Hsp90 inhibition by AT13387 modulates growth factor and cytokine stimulated cell signalling in multiple cell lines.

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The utility of Hsp90 inhibitors that are currently under clinical investigation is limited by their poor pharmaceutical properties and dose limiting toxicities which may be secondary to off-target effects. The identification of a small molecule inhibitor with improved drug-like properties would be of significant value in clinical development. AT13387 a recently identified clinical candidate is a potent (800pM), novel, small molecule inhibitor of Hsp90 discovered by high-throughput X-ray crystallography and fragment-derived chemistry. The effects of Hsp90 inhibition on tumour related signal transduction pathways in multiple growth factor and cytokine stimulated cell lines have been analyzed. AT13387 downregulated multiple Hsp90 client proteins involved in growth and survival of myeloma and breast cancer cell lines. In order to test if Hsp90 clients were down-regulated sufficiently to affect key cell signalling pathways, cells were stimulated with growth factors such as epidermal growth factor and phorbol ester and cytokines such as interleukin 6 in the presence or absence of AT13387, and the phosphorylation status of specific signalling pathway molecules was assessed. Constitutive, as well as growth factor and cytokine-induced phosphorylation of AKT, ERK and Stat3 were all inhibited by AT13387 in multiple tumour cell lines. There were cell line dependent differences in pathway activation by the various stimuli tested. Cell type-dependent differences in Hsp90 client modulation were also observed. AT13387 also caused a decrease in multiple myeloma cell adhesion to cultured bone marrow stromal cells and resulted in induction of apoptosis. AT13387 treatment resulted in cell cycle arrest of the Her-2 overexpressing breast cancer line Bt474 at an IC50 of 22 nM. AT13387 treatment also induced a cell cycle arrest in multiple tumour cell lines and gave good in vivo efficacy in a Bt474 mouse xenograft model. In conclusion the Hsp90 inhibitor AT13387 negatively regulated multiple signal transduction pathways in growth factor and cytokine stimulated cell lines, an effect which was correlated with inhibition of survival and or growth of these tumor lines.