Novel humanized loss-of-function NF1 mouse model of juvenile myelomonocytic leukemia Open Access

• CRISPR/Cas9-mediated NF1 loss-of-function in human hematopoietic cells induced juvenile myelomonocytic leukemia (JMML) in vivo.

• Our model accurately recapitulates key disease features that are hallmarks of disease in JMML patients and patient-derived xenografts.

Juvenile myelomonocytic leukemia (JMML) is a fatal pediatric cancer characterized by classical features such as splenomegaly, monocytosis, and GM-CSF hypersensitivity, with RAS pathway mutations being the major drivers. Mutations causing loss-of-function of the Neurofibromin1 gene (NF1LOF) occur in ~20% of JMML patients. NF1LOF drives upregulation of RAS/MAPK/PI3K pathways that lead to aggressive proliferation/differentiation of immature myeloid cells. Hematopoietic stem cell transplantation is the only curative option, but relapse occurs in ~50% of patients, indicating an urgent need for novel and targeted therapeutic strategies. However, low patient sample availability and a lack of reliable disease models have made it difficult to study and treat JMML. Using CRISPR/Cas9, we have generated NF1LOF in human umbilical cord blood-derived hematopoietic stem and progenitor cells (HSPCs). We achieved a high gene knockout rate of ~89% and concomitant loss of NF1 protein in the modified HSPCs. Importantly, NF1LOF cells displayed marked GM-CSF hypersensitivity in in vitro colony-forming unit assays - mirroring JMML; when transplanted into NSG-SGM3 mice, they caused rapid lethality, (median survival of 32 days), myeloid expansion, tissue infiltration (spleen, liver, and lungs), and specific upregulation of RAS/MAPK pathway and STAT5 genes, consistent with patient profiles. This first humanized NF1LOF mouse model recapitulates key JMML features, enabling investigation of disease mechanisms and targeted therapies.