c-kit D816V- dependent expression of target genes

Systemic mastocytosis (SM) is a clonal disorder of myelomastocytic progenitors. The disease is characterized by accumulation of neoplastic mast cells (MC) in visceral organs with formation of clusters and aggregates, bone marrow fibrosis, and increased angiogenesis. In a majority of the patients, MC display the c-kit point mutation D816V that is considered to play a key role in the pathogenesis of SM. Thus, c-kit D816V exhibits constitutive tyrosine kinase aetivity and leads to cytokine-independent growth of MC. However, little is known so far about regulated genes and underlying signaling pathways through which the D816V-mutated c-kit receptor leads to enhanced survival of MC, their pathologic accumulation in visceral organs, and the typical changes in the bone marrow microenvironment (fibrosis, increased angiogenesis). During the past few years, we and others have shown that MC in patients with SM express a number of survival- (bcl-2, bcl-xL) and adhesionrelated (CD2) as well as angiogenic (VEGF) molecules. Interestingly, some of these antigens are only detectable in neoplastic MC in SM, but are not expressed in normal MC. Other antigens appear to be overexpressed in neoplastic MC. However, it remains unknown whether these genes are regulated in neoplastic MC in a c-kit D816V-dependent manner. The aims of the present study are to define the rote of the c-kit mutation D816V in abnormal expression of survival-, adhesion-, and angiogenesis-related molecules in neoplastic cells, to characterize respective signaling pathways, and to identify specific pharmacologic inhibitors of c-kit D816V-dependent gene expression. For this purpose, Ba/F3 cells with doxycycline-inducible expression of wild type (WT) c-kit and mutated c-kit (D816V) have been established in our laboratory and will be employed in this project. c-kit D816V-dependent gene expression will be analyzed at the promoter-, mRNA- and protein level. Signaling pathways contributing to c-kit D816V-dependent gene expression will be investigated using specific pharmacologic inhibitors as well as mutated (dominant negative or constitutively activated) forms of important signaling molecules such as Ras, PI3-kinase, Akt, or STAT5. Results obtained with Ba/F3 cells will be re-confirmed for the human mast cell leukemia cell line HMC-1 and for primary neoplastic MC obtained from patients with SM. The role of c-kit D816V-dependent gene expression an survival and accumulation of MC will be analyzed in in vitro experiments as well as in vivo employing a xenotransplant SCID mouse model. The results obtained from the current project are expected to contribute to our knowledge about neoplastic MC in general, and in particular to the understanding of c-kit D816V-dependent mechanisms underlying the pathogenesis of SM. The project may also lead to the identification of novel important molecular targets in neoplastic MC and thereby help in the development of targeted drug therapy interfering with growth and survival of MC in patients wich SM.