Deregulation of Autophagy and Apoptosis in Patients with Myelodysplastic Syndromes: Implications for Disease Development and Progression

Round 1

Reviewer 1 Report

The role of apoptosis in myelodysplastic (MDS) was the focus of many studies in the past decades, and few have elucidated the deregulation of mechanisms in the pathogenesis of MDS. In this study authors investigated together the role of macro-autophagy and apoptosis and proposed their consistent downregulation in the progression of MDS from a low-risk to high-risk malignancy. I feel this study is premature and does not represent a sufficiently broad advance in the field.

 

Major comments.

To my level of understanding, expression (RNA) data add little value in interpreting the role of autophagy and apoptosis in the pathogenesis of Myelodysplastic neoplasms (MDS). In the given context I’m skeptical whether the finds at the transcript level are not necessarily translated to the protein level. Moreover, autophagy and apoptosis are intricate mechanisms regulated by multiple signaling networks through post-translational modifications.

To substantiate their claim, authors should carry out a few biochemical validations (i.e western blot, cell biological microscopy-based approaches) for representative low-/high-risk MDS malignancy samples to quantity autophagy and apoptosis.

 

Minor comments.

Line 200: CTSS gene codes for Cathepsin S, a lysosomal cysteine protease that digests the proteins targeted for lysosomal degradation. Not to my understanding they involve in autophagosome–lysosome fusion. If authors differ, please cite.

 

 

Line 207: SQSTM1 / p62 is a well-established autophagy gene, with few non–canonical roles in apoptosis.

Author Response

We would like to thank all the Reviewers for their constructive comments and suggestions. All changes in the text made according to their suggestions and are highlighted in yellow.

 

Reviewer 1

The role of apoptosis in myelodysplastic (MDS) was the focus of many studies in the past decades, and few have elucidated the deregulation of mechanisms in the pathogenesis of MDS. In this study authors investigated together the role of macro-autophagy and apoptosis and proposed their consistent downregulation in the progression of MDS from a low-risk to high-risk malignancy. I feel this study is premature and does not represent a sufficiently broad advance in the field.

Major comments.

To my level of understanding, expression (RNA) data add little value in interpreting the role of autophagy and apoptosis in the pathogenesis of Myelodysplastic neoplasms (MDS). In the given context I’m skeptical whether the finds at the transcript level are not necessarily translated to the protein level. Moreover, autophagy and apoptosis are intricate mechanisms regulated by multiple signaling networks through post-translational modifications.

To substantiate their claim, authors should carry out a few biochemical validations (i.e western blot, cell biological microscopy-based approaches) for representative low-/high-risk MDS malignancy samples to quantity autophagy and apoptosis.

Reply: We would like to thank the Reviewer for this valuable comment. To address this issue we performed preliminary Western blotting experiments to estimate the protein expression levels of three relevant genes (namely TGM2, BCL2 and LC3-II). Our results are in line with the mRNA expression analysis, showing higher protein levels for TGM2 and BCL2 as well as a lower level for LC3-II in MDS patients versus the healthy controls.

Minor comments.

Line 200: CTSS gene codes for Cathepsin S, a lysosomal cysteine protease that digests the proteins targeted for lysosomal degradation. Not to my understanding they involve in autophagosome–lysosome fusion. If authors differ, please cite.

Reply: We fully agree with the Reviewer’s comment and apologize for overlooking this. The relevant part of the text was modified accordingly.

Line 207: SQSTM1 / p62 is a well-established autophagy gene, with few non–canonical roles in apoptosis.

Reply: We apologize for this mistake. The SQSTM1 gene was moved to the category of autophagic genes and the relevant parts of the text have been modified accordingly.

