A subset of gastrointestinal stromal tumors (GISTs) lack gain-offunction mutations in c-KIT and PDGFR␣. These so-called wild-type (WT) GISTs tend to be less responsive to imatinib-based therapies and have a poor prognosis. We identified amplification of IGF1R in a SNP analysis of GIST and thus studied its potential as a therapeutic target in WT and mutant GIST. Expression of IGF1R and downstream effectors in clinical GIST samples was examined by using immunoblots and immunohistochemistry. The roles of IGF1R signaling in GIST and viability were analyzed by using NVP-AEW541, an inhibitor of IGF1R, alone and in combination with imatinib, or via siRNA silencing of IGF1R. IGF1R was strongly overexpressed, and IGF1R amplification was detected at a significantly higher frequency in WT GISTs, including a pediatric WT GIST, compared with mutant GISTs (P ‫؍‬ 0.0173 and P ‫؍‬ 0.0163, respectively). Inhibition of IGF1R activity in vitro with NVP-AEW541 or down-regulation of expression with siIGF1R led to cytotoxicity and induced apoptosis in GIST cell lines via AKT and MAPK signaling. Combination of NVP-AEW541 and imatinib in GIST cell lines induced a strong cytotoxicity response. Our results reveal that IGF1R is amplified and the protein is overexpressed in WT and pediatric GISTs. We also demonstrate that the aberrant expression of IGF1R may be associated with oncogenesis in WT GISTs and suggest an alternative and/or complementary therapeutic regimen in the clinical management of all GISTs, especially in a subset of tumors that respond less favorably to imatinib-based therapy.pediatric GIST ͉ tyrosine kinase inhibitors ͉ imatinib mesylase ͉ adult wild-type GIST ͉ NYP-AEW541

Hepatitis delta virus (HDV) genome replication requires the virus-encoded small delta protein (␦Ag).During replication, nucleotide sequence changes accumulate on the HDV RNA, leading to the translation of ␦Ag species that are nonfunctional or even inhibitory. A replication system was devised where all ␦Ag was conditionally provided from a separate and unchanging source. A line of human embryonic kidney cells was stably transfected with a single copy of cDNA encoding small ␦Ag, with expression under tetracycline (TET) control. Next, HDV genome replication was initiated in these cells by transfection with a mutated RNA unable to express ␦Ag. Thus, replication of this RNA was under control of the TET-inducible ␦Ag. In the absence of TET, there was sufficient ␦Ag to allow a low level of HDV replication that could be maintained for at least 1 year. When TET was added, both ␦Ag and genomic RNA increased dramatically within 2 days. With clones of such cells, designated 293-HDV, the burst of HDV RNA replication interfered with cell cycling. Within 2 days, there was a fivefold enhancement of G 1 /G 0 cells relative to both S and G 2 /M cells, and by 6 days, there was extensive cell detachment and death. These findings and those of other studies that are under way demonstrate the potential applications of this experimental system.

Purpose: Gastrointestinal stromal tumors (GIST) are characterized by expressing a gainof-function mutation in KIT, and to a lesser extent, PDGFR. Imatinib mesylate, a tyrosine kinase inhibitor, has activity against GISTs that contain oncogenic mutations of KIT. In this study, KIT and PDGFRa mutation status was analyzed and protein modeling approaches were used to assess the potential effect of KIT mutations in response to imatinib therapy. Experimental Design: Genomic DNA was isolated from GIST tumors. Exons 9, 11, 13, and 17 of c-KIT and exons 12, 14, and 18 of PDGFRa were evaluated for oncogenic mutations. Protein modeling was used to assess mutations within the juxtamembrane region and the kinase domain of KIT. Results: Mutations in KITexons 9,11, and 13 were identified in GISTs with the majority of changes involving the juxtamembrane region of KIT. Molecular modeling indicates that mutations in this region result in disruption of the KITautoinhibited conformation, and lead to gain-of-function activation of the kinase. Furthermore, a novel germ-line mutation in KIT was identified that is associated with an autosomal dominant predisposition to the development of GIST. Conclusions: We have used protein modeling and structural analyses to elucidate why patients with GIST tumors containing exon11mutations are the most responsive to imatinib mesylate treatment. Importantly, mutations detected in this exon and others displayed constitutive activation of KIT. Furthermore, we have found tumors that are both KITand PDGFRa mutation negative, suggesting that additional, yet unidentified, abnormalities may contribute to GIST tumorigenesis.

Cell signaling plays a central role in the etiology of cancer. Numerous therapeutics in use or under development target signaling proteins; however, off-target effects often limit assignment of positive clinical response to the intended target. As direct measurements of signaling protein activity are not generally feasible during treatment, there is a need for more powerful methods to determine if therapeutics inhibit their targets and when off-target effects occur. We have used the Bayesian Decomposition algorithm and data on transcriptional regulation to create a novel methodology, Differential Expression for Signaling Determination (DESIDE), for inferring signaling activity from microarray measurements. We applied DESIDE to deduce signaling activity in gastrointestinal stromal tumor cell lines treated with the targeted therapeutic imatinib mesylate (Gleevec). We detected the expected reduced activity in the KIT pathway, as well as unexpected changes in the p53 pathway. Pursuing these findings, we have determined that imatinib-induced DNA damage is responsible for the increased activity of p53, identifying a novel off-target activity for this drug. We then used DESIDE on data from resected, post-imatinib treatment tumor samples and identified a pattern in these tumors similar to that at late time points in the cell lines, and this pattern correlated with initial clinical response. The pattern showed increased activity of ETS domain-containing protein Elk-1 and signal transducers and activators of transcription 3 transcription factors, which are associated with the growth of side population cells. DESIDE infers the global reprogramming of signaling networks during treatment, permitting treatment modification that leverages ongoing drug development efforts, which is crucial for personalized medicine. [Cancer Res 2009;69(23):9125-32]

