SCIENCE UP THE WORLD
15TH INTERNATIONAL STUDENT CONGRESS OF MEDICAL SCIENCES
JUNE 3RD - 6TH 2008
Abstract Marjolein Donker
Genes related to mast cell apoptosis in response to tyrosine kinase inhibition1,2Marjolein L. Donker, 1Hanneke C. Kluin-Nelemans, 2Cem Akin
1Department of Hematology, University Medical Center Groningen, Groningen, The Netherlands, 2Department of Allergy and Immunology, University of Michigan, Ann Arbor, MI, USA (United States of America)
Introduction
In the majority of systemic mastocytosis patients, the D816V mutation in the c-kit proto-oncogene is detected. This mutation causes ligand-independent activation of its protein product, the tyrosine kinase receptor KIT, and is associated with enhanced survival of neoplastic mast cells. Since KIT activation is a pivotal event in mastocytosis, specific tyrosine kinase inhibitors, which may inhibit the survival of neoplastic mast cells, are likely to become an attractive therapeutic option.
We studied the apoptotic response of mast cells with activating c-kit mutations to several tyrosine kinase inhibitors in vitro. To elucidate molecular mechanisms underlying mast cell apoptosis in response to specific tyrosine kinase inhibition, expression of both gene profiles and individual genes was investigated. In addition, these identified downstream targets may be attractive targets for new therapies.
Material and Methods
Mastocytosis-derived cell lines HMC-1.1D816V- and HMC HMC-1.2D816V+, carrying activating c-kit mutations and CD34+ derived wild-type mast cells were incubated with tyrosine kinase inhibitors imatinib, AMN107, PKC412 and dasatinib. Cell growth inhibitory effects were quantified by cell counts using trypan blue exclusion after culturing with various concentrations (0-1μM) of drugs for 24, 48 and 72 hours. Apoptosis was assessed by Annexin V flow cytometry. Differentially expressed genes were identified by micro array analysis, using RNA isolated from HMC-1.1D816V- and HMC-1.2D816V+ cells after treatment with no drug, with 1μM of imatinib or 1μM PKC412 for 24 hours. Micro array results were validated by RT-PCR.
Results
All tyrosine kinase inhibitors are able to induce apoptosis in HMC-1.1D816V- cells, but only PKC412 and dasatinib were effective in cells carrying the activating D816V c-kit mutation. The majority of differentially expressed genes are involved in signal transduction, cell growth and protein metabolism. Selected genes of interest include FSH primary response, leukemia inhibitory factor, adult retina protein, adrenomedullin and neurophilin 1.
Conclusion
PKC412 and dasatinib are the most effective tyrosine kinase inhibitors studied to induce apoptosis in mast cells with the activating c-kit mutation D816V, as present in systemic mastocytosis. By identifying differentially expressed genes of interest and clusters of genes in mast cells with activating c-kit mutations in response to treatment with these drugs, putative therapeutic targets for treatment of systemic mastocytosis are identified. These targets include neurophilin 1 and leukemia inhibitory factor, which appear to be related to mast cell survival and proliferation. Both tyrosine kinase inhibitors and new drugs targeting the identified genes may become attractive future therapeutics in systemic mastocytosis.
Keyword(s): mastocytosis, tyrosine kinase inhibitors, gene expression
