Gastrointestinal stromal tumors (GISTs) are rare but deadly mesenchymal tumors accounting for (2,000 cases in the United States per year. This type of sarcoma is typically characterized by the presence of constitutively activated KIT, the receptor tyrosine kinase encoded by the c-KIT proto-oncogene. This observation has been successfully exploited in phase I and II trials, leading to FDA approval of Imatinib Mesylate (IM) (Gleevec() for patients with unresectable and/or metastatic GIST. The biological mechanisms of IM as well as its downstream molecular effects are for the most part unknown. In an attempt to further elucidate the mechanism of IM, we employed a DNA microarray-based approach to identify genetic changes and signaling pathways that were altered in response to IM in a GIST cell line. We identified a total of 148 genes or ESTs (out of 10,367) that were differentially regulated; 7 known genes displayed a sustained durable response over all time courses following treatment. The significantly down-regulated genes included Sprouty4A (SPRY4A), a member of a family of proteins that inhibit signaling by receptor tyrosine kinases. The only up-regulated gene, MAFbx, encodes for a protein that plays a role in muscle atrophy and is a member of the SCF family of E3 ubiquitin ligases. On a functional level, we demonstrated that IM rapidly inhibited autophosphorylation of KIT, and subsequent activation of AKT and ERK1/2 without affecting the total level of these proteins. Of interest our studies also found that differential expression of these response genes involved activation of MAPK-dependent as well as AKT- and MAPK-independent pathways. In an attempt to correlate these in vitro findings to clinical data, we examined GIST needle core biopsy specimens taken from patients before and after IM administration from patients participating in the CSTI571-B2222 Phase II trial and demonstrated that expression levels of SPRY4 and MAFbx correlated well with clinical response. In the studies proposed, we will expand our gene profiling studies to evaluate new clinical GIST biopsies from patients on a multi-institutional RTOG clinical trial S-0132. Patients participating in this trial will have tumor biopsies before IM treatment and definitive resection of the disease at 8 weeks after the initiation of IM. Tissues will thus be available for these profiling studies to evaluate patterns of expression following IM therapy, which will be correlated with clinical outcome and the KIT or PDGFR-alpha mutational status. Next, we will build on the results of the gene profiling studies as well as our strong preliminary data to further elucidate the role of c-KIT in the pathogenesis of GISTs and determine if AKT is an essential down-stream mediator of IM response. Finally we will investigate the role of SPRY4A in the development of GISTs and the therapeutic response to IM by evaluating its ability to negatively regulate KIT signaling. The overall goal of this project is to identify important genetic markers of clinical response in order to better elucidate the molecular mechanisms of action of IM with the ultimate goal of improving the treatment of GISTs.
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