HSP90 Inhibitor PU-H71 in Combination with BH3-Mimetics in the Treatment of Acute Myeloid Leukemia
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors explore the potential of PU-H71, an HSP90 inhibitor, in acute myeloid leukemia cell lines and primary cultures, as a single agent and in combination with S63845 (MCL1 inhibitor) and venetoclax (BCL-2 inhibitor). They also characterize its mechanisms of action and identify possible response biomarkers. The manuscript is well-written, and the methodology used clearly addresses the identified problem. However, some concerns need to be discussed and better explained.
Major issues:
1) The authors present de IC50 values for the three anticancer drugs evaluated in this study. Which method was used to calculate the IC50 values? Did the authors perform dose-response curves for S63845 and venetoclax, as well as for PU-H71?
2) Regarding the combination therapy studies, what criteria were used to choose the different concentrations for each of the three drugs used? For example, different concentrations of venetoclax were used for the different cell lines (100nM for MOLM-13 and ML-2, and 1µM for SKM-1, OCI-AML3, MOLM-16, and PL21).
3) Moreover, the use of different concentration units for the same drugs makes the results very confusing throughout the text and figures. Please, consider using all the concentrations in nM or µM.
4) In section 3.2. (lines 172-185), the authors mention the synergistic or additive effects of the different combination therapies when referring to Figure 3. Can you make those affirmations based only on Figure 3?
5) In Table 3 (page 7), the authors present the results of the combination indexes calculated on Compusyn software as a range of values. However, absolute values should be indicated instead.
6) In section 3.3, line 215, it is referred that the protein levels were determined in two cell lines; however, only present the results for MOML-13 in Figure 4. Cell cycle and mRNA expression analysis are only shown for three cell lines. Moreover, in Figure 5, results for the MOLM-13 cell line are only presented. It is essential to explain why these mechanisms were not evaluated in all cell lines and why only perform most of them in MOLM-13?
7) In lines 248-250 (section 3.5), the authors sorted patient samples into three response groups (SR, IR, and NR). What were the criteria used to divide the samples into these groups?
Minor issues:
1) The quality of the Figures should be improved if possible. The Figure legends should be more uniform (e.g., in the Figure 3 legend, there is no reference to the p values, as is in all the following legends).
2) The authors should review the manuscript for inconsistencies (e.g., in line 20, the HSP90 inhibitor is referred to as PU-H-71)
3) The nomenclature of genes and proteins must be carefully revised throughout the manuscript (e.g., BCL-2 instead of Bcl-2, which represents a mouse protein).
4) In section 2.2. AML Cell Lines (lines 93-100), SKM-1 cell line is not described.
5) In line 131, remove the repeated expression “on GraphPad Prism software”.
6) In Figure 1 (page 5), considering that multiple measurements were performed, standard deviation values should be represented in the graph.
7) There is no section 3.4. The authors go from section 3.3. (page 7, line 207) to section 3.5. (page 9, line 238).
Author Response
Reviewer 1 cimb-2552398
The authors explore the potential of PU-H71, an HSP90 inhibitor, in acute myeloid leukemia cell lines and primary cultures, as a single agent and in combination with S63845 (MCL1 inhibitor) and venetoclax (BCL-2 inhibitor). They also characterize its mechanisms of action and identify possible response biomarkers. The manuscript is well-written, and the methodology used clearly addresses the identified problem. However, some concerns need to be discussed and better explained.
Response: We thank the reviewer for the detailed comments and suggestions. We reworked the data and presented it differently, with several figures added to the supplement. We believe that the manuscript has been substantially improved.
Major issues:
1) The authors present de IC50 values for the three anticancer drugs evaluated in this study. Which method was used to calculate the IC50 values? Did the authors perform dose-response curves for S63845 and venetoclax, as well as for PU-H71?
Response: The concentration of the drug resulting in 50% inhibition of cell viability (IC50) was calculated using three-parameter logistic curve fitting. This sentence has been added in section 2.3. IC50 values for S63845 and venetoclax were calculated, similar to a previous study (Seipel et al., Int J Mol Sci 2022, 23, 12587, doi:10.3390/ ijms232012587). With respect to S63845 and venetoclax, MOLM-13 cells were most susceptible with IC50 values of 0.02 and 0.1 micromolar, respectively, while MOLM-16 cells were most resistant with IC50 values of 10 micromolar. This information has been added in section 3.1.