Most gastrointestinal stromal tumors (GISTs) possess a gainof-function mutation in c-KIT. Imatinib mesylate, a smallmolecule inhibitor against several receptor tyrosine kinases, including KIT, platelet-derived growth factor receptor-A, and BCR-ABL, has therapeutic benefit for GISTs both via KIT and via unknown mechanisms. Clinical evidence suggests that a potential therapeutic benefit of imatinib might result from decreased glucose uptake as measured by positron emission tomography using 18-fluoro-2-deoxy-D-glucose. We sought to determine the mechanism of and correlation to altered metabolism and cell survival in response to imatinib. Glucose uptake, cell viability, and apoptosis in GIST cells were measured following imatinib treatment. Lentivirus constructs were used to stably express constitutively active AKT1 or AKT2 in GIST cells to study the role of AKT signaling in metabolism and cell survival. Immunoblots and immunofluorescent staining were used to determine the levels of plasma membrane-bound glucose transporter Glut4. We show that oncogenic activation of KIT maximizes glucose uptake in an AKT-dependent manner. Imatinib treatment markedly reduces glucose uptake via decreased levels of plasma membranebound Glut4 and induces apoptosis or growth arrest by inhibiting KIT activity. Importantly, expression of constitutively active AKT1 or AKT2 does not rescue cells from the imatinibmediated apoptosis although glucose uptake was not blocked, suggesting that the potential therapeutic effect of imatinib is independent of AKT activity and glucose deprivation. Overall, these findings contribute to a clearer understanding of the molecular mechanisms involved in the therapeutic benefit of imatinib in GIST and suggest that a drug-mediated decrease in tumor metabolism observed clinically may not entirely reflect therapeutic efficacy of treatment. (Cancer Res 2006; 66(10): 5477-86)

Gastrointestinal stromal tumors (GISTs) generally harbor activating mutations in KIT or PDGFRA. Mutations in these receptor tyrosine kinases lead to dysregulation of downstream signaling pathways that contribute to GIST pathogenesis. GISTs with KIT or PDGFRA mutations also undergo secondary cytogenetic alterations that may indicate the involvement of additional genes important in tumor progression. Approximately 10-15% of adult and 85% of pediatric GISTs do not have mutations in KIT or in PDGFRA. Most mutant adult GISTs display large-scale genomic alterations, but little is know about the mutation-negative tumors. Using genome-wide DNA arrays, we investigated genomic imbalances in a set of 31 GISTs, including 10 KIT/ PDGFRA mutation-negative tumors from 9 adults and 1 pediatric case and 21 mutant tumors. While all 21 mutant GISTs exhibited multiple copy number aberrations, notably losses, 8 of the 10 KIT/PDGFRA mutation-negative GISTs exhibited few or no genomic alterations. One KIT/ PDGFRA mutation-negative tumor exhibiting numerous genomic changes was found to harbor an alternate activating mutation, in the serine-threonine kinase BRAF. The only other mutationnegative GIST with significant chromosomal imbalances was a recurrent metastatic tumor found to harbor a homozygous deletion in chromosome 9p. Similar findings in several KIT-mutant GISTs identified a minimal overlapping region of deletion of ~0.28 Mbp in 9p21.3 that includes only the CDKN2A/2B genes, which encode inhibitors of cell-cycle kinases. These results suggest that GISTs without activating kinase mutations, whether pediatric or adult, generally exhibit a much lower level of cytogenetic progression than that observed in mutant GISTs.

Imatinib mesylate (STI571) is an oral 2-phenylaminopyrimidine derivative that acts as a selective inhibitor against several receptor tyrosine kinases and has been viewed as one of the therapeutic success stories of the 21st century. Imatinib was first shown to inhibit the causative molecular translocation in chronic myelogenous leukemia, BCR-ABL. Because imatinib could also inhibit the activity of KIT, a 145-kD transmembrane glycoprotein, and because gastrointestinal stromal tumors (GISTs), the most common mesenchymal tumors of the digestive tract, are characterized by expression of a gain-of-function mutation in KIT, imatinib was used in therapeutic trials of GISTs beginning in 1999. The initial success has now resulted in more widespread use of imatinib for the treatment of patients with GIST. Molecular genetic studies have shown that most GISTs possess a KIT mutation in exon 9, 11, 13, or 17. Clinically, GIST patients with KIT exon 11 mutations (ie, the juxtamembrane region) are the most prevalent and sensitive to imatinib. In addition to the inhibitory effect on KIT, imatinib also inhibits the activity of mutant platelet-derived growth factor receptor-alpha (PDGFRalpha) found in a subset of GIST. What is becoming evident is that there are patients with GIST who lack mutations in KIT or PDGFRalpha, or possess "imatinib-resistant" mutations (such as exon 17 mutations in KIT and exon 18 mutations in PDGFRalpha). These patients typically do not respond well to imatinib therapy. Therefore, identifying additional genetic factors that contribute to the pathogenesis of GIST, independent of KIT and PDGFRalpha, will be important in developing additional anti-GIST therapies. As one might suspect from previous experiences with antitumor therapies, primary and secondary resistance to imatinib is also becoming a major clinical problem in the treatment of this disease. Therefore, new drugs that can serve as alternative therapies in imatinib-resistant patients with GIST or that can be used in combination with imatinib will be needed. As with most recent efforts to derive novel molecular target therapies to treat cancer, improved therapy of GIST will continue to benefit from advances in the molecular characterization of this disease.

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