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Updated: Jul 13, 2026
Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors
Published on: January 20, 2023
Akt shows variable sensitivity to an Hsp90 inhibitor depending on cell context
Maria A Theodoraki1, Mary Kunjappu, David W Sternberg
1Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine, New York, NY 10029, USA.
Abstract:
Hsp90 inhibitors are currently in clinical trials for cancer therapy based on their ability to promote proteasomal degradation of oncogenic protein kinases and nuclear receptors. Results from recent studies suggest that cancer cells are more sensitive to these inhibitors than cells from healthy tissues. We analyzed an immortalized cell line Ba/F3 for sensitivity to the Hsp90 inhibitor geldanamycin in the absence and presence of the oncogenic tyrosine fusion kinase NPM-ALK expressed from a retroviral vector. Our results showed that NPM-ALK expression makes Akt and Cdk4 more resistant to degradation in the presence of geldanamycin, and there was a slightly reduced amount of apoptosis. The mechanism underlying the effect of NPM-ALK on Akt stability was probed by comparison of the turnover of the kinase after translation inhibition and geldanamycin treatment. We observed that Akt was degraded more rapidly in the presence of GA than upon translation inhibition without NPM-ALK expression. This suggests that NPM-ALK protects the mature kinase. Furthermore, Akt failed to bind to the Cdc37 chaperone in cells expressing NPM-ALK, which also correlates with increased Akt stability.
Insights
The oncogenic tyrosine kinase NPM-ALK confers resistance to Hsp90 inhibitors like geldanamycin by stabilizing Akt and Cdk4. This resistance mechanism involves impaired chaperone binding, impacting cancer therapy strategies.
Area of Science:
- Oncology
- Molecular Biology
- Biochemistry
Background:
- Heat shock protein 90 (Hsp90) inhibitors are investigated for cancer therapy due to their role in degrading oncogenic proteins.
- Cancer cells exhibit greater sensitivity to Hsp90 inhibitors than normal cells.
Purpose of the Study:
- To investigate the effect of the oncogenic tyrosine fusion kinase NPM-ALK on cellular sensitivity to the Hsp90 inhibitor geldanamycin.
- To elucidate the molecular mechanisms by which NPM-ALK influences protein stability and apoptosis in the presence of Hsp90 inhibition.
Main Methods:
- Analysis of Ba/F3 immortalized cell line sensitivity to geldanamycin.
- Expression of NPM-ALK oncogenic tyrosine fusion kinase via retroviral vector.
- Assessment of Akt and Cdk4 protein degradation, apoptosis levels, and Akt-Cdc37 chaperone binding.
Main Results:
- NPM-ALK expression rendered Akt and Cdk4 more resistant to geldanamycin-induced degradation.
- A slight reduction in apoptosis was observed in cells expressing NPM-ALK.
- Akt degradation was slower upon translation inhibition compared to geldanamycin treatment in NPM-ALK expressing cells, indicating NPM-ALK protects mature Akt.
- Akt failed to bind to the Cdc37 chaperone in the presence of NPM-ALK, correlating with increased Akt stability.
Conclusions:
- The oncogenic tyrosine fusion kinase NPM-ALK confers resistance to Hsp90 inhibition by stabilizing key client proteins like Akt and Cdk4.
- This stabilization appears to be mediated by impaired interaction between Akt and the Cdc37 chaperone.
- These findings have implications for the efficacy of Hsp90 inhibitors in cancers driven by NPM-ALK.
