Identification and Characterization of Signaling Pathways and Mediators Regulating Mast Cell-Related Disorders

National Institute Of Allergy And Infectious Diseases

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Abstract

Mast cells are effector and regulatory cells involved in the pathogenesis of allergic inflammation and anaphylaxis (reviewed in PMID: 36958519). Despite the increased incidence and awareness of allergic diseases and other diseases involving mast cells, the strategies to intercept mast cell activation and growth remain limited. In addition to the high affinity receptor for IgE, mast cells express a variety of receptors that when activated, they may induce the release of allergic and inflammatory mediators, promote the recruitment and differentiation of mast cell progenitors, and/or regulate the survival of mast cells in tissues. The consequent increases in mast cell mediators or the accumulation of mast cells in tissues are considered drivers of allergic processes and other mast cell-driven disorders. One of such receptors is the tyrosine kinase receptor KIT. KIT mediates mast cell differentiation, growth, survival and regulation of mast cell activation. Gain-of function mutations that result in the constitutive activation of KIT are associated with systemic mastocytosis (SM), a disorder characterized by the abnormal accumulation of neoplastic mast cells in tissues. The identification and characterization of receptors that activate or inhibit mast cell responses, their biological roles and signaling mechanisms (objective 1); as well as novel mediators of intercellular communication that contribute to the presentation of mast cell-related disorders (objective 2), will provide insight into the etiology and pathogenesis of these human illnesses, and potentially provide new targets for treatment. Relating to objective 1: In systemic mastocytosis (SM), neoplastic accumulation of mast cells associates with genetic variants, commonly the missense mutation D816V in KIT, which causes ligand-independent activation of the receptor, oncogenic signaling and abnormal mast cell responses. The mast cell line HMC-1.2 harboring two KIT mutations (D816V and V560G) is a common and widely used preclinical model to explore the functional and pharmacological attributes of neoplastic human mast cells. However, patients with SM present only with D816V-KIT, and the functional interactions of these double oncogenic KIT mutations present in HMC-1.2 cells are unknown. This prompted us to generate, using CRISPR/Cas9 engineering, a mast cell line derived from the parental HMC-1.2 cell line with a single D816V-KIT mutation that would better represent the properties of neoplastic mast cells found in patients with SM. Transcriptome analyses indicated reduced activity in pathways involved in survival, cell-to-cell adhesion, and neoplasia in HMC-1.3 compared to HMC-1.2 cells, with differences in expression of molecular components and cell surface markers. Consistently, subcutaneous inoculation of HMC-1.3 into mice produced significantly smaller tumors than HMC-1.2 cells, and in colony assays, HMC-1.3 formed less numerous and smaller colonies than HMC-1.2 cells. HMC-1.3 cells were also more susceptible than HMC-1.2 cells to various pharmacological inhibitors used clinically for treatment of advanced SM. Our study thus reveals that the additional V560G-KIT oncogenic variant in HMC-1.2 cells modifies transcriptional programs induced by D816V-KIT, confers a survival advantage, alters sensitivity to interventional drugs, and increases the tumorigenicity, and thus engineered huMCs with a single D816V-KIT variant may represent an improved preclinical model for mastocytosis. The study was published in FY2023 (PMID: 37025992). Relating to objective 2: Extracellular vesicles (Evs) are nano-sized vesicular structures secreted by various cell types into body fluids. EVs are considered vehicles for cell-to-cell communication as they shuttle distinct biological molecules taken up by recipient cells. In research we conducted in FY2022 and earlier, we reported that EVs released by neoplastic mast cells and those found in the serum of from patients with SM (SM-EVs) can alter the function of non-hematopoietic cells. Specifically, we found that : 1) Functionally active KIT shuttled in SM-EVs is taken up by hepatic stellate cells inducing a fibrotic phenotype in vitro and in mouse livers, potentially linking SM-EVs and liver fibrosis in SM, and 2) SM-EVs contain miRNA species taken up by osteoblasts that inhibit differentiation and maturation programs of osteoblasts into osteoclasts and cause reduced trabecular bone density, suggesting a role for SM-EVs in the pathogenesis of bone disease, one of the most frequent and debilitating complications in patients with SM. Despite potential contributions to diseases, KIT-containing EVs have not been thoroughly characterized. In FY2022-FY2023, we isolated and characterized KIT-EV subpopulations released by neoplastic human mast cells using an immunocapture approach that selectively isolates EVs containing KIT. Immunocapture of EVs on KIT antibody-coated electron microscopy affinity grids allowed to assess the morphology and size of KIT-EVs. We found that KIT-EVs are numerous among all EVs secreted by neoplastic mast cells and represent an heterogenous group of microvesicle- and exosome-like EVs. Immunoblot and proteomic analysis revealed KIT-EVs are also enriched in proteins involved in signaling, immune responses, and cell migration pathways. The study suggests that oncogenic KIT can disseminate by shuttling in heterogeneous EVs with potentially diverse biological function. The developed selective KIT-EV immunocapture will enable the enrichment of specific huMC-derived EVs from complex human biosamples and facilitate an understanding of their in vivo functions, and has potential to serve as biomarkers of specific pathologies (PMID: 36239715). Given that neoplastic MCs with constitutively active KIT secrete exceptionally large quantities of EVs and that patients with oncogenic KIT-driven diseases have higher concentrations of circulating EVs than healthy controls, in a follow-up study sent for publication recently we investigated a potential role for KIT receptor activation in the regulation of EV secretion. Our study demonstrates that constitutive KIT activation caused by gain-of-function mutations, as well as ligand-mediated KIT activation, induce the release of EVs in a KIT activity- and signaling-dependent manner. The stimulated sEV release was reversed by KIT inhibitors or by inhibition of signaling molecules downstream of KIT activation. This study highlights that the secretion of sEVs by mast cells can be controlled by cellular pathways.The finding that sEV secretion is stimulated by signaling cascades that also transduce other functional responses by KIT provides impetus to the concept of EVs being part of KIT-associated biological functions in the microenvironment and/or regulation of KIT availability and signaling.