2) Regarding the combination therapy studies, what criteria were used to choose the different concentrations for each of the three drugs used? For example, different concentrations of venetoclax were used for the different cell lines (100nM for MOLM-13 and ML-2, and 1µM for SKM-1, OCI-AML3, MOLM-16, and PL21).
Response: Drug concentrations in the combination studies were chosen to correspond to minimally effective concentrations in single compound assays determined in initial titration. This sentence has been added in section 3.2.
3) Moreover, the use of different concentration units for the same drugs makes the results very confusing throughout the text and figures. Please, consider using all the concentrations in nM or µM.
Response. Drug concentrations in the cell viability assays in AML cell lines (Fig. 1 and 2) are micromolar. Drug concentrations in cytometry and gene expression studies in AML cell lines (Fig. 3 and 4) as well as cell viability assays in AML patient samples are nanomolar.
4) In section 3.2. (lines 172-185), the authors mention the synergistic or additive effects of the different combination therapies when referring to Figure 3. Can you make those affirmations based only on Figure 3?
Response: For the calculation of combination indexes, three dosages of PU-H71 and two dosages of the BH3 mimetics were applied alone and in combination. Combination indexes were calculated on Compusyn software (version 1.0; ComboSyn, Inc., Paramus, NJ, USA). The data in Figure 3 presents enhanced effects in the combination treatments. The text in section 3.2. was changed accordingly, with enhanced effects on cell viability in the combination treatments.
5) In Table 3 (page 7), the authors present the results of the combination indexes calculated on Compusyn software as a range of values. However, absolute values should be indicated instead.
Response: Combination indexes were calculated for every single experiment. A limited number of repeat experiments were done for every combination in the different cell lines. Combination index values were calculated for every experiment. The resulting combination index values were within the indicated range of values. In order to indicate a precise value, a larger number of repeat experiments would be required.
6) In section 3.3, line 215, it is referred that the protein levels were determined in two cell lines; however, only present the results for MOLM-13 in Figure 4. Cell cycle and mRNA expression analysis are only shown for three cell lines. Moreover, in Figure 5, results for the MOLM-13 cell line are only presented. It is essential to explain why these mechanisms were not evaluated in all cell lines and why only perform most of them in MOLM-13?
Response: Cytometry analysis of cell cycle and annexin V expression was done in the susceptible cell lines MOLM-13, OCI-AML3, ML-2 and SKM-1. mRNA expression was analyzed in MOLM-13, OCI-AML3 and SKM-1 cells. Protein levels were determined in MOLM-13 and OCI-AML3 cells. Results gained from experiments with MOLM-13 cells were most significant and consistent. We now present a more detailed analysis of the protein quantitation in both MOLM-13 and OCI-AML3 cells in the new supplemental Figure S1. We included cytometry results of other cell lines which are now presented in the new supplemental Figure S2.
7) In lines 248-250 (section 3.5), the authors sorted patient samples into three response groups (SR, IR, and NR). What were the criteria used to divide the samples into these groups?
Response: Criteria are based on comparison to responses of normal cells isolated from healthy donors which had a normal response, by definition. The NR response group all had minimal (normal) response. The SR group had responses which were significantly more substantial than the IR and NR groups. The IR group had responses in between the NR and SR group.
Minor issues:
1) The quality of the Figures should be improved if possible. The Figure legends should be more uniform (e.g., in the Figure 3 legend, there is no reference to the p values, as is in all the following legends).
Response: Reference to the p values has been added to Figure 2 and 3 legends.
2) The authors should review the manuscript for inconsistencies (e.g., in line 20, the HSP90 inhibitor is referred to as PU-H-71)
Response: All instances referring to PU-H71 have been amended.
3) The nomenclature of genes and proteins must be carefully revised throughout the manuscript (e.g., BCL-2 instead of Bcl-2, which represents a mouse protein).
Response: We thank the reviewer for pointing out the protein nomenclature guidelines. We have amended all sites referring to human BCL2 or MCL1 protein.
4) In section 2.2. AML Cell Lines (lines 93-100), SKM-1 cell line is not described.
