Exploring the Role of Macrophage Marker CD68 in Pediatric Acute Myeloid Leukemia

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Summary

This manuscript investigates CD68 expression in pediatric AML reporting correlations with KMT2A-rearrangements and inv(16) and worse event-free survival. Functional studies suggest CD68 promotes proliferation. The topic is clinically relevant given the need for new therapeutic targets. However, concerns about experimental controls and data interpretation should be addressed.

Major comments

Functional assays require additional controls (Figure 4)

1. The proliferation assay used measures metabolic activity rather than cell number. To support the conclusion that CD68 KD reduced proliferation, I recommend distinguishing whether cells proliferate slower or die more. Absolute viable cell counts using flow cytometry and/or Annexin V/PI staining would clarify this.
2. The cytarabine results are somewhat surprising. CD68 KD cells proliferate slower yet show increased sensitivity. Typically, slowly proliferating cells are spared by such agents. Can the authors comment on this? Can authors show that with a different chemotherapeutic like doxorobucin?
3. I think comparing CD68 knock-down in MLL-rearranged THP-1 with OE in APL-derived HL-60 cells limits direct comparison. Can the authors perform overexpression in THP-1 cells? This way, it stays relevant to MLL-rearranged AML and the conclusions are more solid.

Cell cycle interpretation needs clarification (Figure 4E-F)

1. The Results state knock-down cells are "blocked in S-phase" but the Discussion mentions "blocking at G1/S". These are mechanistically different. S-phase accumulation suggests S-phase arrest (cells entered but cannot complete replication), possibly indicating replication stress. BrdU/EdU incorporation or γH2AX staining could distinguish actively replicating from stalled cells.
2. Please clarify what "<G1" and ">G2" represent? Representative flow plots with gating strategy would help readers assess data quality.

Missing details in figure legends. Several details are missing. Biological vs. technical replicate numbers. Figure 4 lacks n values and statistical tests. How CPM cutoffs for survival analyses were determined (median split? optimal cutpoint?).

Minor comments

1. Flow cytometry (Figure 3): The authors note high CD68 in normal monocytes and acknowledge potential toxicity. Could they expand on expected monocyte/macrophage recovery timelines, given relevance to clinical feasibility?
2. RNA-seq findings: The asymmetry of 1,422 upregulated vs. 43 downregulated genes in KD is striking. The Discussion mentions CD68 may act as a "transcriptional regulator" but CD68 is a lysosomal protein. Is there evidence for nuclear localization.
3. Immune biology: While the authors demonstrate cell-intrinsic effects of CD68 in AML cells, it remains unclear whether CD68 expression also directly contributes to immune suppression by AML cells themselves, given the established role of CD68+ M2-like macrophages and myeloid-derived suppressor cells in creating immunosuppressive microenvironments. Co-culture assays with PBMCs could determine whether CD68-knockdown AML cells exhibit reduced suppression of T cell proliferation or effector function compared to controls. At minimum, the discussion should address whether CD68 may play an immunomodulatory role in AML beyond its effects on leukemia cell proliferation.

                                            

Author Response

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Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Camp et al. present data showing that the macrophage marker CD68 is upregulated in pediatric acute myeloid leukemia (pedAML) relative to normal hematopoietic stem cells, and they report associations of CD68 expression with clinical features and outcome. The topic is clinically relevant and the dataset is of interest; however, the manuscript in its current form has several substantial limitations that must be addressed before it can be considered for publication.

Major concerns:

1. The manuscript does not provide sufficient evidence that CD68 acts as a proto-oncogene in pedAML. I recommend the authors perform an overlap analysis of RNA-seq (or comparable transcriptomic) data from the CD68 knockdown model (THP-1) and the CD68 overexpression model (HL-60) to identify concordant candidate downstream effectors. Candidate overlapping genes should then be validated (qPCR and, where feasible, functional perturbation) in additional AML cell lines to support mechanistic claims.
2. Functional conclusions are currently based on knockdown in a single cell line (THP-1) and overexpression in a single cell line (HL-60). To exclude cell-line–specific artifacts, key functional assays (proliferation, apoptosis, colony formation) should be replicated in at least two additional, genetically distinct AML cell lines. If primary patient samples or patient-derived xenograft material are available, inclusion of such data would strengthen translational relevance.
3. The Discussion should more fully compare the present results with relevant prior studies (for example, the works the authors cite such as PMID:35123607 and PMID:7669655). The authors should discuss plausible mechanisms by which CD68 could promote oncogenesis in AML.

In conclusion. The study addresses an important question, but at present the mechanistic claims are not sufficiently supported and the experimental validation is limited. Addressing the major concerns above—particularly the RNA-seq overlap/validation and replication in additional AML models—would substantially strengthen the manuscript.

Author Response

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Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Overall, this manuscript addresses an interesting question regarding the role of CD68 in pediatric AML, and the correlative data in patient samples are valuable. However, several critical aspects of the functional work need strengthening before the study can support the conclusion that CD68 represents a potential therapeutic target in AML.

1. Expand functional validation using macrophage-depleting or macrophage-inhibitory strategies
Since CD68 is a macrophage-associated marker and not a classical druggable protein, the authors should evaluate compounds known to reduce macrophage activity or deplete macrophages. For example, clodronate liposomes, which selectively deplete macrophages, could be tested to determine whether suppressing macrophage-like features affects AML cell growth or drug response. Importantly, these experiments should be performed in at least two  cell lines to validate that the findings are not cell line-specific.

2. Compare knockdown and overexpression effects in matched systems
Figure 4 compares knockdown in THP-1 cells with overexpression in HL-60 cells. These are 2 distinct cell models, which complicates interpretation. Ideally, the authors should show CD68 knockdown and overexpression side-by-side in the same cell line. This would more clearly demonstrate whether CD68 exerts consistent proto-oncogenic effects.

3. Include cytospin analyses to assess phenotypic changes
Given that CD68 is linked to phagocytic and macrophage-related programs, cytospin and morphological changes would be highly informative. Morphological assessment after CD68 knockdown, overexpression, or treatment (e.g., cytarabine) could reveal differentiation or phenotypic changes that are currently missing from the manuscript. This would be an important addition for understanding the biological relevance of CD68 and for determining any synergy exists between cytarabine treatment and CD68 knockdown

4. Need for stronger phenotypic and treatment-response effects
While the patient sample data are compelling, the functional experiments in cell lines show modest effects. To justify consideration of CD68 as a therapeutic target in AML, the manuscript would benefit from clearer and stronger phenotypic changes, either through more robust CD68 modulation, additional drug sensitivity assays, or deeper mechanistic exploration.

Author Response

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Author Response File: Author Response.pdf