Low Renalase Levels in Newly Diagnosed CML: Dysregulation Sensitive to Modulation by Tyrosine Kinase Inhibitors

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Please, see all the comments in the attached file.

In addition, I think that the title “Assessment of renalase in chronic myeloid leukemia and within onco-inflammatory mechanisms”, should be changed. 

I do not find an interesting title. Maybe the authors could use the results to find a better title....like " CML patients present low levels of Renalase enzyme: Implications in......

Thank you.

 

 

 

 

Comments for author File: Comments.pdf

Author Response

Dear Editors and Reviewers,

Thank you for giving us the opportunity to submit a revised manuscript with the modified title “Low renalase levels in newly diagnosed CML: dysregulation sensitive to modulation by tyrosine kinase inhibitors” to your journal Pathophysiology. We thank you for the time and effort you dedicated to reading and critically appraise our manuscript. We did our best to incorporate the suggested changes by the reviewers. We marked text changes with red font within the revised manuscript. Here is a point-by-point response to the reviewers’ comments and concerns.

We thank the reviewer for the time and effort dedicated to reading and providing us with critical and insightful comments and suggestions. We addressed all the comments carefully. In addition, we corrected the literature and included the suggested reference.

1) Introduction section

Comment 1.1. It is well known that among hematologic malignancies, the classic Philadelphia-negative chronic myeloproliferative neoplasms (MPNs) are considered a model of inflammation-related cancer development. But I did not see any reference in introduction about CML and oncoinflammation. Authors need to improve this statement in introduction. The all four references cited in the first page, line 4 refer to myelodysplastic, acute myeloid leukemia and classic Philadelphia-negative chronic myeloproliferative neoplasms, not CML.

Response 1.1. Thank you for your insight. We corrected the literature by providing adequate references. Therefore, instead of reference 2 (Masselli et al) we added a reference:

Gallipoli, P.; Pellicano, F.; Morrison, H.; Laidlaw, K.; Allan, E. K.; Bhatia, R.; Copland, M.; Jørgensen, H. G.; Holyoake, T. L. Autocrine TNF-alpha production supports CML stem and progenitor cell survival and enhances their proliferation. Blood 2013, 122, 3335–3339. doi: 10.1182/blood-2013-02-485607

[.. We observed that CML stem/progenitor cells (SPCs) produce tumor necrosis factor-α (TNF-α) in a kinase-independent fashion and at higher levels relative to their normal counterparts. We therefore investigated the role of TNF-α and found that it supports survival of CML SPCs by promoting nuclear factor κB/p65 pathway activity and expression of the interleukin 3 and granulocyte/macrophage-colony stimulating factor common β-chain receptor. Furthermore, we demonstrate that in CML SPCs, inhibition of autocrine TNF-α signaling via a small-molecule TNF-α inhibitor induces apoptosis. Moreover TNF-α inhibition combined with nilotinib induces significantly more apoptosis relative to either treatment alone and a reduction in the absolute number of primitive quiescent CML stem cells.]

And instead of reference 4 (Camacho et al) we added a reference:

Welner, R.S.; Amabile, G.; Bararia, D.; Czibere, A.; Yang, H.; Zhang, H.; Pontes, L. L.; Ye, M.; Levantini, E.; Di Ruscio, A.; Martinelli, G.; Tenen, D. G. Treatment of chronic myelogenous leukemia by blocking cytokine alterations found in normal stem and progenitor cells. Cancer Cell 2015, 27, 671-81. doi: 10.1016/j.ccell.2015.04.004.

[.. Interestingly, the normal bystander cells acquired gene expression patterns resembling their malignant counterparts. Therefore, much of the leukemia signature is mediated by extrinsic factors. Indeed, IL-6 was responsible for most of these changes... From our analysis of leukemic-exposed HSCs and KSL/progenitors, we thought that the inflammatory environment might mediate many of the observed changes. From our model, we measured serum cytokine levels (Figure 4C). Consistent with data from others (Reynaud et al., 2011; Zhang et al., 2012), several inflammatory cytokines, including GM-CSF, IL-6 and IL-1α were significantly increased during leukemogenesis.. ]

We also relocated the reference 27 (Shen et al) as it supports the statement;

Shen N, Liu S, Cui J, Li Q, You Y, Zhong Z, Cheng F, Guo AY, Zou P, Yuan G, Zhu X. Tumor necrosis factor α knockout impaired tumorigenesis in chronic myeloid leukemia cells partly by metabolism modification and miRNA regulation. Onco Targets Ther. 2019;12:2355-2364. doi: 10.2147/OTT.S197535.

