DSB DNA and holiday junctions, where protein trapped molecules have been reported (reviewed in PMID: 30962000).

Answer: The Holliday junction is evoked several times in the manuscript as a particular case of FWJ. For the sake of clarity, this is now specified in the caption of the Figure 7 (now Figure 8). We thank the Referee for highlighting this review (Xia et al., Trends Genet. 2019) as it fits perfectly within the scope of our review. It has now been added as Ref. 405 (along with the initial Sci. Adv. 2016 article (same authors) as Ref. 406), and the text modified (page 40) as follows: “In vitro and in E.coli, hexapeptides were capable of selectively binding the FWJ of a Holliday junction (a crucial intermediate during HR repair of both DSBs and replication-dependent ssDNA gaps, as demonstrated by live cell imaging and HJ-ChIP-seq)405,406, thus abrogating the action of Holliday junction-processing enzymes by displacing RecG helicase and inhibiting RuvABC resolvase (…)”.

5. recombination (HR and NHEJ) – NHEJ is not a recombination mechanism so this wording should be changed. (pg 3).

Answer: corrected.

6. Figure 1 – It is known that several of the DNA-protein crosslinking agents, especially topoisomerase poisons, induce DSBs as a consequence of their formation. This figure lacks this important concept so the authors should consider modifying to include this concept.

Answer: We agree. Figure 1 has been modified accordingly.

7. pg 6, may want to include meiotic recombination and SPO11 site-specific breaks in the section with RAG and AID.

Answer: agree. The text has been modified as follows (pages 6-7): “This is the case for antibody diversification by V(D)J and class-switch recombination (CSR) in which RAG nuclease for V(D)J, and AID for CSR promote sites-specific DSBs, and for meiotic recombination which relies on SPO11 for genome-wide DSB formation to promote recombination between homologous chromosomes73–75.”

8. Figure 3. There is growing evidence that PARP1 is involved in DSB repair. The authors should considering discussing this and showing it in Figure 3.

Answer: Done. Figure 3 has been completely reorganised to comply with these corrections, and the text modified accordingly (page 11), as follows “Importantly, PARP1 is also activated by DSBs where it promotes the recruitment of NHEJ and HR factors33,109”.

9. ATM is also activated by oxidative stress which could be mentioned.

Answer: We agree. The text has been modified as follows (pages 13-14): “It is noteworthy that ATM can also be directly activated by oxidative stress via oxidation of some of its cysteines71”.

10. A major pathway involved in PARPi resistance is through replication fork stabilization, independent from HR, in BRCA-deficient cells.

Answer: Done. The text has been modified as follows (page 17): “However, various resistance mechanisms to PARP inhibitors have been described, including restoration of HR, via inactivation of the junction endonuclease MUS81 or the N-methyltransferase Enhancer of zeste homolog 2 (EZH2) for example109,166,167”.

11. pg 18 DNA base lesion repairs – should be repaired

Answer: Corrected.

12. pg 19. It is worth making it clear that quadruplex-forming sequences have the potential to fork G4 but don’t necessarily do in cells and at all times. There are likely many processes that regulate the actual forming of G4 in cells at these sequences. This is an important concept to make clear, especially to the non-specialist.

Answer: This is a clever remark. The text has been modified as follows (page 20): “Initially only thought of as an in vitro oddity, G4s are now considered key players in cellular processes: recent sequencing-based methods have demonstrated that thousands of quadruplex-forming sequences (QFS) are present in our genome, >700 000 by G4-seq183. These sequences are massively maintained in an unfolded state as exemplified by the detection of only >10 000 G4s by G4 ChIP-seq184 and ca. 3 000 G4s by the fluorophore SiR-PyPDS (see part 3c)185 in live cells, with a strong correlation between individual G4 formation and the transcriptional activity of the gene they fold from. This originates in the various mechanisms the cells have evolved to regulate G4 formation, among which the G4 helicases are currently being actively studied (see part 3b). Interestingly, the PQS distribution is not random as they are significantly enriched in key regulatory regions including gene promoters, replication origins and telomeres10,186”.

13. pg. 20 – earliest of evidence – earliest evidence

Answer: Corrected.

