Adult B-Cell Acute Lymphoblastic Leukaemia Antigens and Enriched Pathways Identify New Targets for Therapy

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this manuscript, the authors Mohamed et. al. have identified a number of proteins and cell behaviors that are affected in acute lymphoblastic leukemia. Although, the manuscript is timely, well researched and informative, in the current form, the review can benefit from certain elements.

1. In Figure 1, it should be “Confirmation OF transcription by qPCR…”. The “of” is missing.
2. The caption for Figure 3 which also includes Table 3 is a bit confusing. It is recommended that you split it rather than keeping it Figure 3A and 3B.
3. Make sure the font type and size are same for all figures of the manuscript.
4. For Figure 4, the statistics used should be mentioned in the caption. This should also include the mention of the number of replicates for each experiment.
5. Also, please mention the name of the gene of top of each graph. This increases the ease of reading the manuscript.
6. In Figure 4B, how many immunolabeling samples were used for the study. If it is representative i.e. just one sample, then it should be specified. Images from different regions from immunolabelling samples should be provided in the Supplementary to determine a pattern.

Author Response

Reviewer 1 In this manuscript, the authors Mohamed et. al. have identified a number of proteins and cell behaviors that are affected in acute lymphoblastic leukemia. Although, the manuscript is timely, well researched and informative, in the current form, the review can benefit from certain elements.

1. In Figure 1, it should be “Confirmation OF transcription by qPCR…”. The “of” is missing.

Corrected

2. The caption for Figure 3 which also includes Table 3 is a bit confusing. It is recommended that you split it rather than keeping it Figure 3A and 3B.

The formatting had mixed the Table, figure and figure legend up. This has now been corrected. Figure 3A and 3B have become Figure 3 and 4 respectively.

3. Make sure the font type and size are same for all figures of the manuscript.

We have done our best to equalise the size of the font between figures but this is difficult as they were created using different programs and the figures themselves have different amounts of white space available for text. We are hoping once accepted the post-acceptance editorial team can help us achieve a better layout and indicate appropriate figure and font sizes.

4. For Figure 4, the statistics used should be mentioned in the caption. This should also include the mention of the number of replicates for each experiment.

Detailed on line 391 - One way ANOVA for qPCR, and line 397 for Dunnett for Immunoblotting.

5. Also, please mention the name of the gene of top of each graph. This increases the ease of reading the manuscript.

Done

6. In Figure 4B, how many immunolabeling samples were used for the study. If it is representative i.e. just one sample, then it should be specified. Images from different regions from immunolabelling samples should be provided in the Supplementary to determine a pattern.

This is detailed on line 361-2 and indicated in Table 1, but has now been added on line 391 in the Figure legend (now Figure 5B).

Reviewer 2 Report

Comments and Suggestions for Authors

This manuscript presents important findings on the molecular landscape of adult B-cell acute lymphoblastic leukaemia (aB-ALL), with a focus on identifying key antigens and associated pathways that could potentially serve as targets for future treatment strategies. The authors employ a robust approach using multiple high-throughput screening methods to identify proteins and cell behaviors altered in aB-ALL, and their findings regarding the upregulation of specific genes and pathways provide valuable insight into the pathogenesis of this disease.

 

1. The explanation of methods is somewhat dense, and breaking down the different sources and techniques used should be shortened.
2. The study’s limitations are not addressed, and it would be useful to mention any challenges faced during the identification process or gaps in the current knowledge.
3. The conclusions are concise and forward-thinking, suggesting that the identified antigens could be targeted for future immunotherapy. A more detailed outlook on how these findings might influence clinical trials or treatment development would be valuable.
4. The introduction is very long and only the relevant information linked to the discussion should be written.
5. Figure 4B. scale bar are missing.
6. The use of an outdated antigen prioritization model, while noted, limits the robustness of the conclusions. Updating this model or considering alternative methods would improve the reliability of antigen selection.
7. The discussion of novel gene products (UOH-ALL-104, UOH-ALL-105) lacks clarity regarding their functional roles in aB-ALL. More data or insights into their specific contributions would strengthen their proposed value as therapeutic targets.
8. The mechanistic roles of SOX4, ROCK1, TGFβ1, and TEAD4 in aB-ALL are mentioned but not sufficiently explored. A more detailed understanding of how these proteins contribute to the disease process would enhance their potential as therapeutic targets.
9. While small molecule inhibitors, such as GSK269962A, are discussed, there is a lack of clinical trial data to support their effectiveness in treating aB-ALL. Providing data or references from trials would better justify their inclusion in future treatment strategies.
10. The relationship between TGFβ signaling and other cancer-promoting pathways is mentioned but not fully developed. More detailed discussion on how these pathways interact would be important for a deeper understanding of their role in aB-ALL.
11. The challenges associated with targeting proteins like TEAD4 and SMAD3, due to their involvement in homeostasis, are highlighted, but there is insufficient discussion on alternative strategies to mitigate off-target effects, which is crucial for developing these as therapeutic targets.
12. While the need to break immune tolerance in immunotherapy is recognized, the specific strategies to achieve this are not adequately discussed. Further exploration of potential approaches to overcome immune tolerance would improve the practical applicability of the findings.

