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. 2011 Apr 14;6(4):e18552.
doi: 10.1371/journal.pone.0018552.

Multifaceted intervention by the Hsp90 inhibitor ganetespib (STA-9090) in cancer cells with activated JAK/STAT signaling

David A Proia  1 , Kevin P Foley, Tim Korbut, Jim Sang, Don Smith, Richard C Bates, Yuan Liu, Alex F Rosenberg, Dan Zhou, Keizo Koya, James Barsoum, Ronald K Blackman
Affiliations

Multifaceted intervention by the Hsp90 inhibitor ganetespib (STA-9090) in cancer cells with activated JAK/STAT signaling

David A Proia et al. PLoS One. .

Abstract

There is accumulating evidence that dysregulated JAK signaling occurs in a wide variety of cancer types. In particular, mutations in JAK2 can result in the constitutive activation of STAT transcription factors and lead to oncogenic growth. JAK kinases are established Hsp90 client proteins and here we show that the novel small molecule Hsp90 inhibitor ganetespib (formerly STA-9090) exhibits potent in vitro and in vivo activity in a range of solid and hematological tumor cells that are dependent on JAK2 activity for growth and survival. Of note, ganetespib treatment results in sustained depletion of JAK2, including the constitutively active JAK2(V617F) mutant, with subsequent loss of STAT activity and reduced STAT-target gene expression. In contrast, treatment with the pan-JAK inhibitor P6 results in only transient effects on these processes. Further differentiating these modes of intervention, RNA and protein expression studies show that ganetespib additionally modulates cell cycle regulatory proteins, while P6 does not. The concomitant impact of ganetespib on both cell growth and cell division signaling translates to potent antitumor efficacy in mouse models of xenografts and disseminated JAK/STAT-driven leukemia. Overall, our findings support Hsp90 inhibition as a novel therapeutic approach for combating diseases dependent on JAK/STAT signaling, with the multimodal action of ganetespib demonstrating advantages over JAK-specific inhibitors.

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Conflict of interest statement