14. pg 28 – PDS has also been shown to down-regulate SRC and block cancer cell migration PMID: 22306580. This is unlikely to be due to DNA damage per se as DNA damage does not impede cancer cell migration in this same cell line PMID: 26030852. This provides an additional example of a G4 ligands targeting of a cancer-relevant pathway.

Answer: This is absolutely right; the text has been modified accordingly, as follows (page 29): “Beyond the link between G4s and DNA damage, this approach also provided the very first description of the G4 landscape within human cells (via an accurate mapping of the distribution of G4s in the endogenous chromatin context) and highlighted the druggability of the SRC proto-oncogene (involved in multiple pathways that regulate tumor progression),296 thus uncovering a novel G4-mediated anticancer strategy”.

15. A very recent Cell paper from Oliviera et al (Durocher) lab identified PDS as a TOP2 inhibitor. This paper would be worth citing and discussing.

Answer: Agreed. This article was already discussed in the previous version of this manuscript (Ref. 79) notably in the paragraph related to both PDS (cf. page 28) and CX-5461 (cf. page 30).

16. pg 29. The fact that BRCA2-depleted cells are sensitive to G4 compound may also suggest an effect on replication, in addition to repair.

Answer: Done. The text has been modified as follows (page 29): “BRCA2-depleted cells were found far more sensitive to CX-5461 treatment (compared to WT cells), supporting an impact of G4 ligands on replication forks and implicating HR in the repair of the resulting structures”.

17. Conclusion: Given that the genome is organized by chromatin, which will impact DNA structures, it would be interesting to discuss this in the conclusions as chromatin modulators are therapeutic targets and may represent combinatorial treatments that could be considered with DNA structure targeting drugs (ex. see PMID: 25311424).

Answer: This is an interesting remark. We have now discussed this possibility in the manuscript (page 35), as follows: “Advances in genome-wide sequencing techniques such as ChIP-seq and Chem-seq have facilitated the characterisation of genetic and epigenetic modulators331. A combination of specific DNA targeting agents with chromatin-interacting protein modulation (a field also currently undergoing important drug development innovations)332-334 could prove to be another promising strategy in synthetic lethality chemotherapy”.

---- Referee: 2
This is an interesting and timely review that is well-written and easily understandable for a general readership. However, I think that a few minor revisions could improve the manuscript, as outlined below:

Answer: we sincerely thank Referee 2 for her/his very kind comment.

1. The majority of the manuscript is very well written for readers from a variety of scientific fields. However, the initial introduction section seemed like it was written for a lay audience, and this could be improved by revisions to make it more suitable for a scientific audience.

Answer: Agreed. The introduction has been shortened (by 3 lines) to be more straightforward. An example of sentence that has been removed (page 2): “This selectivity results in decreased random interactions with genomic DNA (duplex-DNA) and thus, undesired off-target effects”.

2. While the focus of the review was on G4-DNA structures, the authors did mention other non-B DNA structures (e.g. hairpins, cruciforms, H-DNA, Z-DNA, slipped DNA, etc.) but perhaps did not reference these appropriately. It seems that these other structures were simply mentioned without literature citations to support their roles in genetic instability and disease, for which there are many.

Answer: Done. This part of the manuscript is now better supported thanks to the addition of a series of references in which the impact of other alternative DNA structures on genetic stability is thoroughly described (i.e., Bochman et al. Nat. Rev. Genet. 2012; Wang & Vasquez, DNA Rep. 2014; Choi & Majima, Chem. Soc. Rev. 2011; Kaushal & Freudenreich, Genes Chromosome Canc. 2018). The text has been modified as follows (page 36): “In addition to G4s, a handful of non-B DNA structures have been shown to trigger genetic instability. To date, many examples have been reported and described; some of them will be provided below (R-loops and DNA junctions) but this topic is regularly covered by authoritative reviews that interested readers are invited to refer to 11,17,18,335,338–340”.

3. In the introduction on NER, the authors mention ERCC1-XPF as the structure-specific nuclease that cleaves the DNA at site of damage, but I did not see where they mentioned XPG, which cleaves on the 3' side of the lesion. The authors should revise this section to include both nucleases that are required for NER.

