The Most Common Somatic DDX41 Mutation (p.R525H) Causes Loss of an Essential Function and Is Selected Against in Human iPSC and Leukemia Models
Authors: Kida Junichiro , Fricker Maggie , Stepanchick Emily , Chlon Timothy 
Published: December 2024

Germline mutations in the RNA-helicase gene DDX41 cause predisposition to myeloid malignancy (MDS/AML) with a penetrance of 50% by age 90. More than half of DDX41-mut carriers that develop MDS/AML will acquire a second-hit mutation in the other allele of DDX41, and this mutation frequently causes the amino acid substitution R525H. Additional co-mutations in these patients are few (median 1 per patient) and include common AML-associated mutations such as ASXL1, DNMT3A, and TP53, with none of these being statistically enriched in DDX41-mut patients. Thus, it is thought that the DDX41R525H mutation contributes significantly to the onset of MDS/AML in these patients. We set out to determine the effect of the p.R525H mutation on protein function and on human hematopoiesis.
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. This genetic insult models the most common germline DDX41 mutation, p.D140fs. We attempted to modify the other allele of DDX41 in this cell line to express the p.R525H mutation but could not derive viable cells. To circumvent potential lethality of the acquired mutation, we inserted a Doxycycline-inducible wild-type DDX41 expression cassette in the AAVS1 safe harbor locus of these cells. Then we modified the endogenous allele to cause the p.R525H mutation in trans to the existing frameshift mutation. In this manner, we derived iPSC that would model the most common configuration of DDX41 mutations observed in MDS/AML patients upon the withdrawal of Doxycycline (DOX) from the culture. In undifferentiated iPSC cultures, we observed profound defects in cell proliferation and survival upon DOX withdrawal. Thus, we conclude that DDX41 is required for iPSC proliferation and survival and that the R525H mutant lacks the essential function. To determine the effect of the p.R525H mutation on hematopoietic cells, we differentiated the iPSC in vitro into CD45+, CD34+ hematopoietic progenitor cells (HPC) in the presence of DOX, and then withdrew DOX from the culture and studied the effect of the DDX41R525H/- genotype on HPC function. We found that +DOX cultures produced hematopoietic colonies with similar quality and frequency as control iPSC-derived HPC. In contrast, the -DOX cultures (DDX41R525H/-) produced fewer and smaller colonies with a profound loss of erythroid colonies. To further examine the effect on erythropoiesis, we conducted directed erythroid differentiation of the HPC in the presence or absence of DOX. We found that the -DOX cultures produced fewer differentiated erythroid cells, confirming that the DDX41R525H/- genotype causes defects in erythropoiesis, likely due to reduced proliferation of erythroid progenitor cells.
We used a similar DOX-inducible approach to generate MOLM13 human AML cells with the DDX41R525H/- genotype and found that these cells had profound cell cycle and survival defects. We conclude that DDX41 is essential in proliferative cell models and that the p.R525H mutation causes loss of the essential function.
To examine the effect of the p.R525H mutation on DDX41 activity in vivo, we conducted an APEX proximity-labeling assay for wild-type and p.R525H mutant DDX41 in a human leukemia cell line. This assay identifies proteins that are proximal to the protein of interest in cells. We found that wild-type DDX41 is largely localized near DNA replication factors, transcription and chromatin factors, and splicing factors. For the p.R525H mutant, we found a similar interaction profile with increased enrichment of the same and related factors. This finding indicates that the R525H mutant is localized in the same cellular compartments and interacts with a similar array of proteins. The increased magnitude of enrichment for these factors may indicate that the R525H mutant becomes trapped in chromatin or splicing complexes, perhaps due to its reduced enzymatic activity. We conclude that the p.R525H mutation disrupts some essential cellular process in which DDX41 is involved, which may include transcription, splicing, or R-loop resolution. Since highly proliferative cell models cannot survive with the DDX41R525H/- genotype, it is likely that the p.R525H mutation is confined to slowly proliferative cells in patients and contributes to disease through cell-intrinsic defects on differentiation and cell-extrinsic effects on systemic hematopoiesis.