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Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors
Published on: January 20, 2023
Heat shock protein 90 inhibition in imatinib-resistant gastrointestinal stromal tumor
Sebastian Bauer1, Lynn K Yu, George D Demetri
1Department of Pathology, Brigham and Women's Hospital, Center for Sarcoma and Bone Oncology, Dana-Farber Cancer Institute, and Ludwig Center at Dana Farber/Harvard Cancer Center, Boston, Massachusetts 02115, USA.
Abstract:
Inhibition of KIT oncoproteins by imatinib induces clinical responses in most gastrointestinal stromal tumor (GIST) patients. However, many patients develop imatinib resistance due to secondary KIT mutations. Heat shock protein 90 (HSP90) protects KIT oncoproteins from proteasome-mediated degradation, and we therefore did preclinical validations of the HSP90 inhibitor, 17-allylamino-18-demethoxy-geldanamycin (17-AAG), in an imatinib-sensitive GIST cell line (GIST882) and in novel imatinib-resistant GIST lines that are either dependent on (GIST430 and GIST48) or independent of (GIST62) KIT oncoproteins. 17AAG (>100 nmol/L) inhibited imatinib-sensitive and imatinib-resistant KIT oncoproteins, with substantially reduced phospho-KIT and total KIT expression after 30 minutes and 6 hours, respectively. KIT signaling intermediates, including AKT and mitogen-activated protein kinase, were inactivated by 17-AAG in the KIT-positive GIST lines, but not in the KIT-negative GIST62. Likewise, cell proliferation and survival were inhibited in the KIT-positive GISTs but not in GIST62. These findings suggest that 17-AAG biological effects in KIT-positive GISTs result mainly from KIT oncoprotein inhibition. The dramatic inactivation of imatinib-resistant KIT oncoproteins suggests that HSP90 inhibition provides a therapeutic solution to the challenge of heterogeneous imatinib resistance mutations in GIST patients.
Insights
Heat shock protein 90 (HSP90) inhibition with 17-AAG effectively targets imatinib-resistant gastrointestinal stromal tumors (GIST) by downregulating KIT oncoproteins. This preclinical study shows HSP90 inhibition as a promising strategy for overcoming imatinib resistance in GIST.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Imatinib therapy is effective for gastrointestinal stromal tumors (GIST) but often fails due to secondary KIT mutations causing resistance.
- Heat shock protein 90 (HSP90) stabilizes KIT oncoproteins, protecting them from degradation and contributing to imatinib resistance.
- Novel therapeutic strategies are needed to overcome imatinib resistance in GIST patients.
Purpose of the Study:
- To evaluate the preclinical efficacy of the HSP90 inhibitor 17-allylamino-18-demethoxy-geldanamycin (17-AAG) against imatinib-sensitive and imatinib-resistant GIST.
- To investigate the effects of 17-AAG on KIT oncoprotein expression and signaling pathways in GIST cell lines.
- To determine if HSP90 inhibition can overcome imatinib resistance mediated by diverse KIT mutations.
Main Methods:
- Preclinical validation of 17-AAG in imatinib-sensitive (GIST882) and imatinib-resistant (GIST430, GIST48, GIST62) GIST cell lines.
- Assessment of KIT oncoprotein levels (phospho-KIT and total KIT) and signaling intermediates (AKT, MAPK) following 17-AAG treatment.
- Evaluation of cell proliferation and survival in response to 17-AAG in KIT-positive and KIT-negative GIST cells.
Main Results:
- 17-AAG (>100 nmol/L) significantly inhibited both imatinib-sensitive and imatinib-resistant KIT oncoproteins.
- Substantial reductions in phospho-KIT and total KIT expression were observed within 6 hours of 17-AAG treatment.
- 17-AAG effectively inactivated KIT signaling intermediates and inhibited proliferation/survival in KIT-positive GIST lines, including those resistant to imatinib.
Conclusions:
- HSP90 inhibition by 17-AAG demonstrates potent anti-GIST activity, primarily through the downregulation of KIT oncoproteins.
- 17-AAG effectively targets imatinib-resistant KIT oncoproteins, suggesting its potential as a therapeutic agent for overcoming imatinib resistance in GIST.
- Targeting HSP90 represents a viable therapeutic strategy to address the challenge of heterogeneous imatinib resistance mutations in GIST patients.
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