Reviewer 2 Report

This is a really well-written manuscript on a very interesting topic with research and clinical relevance in Myelodysplastic syndromes (MDS). However, there are some concerns that need to be addressed by the authors:

1. Correlation of array analysis and qPCR results between populations of mononuclear cells derived from bone marrow (BM) of MDS patients and mononuclear cells derived from peripheral blood (PB) of healthy donors should be further discussed (lines 448-449, discussion section) and validated with literature references. BM and PB mononuclear cells include diverse cell populations that both comprise hematopoietic stem and progenitor cells (HSC/HPC), among other cell types, but with significantly different ratios (doi: 10.1111/j.1423-0410.2008.01138.x.). Also, MDS patients of the higher-risk group very often present phenotypic and functional cell alterations in hematopoietic stem cell (HSC) compartment of BM, which is the main cell subgroup targeted for gene expression profile alterations in MDS. The point that is raised by this query is that the number of HSCs circulating in peripheral blood (PB) is very low and becomes sufficient only after induction via administration of granulocyte colony-stimulating factor (G-CSF), in a process termed mobilization. Authors should mention if healthy controls were treated with G-CSF to enrich HSCs prior to PB collection.

2. Authors should discuss the wide range (20 years) of age between MDS patient cohort (median age 75years) and healthy cohort (55 years). Younger healthy donors could possibly be excluded from the study and therefore, increase the median age of this cohort.

3. Ethical approvals from ethics committees were reported, but additionally the project identification code must be filed in.

4. Paragraphs 3.1.1 and 3.1.2. should be merged. They both refer to the same experimental procedure for 10 MDS over 3 healthy controls subjected to PCR-based gene profiling and results are displayed in table 2 and figure 1.

5. Define the selection criteria set for the complement qPCRs analysis of genes related to autophagy and apoptosis (paragraph 3.2). Considering results from table 2, genes displaying the highest deviation in fold change were not always depicted.

6. Discussion should mostly propound the downregulated genes with statistical significance, which were highlighted from the different kinds of analysis techniques performed in this study. As it is, upregulated expression of TGM2 gene is underlined to be of great importance, however, corresponding p-values (table 3) are insignificant. 

Minor concerns:

1. Introduction section, lines 41-42: “….which do not have a known etiology,….”. This is not accurate. Authors might mean “clearly defined etiology”.

2. Introduction section, line 78: “….cells from patients……”. Define the kind of cells mentioned.

3. Introduction section, lines 81-82: Rephrase the sentence to obtain a clear meaning.

Author Response

We would like to thank all the Reviewers for their constructive comments and suggestions. All changes in the text made according to their suggestions and are highlighted in yellow.

Reviewer 2

This is a really well-written manuscript on a very interesting topic with research and clinical relevance in Myelodysplastic syndromes (MDS). However, there are some concerns that need to be addressed by the authors:

1. Correlation of array analysis and qPCR results between populations of mononuclear cells derived from bone marrow (BM) of MDS patients and mononuclear cells derived from peripheral blood (PB) of healthy donors should be further discussed (lines 448-449, discussion section) and validated with literature references. BM and PB mononuclear cells include diverse cell populations that both comprise hematopoietic stem and progenitor cells (HSC/HPC), among other cell types, but with significantly different ratios (doi: 10.1111/j.1423-0410.2008.01138.x.). Also, MDS patients of the higher-risk group very often present phenotypic and functional cell alterations in hematopoietic stem cell (HSC) compartment of BM, which is the main cell subgroup targeted for gene expression profile alterations in MDS. The point that is raised by this query is that the number of HSCs circulating in peripheral blood (PB) is very low and becomes sufficient only after induction via administration of granulocyte colony-stimulating factor (G-CSF), in a process termed mobilization. Authors should mention if healthy controls were treated with G-CSF to enrich HSCs prior to PB collection.

Reply: We apologize for not being very clear on this matter. In order to assess the impact of sample origin (BM versus PB) we performed a comparative analysis between paired BM and PB samples from 3 MDS patients. We did not find any significant differences between the paired samples in any of the analyzed cases and, thus, proceeded with the experimental design of the project as it is evident in the manuscript. This description of these experiments was added to the manuscript.

2. Authors should discuss the wide range (20 years) of age between MDS patient cohort (median age 75years) and healthy cohort (55 years). Younger healthy donors could possibly be excluded from the study and therefore, increase the median age of this cohort.