Author Response

Reviewer 2 This manuscript presents important findings on the molecular landscape of adult B-cell acute lymphoblastic leukaemia (aB-ALL), with a focus on identifying key antigens and associated pathways that could potentially serve as targets for future treatment strategies. The authors employ a robust approach using multiple high-throughput screening methods to identify proteins and cell behaviors altered in aB-ALL, and their findings regarding the upregulation of specific genes and pathways provide valuable insight into the pathogenesis of this disease.

 

Thank you

 

1. The explanation of methods is somewhat dense, and breaking down the different sources and techniques used should be shortened.

This level of detail has been slightly reduced but a previous reviewer had asked for more details in the methods to allow reproduction of the experiments.

2. The study’s limitations are not addressed, and it would be useful to mention any challenges faced during the identification process or gaps in the current knowledge.

 

We have created an extra section – Section 5 to address the studies limitations.

 

3. The conclusions are concise and forward-thinking, suggesting that the identified antigens could be targeted for future immunotherapy. A more detailed outlook on how these findings might influence clinical trials or treatment development would be valuable.

 

This has been extended as suggested – line 547-551.

 

4. The introduction is very long and only the relevant information linked to the discussion should be written.

 

Introduction has been shortened as suggested.

 

5. Figure 4B. scale bar are missing.

Now added and detailed in the figure legend – line 393.

6. The use of an outdated antigen prioritization model, while noted, limits the robustness of the conclusions. Updating this model or considering alternative methods would improve the reliability of antigen selection.

 

We did discuss this at the time the work was being planned but we didn’t feel we could improve on it sufficiently. The Cheever model was devised at the bequest of the NIH and gathered a panel of 80 experts in the field of immunotherapy to achieve the appropriate breadth and depth of considerations of what was required for tumour antigen targets to be effective in immunotherapeutic strategies. In order to get tumour antigens through clinical trials and help the NIH focus their funding strategy of tumour antigen targeting treatments the model had a weighting that disadvantaged less investigated targets.  Instead of attempting to change the model we instead acknowledged the limitations of it, because it is an effective and well understood system by which tumour antigens for immunotherapy can be prioritised.

 

7. The discussion of novel gene products (UOH-ALL-104, UOH-ALL-105) lacks clarity regarding their functional roles in aB-ALL. More data or insights into their specific contributions would strengthen their proposed value as therapeutic targets.

 

The cDNA sequences identified as UOH-ALL-104, 105 and 106 remain unmapped to known genes and therefore their functional role in health or disease is unknown. This has been clarified in the text – line 410.

 

8. The mechanistic roles of SOX4, ROCK1, TGFβ1, and TEAD4 in aB-ALL are mentioned but not sufficiently explored. A more detailed understanding of how these proteins contribute to the disease process would enhance their potential as therapeutic targets.

 

SOX4 is written about on line 426-431, ROCK1 on 431 – 434 & 436 – 446, TGFb1 on lines 456 – 464 and TEAD4 is now described on lines  447- 455.

 

A visual of the role of these genes in leukaemogenesis is presented in Figure 3 where we show how the pathways they interact with are each involved in promoting leukaemogenesis via the upregulation of anti-apoptotic genes and pro-proliferative genes that causes blasts to continue growing and resist to apoptosis.

 

9. While small molecule inhibitors, such as GSK269962A, are discussed, there is a lack of clinical trial data to support their effectiveness in treating aB-ALL. Providing data or references from trials would better justify their inclusion in future treatment strategies. –

 

No clinical data is currently publicly available but we agree that would be interesting!

 

10. The relationship between TGFβ signaling and other cancer-promoting pathways is mentioned but not fully developed. More detailed discussion on how these pathways interact would be important for a deeper understanding of their role in aB-ALL.

 

This is detailed on lines 456 – 464.

 

11. The challenges associated with targeting proteins like TEAD4 and SMAD3, due to their involvement in homeostasis, are highlighted, but there is insufficient discussion on alternative strategies to mitigate off-target effects, which is crucial for developing these as therapeutic targets.

This has now been discussed on lines 502-504 and tying part of the discussion to concepts introduced in the Introduction.