There are several other possible reference options. If you consider another option better suited, we kindly ask you to propose the best option.

Examples:

1. Giustacchini A., Thongjuea S., Barkas N., Woll P.S., Povinelli B.J., Booth C.A.G., Sopp P., Norfo R., Rodriguez-Meira A., Ashley N., et al. Single-cell transcriptomics uncovers distinct molecular signatures of stem cells in chronic myeloid leukemia. Nat. Med. 2017;23:692–702. doi: 10.1038/nm.4336.
2. Nievergall, E.; Reynolds, J.; Kok, C. H.; Watkins, D. B.; Biondo, M.; Busfield, S. J.; Vairo, G.; Fuller, K.; Erber, W. N.; Sadras, T.; Grose, R.; Yeung, D. T.; Lopez, A. F.; Hiwase, D. K.; Hughes, T. P.; White, D. L. TGF-alpha and IL-6 plasma levels selectively identify CML patients who fail to achieve an early molecular response or progress in the first year of therapy. Leukemia 2016, 30, 1263–1272. doi: 10.1038/leu.2016.34.
3. Reynaud D, Pietras E, Barry-Holson K, Mir A, Binnewies M, Jeanne M, Sala-Torra O, Radich JP, Passegué E. IL-6 controls leukemic multipotent progenitor cell fate and contributes to chronic myelogenous leukemia development. Cancer Cell. 2011;20(5):661-73. doi: 10.1016/j.ccr.2011.10.012.
4. Agarwal, P., Li, H., Choi, K., Hueneman, K., He, J., Welner, R. S., Starczynowski, D. T., & Bhatia, R. (2021). TNF-α-induced alterations in stromal progenitors enhance leukemic stem cell growth via CXCR2 signaling. Cell reports, 36(2), 109386. doi: 10.1016/j.celrep.2021.109386
5. Đikić D, Bogdanović A, Marković D, Mitrović-Ajtić O, Subotički T, Diklić M, Vukotić M, Dragojević T, Živković E, Santibanez JF, Čokić VP. Inflammation Promotes Oxidative and Nitrosative Stress in Chronic Myelogenous Leukemia. Biomolecules. 2022;12(2):247. doi: 10.3390/biom12020247.

Comment 1.2. Furthermore, in the line 47, the authors have stated the relationship between oncoinflammation and NF-Kb. I suggest including references about CML and BCR-ABL/NF-κB. For example: The BCR-ABL/NF-κB signal transduction network: a long lasting relationship in Philadelphia positive Leukemias (doi: 10.18632/oncotarget.11507);

Response 1.2. Thank you very much for the comment and suggestion. We added the suggested reference.

Carrà G, Torti D, Crivellaro S, Panuzzo C, Taulli R, Cilloni D, Guerrasio A, Saglio G, Morotti A. The BCR-ABL/NF-κB signal transduction network: a long lasting relationship in Philadelphia positive Leukemias. Oncotarget. 2016 Oct 4;7(40):66287-66298. doi: 10.18632/oncotarget.11507.

Comment 2.1. 2) Materials and methods section

2.1. Please include a table with each CML patient data, such as: gender, age, TKI intake (type of TKI and dose) and response (BCR::ABL1 levels or molecular response or cytogenetic response); Sokal risk, etc. It is important to know if the patients are in remission pos-TKI therapy.

Response 2.1. Thank you for the suggestion. According to our inclusion criteria, the group of patients on TKI treatment comprised those who were in remission. We mentioned this in the result section: “..was in a complete hematological and cytogenetic remission, most of with the major molecular response (MMR or MR3, BCR-ABL1 values of  ≤ 0.1% IS) present in 80% (n=44) at the last control“.

However, for better understanding, we changed the first two sentences in the Method section to „This prospective cross-sectional case-control investigation included 75 consecutive adult patients diagnosed with CML in stable chronic-phase disease. There are two groups of patients: (1) a group with 55 patients already taking TKI therapy for at least 6 months and who are in hematological and cytogenetic remission, and (2) a group of 20 newly diagnosed CML treatment naïve patients.“

We discussed the suggestion of including a table with patients' data, with 55+ rows, but agreed this wouldn't be practical or usual. Also, it would not be conclusive without a statistical analysis. However, with the abovementioned clarification of the inclusion criteria, it may not be necessary.