Reply: The groups of healthy individuals was divided into 2 groups, one consisting of young individuals (age <55 years) and the other comprising older individuals (age ≥55 years). A comparative analysis was performed between these 2 groups in terms of gene expression patterns based on the data generated by the PCR array. No significant differences were observed among the 2 groups, indicating that the age of the healthy individuals included in our study did not affect the results, at least substantially. A small part of the text describing this action was added to the manuscript.

3. Ethical approvals from ethics committees were reported, but additionally the project identification code must be filed in.

Reply: The project identification code was added to the manuscript.

4. Paragraphs 3.1.1 and 3.1.2. should be merged. They both refer to the same experimental procedure for 10 MDS over 3 healthy controls subjected to PCR-based gene profiling and results are displayed in table 2 and figure 1.

Reply: Paragraphs 3.1.1 and 3.1.2. of the Results section were merged according to the Reviewer’s comment.

5. Define the selection criteria set for the complement qPCRs analysis of genes related to autophagy and apoptosis (paragraph 3.2). Considering results from table 2, genes displaying the highest deviation in fold change were not always depicted.

Reply: We apologize for not being clear regarding this aspect. The relevant part of the text was modified in order to clearly depict the gene selection criteria for the qRT-PCR experiments.

6. Discussion should mostly propound the downregulated genes with statistical significance, which were highlighted from the different kinds of analysis techniques performed in this study. As it is, upregulated expression of TGM2 gene is underlined to be of great importance, however, corresponding p-values (table 3) are insignificant. 

Reply: As we already replied to Reviewer 3, the part of the discussion focusing on TGM2 was significantly reduced and new parts were added to highlight other aspects of our analysis.

Minor concerns:

1. Introduction section, lines 41-42: “….which do not have a known etiology,….”. This is not accurate. Authors might mean “clearly defined etiology”.

Reply: The relevant part of the Introduction was changed according to the Reviewer’s comment.

2. Introduction section, line 78: “….cells from patients……”. Define the kind of cells mentioned.

Reply: The relevant part of the text was modified according to the Reviewer’s comment.

3. Introduction section, lines 81-82: Rephrase the sentence to obtain a clear meaning.

Reply: The relevant part of the Introduction was changed according to the Reviewer’s comment.

Reviewer 3 Report

The manuscript, “Deregulation of autophagy and apoptosis in patients with myelodysplastic syndromes: implications for disease development and progression” by Tsekoura et al. investigated the role of autophagy and apoptosis in the pathogenesis and progression of MDS and systematically assessed the expression levels of a large series of genes involved in autophagy and apoptosis in samples from MDS patients and healthy individuals.

Comments and Suggestions for Authors:

1- Please correct the keywords according to MeSH

2- Please use newer and more recent references.

3- Line130 and 336: Why are some rows in table 1 and 3 gray? Please explain this.

4-Please indent the first line of a new paragraph by hitting the tab key.

 

 

 

 

 

 

 

 

Author Response

We would like to thank all the Reviewers for their constructive comments and suggestions. All changes in the text made according to their suggestions and are highlighted in yellow.

 

Reviewer 3

The manuscript, “Deregulation of autophagy and apoptosis in patients with myelodysplastic syndromes: implications for disease development and progression” by Tsekoura et al. investigated the role of autophagy and apoptosis in the pathogenesis and progression of MDS and systematically assessed the expression levels of a large series of genes involved in autophagy and apoptosis in samples from MDS patients and healthy individuals.

Comments and Suggestions for Authors:

1. Please correct the keywords according to MeSH

Reply: The keywords were corrected according to MeSH

2. Please use newer and more recent references.

Reply: The older references were replaced by more recent ones (highlighted in yellow).

3. Line130 and 336: Why are some rows in table 1 and 3 gray? Please explain this.

Reply: We apologize for overlooking this. Tables 1 and 3 (now Table 2) were changed according to the Reviewer’s comment.

4. Please indent the first line of a new paragraph by hitting the tab key.

Reply: The manuscript was modified according to the Reviewer’s comment.

