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FLOT1 knockdown inhibits growth of AML cells through triggering apoptosis and pyroptosis

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Abstract

Acute myeloid leukemia (AML) is a group of hematological malignancies characterized by clonal proliferation of immature myeloid cells. Lipid rafts are highly organized membrane subdomains enriched in cholesterol, sphingolipids, and gangliosides and play roles in regulating apoptosis through subcellular redistribution. Flotillin1 (FLOT1) is a component and also a marker of lipid rafts and had been reported to be involved in the progression of cancers and played important roles in cell death. However, the role of FLOT1 in AML remains to be explored. In this study, we found that increased expression of FLOT1 was correlated with poor clinical outcome in AML patients. Knockdown of FLOT1 in AML cells not only promoted cell death in vitro but also inhibited malignant cells engraftment in vivo. Mechanically, FLOT1 knockdown triggered apoptosis and pyroptosis. FLOT1 overexpression promoted AML cell growth and apoptosis resistance. Our findings indicate that FLOT1 is a prognostic factor of AML and may be a potential target for AML treatment.

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Data availability

The dataset supporting the conclusions of this article is available in the NCBI’s SRA repository. Accession to cite for the SRA data: PRJNA816556.

Abbreviations

AML:

Acute myeloid leukemia

FLOT1:

Flotillin1

HHT:

Homoharringtonine

ADM:

Doxorubicin

Ara-C:

Cytarabine

Nec-1:

Necrostatin-1

Fer-1:

Ferrostatin-1

Z-VAD:

Z-VAD(OH)-FMK

VX-765:

Belnacasan

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Funding

This work is supported by Key International Cooperation Projects of the National Natural Science Foundation of China (81820108004), Research Project of Jinan Microecological Biomedicine Shandong Laboratory (JNL-2022034C) and the National Natural Science Foundation of China (82170144).

Author information

Authors and Affiliations

  1. Department of Hematology, The First Affiliated Hospital, Zhejiang University School of Medicine, No. 79 Qingchun Road, Hangzhou, Zhejiang, People’s Republic of China

    Shihui Mao, Yu Qian, Wenwen Wei, Xiangjie Lin, Qing Ling, Wenle Ye, Jiajia Pan, Yutong Zhou, Yanchun Zhao, Xin Huang, Jiansong Huang, Chao Hu, Mengjing Li, Jie Sun & Jie Jin

  2. Zhejiang Provincial Key Laboratory of Hematopoietic Malignancy, Zhejiang University, Hangzhou, Zhejiang, People’s Republic of China

    Shihui Mao, Yu Qian, Wenwen Wei, Xiangjie Lin, Qing Ling, Wenle Ye, Jiajia Pan, Yutong Zhou, Yanchun Zhao, Xin Huang, Jiansong Huang, Chao Hu, Mengjing Li, Jie Sun & Jie Jin

  3. Zhejiang Provincial Clinical Research Center For Hematological Disorders, Hangzhou, Zhejiang, People’s Republic of China

    Shihui Mao, Yu Qian, Wenwen Wei, Xiangjie Lin, Qing Ling, Wenle Ye, Jiajia Pan, Yutong Zhou, Yanchun Zhao, Xin Huang, Jiansong Huang, Chao Hu, Mengjing Li, Jie Sun & Jie Jin

  4. Zhejiang University Cancer Center, Hangzhou, Zhejiang, People’s Republic of China

    Shihui Mao, Yu Qian, Wenwen Wei, Xiangjie Lin, Qing Ling, Wenle Ye, Jiajia Pan, Yutong Zhou, Yanchun Zhao, Xin Huang, Jiansong Huang, Chao Hu, Mengjing Li, Jie Sun & Jie Jin

  5. The Affiliated People’s Hospital of Ningbo University, Ningbo, Zhejiang, People’s Republic of China

    Fenglin Li

  6. Jinan Microecological Biomedicine Shandong Laboratory, Jinan, People’s Republic of China

    Jie Jin

Authors
  1. Shihui Mao
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  2. Yu Qian
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  5. Qing Ling
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  15. Jie Sun
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Contributions

Sh M and YQ performed the research. JJ and JS designed the research study. XH, Js H, and Mj L contributed essential reagents and tools. Ww W, Xj L QL, Wl Y, Fl L, Jj P, Yt Z, Yc Z, and CH collected clinical samples and analyzed the data. Sh M and JS wrote the paper.

