Germline DDX41 Mutations Disrupt Hematopoiesis and Microenvironment in Patient-Derived Bone Marrow Organoids

Germline mutations play a critical role in the development and progression of myelodysplastic syndromes (MDS), a group of clonal hematopoietic disorders characterized by ineffective hematopoiesis and higher risk to transform acute myeloid leukemia (AML). A growing list of germline mutations, including DDX41, expands our knowledge of the genetic underpinnings of MDS. Germline mutations in the DDX41 gene increase the risk of MDS at an older age. However, the exact mechanisms underneath the increased risk remains unclear. Human patients are typically heterozygous germline carriers. Mice with heterozygous Ddx41 knockout (KO) often show minimal phenotype. These findings raise challenges in DDX41 mechanistic research.

To overcome such challenges and investigate the impact of DDX41 mutations on hematopoiesis in humans, we utilized induced pluripotent stem cells (iPSCs) and CRISPR-Cas9 technology. We created DDX41 knockout iPSCs pool and generated both homozygous and heterozygous mutant clones. Additionally, peripheral blood mononuclear cells (PBMCs) from a middle-aged female patient with a germline DDX41 mutation were reprogrammed into iPSCs. After validating the characteristic features of the iPSCs, all cell lines were differentiated into bone marrow organoids, which closely mimic the bone marrow hematopoiesis and environment. As a result, we found that homozygous DDX41 mutant organoids showed disrupted hematopoiesis with a significant increase in T and B cells compared to controls. An explanation could be that the knockout of DDX41 could alter the interferon response, influencing lymphocyte development and function. Erythropoiesis is markedly compromised with homozygous knockout of DDX41. Moreover, hematopoietic stem cells (HSCs) were found to be at a higher level in the DDX41 homozygous knockout group. This suggests DDX41’s roles in directly regulating hematopoietic differentiation or indirectly through the signaling pathways that influence the bone marrow niche’s ability to support HSCs.

To further investigate the mechanism of above impact, we performed single-cell RNA sequencing (scRNA-seq) using BM organoids derived from DDX41 mutant patient’s iPSCs, which revealed marked heterogeneity in the cellular composition, with a significant reduction in late erythroid and megakaryocyte clusters in the DDX41 mutant bone marrow organoids. This suggests that DDX41 is essential for the maturation and differentiation of these cell types, which are vital for effective hematopoiesis and platelet formation. The impairment in erythroid and megakaryocyte development highlights the potential of DDX41 mutations to contribute to the ineffective hematopoiesis observed in MDS. Moreover, the elevated levels of hematopoietic stem and progenitor cells in DDX41 mutant bone marrow organoids indicate that DDX41 is crucial for the proper signaling and maintenance of the bone marrow niche. This imbalance could impair the bone marrow's ability to replenish blood cells, thereby exacerbating the cytopenia characteristic of MDS.

Taken together, we have demonstrated our genetically manipulated and patient derived DDX41 mutant bone marrow organoids can closely mimic human DDX41 mutation-associated pathogenesis, providing valuable research resources for mechanistic and therapeutic studies. Our findings in DDX41’s critical role in hematopoiesis and the integrity of the bone marrow niche provide novel directions in mechanism research and therapeutic intervention targets.