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 is ineffective in patients carrying KIT D816V mutation. In collaboration with Deciphera Pharmaceuticals, we thus evaluated inhibitors that target the switch pocket (SP) of KIT by altering the catalytic conformation of KIT, thus inhibiting mast cell proliferation and activation. Two SP inhibitors, DP-2976 and DP-4851, were examined for effects on neoplastic mast cell proliferation and mast cell activation. KIT D816V was blocked by these compounds in transfected 293T cells, HMC 1.1 and 1.2 human mast cell lines, and in CD34+-derived human mast cells activated by stem cell factor. Both inhibitors induced apoptosis in the neoplastic mast cell lines and reduced survival of primary bone marrow mast cells from patients with mastocytosis. Additionally, the SP inhibitors more selectively blocked SCF potentiation of FceRI-mediated mast cell activation. Overall, switch pocket inhibitors thus may provide therapeutic benefit. KIT has two major variants which differ by four amino acids (GNNK- or GNNK+) at the juxta-membrane region of the extracellular domain. We hypothesized that the expression pattern of these variants differ in systemic mastocytosis, and that transcripts containing the KIT D816V mutation segregate preferentially to one GNNK variant. A quantitative real-time PCR assay to assess GNNK- and GNNK+ transcripts from bone marrow mononuclear cells was developed. We found that the GNNK-/GNNK+ copy number ratio positively trended with % neoplastic mast cell involvement and KIT D816V containing transcripts displayed a significantly elevated GNNK-/GNNK+ copy number ratio. Relative expression of the GNNK- variant, but not the GNNK+, correlated with increasing percent of neoplastic mast cell involvement. A mast cell transfection system revealed that the GNNK- isoform of wild type KIT was associated with increased granule formation, histamine content and growth. These data suggest that neoplastic mast cells favor a GNNK- variant predominance, which in turn enhances the activating potential of the KIT D816V mutation and thus may influence therapeutic sensitivity in systemic mastocytosis.

Project Start
Project End
Budget Start
Budget End
Support Year
32
Fiscal Year
2013
Total Cost
$603,643
Indirect Cost
City
State
Country
Zip Code
Siebenhaar, F; von Tschirnhaus, E; Hartmann, K et al. (2016) Development and validation of the mastocytosis quality of life questionnaire: MC-QoL. Allergy 71:869-77
Desai, Avanti; Jung, Mi-Yeon; Olivera, Ana et al. (2016) IL-6 promotes an increase in human mast cell numbers and reactivity through suppression of suppressor of cytokine signaling 3. J Allergy Clin Immunol 137:1863-1871.e6
Metcalfe, Dean D; Pawankar, Ruby; Ackerman, Steven J et al. (2016) Biomarkers of the involvement of mast cells, basophils and eosinophils in asthma and allergic diseases. World Allergy Organ J 9:7
Hartmann, Karin; Escribano, Luis; Grattan, Clive et al. (2016) Cutaneous manifestations in patients with mastocytosis: Consensus report of the European Competence Network on Mastocytosis; the American Academy of Allergy, Asthma & Immunology; and the European Academy of Allergology and Clinical Immunology. J Allergy Clin Immunol 137:35-45
Nemeth, Krisztian; Wilson, Todd M; Ren, Jiaqiang J et al. (2015) Impaired function of bone marrow stromal cells in systemic mastocytosis. Stem Cell Res 15:42-53
Arock, M; Sotlar, K; Akin, C et al. (2015) KIT mutation analysis in mast cell neoplasms: recommendations of the European Competence Network on Mastocytosis. Leukemia 29:1223-32
Carter, Melody C; Clayton, Sarah T; Komarow, Hirsh D et al. (2015) Assessment of clinical findings, tryptase levels, and bone marrow histopathology in the management of pediatric mastocytosis. J Allergy Clin Immunol 136:1673-79.e1-3
Carter, Melody C; Metcalfe, Dean D; Clark, Alicia S et al. (2015) Abnormal bone marrow histopathology in paediatric mastocytosis. Br J Haematol 168:865-73
Bandara, Geethani; Metcalfe, Dean D; Kirshenbaum, Arnold S (2015) Growth of human mast cells from bone marrow and peripheral blood-derived CD34(+) pluripotent hematopoietic cells. Methods Mol Biol 1220:155-62
Cruse, Glenn; Metcalfe, Dean D; Olivera, Ana (2014) Functional deregulation of KIT: link to mast cell proliferative diseases and other neoplasms. Immunol Allergy Clin North Am 34:219-37

Showing the most recent 10 out of 32 publications