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ACUTE MYELOID LEUKEMIA

Targeting a mitochondrial E3 ubiquitin ligase complex to overcome AML cell-intrinsic Venetoclax resistance

Leukemia volume 37, pages 1028–1038 (2023)Cite this article

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Abstract

To identify molecules/pathways governing Venetoclax (VEN) sensitivity, we performed genome-wide CRISPR/Cas9 screens using a mouse AML line insensitive to VEN-induced mitochondrial apoptosis. Levels of sgRNAs targeting March5, Ube2j2 or Ube2k significantly decreased upon VEN treatment, suggesting synthetic lethal interaction. Depletion of either Ube2j2 or Ube2k sensitized AML cells to VEN only in the presence of March5, suggesting coordinate function of the E2s Ube2j2 and Ube2k with the E3 ligase March5. We next performed CRISPR screens using March5 knockout cells and identified Noxa as a key March5 substrate. Mechanistically, Bax released from Bcl2 upon VEN treatment was entrapped by Mcl1 and Bcl-XL and failed to induce apoptosis in March5 intact AML cells. By contrast, in March5 knockout cells, liberated Bax did not bind to Mcl1, as Noxa likely occupied Mcl1 BH3-binding grooves and efficiently induced mitochondrial apoptosis. We reveal molecular mechanisms underlying AML cell-intrinsic VEN resistance and suggest a novel means to sensitize AML cells to VEN.

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Fig. 1: A genome-wide CRISPR-Cas9 screen identifies components of the E3 ubiquitin ligase complex as key modulators of VEN sensitivity.
Fig. 2: Ube2j2 and Ube2k function March5-dependently.
Fig. 3: March5 depletion significantly sensitizes mMA9Cas cells to VEN or A-1155463 treatment.
Fig. 4: A genome-wide CRIPSR screen in March5 KO cells identifies Noxa as a March5 substrate relevant to VEN sensitivity.
Fig. 5: The March5/Noxa axis regulates AML cells sensitivity to Venetoclax by modulating BAX saturation of anti-apoptotic proteins.

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Data availability

The processed data of CRISPR/Cas9 screens are available as Supplementary tables. Raw data will be provided upon request.

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Acknowledgements

We thank the members of the Department of Medicine and Biosystemic Science and the Division of Precision Medicine at Kyushu University for assistance and helpful discussion, and Elise Lamar for critical reading of the manuscript. This work is supported in part by a Grant-in-Aid for Young Scientists (18K16089)(to YS), a Grant-in-Aid for Young Scientists (19K17859), a Research Grant from the KANAE Foundation, the MSD Life Science Foundation, the Yasuda Medical Foundation, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, the Shinnihon Foundation of Advanced Medical Treatment Research, the Takeda Science Foundation (to TY), a Grant-in-Aid for Scientific Research (C)(21K08429), the Shinnihon Foundation of Advanced Medical Treatment Research, the Takeda Science Foundation (to KM), a Grant-in-Aid for Scientific Research (B)(22494899)(to YK), a Grant-in-Aid for Scientific Research (C)(19K05544) (to T Masuda), a Grant-in-Aid for Scientific Research (S)(16H06391) (to KA), a Grant-in-Aid for Scientific Research (A) (17H01567, 20H00540), an AMED under grant number 18063889 and a Grant-in-Aid for Scientific Research (S)(20H05699)(to T Maeda).

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Authors and Affiliations

  1. Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan

    Fumihiko Nakao, Yuichiro Semba, Takuji Yamauchi, Jumpei Nogami, Kensuke Sasaki, Hiroshi Imanaga, Tatsuya Terasaki, Kohta Miyawaki, Yoshikane Kikushige & Koichi Akashi

  2. Division of Precision Medicine, Kyushu University Graduate School of Medical Sciences, Fukuoka, Japan

    Kiyoko Setoguchi, Manaka Miyazaki, Shigeki Hirabayashi, Kohta Miyawaki & Takahiro Maeda

  3. Center for Cellular and Molecular Medicine, Kyushu University Hospital, Fukuoka, Japan

    Yoshikane Kikushige & Koichi Akashi

  4. Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan

    Takeshi Masuda

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  1. Fumihiko Nakao
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Contributions

FN, YS, TY and T Maeda designed CRISPR-Cas9 screen experiments. FN, YS, TY, JN, KA and T Maeda reviewed CRISPR screen data. FN, KS, YS, TY, HI, TT, MM, SH, KM, YK, and T Masuda. executed CRISPR-Cas9 experiments and cell biology and molecular biology experiments. YS and JN analyzed CRISPR screen data. FN and T Maeda wrote the manuscript with help from all authors.

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Correspondence to Takahiro Maeda.

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Nakao, F., Setoguchi, K., Semba, Y. et al. Targeting a mitochondrial E3 ubiquitin ligase complex to overcome AML cell-intrinsic Venetoclax resistance. Leukemia 37, 1028–1038 (2023). https://doi.org/10.1038/s41375-023-01879-z

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