Pathogenic Mechanisms of DDX41 Mutations in 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 (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 (Ddx41^R525H/+), or hemizygous for the Ddx41 R525H mutation (Ddx41^R525H/-), 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 Ddx41^R525H/- 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 Ddx41^R525H/+ mice. By contrast, the mice transplanted with Ddx41^+/- or Ddx41^R525H/+ BM showed significantly reduced WBC counts and anemia in long-term observation in both primary and serial transplantations. Some of the Ddx41^+/- or Ddx41^R525H/+ BM-transplanted mice exhibited MDS-like phenotypes, showing ineffective hematopoiesis with evidence of erythroid dysplasia.

Transcriptome analysis revealed that stem cells (Kit⁺Sca-1^-Lin^low cells) derived from Ddx41^R525H/- 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 Ddx41^R525H/- 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 Ddx41^R525H/- BM-transplanted mice in PB was markedly reduced compared to Ddx41^+/+ BM-transplanted mice. In contrast, Ddx41^+/- and Ddx41^R525H/+ 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 Ddx41^525H/--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 Ddx41^525H/- -derived BM, suggesting that Ddx41^525H/--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.