Epigenetic Modulation of Self-Renewal Capacity of Leukemic Stem Cells and Implications for Chemotherapy

Round 1

Reviewer 1 Report

The paper by Monparler et al. Focuses on the interplay between the effects of IDH mutations, epigenetic modifications and differentiation in AML.

The topic is interesting for those in the field and the paper is generally well-written.

The major issue is that it is not clear if it is a review or a paper with novel data. Reading the abstract it seems that it is a paper with data, but the amount of new data is limited to Figure 3 and too preliminary.

If it is a Review the Authors should remove Figure 3 and focus their discussion on already published works. All the text should be re-organized accordingly, including the Abstract.

Minor points:

Some sentences are not very clear or difficult to follow (e.g. lines 66-68). Please check throughout the manuscript for clarity.

Title of paragraph 5 (line 129) is too generic, it can be substitutes for example with “Epigenetic enzymatic functions that can suppress leukemogenesis”

Author Response

Response to Reviewer 1

Comment: If it is a Review the Authors should remove Figure 3 and focus their discussion on already published works. All the text should be re-organized accordingly, including the Abstract.

Response: We agree with the reviewer and we have deleted Fig 3 and legend Fig 3 on page 5 and revised the comments made to this figure.

REVISIONS

Abstract lines 12:  One aspect of LSCs that is poorly understood is the low frequency of these cells in AML patients. Replace sentence by:“One aspect of LSCs that is poorly understood is their low frequency in the total population of leukemic cells in patients.”

Lines 23-25: “We demonstrate using a colony assay that epigenetic agents that inhibit DNA and histone methylation interact in an additive or synergistic manner to also reduce the proliferative potential of AML cells.”

Replace sentence by:“Epigenetic agents that inhibit DNA and histone methylation exhibit a synergistic antineoplastic action on AML cells.”

Line 53: “This very low frequency can be explained by the fact that after each cell division of LSCs many of the daughter cells lose their self-renewal capacity.”

Replace sentence by: “This very low frequency can be explained by the fact that after each cell division of LSCs most of the daughter cells lose their self-renewal capacity.”

“When IDH is mutated, its catalytic activity is modified so as to convert α-KG to 2-hydroxyglutarate (2-HG), an oncometabolite that by its competition with α-KG inhibits the TET pathway and Jumonji-C domain histone demethylases (JHDM).  (very long and difficult to follow) This inhibition by 2-HG blocks the induction of differentiation of mut-IDH-AML cells by preventing gene activation by DNA demethylation by its inhibition of the TET pathway and by preventing gene activation by histone demethylation by its inhibition of the JHDMs [11] (see Fig 1).”

Lines 62-67 :Replace 2 sentences by 3 sentences:  “The catalytic activity of mut-IDH is modified so as to convert α-KG to 2-hydroxyglutarate (2-HG). 2-HG acts as a competitive inhibitor of α-KG resulting in the inhibition of the catalytic activity of the enzymes of the TET pathway and Jumonji-C domain histone demethylases (JHDMs). This inhibition by 2-HG prevents gene activation by DNA and histone demethylation in mut-IDH-AML cells and blocks their differentiation (see Fig 1).

Line 75: correct spelling of “methylatransferase” to “methyltransferase”

Line 108: change “Additional support that aberrant DNA methylation” to: “Support that aberrant DNA methylation”

Line 128: change “5. Enzymatic Function That Can Suppress Leukemogenesis”

to: Epigenetic Enzymatic Functions That Can Suppress Leukemogenesis”

Lines 146-147: change: “Underexpression of JHDM2B is observed in AML patients suggesting that it can function as a TSG in mleukemic cells” to: “Reduced expression of JHDM2B is observed in AML patients suggesting that it can function as a TSG in leukemic cells”

Line 151: change: “The interaction of between these epigenetic alterations” to: “The interaction between these epigenetic alterations”

Line 169: change: “TSG” to: “TSGs”

Lines 171-173 replace with the following setence: “We observed that 5AZA-CdR in combination with this HMT inhibitor of G9a (EHMT2) exhibited synergistic antileukemic action on AML cells as determined by colony assay [34].”