12. While the need to break immune tolerance in immunotherapy is recognized, the specific strategies to achieve this are not adequately discussed. Further exploration of potential approaches to overcome immune tolerance would improve the practical applicability of the findings.

 

This has now been addressed on lines 497-499, in addition to text on lines 64-68, 511-515 and 595-597.

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

I have a few comments on your work. They are sorted by the appearance in the text, not by their importance.

1. In the simple summary you're writing " Adult acute lymphoblastic leukaemia is a cancer..." (line 18), and right afer it, on line 19 "It mostly affects children...". I'd suggest to delete 'adult' on line 18, as it's confusing at such context.
2. Please, insert the type of cells you'd analyzed on line 38.
3. The sentence on lines 40-43 is hard to understand. Please, rewrite it more clearly.
4. It seems to me, that only one control sample (CD19+ cells, line 144) is not enough to make any conclusions. Do you have any possibility to add some samples in this group?
5. Please, add more detailed explanation for the reason of using the testes cDNA library for the analysis of serum from patients with B-ALL.
6. Please, introduce the HV acronym at first mention (line 175).
7. What do you mean by "eighteen diagnostic gold standard 176 categories"?
8. Be aware, that on page 6 the numeration of the lines had started again. I'm referring to the numbers I see in the text.
9. It seems, that the words are missing in the sentences on lines 74-76 and 85-87. It makes difficult to understand the meaning.
10. On line 116, I believe, you'd meant 'non-canonical' rather than 'non-conical'.
11. Please, do not introduce R/R on line 201 again. You've done it on line 66 already.
12. Some acronyms are introduce without real need, as they had been used only once. Please, check the text for such cases.
13. At last, the conclusions. This paragraph does not conclude your work. It contains some general words and adds nothing to the understanding of the results of your work.  Even in the Abstract conclusions are more informative. Please, rewrite this part of the article.

 

Author Response

Reviewer 3 I have a few comments on your work. They are sorted by the appearance in the text, not by their importance.

 

1. In the simple summary you're writing " Adult acute lymphoblastic leukaemia is a cancer..." (line 18), and right afer it, on line 19 "It mostly affects children...". I'd suggest to delete 'adult' on line 18, as it's confusing at such context.

 

Corrected for clarity

2. Please, insert the type of cells you'd analyzed on line 38.

Detailed on line 38-39

3. The sentence on lines 40-43 is hard to understand. Please, rewrite it more clearly. –

 

Sentence edited for flow.

4. It seems to me, that only one control sample (CD19+ cells, line 144) is not enough to make any conclusions. Do you have any possibility to add some samples in this group?

 

The company that the CD19+ cells were purchased from only had cells from 1 donor. We did seek other companies and whether other donors could be sourced to no avail. However other samples of peripheral blood and bone marrow were obtained from HVs and directly compared to peripheral blood and bone marrow samples from B-ALL patients, collected at the same site. Data on expression was also obtained from a second source of samples – RNA-seq as detailed in the manuscript - lines 167 - 179.

5. Please, add more detailed explanation for the reason of using the testes cDNA library for the analysis of serum from patients with B-ALL.

 

Detailed in lines 150-152

 

6. Please, introduce the HV acronym at first mention (line 175).

 

Line 110, changed “donors” to “volunteers” through out the paper and introduced acronym on line 88

 

7. What do you mean by "eighteen diagnostic gold standard 176 categories"?

This has been explained in more detail – line 172-174

8. Be aware, that on page 6 the numeration of the lines had started again. I'm referring to the numbers I see in the text.

 

This has now been corrected.

9. It seems, that the words are missing in the sentences on lines 74-76 and 85-87. It makes difficult to understand the meaning.

 

This has been corrected now lines 79-80 and removed as part of the requested reduction in the length in the Introduction by another reviewer.

 

10. On line 116, I believe, you'd meant 'non-canonical' rather than 'non-conical'.

 

Corrected.

 

11. Please, do not introduce R/R on line 201 again. You've done it on line 66 already. –

 

Changed as suggested.

12. Some acronyms are introduce without real need, as they had been used only once. Please, check the text for such cases.

All acronyms listed have been checked for repetition and checked for clarity, if there has been repeat of the acronym, it has been removed.

 

13. At last, the conclusions. This paragraph does not conclude your work. It contains some general words and adds nothing to the understanding of the results of your work. Even in the Abstract conclusions are more informative. Please, rewrite this part of the article.

 

Edited to be more conclusive.

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

I appreciate the effort you've made to take into account my remarks. I would suggest making the conclusions much more concise and informative than they are now. However, I don't insist on it. A man is the king in his house.