Phosphoproteomic Profiling of Multiple Myeloma Based on Ex Vivo Drug Sensitivity Resistance Testing Identifies Phosphorylation Signatures Associated with Drug Response

Round 1

Reviewer 1 Report (Previous Reviewer 3)

Comments and Suggestions for Authors

The authors have addressed and clarified all the raised questions, and I have no further comments or suggestions.

Author Response

Thank you for the reviewers' comments.

Reviewer 2 Report (New Reviewer)

Comments and Suggestions for Authors

The authors tested bortezomib- and lenalidomide-sensitive multiple myeloma cells for phosphorylation patterns.

The article is interesting and informative. I have just a few minor comments:

I am curious why the study started with >300 drugs. There are not >300 drugs for multiple myeloma, so I am curious about the rationale for including many of the drugs. The study is then limited to bortezomib and lenalidomide -- what was the rationale for focusing on these two drugs?

The Introduction has some redundancy toward the beginning and the end; for one it is mentioned at both places that multiple myeloma is an incurable disease.

The Figure 2 legend does not have text for 2E.

Section 6 is incorrectly titled as "Patents"

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

The reviewed work is focused on a relevant issue of search for the biomarkers which can be used in clinics for efficient prognosis of a therapeutic response in patients with multiple myeloma (MM). Protein phosphorylation was chosen as an object of the study using mass spectrometry of high resolution. This approach was analyzed in cell lysates of 20 MM patients who were stratified according to their sensitivity to standard chemotherapy including Bortezomib and Lenalidomide revealed ex vivo.

The authors convincingly demonstrated that in the lysates of patients highly sensitive to these medicines the phosphorylation was predominantly observed in proteins related to translation, RNA transport, and RNA linking pathways, while in the lysates of patients resistant to therapy the phosphorylation involved proteins associated with adhesion, as well as with Rap1 and phosphatidylinositol signaling pathways.

So, the results of the work demonstrate the relevance of the study of both the phosphorylation processes and the pool of phosphorylated proteins for understanding the mechanisms of the resistance of MM patients to various therapeutic schemes. The objective of the study is selection of the optimal treatment and the efficient method of prognosis of responses to it.

The article’s content is well reflected in the Abstract.

The Introduction section fully covers the problem though based on a large pool of scientific literature mostly dated to the first and second decades of our century, with rather small quantity of works performed in the recent 5 years. This is due to the fact that there are very few studies of protein phosphorylation related to phosphoproteomic profiling of multiple myeloma despite their obvious relevance in view of the trend for personalization of therapeutic approaches.

In “Materials and Methods” section methods and methodologic aspects of the study are described in detail. Every method can be reproduced by other researchers and in case of accurate performance it will lead to similar results.

The performed studies and obtained data are correctly described in the “Results” section which is provided with a huge pool of illustrative material, both Figures and Tables, supplementing the text.

The “Discussion” section is undoubtedly of a particular interest. The authors present their own opinion concerning the obtained results and compare it with the data and views of other researchers on related issues.

In the Conclusion the authors quite justifiably note that studying biological material from 20 MM patients is not enough for final conclusions and practical guidelines. These results only form a methodological basis for deeper study of the problem in the outlined direction.

The article is written in good scientific English and may be recommended for publication in the Biomolecules in the presented form.

Only as an advice:

It can be recommended to include a Table of abbreviations into the text of the article. Though the authors decipher almost all abbreviations and do it often several times, the separate Table of abbreviations would make the text easier for understanding.

Certainly, this decision should be made by the Editor’s board according to the journal guidelines on the design of submitted articles.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

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

Comments and Suggestions for Authors

This is a well-designed and impactful study that integrates ex vivo Drug Sensitivity and Resistance Testing (DSRT) with phosphoproteomics in primary Multiple Myeloma (MM) cells. The authors have identified key phosphorylation signatures associated with drug response, including FLNA, HSP27, and PRKACA/PRKCB. The methodology is innovative, and the findings offer a promising path toward identifying new therapeutic targets.

However, more comprehensive explanation in the Methods/Results sections detailing the statistical or threshold criteria of dividing groups based on DSRT should be indicated, which is very important for this study.

Author Response

Response to Reviewer 1 Comments

This is a well-designed and impactful study that integrates ex vivo Drug Sensitivity and Resistance Testing (DSRT) with phosphoproteomics in primary Multiple Myeloma (MM) cells. The authors have identified key phosphorylation signatures associated with drug response, including FLNA, HSP27, and PRKACA/PRKCB. The methodology is innovative, and the findings offer a promising path toward identifying new therapeutic targets.

However, more comprehensive explanation in the Methods/Results sections detailing the statistical or threshold criteria of dividing groups based on DSRT should be indicated, which is very important for this study.