Corresponding authors

Correspondence to Jie Sun or Jie Jin.

Ethics declarations

Ethics approval and consent to participate

Human data have been performed in accordance with the Declaration of Helsinki and have been approved by Clinical Research Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University (IIT20220548A). All animal experiments were reviewed and approved by the Laboratory Animal Management and Ethics Committee of Zhejiang Chinese Medical University (ZCMU) (Approval No: IACUC-20220214–11). And all animal experiments were performed following animal use guidelines and ethical approval.

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The authors declare no competing interests.

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Supplementary Information

SFig. 1

A. qPCR and Western blotting analysis of FLOT1 expression in human AML cell lines (n=3). B. qPCR analysis of the FLOT1 mRNA level in OCI-AML2, MV-4-11 and MOLM-13 cells transduced with FLOT1 shRNAs and control vectors (n=3). C Bar graph of gray intensity analysis of Fig. 2A (n=3). (PNG 242 kb)

High resolution image (TIF 25502 kb)

SFig. 2

A. The percentage of cells in the G1, S, or G2 phase detected by flow cytometry in OCI-AML2, MV-4-11 and MOLM-13 cells transduced with FLOT1 shRNAs and control vectors. B. Flow cytometric analysis of annexin V-stained cells in primary AML cells transduced with 2 different FLOT1 shRNAs. For primary patients, the flow cytometric analysis was performed once due to limited samples. C. GSEA analysis results with normalized enrichment score (NES), NOM p-value and false discovery rate (FDR) q-value are shown for pathways enriched in MOLM-13 cells transduced with FLOT1 shRNA when compared with control (n=3). D. Bar graph of gray intensity analysis of Fig. ​3E (n=3).E.Bar graph of gray intensity analysis of Fig. 3F (n=3). (PNG 189 kb)

High resolution image (TIF 30842 kb)

SFig. 3

A.Bar graph of gray intensity analysis of Fig. 4A (n=3). B. Bar graph was gray intensity analysis of Fig. 4B(n=3). C. The extracellular acidification rate (ECAR) in OCI-AML2 and MOLM-13 cells transduced with FLOT1 shRNAs and control vectors(n=3)(up panel). Representative data for ECAR over time for 120 min via fluorescent emission(down panel). (PNG 194 kb)

High resolution image (TIF 24434 kb)

SFig. 4

A. qPCR analysis of the FLOT1 mRNA level in THP-1 cells transduced with FLOT1 overexpression vector (n=3).B. Bar graph of gray intensity analysis of Fig, 5A (n=3). C. The percentage of cells in the G1, S, or G2 phase detected by flow cytometry in THP-1 cells transduced with FLOT1 overexpression vector (n=3). D. Bar graph of gray intensity analysis of Fig. 5D (n=3). E.qPCR analysis of the FLOT1 mRNA level in MOLM-13 cells transduced with FLOT1 overexpression vector and shRNAs vectors (n=3). F. Bar graph of gray intensity analysis of Fig. 5F (n=3). G. Bar graph of gray intensity analysis of Fig. 5H (n=3). H. PARP and FLOT1 protein levels were analyzed by Western blot in OCI-AML2 cell lines treated with Homoharringtonine(HHT), doxorubicin(ADM) or cytarabine(Ara-C) for 24 hrespectively (n=3). (PNG 408 kb)

High resolution image (TIF 29334 kb)

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Mao, S., Qian, Y., Wei, W. et al. FLOT1 knockdown inhibits growth of AML cells through triggering apoptosis and pyroptosis. Ann Hematol 102, 583–595 (2023). https://doi.org/10.1007/s00277-023-05103-x

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