Volume 128, Issue 2, Pages 270-279 (February 2005) Mechanisms of resistance to imatinib mesylate in gastrointestinal stromal tumors and activity of the PKC412 inhibitor against imatinib-resistant mutants  Maria Debiec-Rychter, Jan Cools, Herlinde Dumez, Raf Sciot, Michel Stul, Nicole Mentens, Hilde Vranckx, Bartosz Wasag, Hans Prenen, Johannes Roesel, Anne Hagemeijer, Allan Van Oosterom, Peter Marynen  Gastroenterology  Volume 128, Issue 2, Pages 270-279 (February 2005) DOI: 10.1053/j.gastro.2004.11.020 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Histopathology: CD117 immunostaining of (A) nontreated, metastatic and (B) imatinib-resistant lesions from case 2, showing loss of KIT immunoreactivity in progressive tumor (arrow indicates immunopositive mast cell that served as control). (C) Dual-color interphase FISH on resistant tumor cells from case 2 shows only one or no signals. Digoxigenin-labeled (red signal) BAC RP11-568A2 and biotin-labeled BAC RP11-24O11 (green signal) DNA probes that contain sequences specific for KIT and PDGFRA, respectively, were used for analysis. Interphase FISH on imatinib-resistant cells from cases (D) 23 and (E) 26, using KIT (red signal) in reference to SpectrumOrange-labeled chromosome 4 CEP (green signal) probes, shows an increase in copy number of KIT gene in a form of well-defined amplicons. The same analysis in (F) nontreated, primary GIST versus (G) imatinib-resistant GIST from case 18 showed a change from diploidy to haploidy during treatment. Nuclei are visualized with 4′,6-diamidino-2-phenylindole stain (blue). Gastroenterology 2005 128, 270-279DOI: (10.1053/j.gastro.2004.11.020) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 KIT activation in 10 imatinib-resistant GISTs by Western analysis. Whole cell lysates from imatinib-nontreated, primary GIST (case 7, control) and imatinib-resistant tumor samples were immunoblotted with antibodies for total KIT, phospho-KITY703, and actin (for the assessment of the equivalency of lane loading). Eight resistant tumors showed KIT expression and various levels of constitutive KIT autophosphorylation, while 2 resistant GISTs lost KIT expression. Gastroenterology 2005 128, 270-279DOI: (10.1053/j.gastro.2004.11.020) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Ex vivo sensitivity assay of GIST cells to imatinib or PKC412. Tumor cells derived from cases (A) 19 and (B) 14, which harbored KIT-Ins503AY/V654A and KIT-Δ557-558/T670I, respectively, were incubated ex vivo with indicated concentrations of imatinib, PKC412, or diluent alone for 90 minutes. GIST882 cell line served as control (C). Cells were lysed and subjected to Western blot analysis with anti-p-KIT(Y703) or total anti-KIT antibodies. The autophosphorylation of KIT was not affected by exposure of either primary cell line to imatinib (up to 5 μmol/L). In contrast, PKC412 totally inhibited KIT autophosphorylation of the mutant KIT-Ins503AY/V654A and KIT-Δ557-558/T670I cells at concentrations of 5 μmol/L and 1 μmol/L, respectively. Gastroenterology 2005 128, 270-279DOI: (10.1053/j.gastro.2004.11.020) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 (A) PKC412 inhibits KIT-T670I and PDGFRA-D842V imatinib-resistant mutants in vitro. (B) Dose-response curves of imatinib or PKC412 for Ba/F3 cells expressing KIT-ΔWK557-558/T670I, PDGFRA-D842V, or PDGFRA-ΔDIM842-844 mutations. Gastroenterology 2005 128, 270-279DOI: (10.1053/j.gastro.2004.11.020) Copyright © 2005 American Gastroenterological Association Terms and Conditions