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Myelodysplastic syndrome

A germline HLTF mutation in familial MDS induces DNA damage accumulation through impaired PCNA polyubiquitination

Leukemia volume 33, pages 1773–1782 (2019)Cite this article

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Abstract

Although several causal genes of familial myelodysplastic syndromes (MDS) have been identified, the genetic landscape and the molecular pathogenesis are not totally understood. To explore novel driver genes and their pathogenetic significance, we performed whole-exome sequence analysis of four individuals from a familial MDS pedigree and 10 candidate single-nucleotide variants (C9orf43, CYP7B1, EFHB, ENTPD7, FAM160B2, HELZ2, HLTF, INPP5J, ITPKB, and RYK) were identified. Knockdown screening revealed that Hltf downregulation enhanced colony-forming capacity of primary murine bone marrow (BM) stem/progenitor cells. γH2AX immunofluorescent staining assay revealed increased DNA damage in a human acute myeloid leukemia (AML) cell line ectopically expressing HLTF E259K, which was not observed in cells expressing wild-type HLTF. Silencing of HLTF in human AML cells also led to DNA damage, indicating that HLTF E259K is a loss-of-function mutation. Molecularly, we found that an E259K mutation reduced the binding capacity of HLTF with ubiquitin-conjugating enzymes, methanesulfonate sensitive 2 and ubiquitin-conjugating enzyme E2N, resulting in impaired polyubiquitination of proliferating cell nuclear antigen (PCNA) in HLTF E259K-transduced cells. In summary, our results indicate that a familial MDS-associated HLTF E259K germline mutation induces accumulation of DNA double-strand breaks, possibly through impaired PCNA polyubiquitination.

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Acknowledgements

We would like to express our gratitude to all the patients and family members who participated in the present study. We would like to thank Dr. Masashi Miyauchi, Dr. Sho Yamazaki, Dr. Hideaki Mizuno, Dr. Naoki Nariai, and Dr. Akira Motegi for their great discussion and kind help. We would like to thank Professor Lajos Haracska (Biological Research Centre, Institute of Genetics, Hungary), Professor Kyungjae Myung (School of Life Sciences, Ulsan National Institute of Science and Technology), and Professor David Turner (University of Michigan, Molecular and Behavioral Neuroscience Institute) for their kindness and generosity in providing the plasmids. Finally, we would also like to thank Ms. Keiko Tanaka, Ms. Satomi Muroi, Ms. Kaori Ono, Ms. Fumie Ueki, Ms. Mariko Yamamoto, and Ms. Yoko Hokama for their excellent technical assistance and Ms. Tomoko Aiga for the total support. This work was supported by research funding from JSPS KAKENHI grant numbers JP15J03679 and JP17K16181 to KT, and 2017 Bristol-Myers Squibb KK Research and Medical Education Grants to SA.

Author contributions

KT, M Kawazu, JK, AY, YM, H Maki, SA, H Mano, and M Kurokawa designed the research; KT and M Kawazu performed the experiments; T.U. carried out genomic analysis; H Maki, TT, TK, YN, SA, HH, and AM helped and prepared to collect the samples; HU and KS provided the samples; KT, M Kawazu, JK, AY, YM, and M Kurokawa wrote the paper; and HH, AM, YH, H Mano, and M Kurokawa supervised the research.

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  1. These authors contributed equally: Kensuke Takaoka, Masahito Kawazu

Authors and Affiliations

  1. Department of Hematology and Oncology, The University of Tokyo, Tokyo, Japan

    Kensuke Takaoka, Junji Koya, Akihide Yoshimi, Yosuke Masamoto, Hiroaki Maki, Takashi Toya, Takashi Kobayashi, Yasuhito Nannya, Shunya Arai & Mineo Kurokawa

  2. Department of Medical Genomics, The University of Tokyo, Tokyo, Japan

    Masahito Kawazu

  3. Department of Cellular Signaling, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

    Toshihide Ueno & Hiroyuki Mano

  4. Department of Hematology, Tokyo Medical Center, Tokyo, Japan

    Hironori Ueno

  5. Department of Hematology, Japanese Red Cross Medical Center, Tokyo, Japan

    Kenshi Suzuki

  6. School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan

    Hironori Harada

  7. Department of Pediatrics, St. Luke’s International Hospital, Tokyo, Japan

    Atsushi Manabe

  8. Department of Hematology/Oncology, Gunma Children’s Medical Center, Gunma, Japan

    Yasuhide Hayashi

  9. National Cancer Center Research Institute, Tokyo, Japan

    Hiroyuki Mano

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  1. Kensuke Takaoka
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Correspondence to Mineo Kurokawa.

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Conflict of interest

Research funding: SA (Bristol-Myers Squibb), HH (Celgene, Novartis). Speakers Bureau: JK (Bristol-Myers Squibb), HH (NIPPON SHINYAKU CO., LTD.), M Kurokawa (Bristol-Myers Squibb). The remaining authors declare no competing financial interests.

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Takaoka, K., Kawazu, M., Koya, J. et al. A germline HLTF mutation in familial MDS induces DNA damage accumulation through impaired PCNA polyubiquitination. Leukemia 33, 1773–1782 (2019). https://doi.org/10.1038/s41375-019-0385-0

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