Some of the data are placed in the Result section, such as gender, age, and the TKI type “About half of them received imatinib (45.5%), 400 mg once daily, or nilotinib (54.5%), 300 mg twice daily.”

Comment 2.2. I suggest to include a third study group with CML patients resistant to TKI. Do you still have the samples from CML patients who lost IM response before use nilotinib? All the CML patients were in the chronic phase at the time of the blood collection.

Response 2.2. Thank you very much for the proposition, this is an interesting idea. However, we do not have samples from patients who just lost their response and before they switched to nilotinib. This was a cross-sectional investigation, with samples gained from all patients currently at the clinic - those already taking TKI in remission or newly diagnosed without any treatment.

All the patients were in the chronic phase of CML (no acceleration or blasts were detected).

It would be very interesting, for a future investigation, to follow a group of patients and take serial measurements of RNLS to assess changes in its levels concerning disease course and response to TKI treatment, as well as to observe what happens when a response to TKI is lost.

Comment 2.3. Please, inform the exclusion criteria for the control group. It is important to mention that none of them are using anti-inflammatory drugs.

Response 2.3. Yes, thank you, we added a notion in the sentence describing the control group that they were not taking any medication, in the Methods section. “The control group comprised 20 healthy community-based adult volunteers, not taking any medications, and who were age-matched to the patients.”  

Comment 3.1. 3) Results section; 3.1. The results presented in the Table 2 should be exchanged by a figure using a dot-plot graphic;

Response 3.1. Thank you, we changed Table 2 for a figure (Figure 1) showing a dot-plot graphic of RNLS levels by groups. (The Figure 1 can be seen in the pdf file attached)

Figure 1. Comparison of the RNLS concentration between the groups. * p=0.000 compared to the CML treatment-naïve group.

Comment 3.2. Is there any difference of RNLS levels between CML patients with low and intermediate Sokal score?

Response 3.2. There was no significant difference between the Sokal scores. The result is shown in Table 1 but we also added a sentence “We did not determine a significant difference between RNLS levels according to the Sokal score.”

Comment 3.3. Please, try to perform a correlation analysis between BCR::ABL1 levels and RNLS concentration, with the data you have. I know that you state in the line 147, that you do not have all BCR::ABL1 levels data from patients.

Response 3.3. Yes, it would be interesting to assess this association. Unfortunately, we are not able to provide adequate results on this issue. We receive part of these data as confirmation that levels are or are not below 0.1% BCR::ABL1 level, or 0.001%.

Comment 3.4. Do authors still have the plasma of the patients? Can you do Protein C Reactive measure to state the inflammatory status of the CML patients and controls?

Response 3.4. We performed additional analysis of CRP in the patients' samples. And determined significant correlation with renalase but only in treatment naïve patients. We added this result as sentences: “Although around the upper limit of the reference range, the patient’s CRP levels were significantly higher compared to controls but without marked differences between the groups (Table 1). Still, only the values in treatment naïve CML patients correlated significantly and positively with RNLS concentration (p=0.008, r=0.620).“ Also, we added a note in the Abstract.

The results are also discussed as: „Renalase positively correlated with CRP in newly diagnosed CML patients, reiterating its relationship with inflammation and modulatory effects of TKI. Several studies report RNLS association with markers of inflammation but without consistency, that is, while being positively correlated with CRP in peritoneal dialysis patients, it was negatively correlated with ferritin, WBC, and IL-1 values in COVID-19 [Safdar, Gok Oguz].“

Gok Oguz, E.; Akoglu, H.; Ulusal Okyay, G.; Karaveli Gursoy, G.; Yildirim, T.; Merhametsiz, O.; Cimen, T.; Canbakan, B.; Yeter, E.; Ayli, M.D. (2017). Increased serum renalase in peritoneal dialysis patients: Is it related to cardiovascular disease risk?. Nefrologia : publicacion oficial de la Sociedad Espanola Nefrologia 2017, 37, 189–194. doi:10.1016/j.nefro.2016.11.013

Safdar, B.; Wang, M.; Guo, X.; Cha, C.; Chun, H.J.; Deng, Y.; Dziura, J.; El-Khoury, J.M.; Gorelick, F.; Ko, A.I.; Lee, A.I.; Safirstein, R.; Simonov, M.; Zhou, B.; Desir, G.V. Association of renalase with clinical outcomes in hospitalized patients with COVID-19. PLoS One 2022, 17, e0264178. doi: 10.1371/journal.pone.0264178

Comment 4.1. 4) Discussion Section; 4.1. The authors did not state the relevance of their findings. Why low levels of RNLS are relevant for CML patients? For CML pathogenesis? For CML therapy? To design a new therapy for CML?

