Dear Dr. Liu,

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Kota V Ramana, Ph.D.

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: No

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: No

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Here are the suggestions for improvement:

1.Insufficient evidence for "pan-apoptosis": Only classical apoptosis markers were examined, without validation of pyroptosis or necroptosis.

2.Inconsistent terminology: "pan-apoptosis" and "PANoptosis" are used interchangeably throughout the text.

3.Results section ("Establishment and validation of DC-STAMP overexpression and knockdown THP-1 cell models"): The statement "Western blot further confirmed decreased DC-STAMP protein levels in the KD group (P < 0.01) and an xx-fold increase in the OE group (P < 0.001) relative to NC (Figure 2C)" lacks specific data values.

Reviewer #2: Comments to the author,

DC-STAMP Activates the PI3K/AKT/mTOR Signaling Pathway to Regulate Pan-Apoptosis in Acute Myeloid Leukemia.

The authors have demonstrated that DC-STAMP regulates PI3K signaling, and genetic silencing of DC-STAMP or pharmacological inhibition of downstream PI3K restored normal apoptotic processes.

The paper title states Pan-Apoptosis (simultaneous or combined activation of the three major forms of regulated cell death); however, in the manuscript, the authors have demonstrated only caspase-3 and anti-apoptotic proteins. There is no evidence to suggest that it is pan-cell death.

The authors have used only 1 AML cell line (THP-1) to demonstrate their findings and the proposed conclusions.

Apart from the overexpression, did the authors find any DC-STAMP heterogeneity in the available AML cell lines (low DC-STAMP AML cell lines vs. high DC-STAMP AML cells)? Will the observed OE and KD effects be the same in these scenarios?

The work would have been further strengthened by showing the results of PI3K inhibition in the low DC-STAMP expressing cells or the DC-STAMP KD cells, parallel to the DC-STAMP OE cells, to show the specificity of PI3K inhibition.

Reviewer #3: The authors discuss the possible role of DC-STAMP overexpression in influencing the mTOR/PI3K pathway and leukaemic cell proliferation. They investigate this by creating knockdown and overexpression models, and analysing protein expression and apoptosis by AnnexinV assay.

Lines 144 & 158

The term “confluence” is not usually applicable to suspension cells. Are there authors able to be more specific, using density quantified as cells/mL?

Line 154

Can the authors clarify what si-NC refers to? A non-targeting or scrambled si-RNA would be the appropriate control for the knockdown model. For the overexpression model, an empty vector/backbone plasmid would be the appropriate control.

However, throughout the figures, “NC” was used to compare against the KD and OE lines. If NC refers to “normal cells/control”, then which control? The same control cannot be used for both the KD and OE lines, neither should the parental line be used as control. Please clarify what NC refers to.

Line 166

Did the authors mean DC-STAMP overexpression + LY294002?

Line 169

After the incubation, 10 uL of CCK-8…? The sentence is incomplete.

Line 192

Method should be written in past-tense

Line 200 Western blot

Method should be written in past-tense

Lines 233-238

Are the authors able to comment on the other significantly enriched pathways? Were there any other pathways associated with leukaemia?

Can the authors justify why median was used to separate the groups? If the samples were grouped by top and bottom quartile, would the GSEA results hold true?

How many AML samples were analysed? Was not mentioned in methods.

Line 250

“xx-fold increase in the OE group”. Please clarify. Western blot shows marginal increase in DC-STAMP expression at best (Figure 2C). Relative DC-STAMP expression to…? Likely GAPDH, please specify.

Line 259

Are the authors able to show the viability across 0, 24, 48 to 72 hours?

Figure 3B

y-axis of flow plots: presumably PI is propidium iodide, but what is BL3?

NC plot – it is unusual to see THP-1 cells undergoing heavy apoptosis under standard culture conditions. Can the authors explain?

Label for OE plot is missing.

