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. As discussed in our FY2021 review (Falduto et al. Pharmacol. Ther.), these responses are generally, but not exclusively, mediated through canonical activation of the high affinity IgE receptor (Fc-epsilon-RI). Within atopic individuals, mast cell activation occurs via the engagement of Fc-epsilon-RI after exposure to an antigen (or allergen). Thus, targeting of IgE, Fc-epsilon-RI, and the resulting signaling pathways has been a major focus for the treatment of these diseases. However, other cell surface receptors activated by stimulants in the tissue environment play critical roles in enhancing or inhibiting allergic processes or other mast cell-driven disorders. An example is the tyrosine kinase receptor KIT which mediates important biological processes in mast cells such as differentiation, growth, survival and activation. Mutations that result in constitutive activation of KIT are associated with mastocytosis, a rare disorder characterized by abnormal accumulation of mast cells in tissues and reactions caused by excessive mast cell-derived products. Despite the increased incidence and awareness of allergic diseases and other disorders involving mast cells, the strategies to intercept mast cell activation and growth remain limited. A deeper understanding of receptors that activate or inhibit mast cell responses, their biological roles and signaling mechanisms gives important insight on to the regulation of allergic and non-allergic hypersensitivity reactions and treatment options for these human illnesses. Similarly, identification of mediators of intercellular communication that contribute to the presentation of mast cell-related disorders can provide new targets for treatment. Relating to objective 1: Binding of an antigen to IgE/Fc-epsilon-RI complex, initiates multiple intracellular signaling events that drive mast cell responses, including the secretion of vascular mediators by exocytosis or degranulation and release of inflammatory substances. In collaboration with Dr. Martin (Hospital Clinic of Barcelona), we found that the unconventional long-tailed myosin, MYO1F, localized with cortical F-actin, is an important regulator of human mast cell degranulation induced by IgE or by stimulation of the Mas-related G protein-coupled receptor X2 (MRGPRX2), a receptor associated with pseudoallergic drug reactions. Knockdown of MYO1F reduced degranulation induced by these receptors in association with reduced Cdc42 GTPase activity and reassembly of cortical actin, along with deficient translocation of mitochondria to exocytosis sites and mitochondrial fission. In systemic mastocytosis (SM), neoplastic accumulation of mast cells is found in association with genetic variants in KIT, particularly the missense mutation D816V-KIT that causes ligand-independent activation, oncogenic signaling and abnormal mast cell responses. Thus, therapies blocking KIT signaling are a leading strategy to treat MC proliferative disorders. Albeit relatively successful, these KIT inhibitors have off-target effects and in some patients, complete remission or improved survival time cannot be achieved. In FY2020 through FY2021, in collaboration with Dr. Cruse (N. Carolina State University), we demonstrated that targeting KIT expression can be an effective approach to therapy. We employed chemically stable antisense oligonucleotides that induce exon skipping in KIT pre-mRNA (KitStop). The resulting alternatively spliced transcript contains an immediate STOP codon, encoding a severely truncated and non-functional protein, as demonstrated by a marked reduction in KIT protein expression, signaling and function. Furthermore, in preclinical models of advanced mastocytosis, injection of KitStop substantially reduced tumor burden and metastasis without obvious signs of oligonucleotide-induced toxicity or effects on hematopoiesis/erythropoiesis. These results provide proof of concept for a treatment strategy for mast cell and KIT-associated proliferative disorders and malignancies that are often refractory to current treatments and could have benefit in several allergic diseases, such as atopic dermatitis, allergic asthma and seasonal allergic rhinitis. Additionally, neoplastic mast cells in individuals with mastocytosis express receptors on the cell surface that are usually not present in normal mast cells and have served as markers of transformation. One example is CD25 (the receptor for the cytokine IL-2), which is abnormally and characteristically expressed in neoplastic mast cells or during certain inflammatory conditions. In this FY2021, we concluded a study (currently being re-submitted after revisions) investigating what mediators may be responsible for CD25 expression and the potential function for CD25 in neoplastic mast cells. We identified higher levels of IL-3 in the marrow of patients with mastocytosis and exposure of mast cells to IL-3 early during development, but not at later stages, induced CD25 expression, an effect that seemed specific to IL-3 among other cytokines. Although mast cells were able to bind IL-2 via CD25, IL-2 did not induce detectable signaling probably because mast cells lack one of the indispensable subunits for IL-2 signaling, CD122. However, IL-2 bound to mast cell CD25 fully activated Tregs that express the trimeric IL-2 receptor, suggesting that mast cell CD25 may provide a reservoir of IL-2 then available for cross-presentation and activation of other cell types. Relating to objective 2: During previous years, we found that serum from patients with SM contains elevated concentrations of extracellular vesicles (EVs) with a mast cell signature and which associate with markers of disease severity. EVs are nano-sized vesicular structures secreted by various cell types and generally found in body fluids. EVs have emerged as vehicles for cell-to-cell communication as they enable intercellular exchange by the transfer of distinct biological molecules to recipient cells. We had found that EVs from patients with SM (SM-EVs) induce a fibrotic phenotype in hepatic stellate cells (a recognized as a step in the pathogenesis of liver fibrosis) in vitro and in vivo by transferring functionally active KIT, thus linking patient EVs to liver pathology in mastocytosis. The identification of SM-EVs from neoplastic cells altering non-hematopoietic cells unfolded the concept that SM-EVs may contribute to other presentations in mast cell related diseases. In FY2021, we have completed a study on a role for SM-EVs on bone homeostasis, an environment where mast cells pathologically accumulate. Bone disease, including osteopenia, osteoporosis, osteolytic lesions and osteosclerosis, is one of the most frequent and debilitating complications in patients with systemic mastocytosis. However, the pathogenesis of bone disease in SM is not understood. We found that EVs isolated from patients with SM or EVs released from neoplastic mast cells inhibited differentiation and maturation of osteoblasts and bone formation both in vitro and in vivo. These EVs, taken up by pre-osteoblastic cells, contained miRNA-30a and miRNA-23a that repressed the transcription of RUNX2 and SMAD proteins in pre-osteoblasts, thus regulating osteogenesis and osteoblast differentiation. Correlating with those findings, miRNA-30a and miRNA-23a levels were higher in EVs from patients with SM positively diagnosed for osteopenia/osteoporosis. Consistently, bone marrow cells isolated from patients with bone disease expressed lower mRNA levels of RUNX2. Our investigations may shed light into the contributions of EVs to bone homeostasis by affecting osteoblast functions and the pathogenesis of bone disease in mast cell-related disorders.