The somatic DDX41 hot spot mutation (p.R525H) causes skewed differentiation into plasmacytoid dendritic cells in human iPSC and leukemia models Free

Germline mutations in the RNA-helicase gene DDX41 cause predisposition to myeloid malignancies (MDS/AML) with a penetrance of 50% by age 90. DDX41 germline mutations are heterozygous and predominantly truncating variants, indicative of loss-of-function. More than half of DDX41-mutated carriers that develop MDS/AML will acquire a second-hit mutation in the other allele of DDX41, frequently causing the amino acid substitution p.R525H. Single-cell analysis from a biallelic DDX41-mutated MDS patient showed that the p.R525H mutation was highly enriched in dendritic cells (DCs) but less frequent in other cell types. We hypothesized that p.R525H causes the hematopoietic stem and progenitor cells (HSPCs) to differentiate preferentially into DC.

To precisely model the germline of DDX41-mut patients, we generated human induced pluripotent stem cells (iPSC) with a frameshift mutation in exon 6 of one allele of DDX41, modeling the most common germline mutation. We attempted to modify the other allele of DDX41 to express the p.R525H mutation but could not derive viable cells. To circumvent potential lethality of the acquired mutation, we inserted a Doxycycline (DOX)-inducible wild-type DDX41 expression cassette in the AAVS1 safe harbor locus. Then we modified the endogenous allele to cause the p.R525H mutation in trans to the existing frameshift mutation. In this manner, we derived iPSCs that would model the most common configuration of DDX41 mutations (DDX41^R525H/-) upon the withdrawal of DOX from the culture.

In undifferentiated iPSC cultures, we observed profound defects in cell proliferation and survival upon DOX withdrawal. To determine the effect of the p.R525H mutation on hematopoietic cells, we differentiated the iPSCs into CD34+ HSPCs in the presence of DOX, and then withdrew DOX and studied the effect of the DDX41^R525H/- genotype on HSPC function. In colony formation assays, DDX41^R525H/- cells produced fewer and smaller myeloid and erythroid colonies after DOX withdrawal. In liquid culture, DDX41^R525H/- cells showed cell cycle arrest by EdU incorporation assay and protein synthesis deficit by HPG assay. Thus, we conclude that DDX41 is required for proliferation and survival in iPSC and hematopoietic progenitor cells and that p.R525H lacks the essential function.

Next, we tracked HSPC differentiation in liquid culture containing Flt3 ligand, stem cell factor and thrombopoietin with or without GM-CSF. Identification of DC subsets by flow cytometry was based on the literature (Sontag S, et al. Stem Cells 2017;35(4)). We observed reduced overall differentiation in DDX41^R525H/- cultures, evidenced by maintenance of CD34 positivity. However, among the CD45+CD34- cells, DDX41^R525H/- cells were 3-4 fold more likely to be identified as plasmacytoid DC (pDC) compared to control conditions (+DOX or DDX41^+/- cells). Notably, in DDX41^R525H/- CD45+CD34- cells, overall CD123 and CD304 expression were profoundly upregulated and CD11c was downregulated, indicative of increased pDC character.

We used a similar DOX-inducible approach to generate Molm13 human AML cell line with DDX41^R525H/- and homozygous knockout (DDX41^-/-) genotype and found that these cells had profound survival defects. Both genotypes exhibited DC-like morphological change with extended multiple projections upon DOX withdrawal. In flow cytometry analysis, both genotypes exhibited upregulated Clec9a (conventional DC1 (cDC1) marker) and Clec10a and CD1c (cDC2 marker) upon DOX withdrawal. DDX41^R525H/- cells specifically exhibited pDC features shown by upregulated CD123 by flow cytometry and increased IRF7 by western blotting, while DDX41^-/- had decreased IRF7. Furthermore, we found that DDX41^R525H/- cells specifically showed increased IRF9 and phosphorylated STAT1 and STAT2, which are downstream of interferon receptor activation, indicating that type-I interferon produced by DDX41^R525H/- pDC activates interferon signaling in autocrine/paracrine manner. These results indicate that p.R525H promotes differentiation into pDC, which is not observed in DDX41^-/- cells.

In conclusion, DDX41 p.R525H mutation has bimodal functions in HSPC: loss-of-function for proliferation/survival and gain-of-function for skewed differentiation into pDC. Dysregulated DCs may contribute to the pathogenesis of DDX41-mutated MDS/AML, particularly in light of recent descriptions of AML with pDC differentiation and the broader role of DC dysregulation in MDS/AML.