Response: We thank the Reviewer for their useful comments. We have now revised our manuscript to include a more extensive explanation of the Drug Sensitivity and Resistance Test platform in the Methods and Results section with appropriate refence (Pemovska et al. Cancer Discovery. 2013;3(12):1416-29. doi: 10.1158/2159-8290.CD-13-0350) to the scoring criteria of the chemosensitive/chemoresistance patient Groups (Group 1-Group 4). [This change can be found in Page 4, Lines 167-190]

Reviewer 2 Report

Comments and Suggestions for Authors

In this manuscript, the Authors have performed a phosphoproteomic analysis in multiple myeloma primary cells by mass spectrometry and then confirmation by western blotting. Although of interest, this work requires severel improvements. 

1. Clinical characteristics of patients should be better reported: for example, one table with most significant information, including age, sex, M protein type, cytogenetics, R-ISS, TP53 status, treatment and response to treatment. In particular, for those with disease relapse at sampling should be reported at which line of therapy blood was obtained or at least it should be reported which drug refractoriness (e.g. triple refractory).
2. Ex vivo drug sensitivity data should be reported, as well as related methods, drugs, and control used. A similar observation also for data included in Figure 3. Groups should be renamed in a more intuitive manner. 
3. Group 3 included resistant patients (to what?) and most were newly diagnosed, none of them had achieved a CR: this observation suggests that probably those patients were at the beginning high genetic risk or functional high risk MM patients. However, no clinical, laboratory or molecular data were reported.
4. A CR group was not included as control. 
5. There is no association with clinical outcomes and phosphoprotein status, while patients were divided only according to ex vivo drug sensitivity. 
6. GAPDH or other housekeeping proteins should be included in the Western blotting analysis, as well as some baseline-phosphorylated protein.
7. Some Tables could be moved as Supplementary

Author Response

 

 

Response to Reviewer 2 Comments

In this manuscript, the Authors have performed a phosphoproteomic analysis in multiple myeloma primary cells by mass spectrometry and then confirmation by western blotting. Although of interest, this work requires several improvements.

1. Clinical characteristics of patients should be better reported: for example, one table with most significant information, including age, sex, M protein type, cytogenetics, R-ISS, TP53 status, treatment and response to treatment. In particular, for those with disease relapse at sampling should be reported at which line of therapy blood was obtained or at least it should be reported which drug refractoriness (e.g. triple refractory).

Response: We have now revised the tables with the clinical characteristics of the patients to include the R-ISS and the drug refractoriness. Unfortunately, data on TP53 status were not available from the clinical team. In light of Reviewer’s 4 comment, Tables 1 and 2 have now been moved to Supplementary Material.

 

2. Ex vivo drug sensitivity data should be reported, as well as related methods, drugs, and control used. A similar observation also for data included in Figure 3. Groups should be renamed in a more intuitive manner.

Response: We thank the Reviewer for this comment. We have now provided a more extensive description of the Drug Sensitivity and Resistance Test platform [This change can be found in Page 4, Lines 167-190] The platform incorporates a substantial number of drugs (>300) which would be beyond the scope of this manuscript to include in detail. We have also incorporated in our main text (Page 4, Lines 167-190) the appropriate reference ((Pemovska et al. Cancer Discovery. 2013;3(12):1416-29. doi: 10.1158/2159-8290.CD-13-0350) that will direct the reader to additional details of the DSRT platform.

 

3. Group 3 included resistant patients (to what?) and most were newly diagnosed, none of them had achieved a CR: this observation suggests that probably those patients were at the beginning high genetic risk or functional high risk MM patients. However, no clinical, laboratory or molecular data were reported.

Response: We apologize for this omission, and we now clarify that the Grouping of patients to the different groups (Group 1 - Group 4) is based on the ex vivo response of the patients to a panel of >300 drugs employed in the Drug Sensitivity and Resistance Test (DSRT) platform. In addition, we have now provided an extended table with additional clinical data (Supplementary Tables 1 and 2). The cytogenetics data are detailed in Figure 1A.

 

4. A CR group was not included as control.

Response: We agree with the Reviewer that including a CR group as control would be very beneficial. However, none of the patients included in this study achieved CR, hence we are not in a position to include those in this manuscript.

 

5. There is no association with clinical outcomes and phosphoprotein status, while patients were divided only according to ex vivo drug sensitivity.

Response: We thank the Reviewer for this comment. Although this is a very interesting suggestion it is beyond the scope of this manuscript. We are currently working with a larger patient cohort where any association of the phosphoprotein status with clinical outcomes will be extensively investigated.

 

6. GAPDH or other housekeeping proteins should be included in the Western blotting analysis, as well as some baseline-phosphorylated protein.

