The Geldanamycin Derivative DMAG Demonstrates Improved Cytotoxicity and Down-Modulation of Hsp90 Client Proteins Relative to 17-AAG in Chronic Lymphocytic Leukemia (CLL) Cells: Justification for Clinical Trials in CLL.

The heat shock protein Hsp90 functions to stabilize important cell survival- and proliferation-related kinases such as AKT, IKK, c-Src, Raf-1, and cdk9. Cancer cells have activated Hsp90 as compared to normal cells, making this a relevant therapeutic target to eliminate these kinases. The semi-synthetic geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) binds to and inhibits the activity of Hsp90, and previous work demonstrated the ability of 17-AAG to deplete AKT in several cancer types in vitro. A newer geldanamycin derivative, 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (DMAG) has improved pharmacological properties including solubility and oral bioavailability, and was shown to be more effective than 17-AAG in melanoma and pancreatic carcinoma mouse xenograft mouse models. We therefore examined the effects of 17-AAG and DMAG against CD19-positive tumor cells from CLL patients. Cell viability was examined by the MTT assay, and AKT and IKK protein expression was examined by immunoblot analysis. In samples from seven CLL patients, 1.0uM DMAG resulted in 31.5% viability (95% CI: 13.1–50.0%), compared to 61.5% viability (95% CI: 45.0–78.0%) using the same concentration of 17-AAG. In four CLL patient samples treated with 1.0uM DMAG for 24 hours, AKT was decreased an average of 72.5% (95% CI: 57.7–87.3%) relative to the untreated controls. This is in comparison to 1.0uM 17-AAG, which caused a 52.7% decrease in AKT (95%CI: 39.7–65.6%). IKK protein was also decreased at similar levels in all patient samples examined. This data indicates that in CLL cells, DMAG has superior activity both in cytotoxicity and in reduction of relevant Hsp90 client proteins. 17-AAG is currently undergoing Phase I clinical testing in CLL, and DMAG is completing phase I clinical development in solid tumor malignancies. Overall, our data and that of others support clinical development of DMAG in CLL, based on the improved pharmacologic properties, enhanced efficacy relative to 17-AAG, and expected down-regulation of target proteins. In addition, our in vitro observations support using measurement of protein down-regulation of AKT and IKK as pharmacodynamic biomarkers of activity in patients undergoing therapy with these agents.