Abstract 3265A: GIST-associated c-Kit mutants exhibit differential affinities to hsp90 and its co-chaperone Cdc37
Authors: Panke Jutta , Seidler Joerg , Erlbruch Andrea , Bossemeyer Dirk , Lehmann Wolf-Dieter , Hohenberger Peter 
Published: June 2012

Abstract
Background: Hsp90 inhibitors have been introduced to clinical treatment of imatinib-resistant GIST, but their efficacy is dependant on KIT mutations. We analysed the role of the chaperone Hsp90 as stabilizing factor for wild-type and oncogenic, misfolded c-Kit.
Methods: the intracellular part of wild-type and mutated c-Kit (aa544-977) fused with an N-terminal TAP-tag was expressed in HEK293T cells. The clinically most abundant primary and imatinib-resistant secondary c-Kit mutations were selected: V560D; V559D; del557,558; L576P; K642E; V560D+D820A; del557,558+Y823D; D820A. A shortened TAP assay followed by LC-MS/MS was applied for identification of interaction partners. For verification a GST pulldown assay was used. Relative quantifications of mutation-specific interaction partners were performed by quantitative immunoblotting using an antibody raised against the C-terminus of human c-Kit. In addition, two GIST cell lines containing an imatinib-resistant and an imatinib-sensitive c-Kit mutation were investigated by quantitative immunoblotting. The activityof hsp90 inhibitor 17-AAG was studied in whole cell lysates of HEK293T cells expressing selected mutated c-Kit constructs.
Results: Using the TAP-c-Kit constructs for a shortened TAP assay, Hsp90 and its cochaperone Cdc37 were identified as specific interaction partners for the GIST-associated c-Kit mutants. This interaction was confirmed using GST-Cdc37 pulldown analysis.
Differential analysis of c Kit/Hsp90 interaction revealed mutation-specific affinities (up to a relative factor of about 8), with D820A showing the lowest, close-to wild-type affinity, and L576P showing the highest affinity. Among the two GIST cell lines studied, the imatinib-resistant mutant (c-Kit K642E) exhibited a 2.5 fold higher affinity to Hsp90 compared to the imatinib-sensitive mutant (c-Kit V560D+D820A). In both cell lines, the Cdc37 and Hsp90 affinities are observed in parallel. A closely related situation was observed for the interactions of the corresponding TAP-c-Kit constructs. In addition, the influence of the When analysing hsp90 inhibitor 17AAG (17-(allylamino)-17-demethoxygeldanamycin)) incells expressing selected mutated c-Kit constructs, western blot analyses showed significant degradation of total c-Kit relative to untreated controls in all mutants studied. Amongst these, mutant D820A was found to be less sensitive to 17AAG. Wild-type c-Kit showed no sensitivity against 17AAG.
Conclusion: this study demonstrates mutation-specific affinities between Hsp90 and Cdc37 with c-Kit mutants. It also points to a conformity between this phenomenon and the 17AAG effect. These results allow a better understanding of the potential of hsp90 inhibition in GIST therapy and better selection of candidates for treatment.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3265A. doi:10.1158/1538-7445.AM2011-3265A