Response: The Reviewer is correct here. Despite attempting to use standard loading controls such as actin and GAPDH, the concentrations of these proteins varied likely due to the heterogeneity of the patient samples and role of the cytoskeleton in drug resistance in myeloma. This was further supported by altered concentrations of these proteins being detected in our mass spectrometry data. Although no loading controls were available, equal protein concentrations were visually confirmed using the Ponceau stain/densitometry.

 

 

7. Some Tables could be moved as Supplementary

Response: We have now moved Tables 1 and 2 to Supplementary Material

Reviewer 3 Report

Comments and Suggestions for Authors

Multiple myeloma remains an incurable disease, with all patients eventually experiencing relapse and developing drug resistance. Bortezomib and lenalidomide are first-line therapies for MM; however, the proportion of patients refractory to these two agents has been on the rise. Phosphoproteomics is a powerful bioinformatic tool for investigating protein expression and modification patterns in disease states. In this study, the authors applied phosphoproteomics to MM to characterize the protein profiling differences associated with varying sensitivities to bortezomib and lenalidomide. The results are informative and reveal distinct molecular characteristics in MM patients with different drug sensitivity phenotypes. Nevertheless, several aspects of the manuscript require further refinement:​

1. The grouping of samples based on drug sensitivity/resistance is not clearly described. It is confusing that Group 1 (the most sensitive group) comprises all relapsed patients, whereas the resistant group includes a large number of newly diagnosed patients. No rationale is provided to explain this counterintuitive sample distribution.​2. In the current study, quantitative phosphoproteomic mass spectrometry analysis identified a total of 1,473 proteins and 2,945 phosphorylation sites, which were mapped to 2,232 phosphopeptides derived from 690 phosphoproteins. In contrast, proteogenomic data reported by Ramberger et al. (PMID: 38942927) identified over 10,000 proteins and 50,000 phosphopeptides, with 8,336 proteins and 25,131 phosphopeptides quantified in at least half of the samples. The authors should clarify the reasons for this substantial discrepancy in the number of detected proteins and phosphopeptides.​

3. Deletion of 14q32 (del[14q32]) is not a common genetic aberration in MM. However, the data in Figure 1A show 5 positive events out of 20 samples, representing a relatively high frequency (25%). The authors need to provide a detailed explanation for this inconsistency with established literature.​

4. Western blot (WB) figures do not include internal controls (e.g., GAPDH, β-actin) in the same blot.

Author Response

 

Response to Reviewer 3 Comments

Multiple myeloma remains an incurable disease, with all patients eventually experiencing relapse and developing drug resistance. Bortezomib and lenalidomide are first-line therapies for MM; however, the proportion of patients refractory to these two agents has been on the rise. Phosphoproteomics is a powerful bioinformatic tool for investigating protein expression and modification patterns in disease states. In this study, the authors applied phosphoproteomics to MM to characterize the protein profiling differences associated with varying sensitivities to bortezomib and lenalidomide. The results are informative and reveal distinct molecular characteristics in MM patients with different drug sensitivity phenotypes. Nevertheless, several aspects of the manuscript require further refinement:

1. The grouping of samples based on drug sensitivity/resistance is not clearly described. It is confusing that Group 1 (the most sensitive group) comprises all relapsed patients, whereas the resistant group includes a large number of newly diagnosed patients. No rationale is provided to explain this counterintuitive sample distribution.

Response: We thank the Reviewer for their useful comments. We have now revised our manuscript to include a more extensive explanation of the Drug Sensitivity and Resistance Test platform in the Methods and Results section with appropriate refence (Pemovska et al. Cancer Discovery. 2013;3(12):1416-29. doi: 10.1158/2159-8290.CD-13-0350) to the scoring criteria of the chemosensitive/chemoresistance patient Groups (Group 1-Group 4). [This change can be found in Page 4, Lines 167-190]

We also now clarify that the Grouping of patients to the different groups (Group 1 - Group 4) is based on the ex vivo response of the patients to a panel of >300 drugs employed in the Drug Sensitivity and Resistance Test (DSRT) platform.

 

2. In the current study, quantitative phosphoproteomic mass spectrometry analysis identified a total of 1,473 proteins and 2,945 phosphorylation sites, which were mapped to 2,232 phosphopeptides derived from 690 phosphoproteins. In contrast, proteogenomic data reported by Ramberger et al. (PMID: 38942927) identified over 10,000 proteins and 50,000 phosphopeptides, with 8,336 proteins and 25,131 phosphopeptides quantified in at least half of the samples. The authors should clarify the reasons for this substantial discrepancy in the number of detected proteins and phosphopeptides.

Response: We thank the Reviewer for this comment. The Reviewer has correctly identified this disparity between the number of phosphosites identified in our study with those reported by Ramberger et al. Reason for this disparity is the limitations we encountered due to number of plasma cells in our samples as we are working with very limited material. However, to mitigate for this we used TMT which allows individual patient samples to be combined in an effort to maximize protein abundance for subsequent phssphopeptide protein enrichment. We do appreciate that despite  this we are still limited by the number of phosphosites identified. However, in our study we have identified nearly 3,000 phosphosites representing a significant number from limited patient sample quantities.

