Modeling germline DDX41 and/or CHEK2 mutations using patient-derived bone marrow Organoids

Abstract

Abstract Germline mutations in DDX41 and CHEK2 are recurrently linked to myeloid malignancies, yet their early impact on human hematopoiesis remains undefined. To investigate this, we established patient-derived bone marrow organoids by reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs) and subsequently differentiating them into self-organizing bone marrow organoids, a novel 3D in vitro platform that enables concurrent modeling of hematopoietic and stromal compartments in a human context. The model comprises four iPSCs lines based on their mutation status: normal, DDX41 mutation only, CHEK2 mutation only, and DDX41+CHEK2 double-mutant organoids. To investigate the mechanisms underlying the hematopoietic impact of these germline mutations, we performed single-cell RNA sequencing (scRNA-seq) analyses on bone marrow organoids derived from these four groups of iPSCs. These studies revealed distinct transcriptional and lineage-specific alterations across genotypes. In DDX41 mutation-only organoids, we observed impaired maturation of erythroid and megakaryocyte populations, alongside upregulation of cell cycle checkpoint and DNA repair pathways, suggesting defective differentiation under replicative stress. CHEK2 mutant-only organoids exhibited a more inflammatory transcriptional profile with moderate erythroid skewing, but without the profound progenitor depletion seen in DDX41 mutation-associated samples. Notably, double-mutant organoids displayed the most severe phenotype, characterized by aberrant activation of Notch and Wnt/β-catenin signaling, reduced hematopoietic progenitors, and expansion of stromal compartments such as fibroblasts. These findings indicate that DDX41 is critical for terminal erythroid and megakaryocyte maturation, while CHEK2 contributes to inflammatory and proliferative dysregulation. The combined effect of both mutations exacerbates bone marrow niche imbalance and compromises hematopoietic output. These transcriptional and phenotypic findings were further supported by flow cytometry, which independently validated defects in lineage output and cell cycle progression across mutant groups. Taken together, our patient-derived bone marrow organoid model faithfully recapitulates the early hematopoietic and microenvironmental defects associated with germline DDX41 and CHEK2 mutations. By capturing the combined effects of proliferative stress, differentiation blockade, and niche remodeling in a human context, this platform provides a powerful tool for the mechanistic interrogation of myeloid predisposition. Our findings underscore the critical role of DDX41and CHEK2 in maintaining hematopoietic balance and offer novel insights into potential therapeutic vulnerabilities in individuals carrying germline mutations.