Rachana Vinay Patil

Juvenile myelomonocytic leukemia (JMML) is a fatal pediatric cancer characterized by classical features such as splenomegaly, monocytosis, and granulocyte-macrophage colony-stimulating factor (GM-CSF) hypersensitivity, with RAS pathway mutations being the major drivers. Mutations causing loss-of-function of the Neurofibromin 1 gene (NF1LOF) occur in ∼20% of patients with JMML. NF1LOF drives upregulation of RAS/MAPK/PI3K pathways that leads 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.

Despite therapeutic advancements, graft-versus-host disease (GVHD) is a major complication of hematopoietic stem cell transplantation (HSCT). In current models of GVHD, tissue injury induced by cytotoxic conditioning regimens, along with translocation of microbes expressing pathogen-associated molecular patterns, result in activation of host antigen-presenting cells (APCs) to stimulate alloreactive donor T lymphocytes. Recent studies have demonstrated that in many pathologic states, tissue injury results in the release of mitochondria from the cytoplasm to the extracellular space. We hypothesized that extracellular mitochondria, which are related to archaebacteria, could also trigger GVHD by stimulation of host APCs. We found that clinically relevant doses of radiation or busulfan induced extracellular release of mitochondria by various cell types, including cultured intestinal epithelial cells. Conditioning-mediated mitochondrial release was associated with mitochondrial damage and impaired quality control but did not affect the viability of the cells. Extracellular mitochondria directly stimulated host APCs to express higher levels of major histocompatibility complex II (MHC-II), costimulatory CD86, and proinflammatory cytokines, resulting in increased donor T-cell activation, and proliferation in mixed lymphocyte reactions. Analyses of plasma from both experimental mice and a cohort of children undergoing HSCT demonstrated that conditioning induced extracellular mitochondrial release in vivo. In mice undergoing MHC-mismatched HSCT, administration of purified syngeneic extracellular mitochondria increased host APC activation and exacerbated GVHD. Our data suggest that pre-HSCT conditioning results in extracellular release of damaged mitochondria, which increase alloreactivity and exacerbate GVHD. Therefore, decreasing the extracellular release of damaged mitochondria after conditioning could serve as a novel strategy for GVHD prevention.