 

3. Deletion of 14q32 (del[14q32]) is not a common genetic aberration in MM. However, the data in Figure 1A show 5 positive events out of 20 samples, representing a relatively high frequency (25%). The authors need to provide a detailed explanation for this inconsistency with established literature.

Response: We agree the Reviewer that this is an interesting observation. However, it is beyond the scope of this manuscript. We are currently working with a larger patient cohort where any association of the phosphoprotein status with cytogenetics and clinical outcomes will be extensively investigated.

 

4. Western blot (WB) figures do not include internal controls (e.g., GAPDH, β-actin) in the same blot.

Response: The Reviewer is correct here and this point was also raised by Reviewer 2. Despite attempting to use standard loading controls such as actin and GAPDH, the concentrations of these proteins varied likely due to the heterogeneity of the patient samples and role of the cytoskeleton in drug resistance in myeloma. This was further supported by altered concentrations of these proteins being detected in our mass spectrometry data. Although no loading controls were available, equal protein concentrations were visually confirmed using the Ponceau stain.

Reviewer 4 Report

Comments and Suggestions for Authors

see file in attachment

Comments for author File: Comments.pdf

Comments on the Quality of English Language

no comment

Author Response

Response to Reviewer 4 Comments

The manuscript by Dunphy et al deals with a new type of profiling: phosphoproteomics profiling to correlate profiles to drug resistance (bortezomib and lenalidomide) in myeloma cells of patients with various stage. This a study on the field to define individualized treatments with drug profiling. This strategy of personalized treatment requires either better drug targeting either better profiling of patients malignant cells. The approach by phophoproteomics and myeloma is original and welcomed in a disease were treatments have strong but transient effects. The manuscript is detailed, sometimes hard to read but well explained.

1. The discussion section appears in disproportion considering the scientific results. The results were obtained from only 20 patients, so small subgroups between diagnosis relapse and should have been carefully interpretated. So the discussion section should be shortened to only the essential key messages. It wouldn’t be a review of literature.

Response: We have now amended our manuscript to provide a more focused and concise Discussion section.

 

2. The reference on myeloma (IWG of 2014) should be updated: it is the reference for classification and CRAB criteria but there is another classification of heamatologic diseases (lymphoid part) and numerous update reviews ware published the authors will do choose another one at the era of immunotherapy (after 2021)

Response: We thank the Reviewer for this suggestion. We have now updated the reference for the Myeloma criteria to include: Moreau, P.; Kumar, S. K.; San Miguel, J.; Davies, F.; Zamagni, E.; Bahlis, N.; Ludwig, H.; Mikhael, J.; Terpos, E.; Schjesvold, F. Treatment of relapsed and refractory multiple myeloma: recommendations from the International Myeloma Working Group. The Lancet Oncology 2021, 22 (3), e105-e118.

 

3. With regard to the demonstration, that required many experiments and figures, the reader could be swamped; so, I strongly suggest the authors to choose to move some experiments and figures to a supplementary material

Response: We have now moved Tables 1 and 2 to the Supplementary Material section.

 

4. Table 1 and 2 can easy be fused in one

Response: These two tables have been moved to the Supplementary Material section.

 

5. Tables 4 to 6 the authors have to choose either to move the top lists restricted to sensitive or more interesting to resistance to bortezomib and lenalidomide and move others in the supplementary material. Alternatively move all but results remain as text (shortened)

Response: We thank the Reviewer for their comment. However, from a biological perspective it is important to include these Tables in the main manuscript body as they provide an understanding of both, resistance and sensitivity to the different drugs in addition to the underlying biological mechanisms. Furthermore, from a personalized perspective, identifying biomarker of both responses (resistance/sensitivity) would be an important criterion as to which biomarkers are put forward for clinical validation.

 

6. Section 3.7 on western blotting: move to supplementary material but results remains in the text

Response: Given the comments raised by Reviewers 2 and 3 on the western blotting, we believe that it is more beneficial if it remains within the main body of the manuscript. We have however moved Tables 1 and 2 in the Supplementary Material section and have in addition reduced the length of the Discussion section in an effort to to provide a more concise and focused manuscript.

 

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors have addressed all concerns.

Reviewer 4 Report

Comments and Suggestions for Authors

Following my report on version 1 the revised version shows great improvments.

 the author's reply  by a point to point  actions in the manuscript meet all my  suggestions. Though , I cannot write more than acceptance.

Comments on the Quality of English Language

no comment - could be validate by a native English speaker , but in my opnion text  is clear some sentences are quite long