Answer: Sorry for this oversight. The text has now been corrected as follows (page 9): “then, an endonuclease complex comprising the xeroderma pigmentosum group F (XPF) and excision repair cross-complementation group 1 (ERCC1) proteins incises on the 5’ end of the bubble while the xeroderma pigmentosum group G (XPG) endonuclease incises at the 3’ end, releasing an oligonucleotide carrying the DNA lesion”.

---- Referee: 3
This is an extremely well written and original review, which presents in a very accessible way the basics of DNA-damage lesions and DDR machineries available in the cells to repair them, putting those into context of their function within standard chemotherapy and projecting a vision towards the targeting of alternative DNA-structural features, particularly G4s, that could be used to develop novel targeted therapies or synthetic lethality strategies. The narrative of the document is flowing and I was particularly impressed by the accuracy in the chronology of the key discoveries illustrated in the manuscript. I feel that this review will be of particular interest and use to students who can use it as document where all the key information about DNA-targeting and response are located, but also established researchers novel to this area. Overall, I am very supportive for publication of this review in RSC Chemical Biology.

Answer: we sincerely thank Referee 3 for her/his very kind words and helpful comments.

I have only few very minor comments for the authors to address prior publication:

1) In section 3a (Prevalence of G4s in the human genome), it would be helpful to have a figure/scheme of a G4 to guide non expert reader through the section and follow the description of what a G-tetrad is an how an assembled G4-structure looks like

Answer: We totally agree. A new figure has now been included (figure 5, see the PDF version of this letter) in which the formation of a G4 structure from a G-rich strand is schematically depicted, and the text has been modified accordingly (pages 19-20), as follows: “Contiguous G-stretches within a strand of ssDNA come together into contiguous planar G-quartets, which stack on top of each through π-system interactions, with each G-quartet being stabilised by a central physiological cation (K+, Na+) to form a G4 structure that can be classified as antiparallel-, hybrid- and parallel-type G4, depending on the polarity of the strands (figure 5). Of note, this topological diversity leads to a variety of intervening loops, which can be diagonal, lateral and reversal loops (figure 5)”.

2) In the Pyridostatin section when discussing about reference 88 and the ability of PDS to induce a DNA damage response in the form of gH2AX, it would be worth mentioning briefly also that this study was used to create the first genomic map of G4 in a chromatin context looking at gH2AX sites generated by the ligand. This is important to show that not only the DDR eliciting properties of the molecules where characterised but also used to map (for the first time at the time) the distribution of G4s in chromatin, which will important to underline as a chemical-biology approach and also for its relevance in the context of G4 investigation.

Answer: This is absolutely right; the text has been modified accordingly, as follows (page 29): “Beyond the link between G4s and DNA damage, this approach also provided the very first description of the G4 landscape within human cells (via an accurate mapping of the distribution of G4s in the endogenous chromatin context) and highlighted the druggability of the SRC proto-oncogene (involved in multiple pathways that regulate tumour progression),296 thus uncovering a novel G4-mediated anticancer strategy”.

3) Page 17 first line "clinics" should be clinic. This is the only typo I spotted but is worth giving a second round of checking given is a long document and some more typos might be present.

Answer: done.

20-Sep-2020

Dear Prof Monchaud:

Manuscript ID: CB-REV-08-2020-000151.R1
TITLE: DNA folds threaten genetic stability and can be leveraged for chemotherapy

Thank you for submitting your revised manuscript to RSC Chemical Biology. After considering the changes you have made, I am pleased to accept your manuscript for publication in its current form. I have copied any final comments from the reviewer(s) below.

You will shortly receive a separate email from us requesting you to submit a licence to publish for your article, so that we can proceed with publication of your manuscript.

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With best wishes,

Dr Gonçalo Bernardes
Associate Editor, RSC Chemical Biology

Reviewer 3

The authors have successfully addressed my comments and I am supportive of publication of this review in the current state.

Reviewer 1

The authors have provided a revised manuscript that has strengthened what was already a very good review. This article is now ready for publication.