Systemic mastocytosis (SM), a myeloproliferative disorder with variable clinical manifestations, is associated in most cases with the D816V mutation in KIT. The identification of the KIT D816V mutation in patients with systemic mastocytosis has gained a major prognostic significance in the last several years, largely because of the availability of tyrosine kinase receptor inhibitors such as imatinib. However, imatinib was shown to be ineffective in patients carrying KIT D816V mutation. This past year we made significant progress in evaluating novel therapeutics and identifying genetic/biological signatures for systemic mastocytosis. RAS proteins are small membrane associated GTPases that play a pivotal role in signal transduction events regulating cell proliferation, differentiation and survival. In murine models, activating mutations in RAS have produced a phenotype consistent with aggressive systemic mastocytosis. With this in mind, 44 patients with KIT-D816V systemic mastocytosis were evaluated for coexisting NRAS, KRAS, HRAS or MRAS mutations by Sanger sequencing. Activating NRAS mutations were identified in 2 of 8 patients with advanced disease. NRAS mutations were not found in patients with indolent systemic mastocytosis. To better understand the clonal evolution of mastocytosis, we evaluated the cell compartments impacted by the NRAS and KIT mutations. Clonal mast cells harbored both mutations. KIT-D816V was not detected in bone marrow CD34+ progenitors, whereas the NRAS mutation was present. These findings suggest that NRAS mutations may have the potential to precede KIT-D816V in clonal development. Unlike other mature lineages, mast cell survival is dependent on KIT and the presence of these two activating mutations may have a greater impact on the expansion of this cell compartment and in resultant disease severity. Most KIT tyrosine kinase inhibitors target the ATP binding domain which is often conserved between various tyrosine kinases. This therapeutic approach has resulted in off target inhibition with significant side effect profiles. In collaboration with Deciphera Pharmaceuticals, we evaluated novel KIT switch pocket inhibitors on mast cell proliferation and activation. Switch pocket interactions control the conformation and activity of kinases and are generally more kinase specific. We found that neoplastic human mast cell lines harboring the KIT D816V mutation were significantly inhibited by the switch pocket inhibitors through the induction of apoptosis. Both SCF mediated phosphorylation of wild type KIT and autophysphorylation of KIT D816V were completely inhibited at low nMolar ranges. Moreover, ex vivo studies with neoplastic mast cells from mastocytosis patients were significantly sensitive to these compounds. Overall, switch pocket inhibitors appear to offer a novel and potent KIT inhibition profile whose selectivity and dual suppression of SCF enhanced mast cell activation and KIT D816V neoplastic proliferation may provide significant therapeutic benefits Identifying novel clinical markers in mastocytosis may have prognostic and therapeutic relevance. Eosinophilia is frequently observed in systemic mastocytosis and the significance is poorly understood. Interleukin (IL)-5 plays a central role in the development and maintenance of eosinophilia and eosinophil activation. With this in mind, we decided to assess surface and soluble IL-5R levels in patients with eosinophilia and/or mastocytosis in collaboration with Drs. Klion and Nutman. Surface IL-5R expression was assessed by flow cytometry in blood and/or bone marrow from subjects with eosinophilia (n=39), systemic mastocytosis (n=8) and normal volunteers (n=28). Soluble IL-5R (sIL-5R) was measured in a cohort of 177 untreated subjects and correlated with eosinophilia, eosinophil activation, serum tryptase and cytokine levels. Whereas IL-5R expression on eosinophils inversely correlated with eosinophilia, serum levels of sIL-5R increased with eosinophil count and serum IL-5 levels. Of interest, sIL-5R was significantly elevated in patients with systemic mastocytosis without eosinophilia. Although sIL-5R levels correlated with serum tryptase levels in these patients, eosinophil activation, assessed by CD69 expression on eosinophils and serum eosinophil-derived neurotoxin levels, was increased compared to normal subjects. These data may have important implications with respect to the use of novel therapeutic agents targeting IL-5 and its receptor in patients with mastocytosis. Future research efforts to better understand the contribution of other cell lineages, such as mesenchymal stem cells, to mast cell disease are currently underway. In addition, a range of molecular approaches to identify coexisting genetic events are in progress including candidate gene sequencing, whole genome platforms and gene regulation studies.
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