IFNα Inhibits JAK2V617F-Driven Neoplastic Vasculogenesis and Endothelial-to-Mesenchymal Transition in a 3D iPSC-Based Bone Marrow Niche Model

The vascular niche plays a crucial role in regulating hematopoiesis, and the presence of JAK2V617F endothelial cells (EC) in myeloproliferative neoplasms (MPN) underscores its therapeutic significance. Leveraging patient-specific induced pluripotent stem cells (iPSC) harboring JAK2 ^WT or the MPN-driver JAK2V617F (heterozygous, JAK2V617F ^HET or homozygous, JAK2V617F ^HOM ), we developed a scalable 3D bone marrow (BM)-niche mimicking model to study neoplastic vasculogenesis with precise control over cellularity and genetics. Global RNA-sequencing revealed zygosity-dependent phenotypic differences in EC, with JAK2V617F ^HET iPSC-derived EC (iEC) shifting toward a pro-mesenchymal phenotype via endothelial-to-mesenchymal transition (EndMT), while JAK2V617F ^HOM iEC displayed dysregulated translation machinery. EndMT was exacerbated in JAK2V617F ^HET iEC-mesenchymal stromal cells 3D cocultures exposed to inflammatory cytokines and was effectively inhibited by the tyrosine kinase inhibitors ruxolitinib and nintedanib. Notably, interferon-alpha (IFNα) showed potential in reducing EndMT, with its anti-EndMT effect validated in vivo in murine and MPN patient samples. Single-cell RNA sequencing revealed that JAK2V617F hematopoietic cells further amplified EndMT, emphasizing the interplay between the vascular niche and hematopoietic compartments. To our knowledge, this is the first study to report that IFNα’s anti-fibrotic and anti-angiogenic effects may be partly mediated through EndMT inhibition. Our 3D coculture model offers a powerful platform for mechanistic studies of human hematopoiesis.