Ganetespib  (Synonyms: STA-9090)

Exponentially growing cells are processed in lysis buffer (20 mM HEPES, pH 7.4, 1 mM EDTA, 5 mM MgCl₂, 100 mM KCl) and incubated with increasing concentrations of 17-AAG or ganetespib for 30 min at 4°C, and incubated with biotin-GM linked to Dynabeads MyOne Streptavidin T1 magnetic beads for 1 h at 4°C. Beads are washed three times in lysis buffer and heated for 5 min at 95°C in SDS-PAGE sample buffer. Samples are resolved on 4-12% Bis-Tris gradient gel and Western blots are performed using an anti-HSP90 antibody.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Cells are grown in 96-well plates based on optimal growth rates determined empirically for each line. Twenty-four hours after plating, cells are treated with the indicated compounds or controls for 72 hours. AlamarBlue is added (10% v/v) to the cells, and the plates are incubated for 3 hours and, then, subjected to fluorescence detection. For the comparative viability/apoptosis assay, NCI-H1975 cells are treated with escalating concentrations of ganetespib for the indicated time periods and subjected to viability analysis via CellTiter Fluor and apoptosis via Caspase Glo 3/7.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice: NCI-H1975 or HCC827 cells are cultured as above and 0.5-1×10⁷ cells are mixed with 50% RPMI 1640/50% Matrigel and subcutaneously injected into the flanks of SCID mice. For efficacy studies, animals with 100-200 mm³ tumors are then randomized into treatments groups of eight. Tumor volumes (V) are calculated by the equation V=0.5236×L×W×T (Length, width, and thickness). Animals are treated by intravenous bolus tail vein injection at 10 mL/kg with ganetespib formulated in 10/18 DRD (10% DMSO, 18% Cremophor RH 40, 3.6% dextrose and 68.4% water). As a measurement of in vivo efficacy, the relative size of treated and control tumors [(%T/C) value] is determined from the change in average tumor volumes of each drug-treated group relative to the vehicle-treated group, or itself in the case of tumor regression. Body weights are monitored daily. For biomarker studies, mice bearing NCI-H1975 xenografts are treated with either a single dose of vehicle or ganetespib, or with 5 daily doses of vehicle or ganetespib, in groups of 3 or 8, and harvested at various time points. Tumors are excised and flash frozen in liquid nitrogen for preparation of protein lysates or fixed in 10% neutral buffered formalin for immunohistochemistry.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Shimamura T, et al. Ganetespib (STA-9090), a Non-Geldanamycin HSP90 Inhibitor, has Potent Antitumor Activity in In Vitro and In Vivo Models of Non-Small Cell Lung Cancer. Clin Cancer Res. 2012 Jul 17.  [Content Brief]

  [2]. Ying W, et al. Ganetespib, a unique triazolone-containing Hsp90 inhibitor, exhibits potent antitumor activity and a superior safety profile for cancer therapy. Mol Cancer Ther. 2012 Feb;11(2):475-84.  [Content Brief]

  [3]. London CA, et al. Phase I evaluation of STA-1474, a prodrug of the novel HSP90 inhibitor ganetespib, in dogs with spontaneous cancer. PLoS One. 2011;6(11):e27018.  [Content Brief]

  [4]. Proia DA, et al. Multifaceted intervention by the Hsp90 inhibitor ganetespib (STA-9090) in cancer cells with activated JAK/STAT signaling. PLoS One. 2011 Apr 14;6(4):e18552.  [Content Brief]

  [5]. Stewart E, et al. Identification of Therapeutic Targets in Rhabdomyosarcoma through Integrated Genomic, Epigenomic, and Proteomic Analyses. Cancer Cell. 2018 Sep 10;34(3):411-426.e19.  [Content Brief]