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Imatinib resistance and microcytic erythrocytosis in a KitV558?;T669I/+ gatekeeper-mutant mouse model of gastrointestinal stromal tumor.

ABSTRACT: Most gastrointestinal stromal tumors (GISTs) harbor a gain-of-function mutation in the Kit receptor. GIST patients treated with the tyrosine kinase inhibitor imatinib frequently develop imatinib resistance as a result of second-site Kit mutations. To investigate the consequences of second-site Kit mutations on GIST development and imatinib sensitivity, we engineered a mouse model carrying in the endogenous Kit locus both the Kit(V558?) mutation found in a familial case of GIST and the Kit(T669I) (human KIT(T670I)) "gatekeeper" mutation found in imatinib-resistant GIST patients. Similar to Kit(V558/+) mice, Kit(V558;T669I/+) mice developed gastric and colonic interstitial cell of Cajal hyperplasia as well as cecal GIST. In contrast to the single-mutant Kit(V558/+) control mice, treatment of the Kit(V558;T669I/+) mice with either imatinib or dasatinib failed to inhibit oncogenic Kit signaling and GIST growth. However, this resistance could be overcome by treatment of Kit(V558;T669I/+) mice with sunitinib or sorafenib. Although tumor lesions were smaller in Kit(V558;T669I/+) mice than in single-mutant mice, both interstitial cell of Cajal hyperplasia and mast cell hyperplasia were exacerbated in Kit(V558;T669I/+) mice. Strikingly, the Kit(V558;T669I/+) mice developed a pronounced polycythemia vera-like erythrocytosis in conjunction with microcytosis. This mouse model should be useful for preclinical studies of drug candidates designed to overcome imatinib resistance in GIST and to investigate the consequences of oncogenic KIT signaling in hematopoietic as well as other cell lineages.

SUBMITTER: Bosbach B

PROVIDER: S-EPMC3427109 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Imatinib resistance and microcytic erythrocytosis in a KitV558Δ;T669I/+ gatekeeper-mutant mouse model of gastrointestinal stromal tumor.

Bosbach Benedikt B Deshpande Shayu S Rossi Ferdinand F Shieh Jae-Hung JH Sommer Gunhild G de Stanchina Elisa E Veach Darren R DR Scandura Joseph M JM Manova-Todorova Katia K Moore Malcolm A S MA Antonescu Cristina R CR Besmer Peter P

Proceedings of the National Academy of Sciences of the United States of America 20120531 34

Most gastrointestinal stromal tumors (GISTs) harbor a gain-of-function mutation in the Kit receptor. GIST patients treated with the tyrosine kinase inhibitor imatinib frequently develop imatinib resistance as a result of second-site Kit mutations. To investigate the consequences of second-site Kit mutations on GIST development and imatinib sensitivity, we engineered a mouse model carrying in the endogenous Kit locus both the Kit(V558Δ) mutation found in a familial case of GIST and the Kit(T669I) ...[more]

PMID: 22652566

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Project description:Imatinib mesylate (imatinib) is the primary agent of choice used to treat gastrointestinal stromal tumors (GIST). However, drug resistance to imatinib poses a major obstacle to treatment efficacy. In addition, the relationship between imatinib resistance and glycolysis is poorly understood. Glucose transporter (GLUT)-1 is a key component of glycolysis. The present study aimed to assess the potential relationship between components in the glycolytic pathway and the acquisition of imatinib resistance by GIST cells, with particular focus on GLUT-1. An imatinib-resistant GIST cell line was established through the gradual and continuous imatinib treatment of the parental human GIST cell line GIST-T1. The expression of glycolysis-related molecules (GLUT-1, hexokinase 2, pyruvate kinase M2 and lactate dehydrogenase) was assessed in parental and imatinib-resistant cells by western blotting, reverse transcription-quantitative PCR and glucose and lactate measurement kits. In addition, clinical information and transcriptomic data obtained from the gene expression omnibus database (GSE15966) were used to confirm the in vitro results. The potential effects of GLUT-1 inhibition on the expression of proteins in the glycolysis (GLUT-1, hexokinase 2, pyruvate kinase M2 and lactate dehydrogenase) and apoptosis pathways (Bcl-2, cleaved PARP, caspase-3 and caspase-9) in imatinib-resistant cells were then investigated following gene silencing and treatment using the GLUT-1 inhibitor WZB117 by western blotting. For gene silencing, the mature siRNAs for SLC2A1 were used for cell transfection. Annexin V-FITC/PI double-staining followed by flow cytometry was used to measure apoptosis whereas three-dimensional culture experiments were used to create three-dimensional spheroid cells where cell viability and spheroid diameter were measured. Although imatinib treatment downregulated GLUT-1 expression and other glycolysis pathway components hexokinase 2, pyruvate kinase M2, and lactate dehydrogenase in parental GIST-T1 cells even at low concentrations. By contrast, expression of these glycolysis pathway components in imatinib-resistant cells were increased by imatinib treatment. WZB117 administration significantly downregulated AKT phosphorylation and Bcl-2 expression in imatinib-resistant cells, whereas the combined administration of imatinib and WZB117 conferred synergistic growth inhibition effects in apoptosis assay. WZB117 was found to exert additional inhibitory effects by inducing apoptosis in imatinib-resistant cells. Therefore, the present study suggests that GLUT-1 is involved in the acquisition of imatinib resistance by GIST cells, which can be overcome by combined treatment with WZB117 and imatinib.

Dataset Information

Imatinib resistance and microcytic erythrocytosis in a KitV558?;T669I/+ gatekeeper-mutant mouse model of gastrointestinal stromal tumor.

Publications

Imatinib resistance and microcytic erythrocytosis in a KitV558Δ;T669I/+ gatekeeper-mutant mouse model of gastrointestinal stromal tumor.

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