The Molecular Actions of Imatinib Mesylate in GISTs

描述（由申请人提供）：胃肠道间质瘤（gist）是一种罕见但致命的间质肿瘤，在美国每年约有2000例。这种类型的肉瘤的典型特征是存在组成型激活KIT，即由c-KIT原癌基因编码的受体酪氨酸激酶。这一观察结果已在I期和II期试验中成功利用，导致FDA批准甲磺酸伊马替尼（Imatinib Mesylate，简称Gleevec）用于不可切除和/或转移性GIST患者。IM的生物学机制及其下游分子效应在很大程度上是未知的。为了进一步阐明IM的机制，我们采用了一种基于DNA微阵列的方法来鉴定胃肠道间质瘤细胞系中因IM而改变的遗传变化和信号通路。我们共鉴定出148个基因或ESTs（10,367个）受到差异调控；7个已知基因在治疗后的所有时间过程中表现出持续持久的反应。显著下调的基因包括Sprouty4A (SPRY4A)，这是一个抑制受体酪氨酸激酶信号传导的蛋白家族的成员。唯一上调的基因MAFbx编码一种在肌肉萎缩中起作用的蛋白质，是E3泛素连接酶SCF家族的成员。在功能水平上，我们证明了IM快速抑制KIT的自磷酸化，随后抑制AKT和ERK1/2的激活，而不影响这些蛋白的总水平。有趣的是，我们的研究还发现，这些应答基因的差异表达涉及mapk依赖性通路以及AKT-和mapk -非依赖性通路的激活。为了将这些体外结果与临床数据联系起来，我们检查了参加CSTI571-B2222 II期试验的患者在IM给药前后的GIST针芯活检标本，并证明SPRY4和MAFbx的表达水平与临床反应密切相关。在拟议的研究中，我们将扩大我们的基因谱研究，以评估来自多机构RTOG临床试验S-0132的患者的新的临床GIST活检。参加该试验的患者将在IM治疗前进行肿瘤活检，并在IM开始后8周进行最终切除。因此，组织将可用于这些分析研究，以评估IM治疗后的表达模式，这将与临床结果和KIT或pdgfr - α突变状态相关。接下来，我们将基于基因谱研究的结果以及我们强有力的初步数据，进一步阐明c-KIT在gist发病机制中的作用，并确定AKT是否是IM反应的重要下游介质。最后，我们将通过评估SPRY4A负性调节KIT信号的能力来研究SPRY4A在gist发展和IM治疗反应中的作用。本项目的总体目标是识别临床反应的重要遗传标记，以便更好地阐明IM的分子作用机制，最终达到改善gist治疗的目的。

DESCRIPTION (provided by applicant): 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.