Reviewer 4 Report

In this manuscript, the authors assessed the role of autophagy and apoptosis in myelodysplastic neoplasms (MDS). With a combination of PCR array and qPCR, the authors analyzed the expression profiles of genes involved in autophagy and apoptosis, in patients with varied malignancy versus control individuals. Interestingly, both analyses showed that the expression levels of distinct genes in the autophagy and apoptosis pathways were significantly impacted in patient samples. Furthermore, the authors identified a few gene targets that consistently correlates with malignancy of MDS, providing promising molecular targets as diagnostic biomarkers or therapeutic targets. Overall, this manuscript advances the knowledge of the mechanism of MDS pathogenesis, and the experiments are well designed and well documented. However, the relatively small sample size and small number of genes tested limits the strength of the conclusions and implications. Additionally, without any functional analysis, it is difficult to tease out causality and provide fundamental insights into the mechanisms.

 

Given the scope of the manuscript, I would recommend acceptance of the manuscript after the authors address the following comments.

1. The authors should compile and plot the data in Table 2 more succinctly, and put the table with numbers in the supplementary if needed. An effective way to plot such data is a classic volcano plot, with log fold change on the x axis and -log(p.value) on the y axis. Dots can be color coded based on whether it belongs to the autophagy or apoptosis gene set.

2. In Figure 1, the authors should use more discernible colors to label the three groups of genes. Additionally, for better clarity and easier interpretation, instead of the current plots, the authors can plot the log fold change values (patient vs ctrl) of each gene, with values for high-risk group on one axis vs values for low-risk group on the other.

3. In Figure 2, the authors should describe in detail what the color scheme represents. Do the “min”, “avg”, “max” represents statistics calculated from all genes tested? If possible, the figure should also be presented in higher resolution.

4. Line 278-283: the authors found a small gene set showed low expression levels in all samples, even in the control group, and autophagy genes are slightly enriched. Why does this suggest that “autophagy may be more involved in MDS progression”, given that they are also expressed at low levels in the control group?

5. For qPCR analysis, samples should be run in at least three replicates, and standard deviation should be reported. The authors should also describe how p-value is calculated in Table 3 (what statistical analysis is performed).

6. Line 316-318, please show the data to support this claim.

7. For Figure 3, the authors should remove the 3D effects and shading to improve clarity of the plots. The authors should group the columns in panel B based on MDS groups (just as in A), or consider combining panel A and B into one bar graph to make plots consistent and easier to interpret. Alternatively, the authors can plot the log fold change of each gene, with values for high-risk on one axis and low-risk on the other, as suggested in comment #2.

8. The authors reported that only BCL2 gene expression is inconsistent between two experimental approaches. The authors should discuss what could be the reasons for this. Is this because the two techniques are detecting different splicing variants of BCL2?

9. Although the identification of elevated levels of TGM2 in MDS patients is intriguing, the discussion centered around TGM2 (page 17) should be downplayed significantly for the following reasons. 1) The increase of TGM2 levels in MDS patients is mild; 2) the claim that TGM2 is an autophagy regulator lacks strong support, and whether it directly regulates autophagy is not known; 3) cited literature showed conflicting results on whether TGM2 induces or inhibits autophagy, limiting its potential as a target for direct manipulation.

10. The authors should validate the protein expression levels of interesting gene targets, at least those showed the strongest fold change in mRNA expression.

 

Minor points:

1. Move Table 1 into supplementary, and provide a summarized table based on IPSS-R category.

2. Line 255: “indicating an enrichment of genes involved in…”. There are only slightly more genes in autophagy than apoptosis, and concluding that there is an enrichment is inaccurate. Same for line 280-281.

 

Author Response

We would like to thank all the Reviewers for their constructive comments and suggestions. All changes in the text made according to their suggestions and are highlighted in yellow.

