Targeting HMGB1 in the Treatment of Non-Small Cell Lung Adenocarcinoma

Round 1

Reviewer 1 Report

The Authors have addressed my concerns. I endorse publication.

Reviewer 2 Report

The authors have addressed the questions in a reasonable way. I recommend accepting the manuscript for publication.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

The stated aim of this review is to summarise knowledge concerning post translational modification of HMGB1 with a view to targeting its immunomodulatory properties for the purpose of improving lung cancer immunotherapies, the latter in particular focusing on HDAC inhibitors. It makes a promising start, using a search strategy to screen available literature databases for relevant full text research articles, identifying an initial 490 that were reduced to 28 following sequential independent review. The articles further divided into use of HDAC inhibitors or ethyl pyruvate or glycyrrhizin as the HGMB1 inhibitors and whether HMGB1 levels were increased or decreased. Thereafter, the review is a mixture of specifics and generalities, mostly relevant and some not, which in combination do not give sufficient background as to the mechanisms of action at work and how these would be effectively targeted.

In 3.1, for example, the nuclear protein role of HGMB1 is described and its effect on 3D structure of chromatin and a few associated proteins. There are no details on how this is brought about or how it is modulated. Given that as discussed in 3.2, its pro inflammatory role depends on its movement from the nucleus, the is an omission because it is one area where intervention could be beneficially targeted. On the other hand, the interaction of HGMB1 with TP53 is described as an anecdotal point, without any discussion of how this may be relevant to the main subject.

In 3.2, the HMGB1 pro inflammatory section, it is pointed out that under certain conditions (not adequately discussed), HGMB1 is transported into the cytoplasm and the extracellular space. Post translational modification, in particular acetylation of lysines in 2 nuclear localisation signals is a factor. This raises questions including, which lysines, which are the most important, are the effects additive, all or any of which could be significant for intervention. It is mentioned that conventional ER secretory mechanisms appear to be not important for HGMB1 with no discussion of what takes their place. The authors are not unaware of the significance of details because the importance of the redox state of HGMB1 is discussed and the critical stein residues are identified.

In 3.3 the role of HGMB1 as a cell surface receptor ligand, leading to pro inflammatory signal transduction through the release of pro-inflammatory cytokines. However, the activation of certain MAPKs is mentioned but it is not clear which ones or how the pathway leads to cytokine release.

Section 3.4 provides a better model for the levels of detail that are useful.

Inevitably, the discussion is a brief summary of the preceding material since insufficient hooks are presented in the main body to develop in detail the immunomodulatory opportunities exploiting HGMB1. Having identified, key references, perhaps more use of the relevant, background literature concerning properties and mode of action of HGMB1 could have been made, in particular mechanistic studies.

Author Response

Thank you for your help comments and critiques. Below we have summarized what was done to address each point made in the previous review. I hope that you find the changes improve on the original submission. (Review comments are bold, and author responses are bulleted.)

In 3.1, for example, the nuclear protein role of HGMB1 is described and its effect on 3D structure of chromatin and a few associated proteins. There are  Given that as discussed in 3.2, its pro inflammatory role depends on its movement from the nucleus, the is an omission because it is one area where intervention could be beneficially targeted. On the other hand, the interaction of HGMB1 with TP53 is described as an anecdotal point, without any discussion of how this may be relevant to the main subject.

• To address this point we have de-emphasized the nuclear function of HMGB1 as it is not central to our focus in the review. We have thus combined section 3.1 and 3.2. We have also discussed the concept of targeting nuclear export of HMGB1 as a potential strategy especially as it related to modifications of lysine residues.

In 3.2, the HMGB1 pro inflammatory section, it is pointed out that under certain conditions (not adequately discussed), HGMB1 is transported into the cytoplasm and the extracellular space. Post translational modification, in particular acetylation of lysines in 2 nuclear localisation signals is a factor. This raises questions including, which lysines, which are the most important,  , all or any of which could be significant for intervention. It is mentioned that conventional ER secretory mechanisms appear to be not important for HGMB1 with no discussion of what takes their place. The authors are not unaware of the significance of details because the importance of the redox state of HGMB1 is discussed and the critical cysteine residues are identified.

• We have broadened the discussion of cellular mechanism that lead to release of HMGB1. We have also included additional references and text indicating the nature and importance of the various lysines in HMGB1 NLS regions. We have also provided reference for the most recently defined mechanism for HMGB1 release from cells, which associated with NLRP3 inflammasome activation.

