Effects of Tanespimycin On Glucocorticoid Receptor Translocation.

Tanespimycin (BMS-722782) exhibits antitumor activity in diverse models of hematologic malignancies and solid tumors by suppressing the chaperoning activity of heat shock protein 90 (Hsp90) including its ability to preserve the proper three-dimensional structure and intracellular trafficking of its client proteins. However, not all potential client proteins are affected to the same degree by Hsp90 inhibitors. Tanespimycin is in phase 3 clinical development with bortezomib for the treatment of multiple myeloma (MM). Because glucocorticoids form the backbone of many anti-myeloma regimens, this preclinical study examined the effect of tanespimycin on the glucocorticoid receptor (GR), a chaperone protein of Hsp90.

Our objective is to evaluate whether tanespimycin impedes the nuclear translocation of GR in the presence or absence of GR ligand stimulation and examine the anti-myeloma activity of tanespimycin combinations with glucocorticoids. COS-7 cells transiently transfected with YFP-GR (fluorescently tagged GR) were incubated in the presence or absence of GR ligands (eg, prednisolone), tanespimycin, combinations thereof, or DMSO control. The cellular location of GR (cytoplasm or nucleus) was evaluated using a confocal high content imager. The in vitro anti-MM activity of combinations of tanespimycin with dexamethasone was evaluated by MTT survival assays.

With prednisolone (100 nM; 30 min) stimulation, GR rapidly translocates to the nucleus, consistent with previous observations. Tanespimycin did not induce GR nuclear translocation, but resulted in heterogeneous cytoplasmic aggregates of GR. Pretreatment of COS-7/YFP-GR cells with tanespimycin for 30 minutes inhibited prednisolone-induced (5 nM; 2 h and 4 h) GR nuclear translocation at higher tanespimycin concentrations (eg, 1.25 μM), but partial GR translocation was observed at lower (4.8 nM) concentrations. However, complete ligand-induced GR nuclear translocation was observed when COS-7/YFP-GR cells were pretreated with either prednisolone or dexamethasone (dose ranges 10 μM–0.019 nM; 30 min) prior to the addition of clinically relevant tanespimycin concentrations (200 nM; 1, 2, 6, and 24 h). In vitro cell viability assays with the human MM cell line MM.1S were consistent with these observations. Concurrent administration of dexamethasone (40–80 nM; 72 h) and tanespimycin (0.5–1 μM) to MM cells did not exhibit antagonistic interaction. Similarly, no antagonistic interaction was observed when dexamethasone pretreatment was followed by tanespimycin. Interestingly, for some of the experimental conditions of these combinations, at least additive effects on suppression of MM cell survival were observed.

These results suggest that appropriate sequencing of tanespimycin and GR ligands can avoid any theoretical antagonistic effect of Hsp90 inhibition on GR nuclear translocation, thus providing a framework for incorporation of dexamethasone or other glucocorticoids into tanespimycin-based regimens.

Mitsiades:Millennium, Novartis, BMS, Merck, Kosan, Pharmion: Honoraria; Amgen, AVEO, EMD Serono and Sunesis: Research Funding; Millennium, Novartis, BMS, Merck, Kosan, Pharmion: Consultancy; PharmaMar: Patents & Royalties. Anderson:Celgene, Novartis, Millennium, BMS: Consultancy; Celgene, Novartis, Millennium, BMS: Research Funding; Celgene, Novartis, Millennium, BMS: Honoraria. Richardson:Celgene: Speakers Bureau; Millennium Pharmaceuticals, Inc.: Research Funding, Speakers Bureau.