Competing Interests: All authors are or were employees of Synta Pharmaceuticals, Inc. and all except R.C. Bates and A.F. Rosenberg hold either stock or options in Synta Pharmaceuticals, Inc. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Effects of ganetespib on tumor cell viability.
(A) SET-2, HEL92.1.7, MV4-11, NCI-H1975 and DU145 cells were treated with ganetespib or 17-AAG over a broad dose range (0.0001 to 1 uM) for 72 h and cell viability assessed by Alamar blue staining. (B) Ganetespib exhibits more durable inhibition of JAK/STAT signaling compared to P6. HEL92.1.7 cells were cultured in the presence of 250 nM ganetespib or 1000 nM P6 and harvested between 0 and 48 h. Expression levels of the indicated proteins were determined by western blot. (C) Ganetespib is significantly more potent than 17-AAG. SET-2 cells were dosed with the indicated concentrations of ganetespib or 17-AAG for 24 h and analyzed to determine JAK/STAT protein and target levels using the antibodies indicated.
Figure 2
Figure 2. Inhibition of JAK2/STAT signaling by ganetespib in solid tumors.
(A) Client protein downregulation in NSCLC. NCI-H1975 cells were dosed with the indicated concentrations of ganetespib for 24 h and their cell lysates analyzed to determine JAK/STAT and Hsp90 client protein levels using the antibodies indicated. (B) Ganetespib blocks IL-6 induced and constitutive STAT3 activity in NSCLC cells. HCC827 lung cancer cells were treated with increasing concentrations of ganetespib or P6 for 24 h followed by a 15 min stimulation with or without 50 ng/ml human recombinant IL-6. The levels of JAK2, total and phospho-STAT3, and PIM2 were analyzed by western blot. GAPDH is included as a loading control. (C) Client protein degradation in prostate cancer cells. DU145 cells were dosed with graded concentrations of ganetespib for 24 h and cell lysates subject to western blot to determine JAK/STAT and target protein levels using the antibodies indicated. (D) Functional Hsp90 is required for JAK2, but not JAK1, stability in DU145 cells. DU145 cells were treated with DMSO (control, C), 15 nM, 60 nM or 240 nM ganetespib for either 24 or 48 h and lysates probed by western blot with the indicated antibodies.
Figure 3
Figure 3. Ganetespib inhibits JAK/STAT target and cell cycle gene expression.
(A) Comparative effects of ganetespib and P6 on HEL92.1.7 tumor cell viability. HEL92.1.7 cells were treated with ganetespib or P6 over a broad dose range (0.0001 to 10 uM) for 72 h and cell viability assessed by Alamar blue. (B) Temporal and dose-dependent effects on JAK/STAT targets by ganetespib and P6. HEL92.1.7 cells were treated with ganetespib or P6 for 4 and 24 h and cell lysates subject to western blot to determine JAK2/STAT and target protein levels using the indicated antibodies. (C) Affymetrix GeneChip analysis of cells treated with ganetespib and P6. HEL92.1.7 cells treated with 250 nM ganetespib for 24 h or 1000 nM P6 for 4 h. Gene expression levels in DMSO treated (i.e. vehicle control) cells (X-axis) are graphed against those of drug treated cells (Y-axis). (D) Venn diagram of number of genes differentially regulated by ganetespib and P6.
Figure 4
Figure 4. Ganetespib modulates cell cycle protein expression and induces growth arrest.
(A) HEL92.1.7 cells were treated with 250 nM ganetespib or 1000 nM P6 (or DMSO as a control) and cell cycle distribution determined by flow cytometry at 3, 5, 9 and 24 h post-treatment. (B) HEL92.1.7 cells were dosed with ganetespib (250 nM) or P6 (1000 nM) for 48 h. Cells were harvested at the indicated time points and the levels of total and phospho-Cdk1, phospho-Chk2 and GAPDH analyzed by western blot. (C) Kinetics of ganetespib effects on JAK/STAT and cell cycle protein expression. HEL92.1.7 cells were treated with 100 nM ganetespib, harvested at hourly intervals over an 11 h time course, and subject to western blot with the indicated antibodies. (D) MCF-7, GIST882, HPAF and DU145 cells were dosed with graded concentrations of ganetespib for 24 h and analyzed by western blot using the indicated antibodies.
Figure 5
Figure 5. Ganetespib is highly efficacious in vivo in a leukemia survival model expressing activated JAK2V617F.
(A) Kaplan-Meier analysis of overall survival in a leukemia model established by i.v. injection of HEL92.1.7 cells into SCID mice, which resulted in the development of disseminated disease. Beginning one day after tumor cell implantation, ganetespib was i.v. dosed at its HNSTD (25 mg/kg) on a five-times per week schedule for 3 weeks through day 19 (n = 10/group). *P<0.0001; 2-sided log-rank test. (B) Ganetespib dramatically inhibits tumor cell burden in the spinal cord and adjacent bone marrow. Immunohistochemistical staining (H&E) of lumbar spine cross sections from vehicle control (left panels) or ganetespib treated (right panels) animals. Insets are enlarged in the lower panels. Original magnification: 40× in upper panels; 200× in the lower panels.
Figure 6
Figure 6. Ganetespib efficacy and pharmacodynamics in an in vivo leukemia model with constitutively activated STAT5 signaling.
(A) SCID mice were subcutaneously implanted with MV4-11 acute myeloid leukemia cells. Mice bearing established MV4-11 xenografts (100–200 mm3, n = 8 mice/group) were i.v. dosed (arrowheads) with ganetespib at either 25 or 150 mg/kg once weekly for 3 weeks, or at the HNSTD of 25 mg/kg five-times per week, as indicated. % T/C values are indicated to the right of each growth curve and the error bars are the s.e.m. (B) ganetespib inhibits STAT-5 phosphorylation and Cdk1 expression in tumor xenografts in SCID mice. SCID mice bearing MV4-11 tumors (n = 4 mice/group) were treated with vehicle or ganetespib at either 25 mg/kg or 150 mg/kg at the indicated time points between 6 h and 144 h (6 days). Tumors were resected and the levels of p-STAT5, Cdk1, Hsp70 and GAPDH were determined by western blot.
Figure 7
Figure 7. Multimodal activity of Hsp90 inhibition induced by ganetespib.
Ganetespib exerts potent antitumor effects through perturbation of multiple signaling cascades, including the JAK/STAT signaling axis and cell cycle mediators. (A) The JAK/STAT pathway is a principal signaling mechanism for a wide array of cytokines and growth factors. Hyperactivation of this pathway, through ligand activated receptor tyrosine kinases (EGFR or FLT3), cytokine receptor mediated activation of JAK2, or activating mutations such as JAK2V617F, is often associated with oncogenesis. Regulation of the cell cycle involves a number of highly coordinated and essential processes, including checkpoint control and detection/repair of genetic damage, critical for correct progression and cell division. (B) Inactivation of Hsp90 by ganetespib results in the proteasome-mediated degradation of the upstream signaling components (indicated in grey) critical for STAT, MAPK and AKT activation, thereby resulting in growth inhibition. In addition, concomitant downregulation of key cell-cycle regulatory genes induced by ganetespib (as shown in Table 1) results in cell cycle arrest in the G1 and G2/M phases of the cell cycle, and subsequent loss of S phase.
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