Reviewer 4

In this manuscript, the authors assessed the role of autophagy and apoptosis in myelodysplastic neoplasms (MDS). With a combination of PCR array and qPCR, the authors analyzed the expression profiles of genes involved in autophagy and apoptosis, in patients with varied malignancy versus control individuals. Interestingly, both analyses showed that the expression levels of distinct genes in the autophagy and apoptosis pathways were significantly impacted in patient samples. Furthermore, the authors identified a few gene targets that consistently correlates with malignancy of MDS, providing promising molecular targets as diagnostic biomarkers or therapeutic targets. Overall, this manuscript advances the knowledge of the mechanism of MDS pathogenesis, and the experiments are well designed and well documented. However, the relatively small sample size and small number of genes tested limits the strength of the conclusions and implications. Additionally, without any functional analysis, it is difficult to tease out causality and provide fundamental insights into the mechanisms.

Given the scope of the manuscript, I would recommend acceptance of the manuscript after the authors address the following comments.

1. The authors should compile and plot the data in Table 2 more succinctly, and put the table with numbers in the supplementary if needed. An effective way to plot such data is a classic volcano plot, with log fold change on the x axis and -log(p.value) on the y axis. Dots can be color coded based on whether it belongs to the autophagy or apoptosis gene set.

Reply: Table 2 was moved to the supplementary material (now Supplementary Table 3). Figure 1 was modified according to the Reviewer’s comment (volcano plots with required axes).

2. In Figure 1, the authors should use more discernible colors to label the three groups of genes. Additionally, for better clarity and easier interpretation, instead of the current plots, the authors can plot the log fold change values (patient vs ctrl) of each gene, with values for high-risk group on one axis vs values for low-risk group on the other.

Reply: Different colors were used (green, black and red) for the different gene categories. Different plots were included for each specific comparison.

3. In Figure 2, the authors should describe in detail what the color scheme represents. Do the “min”, “avg”, “max” represents statistics calculated from all genes tested? If possible, the figure should also be presented in higher resolution.

Reply: “min”, “avg” and “max” values are now explained in the Figure legend. The color code is based on the “raw” gene expression values, which is the default option to depict this type of data.

4. Line 278-283: the authors found a small gene set showed low expression levels in all samples, even in the control group, and autophagy genes are slightly enriched. Why does this suggest that “autophagy may be more involved in MDS progression”, given that they are also expressed at low levels in the control group?

Reply: This particular sentence was removed from the manuscript according to the comment made by Reviewer 4 (please see below).

5. For qPCR analysis, samples should be run in at least three replicates, and standard deviation should be reported. The authors should also describe how p-value is calculated in Table 3 (what statistical analysis is performed).

Reply: As mentioned in the Material and Methods section, each sample in the qPCR analysis was run in duplicate. A supplementary table was added (Supplementary table S4) including the SD values for the sample technical replicates. Regarding the estimation of the p-values in Table 3 (now Table 2), an explanation is now given below the table.

6. Line 316-318, please show the data to support this claim.

Reply: We apologize, however according to our version of the manuscript lines 316-318 we did not make any claims. We would be more than happy to review that part if the Reviewer could point the exact sentence.

7. For Figure 3, the authors should remove the 3D effects and shading to improve clarity of the plots. The authors should group the columns in panel B based on MDS groups (just as in A), or consider combining panel A and B into one bar graph to make plots consistent and easier to interpret. Alternatively, the authors can plot the log fold change of each gene, with values for high-risk on one axis and low-risk on the other, as suggested in comment #2.

Reply: Figure 3 was modified according to the Reviewer’s comments.

8. The authors reported that only BCL2 gene expression is inconsistent between two experimental approaches. The authors should discuss what could be the reasons for this. Is this because the two techniques are detecting different splicing variants of BCL2?

Reply: We would like to thank the Reviewer for this observation. We fully agree that the difference in the level of BCL2 mRNA expression between the 2 experimental approaches is quite large. In the case of the qPCR experiments, detection concerned all different splicing variants of BCL2. However, in regard to the assay experiment this information is not provided by the manufacturer (QIAGEN). Hence, only speculations can be made regarding this issue. A relevant sentence was added to the manuscript.