The appears to be little/no cells in Q3 (early apoptosis) in all NC, KD and OE, which is highly unusual. Again, it would be useful to show change in viability (cells shifting from Q4 to Q3 to Q2) over 0, 24, 48 and 72 hours.

Lines 266-271

It is premature to speculate this here. Suggest moving this paragraph after the Western data with apoptosis markers have been introduced and discussed.

Figure 4A

Can the authors also include immunofluorescence for p-PI3K in the panel, together with p-Akt and p-mTOR?

Lines 288-294

Figure 4B from the western blot does not seem to support the claims that KD show reduced expression and OE show increased expression of p-PI3K, p-Akt and p-mTOR. Quantitation by densitometry and the resulting P-values are debatable. To state that the data “confirms that OE activates and KD suppresses” PI3K/Akt/mTOR pathway based on the figure may be an overreach.

Lines 294-298

The authors discuss the qRT expression in Figure 4C, but the western data in Figure 4B is not consistent with this. In the blot, KD does not show Bcl-2 downregulation and Bax, caspase-3 upregulation compared to NC. The differences appear marginal at best. Likewise, OE does not show Bcl-2 upregulation, however it does show Bax and caspase-3 downregulation in the blot.

Lines 301-303 (and lines 266-271)

Overall, the data does not appear to support these statements in a convincing manner.

Lines 305-306, 317-320

The authors propose that inhibiting DC-STAMP overexpressing cells using the PI3K inhibitor LY294002 reduces proliferation. However, the authors have not established that total PI3K or p-PI3K is at all increased in their OE model (immunofluorescence data is not shown and western blot show marginal differences).

Additionally, the authors must show NC + LY294002 as an appropriate control in Figure 5. If the inhibitor decreases the phosphorylation of PI3K, mTOR and Akt in the NC (as suspected it will) in a similar manner to OE, then it cannot be concluded that “targeting the downstream PI3K node effectively counteracts the pro-leukaemogenic effects of DC-STAMP overexpression (lines 330-331)”, because this inhibition is not necessarily unique to high DC-STAMP expressing cells.

Line 321

“treatment restored cell viability”. Did the authors mean reduced? A proliferation curve with treatment over time may be a more appropriate data to show here.

Figure 5 A-D

NC + LY294002 should be included as the appropriate control. Can the authors clarify what NC refers to here? Parental line transduced with empty vector/backbone plasmid is the appropriate control.

Figure 5B

p-PI3K should be included in the immunofluorescence panel.

Lines 322-324

Figure 5C does not show “pro-apoptotic Bax and caspase-3 in OE restored to levels comparable to wildtype cells” in OE+PI3K-LY.

Lines 327-333

The authors claim that “DC-STAMP expression regulates PANoptosis in AML” but have not provided evidence for the involvement of pyroptosis, necroptosis, PANoptosome complex, etc, and data shown for apoptosis is not strong.

“leading to apoptosis resistance and drug tolerance” also claimed but not supported. Authors could try to show that their OE model is venetoclax resistant, transduce their OE vector into a venetoclax-resistant THP1 line or show that venetoclax-resistant THP1 has high DC-STAMP expression.

Suggest for authors to draw more modest conclusions based on data presented for their manuscript.

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what does this mean? ). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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<p>DC-STAMP Activates the PI3K/AKT/mTOR Signaling Pathway to Regulate PANoptosis in Acute Myeloid Leukemia

PONE-D-25-38312R1

Dear Dr. Liu,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Kota V Ramana, Ph.D.

Academic Editor

PLOS One

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #2: Yes

**********

Reviewer #1: I have carefully reviewed the revised manuscript and would like to sincerely thank you for your thorough and diligent responses to the previous round of review comments. With the current revisions, the manuscript has shown significant improvement in logical coherence, data integrity, and clarity of expression, and now meets the standards for publication. Congratulations on completing this excellent research, and thank you for your professional and constructive engagement throughout the peer review process.

Reviewer #2: (No Response)

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what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #2: No

**********