Response 4.1. Thank you for drawing our attention to this issue. We changed the text at the end of the Discussion Section, elaborating on the relevance of our findings. 

“Our research provides new information in the field of hematological malignancies. The relevance of our study is reflected in the confirmation that RNLS is altered in this malignancy as well. However, unlike malignancies of another nature, RNLS plasma levels are significantly decreased. Treatment-naïve CML patients display lower values than those on TKI treatment suggesting RNLS alteration is sensitive to modulation by TKI.

Chronic myeloid leukemia is a dynamic and complex disease with pathogenic inputs coming from the oncogene, microenvironment, metabolism, miRNA, etc. The functions of RNLS, as a multifaced molecule, may perhaps be exploited altogether in this intertwined nature of CML. It can be seen as one of the central elements whose modulation may affect several of these routes at the same time. In future perspectives, RNLS can be employed in diagnostic, prognostic, or even therapeutic options.”

Comment 4.2. Is there any relation between RNLS levels and TKI response?

Response 4.2. We can not assess RNLS relation to TKI response, because we do not have blood samples for these patients before they started TKI treatment.

Comment 5. In addition, I think that the title “Assessment of renalase in chronic myeloid leukemia and within onco-inflammatory mechanisms”, should be changed.  I do not find an interesting title. Maybe the authors could use the results to find a better title....like " CML patients present low levels of Renalase enzyme: Implications in...... Thank you.

Response 5. Thank you for the suggestion. We changed the title to: “Low renalase levels in newly diagnosed CML: dysregulation sensitive to modulation by tyrosine kinase inhibitors”.

I hope you will find this title more interesting.

Kind regards. Sincerely, Corresponding author

 

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Comments to the Author

In the manuscript entitled “Assessment of renalase in chronic myeloid leukemia and within onco-inflammatory mechanisms”, the authors presented a study related to renalase (RNLS) level in the blood, in patients with chronic myeloid leukemia (CML) in the chronic phase, treatment naive and under tyrosine kinase inhibitors (TKI) treatment (imatinib or nilotinib).

Overall, the paper is well-written. It is informative and interesting to read.

There are a rationale for asessment of renalase in CML, given that the signaling pathways that are dysregulated in CML, are relevant for RNLS.  Renalase expression is changed in cancer, and  not evaluated in hematologic malignancies. The topic is very interesting. The most important results are: 1) there are significantly decreased RNLS plasma levels in patients with CML compared to controls as well as lower levels in treatment naive than patients  taking TKI; 2) RNLS alteration seems to be sensitive to modulation by TKI; 3) a significant negative correlation was present between the RNLS concentration and total leukocyte count and neutrophil count in the blood.

It should be noted that neutrophils are part of the malignant clone in CML.

Some suggestions are listed below:

 

Title:

I suggest a slight change of a title:

“Assessment of renalase in chronic myeloid leukemia and within onco-inflammatory mechanisms”

for example:

“Assessment of renalase in chronic myeloid leukemia: dysregulated expression sensitive to modulation by tyrosine kinase inhibitors”

(or something else, it is just a suggestion, considering that the paper does not examine onco-inflammatory mechanisms in CML).

 

Abstract:

I suggest that the sentence:

“Also, RNLS signaling network affects mitochondrial energy production, making it a susceptible target, especially considering the metabolic adaptations of leukemia stem cells, which all together entail further investigations”.

should be deleted from the abstract, considering that it was not the subject of their examination.

 

The authors themselves presented the advantages and disadvantages of their study.

Disadvantages:

Authors: “There are several limitations of our study. First, this is a single-center clinical investigation and included a relatively small number of patients. The study included only CML patients, however, there is a rational need to explore a RNLS role in Ph(-) MPNs and bone marrow fibrosis cases, given the RNLS anti-fibrotic effects. We did not perform any in vitro or in vivo experiments, which undoubtedly need to be implemented to delineate the pathophysiological mechanisms and relevance of RNLS in CML.”

