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

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, the NF1LOF cells displayed marked hypersensitivity to GM-CSF in in vitro colony-forming unit assays, mirroring a distinctive trait seen in JMML patient cells. Humanized NSG-SGM3 mouse neonates transplanted with NF1LOF HSPCs had a median survival of 32 days, and displayed abrogation of lymphocytes, accumulation of myeloid cells and extensive leukemia infiltration in spleen, liver, and lungs, consistent with those observed in human JMML patients. This is the first successful development of a humanized mouse model of NF1LOF that recapitulates many of the features of JMML thus providing a novel system to investigate JMML progression and interrogate targeted treatment strategies.