PONE-D-21-30344VCP inhibition induces an unfolded protein response and apoptosis in human acute myeloid leukemia cellsPLOS ONE

Dear Dr. Wagner,

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Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: The authors performed a series of in vitro experiments to demonstrate the effects of VCP

( valosin-containing protein ) inhibitor CB-5083 on the human leukemia cell lines THP-1 and

MV4-11. They have successfully showed that CB-5083 impaired the cell growth and viability

of these leukemia cells and promoted cellular apoptosis via accumulation of ubiquitylated

proteins and activation of unfolded protein response (UPR). They have also observed that the

combination of VCP inhibitor and cytarabine or the Bcl-2 inhibitor venetoclax may exert

synergistic effects on these leukemia cell lines. Despite the leukemia cell may develop resistance

to CB-5083 by VCP gene mutation after a longer exposure , the VCP inhibitor, especially in

combination with other agents, still provide a promising therapeutic strategy for treatment of

AML patients.

This study has been elaborately designed and carefully conducted. All techniques explored in

this study are described in detail. The data presented in figures and tables are clear, sound and

persuasive. My comments and suggestions are as the follows:

1, As shown in Fig 5, CB5083 combined with cytarabine or venetoclax synergistically impaired

the growth and reduced the viability of AML cells, but no data support if these combinations can

induce apoptosis of AML cells in synergy. Did these combinations do so?

2, If the combination of CB5083 with cytarabine or venetoclax did synergistically induce

apoptosis of the AML cells, could it be an effective way to overcome the resistance to CB5083?

3, Compare the Western blot results in Fig1C to that in Fig 6. In Fig1C the amount of Ero-1α

increased as the MV4-11 treated with CB5083 at 200nM increased to 500nM, while in Fig 6 the

high level of Ero-1α gradually decreased in the CB5083-resistant MV4-11 treated with CB5083

from 200nM to 500nM. Could the authors explain a little bit on this issue?

4, As shown in Fig 6, the CB5083-resistant MV4-11 cells led by VCP gene mutation exhibited

permanent ER stress, but no apoptosis occurred. Please briefly discuss the possible mechanism of

this phenomena in DISCUSSION, especially on the relationship among UPR, ER stress and

apoptosis.

5, The origin of human BM stromal cell line HS-5 should be mentioned in MTERIALS AND

METHODS

I like to recommend accept this manuscript for publication after the authors revise or explain on

these minor points mentioned above.

Reviewer #2: This is an interesting study and the authors have collected a unique dataset using cutting

edge methodology. The paper is generally well written and structured. An excellent report on very thorough research. The literature review was thorough, the methodology was painstakingly thorough.

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Reviewer #1: Yes: Zi-xing Chen

Reviewer #2: No

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VCP inhibition induces an unfolded protein response and apoptosis in human acute myeloid leukemia cells

PONE-D-21-30344R1

Dear Dr. Wagner,

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.

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Kind regards,

Anilkumar Gopalakrishnapillai

Academic Editor

PLOS ONE