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952 Pathogenic Mechanisms of DDX41 Mutations in the Development of Myeloid Malignancies

Program: Oral and Poster Abstracts
Type: Oral
Session: 602. Myeloid Oncogenesis: Basic: Mechanisms and Models of Myeloid Malignancy
Hematology Disease Topics & Pathways:
Research, Fundamental Science
Monday, December 11, 2023: 5:15 PM

Ayana Kon1*, Masahiro Marshall Nakagawa, MD, PhD1, Keisuke Kataoka, MD, PhD2,3, Nobuyuki Kakiuchi, MD, PhD1,4*, Hideki Makishima, MD, PhD1, Manabu Nakayama, MD, PhD5*, Haruhiko Koseki, MD, PhD6*, Yasuhito Nannya, MD, PhD1,7 and Seishi Ogawa1,8

1Department of Pathology and Tumor Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
2Division of Hematology, Department of Medicine, Keio University School of Medicine, Tokyo, Japan
3Division of Molecular Oncology, National Cancer Center Research Institute, Tokyo, Japan
4The Hakubi Center for Advanced Research, Kyoto University, Kyoto, Japan
5Chromosome Engineering Team, Department of Technology Development, Kazusa DNA Research Institute, Chiba, Japan
6Laboratory for Developmental Genetics, RIKEN Center for Integrative Medical Sciences, Yokohama, Japan
7Division of Hematopoietic Disease Control, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
8Center for Hematology and Regenerative Medicine, Department of Medicine (MedH), Karolinska Institutet, Huddinge, Sweden

DDX41 is a newly identified leukemia predisposition gene encoding an RNA helicase, whose germline mutations are tightly associated with late-onset myeloid malignancies. Importantly, germline DDX41 mutations were also found in as many as ~7 % of sporadic cases of high-risk MDS, conferring the largest germline risk for myeloid malignancies. In typical cases, a germline loss-of-function allele (most commonly p.A500fs or p.D140fs, depending on the ethnicity) is compounded by a somatic missense mutation affecting the helicase domain in the remaining allele (p.R525H). However, the molecular mechanism by which DDX41 mutations lead to myeloid neoplasms have not fully been elucidated.

To clarify the role of these distinct DDX41 alleles, we generated mice models carrying either or both of conditional/constitutive Ddx41 knock-out (KO) and conditional R525H knock-in (KI) alleles. Vav1-Cre mediated homozygous deletion of Ddx41 resulted in embryonic lethality, suggesting that Ddx41 is indispensable for normal hematopoiesis. Next, by crossing these mice and further breeding with Rosa26-CreERT2 transgenic mice, we engineered mice that were wild-type for Ddx41 (Ddx41+/+), heterozygous Ddx41 KO (Ddx41+/-), homozygous Ddx41 KO (Ddx41-/-), heterozygous for the Ddx41 R525H mutation (Ddx41R525H/+), or hemizygous for the Ddx41 R525H mutation (Ddx41R525H/-), in which expression of the mutant allele was induced by tamoxifen administration.

First, we assessed cell intrinsic effects of these Ddx41 alleles, using noncompetitive transplantation experiments. Shortly after tamoxifen administration, most of the recipient mice that were transplanted with BM from Ddx41-/- or Ddx41R525H/- mice died within a month after CreERT2 induction due to severe BM failure (BMF), which was not observed in mice transplanted with BM from Ddx41+/+, Ddx41+/- or Ddx41R525H/+ mice. By contrast, the mice transplanted with Ddx41+/- or Ddx41R525H/+ BM showed significantly reduced WBC counts and anemia in long-term observation in both primary and serial transplantations. Some of the Ddx41+/- or Ddx41R525H/+ BM-transplanted mice exhibited MDS-like phenotypes, showing ineffective hematopoiesis with evidence of erythroid dysplasia.

Transcriptome analysis revealed that stem cells (Kit+Sca-1-Linlow cells) derived from Ddx41R525H/- BM-transplanted mice exhibited a significant upregulation of genes involved in innate immunity, including interferon stimulated genes, compared with stem cells derived from Ddx41+/+ BM-transplanted mice. In addition, snoRNA and ribosomal genes were significantly deregulated in stem cells from Ddx41-/- and Ddx41R525H/- BM-transplanted mice, which could result in abnormal ribosome biogenesis and protein synthesis in Ddx41 mutant cells.

We also assessed the reconstitution capacity of whole BM cells from different Ddx41 mutant mice in competitive transplantation experiments. The donor chimerism of Ddx41-/- and Ddx41R525H/- BM-transplanted mice in PB was markedly reduced compared to Ddx41+/+ BM-transplanted mice. In contrast, Ddx41+/- and Ddx41R525H/+ BM-transplanted mice did not show significant changes in competitive bone marrow reconstitution compared to Ddx41+/+ BM-transplanted mice. Given that the MDS clones bearing DDX41 R525H somatic alleles are typically observed as a small subclone in patients, we next co-transplanted Ddx41+/+- and Ddx41525H/--derived BM cells with Ddx41+/+- or Ddx41+/--derived BM cells at the ratio of 1:9. Recipient mice showed significantly reduced WBC counts when Ddx41+/+- or Ddx41+/- were co-transplanted with Ddx41525H/- -derived BM, suggesting that Ddx41525H/--derived hematopoietic cells have negative effect on normal hematopoiesis.

In summary, compound biallelic loss-of function and R525 alleles led to severe BM failure. Monoallelic Ddx41 loss-of function and R525H knock-in alleles, by contrast, are compatible with hematopoiesis but associated with an impairment of hematopoiesis and the development of MDS with ageing, where activated innate immunity, impaired RNA metabolism and ribosome functions may play important roles.

Disclosures: Kataoka: Alexion Pharmaceuticals: Honoraria; Mundipharma: Honoraria; Chordia Therapeutics: Research Funding; Otsuka Pharmaceutical: Other: Scholarship, Research Funding; Eisai: Honoraria, Other: Scholarship; Sumitomo Pharma: Honoraria, Other: Scholarship; Kyowa Kirin: Honoraria, Other: Scholarship; Takeda Pharmaceutical: Honoraria, Other: Scholarship, Research Funding; Janssen Pharmaceutical: Honoraria; Asahi Kasei Pharma: Other: Scholarship; Kyorin Pharmaceutical: Honoraria; AstraZeneca: Honoraria; Novartis: Honoraria; Chugai Pharmaceutical: Honoraria, Other: Scholarship, Research Funding; AbbVie: Honoraria; Sysmex: Honoraria; Meiji Seika Pharma: Honoraria, Research Funding; Sanofi: Honoraria; SymBio Pharmaceuticals: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Nippon Shinyaku: Honoraria, Other: Scholarship; Daiichi Sankyo: Honoraria, Other: Scholarship; Incyte Corporation: Honoraria; Ono Pharmaceutical: Honoraria; Shionogi: Other: Scholarship; Teijin Pharma: Other: Scholarship; Japan Blood Products Organization: Other: Scholarship; Mochida Pharmaceutical: Other: Scholarship; JCR Pharmaceuticals: Other: Scholarship; Nippon Kayaku: Other: Scholarship. Nannya: Daiichi Sankyo Company Limited: Research Funding; Amelieff Corporation: Speakers Bureau; Otsuka Pharmaceutical Co., Ltd: Speakers Bureau.

*signifies non-member of ASH