Impacts of Monoallelic and Biallelic DDX41 Mutations on Hematopoiesis and the Development of Myeloid Malignancies

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 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 alleles. 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 (Ddx41^R525H/+), or hemizygous for the Ddx41 R525H mutation (Ddx41^R525H/-), in which expression of the mutant allele was induced by tamoxifen administration.

In noncompetitive BM transplantation, most of the recipient mice that were transplanted with BM from Ddx41^-/- or Ddx41^R525H/- mice died within a month after CreERT2 induction due to severe BM failure, which was not observed in mice transplanted with BM from Ddx41^+/+, Ddx41^+/- or Ddx41^R525H/+ mice. Transcriptome analysis revealed that stem cells (Kit⁺Sca-1^-Lin^low cells) derived from Ddx41^R525H/- BM-transplanted mice exhibited a significant upregulation of ribosomal genes and several snoRNA genes compared with those derived from Ddx41^+/+ BM-transplanted mice, which could result in abnormal ribosome biogenesis. In addition, Ddx41^R525H/--derived Gr1+CD11b+ myeloid cells showed a significant upregulation of genes involved in cGAS-STING signaling pathways compared with Ddx41^+/+-derived myeloid cells. However, the survival and cytopenia and the ribosome biogenesis in the stem cells in Ddx41^R525H/- BM-transplanted mice were improved only marginal when the intact Sting alleles were deleted. These results suggest that the impact of the cGAS-Sting signaling on these phenotypes was, if ever, very small, highlighting the role of Sting-independent mechanisms.

In long-term observations, mice transplanted with Ddx41^+/- or Ddx41^R525H/+ BM exhibited significantly lower white blood cell counts and anemia, both in primary and subsequent transplantations, although they did not exhibit significant transcriptional changes relative to the wild-type control animals until just before the onset of disease. Some of these mice developed MDS-like phenotypes, including ineffective hematopoiesis and erythroid dysplasia. Stem cells from these mice showed abnormal ribosome biogenesis and reduced expression of interferon response genes. It may be that clones that adapted to the inflammatory environment were gradually selected and contribute to their clonal advantage.

Given that the MDS clones with the DDX41 R525H somatic allele are commonly observed as a small subclone in patients, we next co-transplanted Ddx41^+/+- and Ddx41^525H/--derived BM cells with Ddx41^+/+- or Ddx41^+/--derived BM cells at the ratio of 1:9. The recipient mice showed significantly reduced WBC counts when Ddx41^+/+- or Ddx41^+/- were co-transplanted with Ddx41^525H/- -derived BM, suggesting that Ddx41^525H/--derived hematopoietic cells have negative effect on normal hematopoiesis.

In order to assess the crosstalk between biallelic Ddx41 mutant cells and their microenvironment cells harboring monoallelic Ddx41 mutations, we co-transplanted Ddx41^+/+- or Ddx41^525H/--derived BM cells with Ddx41^+/--derived BM cells into the constitutive heterozygous Ddx41 KO mice. The phenotypic and molecular characteristics of these mice are under investigation.

In summary, conditionally introduced compound loss-of function and R525 alleles caused severe BM failure, whereas heterozygous Ddx41 loss-of function and R525H knock-in alleles are compatible with hematopoiesis, although associated with impaired hematopoiesis and the development of MDS with aging, where an attenuated inflammatory response and ribosome functions may play important roles.

Kataoka:Celgene: Honoraria; AbbVie: Honoraria; Sanofi: Honoraria; JCR Pharmaceuticals: Research Funding; Sumitomo Dainippon Pharma: Honoraria, Research Funding; Chordia Therapeutics: Research Funding; Asahi Kasei Pharma: Research Funding; Eisai: Honoraria, Research Funding; Teijin Pharma: Research Funding; Meiji Seika Pharma: Honoraria; Novartis: Honoraria; Kyowa Kirin: Honoraria, Research Funding; Ono Pharmaceutical: Honoraria, Research Funding; Shionogi: Research Funding; Asahi Genomics: Current equity holder in private company; Takeda Pharmaceutical: Honoraria, Research Funding; Mochida Pharmaceutical: Research Funding; Japan Blood Products Organization: Research Funding; Otsuka Pharmaceutical: Honoraria, Research Funding; Chugai Pharmaceutical: Honoraria, Research Funding; SymBio Pharmaceuticals: Honoraria; AstraZeneca: Honoraria; Pfizer: Honoraria; Nippon Shinyaku: Honoraria; Daiichi Sankyo: Honoraria; Alexion Pharmaceuticals: Honoraria; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceutical: Honoraria. Kakiuchi:Sumitomo Pharma: Research Funding. Makishima:Nippon Shinyaku: Research Funding. Nannya:Astra-Zeneca: Speakers Bureau; Otsuka Pharmaceutical: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Pfizer: Speakers Bureau; Novartis: Speakers Bureau; KyowaHakko Kirin: Speakers Bureau; Daiichi Sankyo: Speakers Bureau; Takeda Pharmaceutical: Speakers Bureau; Bristol Meyer Squibb: Speakers Bureau; Bristol Meyer Squibb: Consultancy; Otsuka Pharmacutical: Consultancy; Bristol Meyer Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Ogawa:Otsuka Pharmaceutical Co., Ltd.: Research Funding; Nanpuh Hospital: Other: Endowed chair; Nihonshinyaku Co., Ltd.: Other: Donation; Nakatani Foundation: Speakers Bureau; The Mitsubishi Foundation: Speakers Bureau; Asahi Genomics Inc: Current equity holder in publicly-traded company; Chordia Therapeutics Inc.: Consultancy, Other: Endowed chair, Research Funding; Eisai Co., Ltd.: Consultancy, Research Funding.