This sentence contains a new reference {34]: Momparler RL, Côté S, Momparler LF. Enhancement of the antileukemic action of 5-aza-2’-deoxycytidine and 3-deazaneplanocin-A by inhibition of histone methyltransferase G9a (EHMT2) and by vitamin C (unpublished; manuscript in preparation).

Lines 189-190: revised to:  The triple combination of 5AZA-CdR, DZNep and BIX-01294 exhibited greater antineoplastic action against the leukemic cells than any of the double combinations [34].

Lines 208-210: delete “This hypothesis is supported by the data exhibited in Fig. 3 that indicate that the combinations of epigenetic agents that inhibit DNA and histone methylation exhibit a very positive antileukemic interaction.”

Replace by the following sentence:“This hypothesis is supported by the remarkable antineoplastic synergy on leukemic cells exhibited by combinations of epigenetic agents that inhibit DNA and histone methylation [30,31].”

Line 222: change: “self-renewal they have” to: “self-renewal, they have to”

Line 233-239: revised paragraph The remarkable antileukemic synergy observed between 5AZA-CdR and DZNep was first observed in human HL-60 myeloid leukemia cells [30]. An important question is whether this novel epigenetic therapy has potential to be effective against other types of malignancies. This antineoplastic interaction was also observed in murine L1210 lymphoid leukemia cells [30] and in human A549 lung carcinoma cells [42]. These observations suggest that these epigenetic interactions also occur in other cancers. This signifies that the combination of 5AZA-CdR and DZNep may also have the potential to be effective therapy for other types of malignancies

Reference 40 up date with complete citation

[40] Momparler RL, Côté S, Marquez VE, Momparler LF. Comparison of the antineoplastic action of 3-deazaneplanocin-A and inhibitors that target the catalytic site of EZH2 histone methyltransferase. Cancer Rep Rev2020;3:1-4.

Reviewer 2 Report

The topic of this article appears to be scientifically interesting and sound. However, the additional presentation of study results using various leukemic cells lines would be needed because the data presented in Fig 3 of this article was deduced from HL-60 cells originated from acute promyelocytic leukemia cells which did not represent all of the characteristics in acute myeloid leukemia cells. 

Author Response

Response to Reviewer 2

Comment:The topic of this article appears to be scientifically interesting and sound. However, the additional presentation of study results using various leukemic cells lines would be needed because the data presented in Fig 3 of this article was deduced from HL-60 cells originated from acute promyelocytic leukemia cells which did not represent all of the characteristics in acute myeloid leukemia cells.

Response:We agree with this comment. However, it should be mentioned the synergistic interaction between 5AZA-CdR and DZNep has been observed in murine L1210 leukemia cells, a cell line of lymphoid origin (ref 30). In addition, the synergistic interaction between these epigenetic agents occurs in human 549 lung carcinoma cells (ref 41). These observations suggest that the interesting interactions between inhibitors of DNA and histone methylation also occurs in other types of malignancy, not only in AML. This signifies that the combination of 5AZA-CdR and DZNep has the potential to be effective therapy for different types of malignancies.

Insertion of additional text to the Discussion:

            The remarkable antileukemic synergy observed between 5AZA-CdR and DZNep was first observed in human HL-60 myeloid leukemic cells [30,31]. An important question is whether this novel epigenetic therapy has potential to be effective against other types of malignancies. This antineoplastic interaction was also observed in murine L1210 lymphoid leukemia cells [30] and in human A549 lung carcinoma cells [40]. These observations suggest that these epigenetic interactions also take place in other cancers. This signifies that the combination of 5AZA-CdR and DZNep may also have the potential to be effective therapy for other types of malignancies.

The following reference was added to the list of references

Round 2

Reviewer 1 Report

I do not have further major issues, just a couple of text modifications:

Abstract - Line 17: I suggest the following correction: "...an inhibitor OF the TET pathway..."

Line 120: please check this sentence. I suggest the following correction: "...that this oncogenic action of EZH2 LOSS observed..."