The goal of this proposal is to add to the understanding of the mechanisms underlying the pathogenesis of mastocytosis. They have previously shown that essentially all adult and atypical pediatric mastocytosis are characterized by mutations in codon 816 of c-KIT gene causing constitutive activation of its protein product, KIT, a receptor tyrosine kinase. In contrast, most pediatric cases lack known activating c-KIT mutations. Our recent data indicate that activation of the P13K/AKT pathway downstream of KIT is a general feature of all forms of mastocytosis. We thus hypothesize that most, if not all, cases of mastocytosis are caused by over-activation of the KIT signaling pathway, or another pathway that converges with it at or above the P13K/AKT node. We have found that synthetic compounds that effectively inhibit wild-type KIT are ineffective against KIT containing codon 816 activating mutations at concentrations that can be safely achieved in vivo. Since the residue encoded by codon 816 lies in the active site of the receptor kinase, where the compounds presumably bind, we hypothesize that the conformation of the active site of the mutant receptor differs from that of wild-type KIT. We propose to test the two hypotheses by two specific aims. 1. To determine the mechanisms of oncogenesis in c-KIT mutation negative pediatric mastocytosis, genes encoding molecules affecting P13K/AKT signaling will be examined in laser captured lesional mast cells by PCR sequencing for mutations and by microsatellite analysis for loss of heterozygosity. The functional significance of identified defects will be determined by cDNA expression in mast cells in vitro and in vivo. 2. To determine the three-dimensional structure of KIT kinase domain, wild-type and codon 816 mutated KIT kinase domains will be expressed using the Baculovirus/Sf9 system and purified using anion exchange and gel filtration chromatography. The proteins will be crystallized using vapor diffusion in hanging drops. X-ray diffraction of the crystals will be analyzed using the multiwavelength anomalous dispersion procedure for phase determination. The WARP program will be used to build models, and the models will be refined and rebuilt using the program XPLOR and O. Knowing the structures of the wild-type and mutant kinase domains will allow rational development of drugs targeted specifically at a defined cause of mastocytosis.
| Yang, Bing; Wu, Jianqiang; Maddodi, Nityanand et al. (2007) Epigenetic control of MAGE gene expression by the KIT tyrosine kinase. J Invest Dermatol 127:2123-8 |