9. Although the identification of elevated levels of TGM2 in MDS patients is intriguing, the discussion centered around TGM2 (page 17) should be downplayed significantly for the following reasons. 1) The increase of TGM2 levels in MDS patients is mild; 2) the claim that TGM2 is an autophagy regulator lacks strong support, and whether it directly regulates autophagy is not known; 3) cited literature showed conflicting results on whether TGM2 induces or inhibits autophagy, limiting its potential as a target for direct manipulation.

Reply: The part of the discussion regarding TGM2 was made smaller, whereas some new parts were added regarding other aspects of the study that were expanded according to the Reviewers’ comments.

10. The authors should validate the protein expression levels of interesting gene targets, at least those showed the strongest fold change in mRNA expression.

Reply: As already mentioned in our reply to the comment made by Reviewer 1, we performed preliminary Western blotting experiments to estimate the protein expression levels of three relevant genes (namely TGM2, BCL2 and LC3-II). Our results are in line with the mRNA expression analysis, showing higher protein levels for TGM2 and BCL2 as well as a lower level for LC3-II in MDS patients versus the healthy controls.

Minor points:

1. Move Table 1 into supplementary, and provide a summarized table based on IPSS-R category.

Reply: Original Table 1 was moved to the Supplementary material (now Supplementary table S1) according to the Reviewer’s comment. A novel, summarized version of Table 1 was added to the manuscript.

2. Line 255: “indicating an enrichment of genes involved in…”. There are only slightly more genes in autophagy than apoptosis, and concluding that there is an enrichment is inaccurate. Same for line 280-281.

Reply: Relevant sentences were removed from the manuscript according to the Reviewer’s comment.

Round 2

Reviewer 1 Report

The authors have satisfactorily attended to all my concerns, I recommend the publication of the manuscript in its current format.  

 

Author Response

We would like to thank the Reviewer for his/her recommendation.

Reviewer 4 Report

The authors have made great efforts in addressing the concerns in this revised manuscript, and the quality of the manuscript has improved. However, there remains some concerns that I would like the authors to address before I can recommend for publication.

1. Regarding the original comment #6, I was referring to the sentence “… showed highly similar expression patterns among individual patients (data not shown)”, which is on Line 360-363 in the revised version.

2. It is great to see that the authors have performed western blotting to validate protein expression levels of certain gene targets. However, the authors should make side-by-side comparison of all samples (on the same gel) in order to draw any meaningful conclusion. Additionally, the experiments should be repeated for at least three times, and data from all repeats should be included in the plot.

Author Response

Reviewer 4

 

The authors have made great efforts in addressing the concerns in this revised manuscript, and the quality of the manuscript has improved. However, there remain some concerns that I would like the authors to address before I can recommend it for publication.

 

1. Regarding the original comment #6, I was referring to the sentence “… showed highly similar expression patterns among individual patients (data not shown)”, which is in Lines 360-363 in the revised version.

Reply: We thank the Reviewer for clarifying this aspect. As we already mentioned in our previous reply, the Discussion has been extensively modified in order to reduce the part regarding TGM2 and highlighting other aspects of the present study. To better address the Reviewer’s comment an additional piece of text regarding the downregulation of certain autophagic genes showing similar expression patterns among different MDS patients was added to the Discussion (highlighted in yellow).

 

2. It is great to see that the authors have performed western blotting to validate protein expression levels of certain gene targets. However, the authors should make side-by-side comparison of all samples (on the same gel) in order to draw any meaningful conclusion. Additionally, the experiments should be repeated at least three times, and data from all repeats should be included in the plot.

Reply: As mentioned in the previous version of the manuscript, Western blot experiments were run twice without significant differences. The experiment was repeated for a third time, according to the Reviewer’s comment, further corroborating our previous findings (see Figure legend). A new version of Figure 4 (Figure 4A in more specific) was added to the manuscript, where the protein expression levels of all samples are provided side-by-side in order to allow for better comparison. Results depicted in the figure are based on this last, third experiment and the relative protein expression in the plot are based on data from all experiment repeats.

Round 3

Reviewer 4 Report

Thank you for responding to my concerns. I would still recommend presenting the quantification from all three repeats on the plot, or to show mean with standard deviation. Otherwise the readers cannot see the variability of the repeats.