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 (1). Mast cells express a variety of receptors that when activated by their cognate molecules can 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, and play regulatory roles in other pathologies (2,3). 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. 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. 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 new targets for treatment. Relating to objective 1: In SM, neoplastic accumulation of mast cells associates with genetic variants, particularly the missense mutation D816V in KIT. D816V-KIT causes ligand-independent activation of the receptor, oncogenic signaling and abnormal mast cell responses (4). In FY2022 we completed a study (5) in collaboration with Dr. Cruse (N. Carolina State University), demonstrating that treatment with antisense oligonucleotides, designed to induce exon skipping of KIT pre-mRNA and form alternatively spliced transcripts, reduced KIT expression, signaling and function in malignant mast cells. In preclinical models of advanced mastocytosis, injection of these oligonucleotides also substantially reduced tumor burden and metastasis. The study provides proof of concept for a treatment strategy targeting KIT expression instead of its tyrosine kinase activity to reduce mast cell numbers in KIT-associated proliferative disorders and malignancies that are often refractory to current treatment. In mast cell-related disorders, mast cell can be hyperactive and tend to accumulate in tissues. The molecular pathways regulating mast cell survival in homeostasis and disease are not well understood. Glioma-associated oncogene (GLI) proteins are known to regulate cell fate decisions in tissues and in a variety of cells, including hematopoietic cells. In FY2022 we investigated the role for GLI proteins in the control of proliferation and survival of human mast cells (6). GLI1 transcripts were present in primary human mast cells and mast cell lines harboring or not activating mutations in KIT, while GLI2 transcripts were only present in mast cells with KIT mutations, suggesting a role for oncogenic KIT signaling in the regulation of GLI2. Reduction in GLI activity by small molecule inhibitors, or by shRNA-mediated knockdown of GLI1 or GLI2, led to increases in apoptotic cell death in both human and murine mast cells in culture, and a reduction in the number of mast cells when injected into mice. Apoptosis induced by GLI silencing was associated with a downregulation in the expression of KIT and of genes that influence p53 stability and function. Furthermore, GLI silencing also inhibited the proliferation of neoplastic mast cell lines, especially those with fastest growth. This study uncovers a function of GLI proteins in mast cell biology and tissue homeostasis, adding to the understanding of the molecular pathways that regulate mast cell proliferation, survival and death, which is of relevance for developing novel pharmacological strategies to reduce mast cell burden in mast cell-related diseases (reviewed in 7). Neoplastic mast cells present in individuals with SM also show abnormal expression of certain cell surface receptors that in some instances have served as markers of transformation. One example is CD25, the receptor for the cytokine IL-2. During FY2022, we published a study investigating the potential mechanisms for the upregulation of CD25 in human mast cell populations and explored the potential function of CD25 in these cells (8). We demonstrated that the cytokine IL-3, more abundantly produced by bone marrow cells from patients with mastocytosis, causes an upregulation of CD25 in human mast cells, an effect specific to IL-3 among other cytokines. CD25-positive mast cells were able to bind its ligand IL-2, but such interaction was not signaling competent, probably because mast cells lack one of the indispensable subunits for IL-2 signaling, CD122. However, when CD25+ mast cells were pre-exposed to IL2, they were capable of cross-presentation and activation of regulatory T-cells , which contain all the subunits necessary for IL-2 signaling, suggesting that mast cell CD25 provides a reservoir of IL-2 for activation of other cell types. 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. We recently found that serum from patients with SM contains elevated numbers of EVs (SM-EVs) in association with markers of disease severity. In two consecutive studies, we have reported that EVs released by neoplastic mast cells and SM-EVs can alter the function of non-hematopoietic cells, and potentially contribute to the pathogenesis of SM: 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 (PMID: 30352845). 2) In another study in FY2021 (9), we demonstrated that 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, 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 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 for specific pathologies. The study is under review.