I agree with them.

However, I think that the results are significant and intriguing (Authors: “The low RNLS levels were somewhat surprising given the enhanced activity of its inflammatory stimuli, high ROS production (BCR-ABL1-induced via PI3K/Akt, 212 STAT1 and STAT5), and evidence of increased RNLS levels (blood and tissue) in several other malignancies [6,17,22,32,33]. A question is whether the decreased RNLS level is a result of the oncogene-directed transformation, or a secondary consequence of local signaling (mal)adaptations, in transformed or neighboring cells.”), and that the paper should be published. Myeloproliferative neoplasms (MPN) are complex diseases, while renalase are multifunctional protein, synthetized and secreted by multiple cell types. It would be important to see the cellular origin of RNLS in MPN. (Authors: “Dysregulated microenvironment is considered one of the main reasons why current TKI therapies do not secure complete control of the disease [1,2,7-10].”)

This is an important study that has the potential to open the door to a new approach to research in MPN.

Comments on the Quality of English Language

Minor editing of English language required.

Author Response

Dear Editors and Reviewers,

Thank you for giving us the opportunity to submit a revised manuscript with the modified title

“Low renalase levels in newly diagnosed CML: dysregulation sensitive to modulation by tyrosine kinase inhibitors” to your journal Pathophysiology.

We thank you for the time and effort you dedicated to reading and critically appraise our manuscript. We did our best to incorporate the suggested changes by the reviewers. We marked text changes with red font within the revised manuscript. Here is a point-by-point response to the reviewers’ comments and concerns.

Sincerely,

Corresponding author

 

Reply to the Second Reviewer’s comments

We thank the reviewer for the time and effort dedicated to reading and providing us with critical and insightful comments and suggestions. We addressed all the comments carefully.

Comment 1. It should be noted that neutrophils are part of the malignant clone in CML.

Response 1. Thank you for the insight, we included neutrophil count association in the Abstract and added a sentence in the Discussion section emphasizing this issue “An additional negative correlation existed with the neutrophil count, the cells that make a substantial part of the CML clone “.

Comment 2. Title: for example: “Assessment of renalase in chronic myeloid leukemia: dysregulated expression sensitive to modulation by tyrosine kinase inhibitors”

Response 2. Thank you for the suggestion. We changed the title to: “Low renalase levels in newly diagnosed CML: dysregulation sensitive to modulation by tyrosine kinase inhibitors”. I hope you will find this title more interesting.

Comment 3. Abstract: I suggest that the sentence: “Also, RNLS signaling network affects mitochondrial energy production, making it a susceptible target, especially considering the metabolic adaptations of leukemia stem cells, which all together entail further investigations”, should be deleted from the abstract, considering that it was not the subject of their examination.

The authors themselves presented the advantages and disadvantages of their study.

Response 3. We replaced the sentence with another that referenced the relevance of our findings:

„Overall, our research provides new insights into the field of hematological malignancies. Unlike other malignancies studied, RNLS plasma levels are significantly decreased in CML. In future perspectives, RNLS could potentially serve as a diagnostic, prognostic, or therapeutic option for these patients.“

Comment 4. Disadvantages. I agree with them. However, I think that the results are significant and intriguing (Authors: “The low RNLS levels were somewhat surprising ...”), and that the paper should be published. Myeloproliferative neoplasms (MPN) are complex diseases, while renalase are multifunctional protein, synthetized and secreted by multiple cell types.

It would be important to see the cellular origin of RNLS in MPN. (Authors: “Dysregulated microenvironment is considered one of the main reasons why current TKI therapies do not secure complete control of the disease [1,2,7-10].”). It would be important to see the cellular origin of RNLS in MPN.

Response 4. Indeed, it would be interesting to test the status of RNLS in the bone marrow, perhaps exploring ex vivo cell culture, which we will try in the future.

Comment 5. This is an important study that has the potential to open the door to a new approach to research in MPN.

Response 5. Thank you very much for your supporting words and suggestions.

Comment 6. Minor editing of English language required.

Response 6. We performed additional English language checks, using the Grammarly software, and marked those changes in the text with red font letters.

 

Kind regards.

Sincerely,

Corresponding author

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors answered the questions appropriately. I have no further suggestions or concerns.