In 3.3 the role of HGMB1 as a cell surface receptor ligand, leading to pro inflammatory signal transduction through the release of pro-inflammatory cytokines. However, the activation of certain  MAPKs is mentioned but it is not clear  

• We added additional text indicating the role of MAPK pathway activation and specified MAPKs involved and the transcription factor activated as a result. However we tried to focus on the role TLR and RAGE in activation of NfKB as it is more closely associated with inflammation, the focus of our review.

Section 3.4 provides a better model for the levels of detail that are useful. 

• Thank this comment was very helpful in guiding our revisions.

Inevitably, the discussion is a brief summary of the preceding material since insufficient hooks are presented in the main body to develop in detail the immunomodulatory opportunities exploiting HGMB1. Having identified, key references, perhaps more use of the relevant, background literature concerning properties and mode of action of HGMB1 could have been made, in particular mechanistic studies.

• We have attempted to incorporate some additional references in order to address this particular critique.

Reviewer 2 Report

The manuscript titled ' Targeting HMGB1 in the treatment of non-small cell lung adenocarcinoma is an interesting review on basis of immunotherapy in lung cancer. However, few minor points would improve the manuscript.

Comments:

1. A line or two in the introduction about molecular targeted therapy in lung cancer.
2. Table 1, study numbers 27 and 28 reference numbers are the same but the study looks different.
3. The structure of HMGB1 protein would be easy for readers to grab the points explained in the results.

Author Response

Thank you for the helpful comments and critiques. Below we have summarized what was done to address the reviewers comments. I hope that these changes have improved on the original submission. Thank you.

The manuscript titled ' Targeting HMGB1 in the treatment of non-small cell lung adenocarcinoma is an interesting review on basis of immunotherapy in lung cancer. However, few minor points would improve the manuscript.

Comments:

A line or two in the introduction about molecular targeted therapy in lung cancer.

Table 1, study numbers 27 and 28 reference numbers are the same but the study looks different.

The structure of HMGB1 protein would be easy for readers to grab the points explained in the results.

• We have made additional comments regarding molecular targeted therapy in the introductory paragraph.
• We have also corrected the issue with table 1 regarding numbering of the references.
• An additional figure was added just after the reference to map out the lysine residues in the 2 NLS regions of HMGB1.

Reviewer 3 Report

In  this review authors summarize what has been published regarding the possible role of targeting HMGB1 for NSCLC treatment.  This review is well organized and clearly written. 

I have only some minor concerns. In details:

• about how the "HMGB1 as a therapeutic target" chapter has been organized:  authors should distinguish models of lung injury (that may offer a therapeutic window for lung cancer prevention/early intervention?) and models of lung cancer. I suggest that they split this chapter in two sub-chapters.
• Since HMGB1 is related to proinflammatory signaling and in interactions with immune system, authors should give some more insights, at least in the discussion section,  on the possible effects of therapeutic intervention, using HMGB1 as a target in combination with immune checkpiont inhibitors. 
• par 22 (end of page 6): [...]ethyl pyruvate suppresses HMGB1/RAGE protein in Non-small cell lung carcinoma (NSCLC)[...]. In my opinion authors should refer to a HMGB1/RAGE axis rather then protein.

Author Response

Thank you for your help comments and critiques of the original submission. We have summarized the steps we took to address the reviewer comments below. I hope that this show an improvement on the original submission. Thank you very much for taking the time to review this resubmission.

In  this review authors summarize what has been published regarding the possible role of targeting HMGB1 for NSCLC treatment.  This review is well organized and clearly written. 

I have only some minor concerns. In details:

• about how the "HMGB1 as a therapeutic target" chapter has been organized:  authors should distinguish models of lung injury (that may offer a therapeutic window for lung cancer prevention/early intervention?) and models of lung cancer. I suggest that they split this chapter in two sub-chapters.
• Since HMGB1 is related to proinflammatory signaling and in interactions with immune system, authors should give some more insights, at least in the discussion section,  on the possible effects of therapeutic intervention, using HMGB1 as a target in combination with immune checkpiont inhibitors. 
• par 22 (end of page 6): [...]ethyl pyruvate suppresses HMGB1/RAGE protein in Non-small cell lung carcinoma (NSCLC)[...]. In my opinion authors should refer to a HMGB1/RAGE axis rather then protein.
• We have made the lung injury paragraph separate from lung cancer portion, as it is a distinct disease state and may suggest an inflammatory condition that could be seen as a precursor to malignant growth.
• We have included references and additional discussion in the text regarding the potential combination of HMGB1 targeted therapies and immune checkpoint inhibitors.
• We have re-phrased the term “HMGB1/Rage protein” to “HMGB1/RAGE axis “ as suggested.