Effects of the KIT K509I Extracellular Activating Mutation on Human Mast Cell Homeostasis

RationaleKIT is a tyrosine kinase receptor which binds stem cell factor (SCF) and is critical to human mast cell (HuMC) development and survival. Recently we identified a patient with systemic mastocytosis harboring a rare heterozygous germline KIT K509I mutation. K509I is located in the 5th immunoglobulin region and such mutations are thought to enhance KIT homodimerization and activation. With this in mind, the effect of the K509I mutation on HuMC development was investigated.MethodsPrimary HuMC's were derived from CD34+ peripheral blood progenitors collected by apheresis. Cells were cultured in IL-3 (week 1) and IL-6 +/- SCF for 8 weeks. HuMC growth and survival was analyzed by MTT and Annexin V apoptosis assays. HuMC morphology was assessed by electron and light microscopy. Multiparameter FACS analysis, β-hexosaminidase release and total cell histamine assays were performed.ResultsK509I progenitors cultured in SCF demonstrated a ten-fold expansion compared to progenitors from healthy controls; and developed into morphologically appearing mature HuMCs. K509I HuMC expansion continued for up to 20 weeks, exceeding normal survival (10-12 weeks); and K509I HuMCs displayed decreased apoptosis upon SCF withdrawal when compared to normal HuMCs. A two-fold increase in FcεRI surface expression, cell histamine content and antigen mediated degranulation was observed in K509I HuMCs. K509I progenitors cultured in the absence of SCF also survived and developed into hypogranular appearing HuMCs.ConclusionsKIT K509I progenitors develop into mature appearing HuMCs with enhanced proliferation, survival and activation. K509I progenitors provide a unique opportunity to study the impact of a KIT germline mutation on HuMC homeostasis. RationaleKIT is a tyrosine kinase receptor which binds stem cell factor (SCF) and is critical to human mast cell (HuMC) development and survival. Recently we identified a patient with systemic mastocytosis harboring a rare heterozygous germline KIT K509I mutation. K509I is located in the 5th immunoglobulin region and such mutations are thought to enhance KIT homodimerization and activation. With this in mind, the effect of the K509I mutation on HuMC development was investigated. KIT is a tyrosine kinase receptor which binds stem cell factor (SCF) and is critical to human mast cell (HuMC) development and survival. Recently we identified a patient with systemic mastocytosis harboring a rare heterozygous germline KIT K509I mutation. K509I is located in the 5th immunoglobulin region and such mutations are thought to enhance KIT homodimerization and activation. With this in mind, the effect of the K509I mutation on HuMC development was investigated. MethodsPrimary HuMC's were derived from CD34+ peripheral blood progenitors collected by apheresis. Cells were cultured in IL-3 (week 1) and IL-6 +/- SCF for 8 weeks. HuMC growth and survival was analyzed by MTT and Annexin V apoptosis assays. HuMC morphology was assessed by electron and light microscopy. Multiparameter FACS analysis, β-hexosaminidase release and total cell histamine assays were performed. Primary HuMC's were derived from CD34+ peripheral blood progenitors collected by apheresis. Cells were cultured in IL-3 (week 1) and IL-6 +/- SCF for 8 weeks. HuMC growth and survival was analyzed by MTT and Annexin V apoptosis assays. HuMC morphology was assessed by electron and light microscopy. Multiparameter FACS analysis, β-hexosaminidase release and total cell histamine assays were performed. ResultsK509I progenitors cultured in SCF demonstrated a ten-fold expansion compared to progenitors from healthy controls; and developed into morphologically appearing mature HuMCs. K509I HuMC expansion continued for up to 20 weeks, exceeding normal survival (10-12 weeks); and K509I HuMCs displayed decreased apoptosis upon SCF withdrawal when compared to normal HuMCs. A two-fold increase in FcεRI surface expression, cell histamine content and antigen mediated degranulation was observed in K509I HuMCs. K509I progenitors cultured in the absence of SCF also survived and developed into hypogranular appearing HuMCs. K509I progenitors cultured in SCF demonstrated a ten-fold expansion compared to progenitors from healthy controls; and developed into morphologically appearing mature HuMCs. K509I HuMC expansion continued for up to 20 weeks, exceeding normal survival (10-12 weeks); and K509I HuMCs displayed decreased apoptosis upon SCF withdrawal when compared to normal HuMCs. A two-fold increase in FcεRI surface expression, cell histamine content and antigen mediated degranulation was observed in K509I HuMCs. K509I progenitors cultured in the absence of SCF also survived and developed into hypogranular appearing HuMCs. ConclusionsKIT K509I progenitors develop into mature appearing HuMCs with enhanced proliferation, survival and activation. K509I progenitors provide a unique opportunity to study the impact of a KIT germline mutation on HuMC homeostasis. KIT K509I progenitors develop into mature appearing HuMCs with enhanced proliferation, survival and activation. K509I progenitors provide a unique opportunity to study the impact of a KIT germline mutation on HuMC homeostasis.