Response: SKM-1 cell line has been added to the Materials and Methods.
5) In line 131, remove the repeated expression “on GraphPad Prism software”.
Response: Duplicate has been removed.
6) In Figure 1 (page 5), considering that multiple measurements were performed, standard deviation values should be represented in the graph.
Response: Standard deviations are represented in new Figure 1.
7) There is no section 3.4. The authors go from section 3.3. (page 7, line 207) to section 3.5. (page 9, line 238).
Response: Chapter numbering has been revised.
Submission Date 26 July 2023
Date of this review 05 Aug 2023 11:08:29
Date of revision: 11 August 2023
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsAn interesting publication by Swiss authors about the role of PU-H71 in the potential treatment of AML.
Here are the comments on the manuscript:
1. the abstract should be redrafted to highlight the most relevant research results and conclusions.
2. I ask authors to check that all abbreviations are explained when they are used for the first time,
3. keywords: there are too many.
4. the introduction is clearly written.
5. the purpose of the paper is clearly defined.
6. materials and methods - generally written carefully, but more patient and ELISA data (CVs, detection rank) should be added.
7. the results are clearly described - congratulations to the authors.
8. discussion - please describe the limitations of your research and what are the clinical consequences of the observations made by the authors.
9. references could be extended:
https://www.mdpi.com/2072-6694/15/3/984
https://www.mdpi.com/1420-3049/25/9/2220?type=check_update&version=2
Author Response
An interesting publication by Swiss authors about the role of PU-H71 in the potential treatment of AML.
Response: We thank the reviewer for the helpful comments.
Here are the comments on the manuscript:
- the abstract should be redrafted to highlight the most relevant research results and conclusions.
Response: The abstract has been redrafted accordingly.
- I ask authors to check that all abbreviations are explained when they are used for the first time.
Response: Introduction of abbreviations have been checked.
- keywords: there are too many.
Response: Keywords were limited to ten.
- the introduction is clearly written.
- the purpose of the paper is clearly defined.
- materials and methods - generally written carefully, but more patient and ELISA data (CV-coefficient of variation should be <20%, detection rank-detection range? should be added.
Response: ELISA assays with detection range of 0.312-20ng/mL and intra-assay CV<10%, has been added to materials and methods.
- the results are clearly described - congratulations to the authors.
- discussion - please describe the limitations of your research and what are the clinical consequences of the observations made by the authors.
Response: In order to address a potential association of treatment response to HSP90 inhibitors with TP53 and PTPN11 mutations in primary AML, and to confirm the response markers CD34, CD117 and CD11b, preclinical studies in larger cohorts are be required. This sentence was added at the end of the discussion.
- references could be extended:
https://www.mdpi.com/2072-6694/15/3/984
Response: New Review Article has been added to the references in the introduction.
Submission Date 26 July 2023
Date of this review 08 Aug 2023 09:12:22
Date of revision 11 August 2023
Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThis work is well performed and clearly presented. It merits to be accepted as it stands with minor revision. The message is somehow expected, but actually the effort of the authors in analysing primary and AML cell lines merits to be considered. Also, the experiments are clear, and the manuscript is readable and informative.
Comments on the Quality of English LanguageEnglish language is good.
Author Response
This work is well performed and clearly presented. It merits to be accepted as it stands with minor revision. The message is somehow expected, but actually the effort of the authors in analysing primary and AML cell lines merits to be considered. Also, the experiments are clear, and the manuscript is readable and informative.
Response: We thank the reviewer for the concise and positive feedback. We have revised the manuscript according to the suggestions of the other reviewers and consider the revised version to be substantially improved.
Comments on the Quality of English Language
English language is good.
Submission Date 26 July 2023
Date of this review 04 Aug 2023 08:15:19
Date of revision 11 August 2023
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors explore the potential of PU-H71, an HSP90 inhibitor, in acute myeloid leukemia cell lines and primary cultures, as a single agent and in combination with S63845 (MCL1 inhibitor) and venetoclax (BCL-2 inhibitor). They also characterize its mechanisms of action and identify possible response biomarkers. They replied to the raised concerns and improved the manuscript.
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have addressed my concerns and improved the quality of the manuscript.
