Surrogate markers of imatinib mesylate response in Gastrointestinal Stromal Tumors (GISTs)

A Frolov^a,c, S Chahwan^a, ZZ Pan^a, J Fletcher^b, O Favorova^c, M von Mehren^a, B Eisenberg^a, AK Godwin^a

^aFox Chase Cancer Center, Philadelphia, PA, Russian Federation, ^bDana-Farber Cancer Institute, Boston, MA, Russian Federation, ^cGraduate Program in the Department of Molecular Biology and Biotechnology, Russian State Medical University, Moscow, Russian Federation

Aim: 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) (GeevecTM) 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 identify surrogate markers of response and 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. Methods: We utilized GIST882, an immortal GIST cell line which possesses a homozygous exon 13 missense mutation (i.e., K642E) in c-KIT and is responsive to IM-induced growth arrest in vitro. cDNA microarrays were constructed from 10,367 human cDNA fragments corresponding to known genes and ESTs from the I.M.A.G.E. consortium library and were interrogated with RNA probes from IM treated GIST cells. Results:. 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 with IM. 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. Conclusions: We demonstrated that two of the identified genes, SPRY4A and MAFbx are likely regulated via the ERK1/2 pathway and are reliable genetic markers of immediate clinical response to IM treatment. This study also emphasizes the potential value of an in vitro cell model to investigate GIST response to IM in vivo, for the purpose of identifying important genetic markers of clinical response, mechanisms of drug action, and possible therapeutic targets.

Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 994 (Signaling pathways - Part II).