Molecular profile and anti-myeloma activity of the novel Hsp90 inhibitor NVP-AUY922

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The heat shock protein of 90kD (Hsp90) has recently been found overexpressed in multiple myeloma (MM) and blockade of Hsp90 function results in MM cell death. However, preclinical investigations have mostly relied on experiments with MM cell lines and have been devoted to a single class of pharmacologic inhibitor, namely derivatives of geldanamycin. Here, we characterize the molecular and biological effects of the novel diarylisoxazole-based Hsp90 inhibitor NVP-AUY922 in a large set of primary myeloma cells and in MM cell lines.
 Dose-effect curves for NVP-AUY922-mediated cell death were established for MM cell lines and freshly isolated CD138+ primary myeloma cells either with or without coculture with bone marrow stromal cells (BMSCs). The molecular effects of drug treatment on Hsp90 client proteins and on the activation status of survival pathways were investigated by Western analysis or by intracellular antibody staining and flow cytometry.
 MM samples segregated into two distict groups concerning their sensitivity for the novel Hsp90 inhibitor: those with steep dose-response curves and profound induction of apoptosis at low nanomolar concentrations and those that were more resilient. The former group comprised the large majority of primary tumor cells tested (31 of 39 samples), as well as five of eight MM cell lines. EC50 and EC90 values for drug-induced cell death ranged from 6 to 16 nM and 8 to 25 nM, respectively. More resilient samples displayed EC50 values below 25 nM, but EC90 values were not reached (up to 50 nM tested). Coculture with BMSCs did not protect either group against the effect of the drug. The molecular signature of NVP-AUY922-treatment was entirely consistent with its effect as Hsp90 inhibitor and led to downregulation of multiple signaling and survival pathways, as shown by decreases in the levels of pathway components (for example, Akt, Raf-1, IkappaB kinase alpha) or their activated forms (phospho-signal transducer and activator of transcription 3, phospho-extracellular signal-regulated kinase 1&2, phospho-glycogen synthase kinase-3 beta, phospho-Akt). Conversely, drug treatment was always accompanied by strong upregulation of Hsp70. There was no correlation between the sensitivity of MM cells for NVP-AUY922 and TP53 mutation status, pre- or post-treatment Hsp70 levels or the activation status of any particular survival pathway. The viability of non-myeloma cells, such as peripheral blood mononuclear cells and BMSCs was much less affected by high (50-100 nM) concentrations of the Hsp90 inhibitor, indicating the scope for establishment of a therapeutic window for the treatment of multiple myeloma.
 NVP-AUY922 is a promising new drug candidate for the treatment of a majority of myeloma patients. The preclinical efficacy of this diarylisoxazole compound against MM cells shows that the pharmacochemical options available for therapies directed at Hsp90 inhibition can be greatly extended.