Introduction
Non-small cell lung cancer (NSCLC) accounts for 85% of all cases of lung cancer, the leading cause of cancer-related deaths worldwide [
1]. This high mortality is associated, in part, to the fact that a majority of patients present with advanced disease at the time of diagnosis with treatment options limited to systemic therapy. Combination chemotherapy with a platinum-based regimen is the foundation of current treatment for patients with advanced NSCLC [
2]. Two-drug combinations consisting of either cisplatin or carboplatin with an additional ‘third-generation’ cytotoxic agent (paclitaxel, docetaxel, gemcitabine, vinorelabine, or pemetrexed) represent the current standard of care for most patients [
3]. Paclitaxel and docetaxel comprise the taxane family of microtubule stabilizers widely used in the treatment of advanced NSCLC. Docetaxel, the only agent that is approved for both first- and second-line treatment of NSCLC [
4], was also the first drug to establish superior efficacy and tolerability over other third-generation agents when used in combination with platinum compounds [
3]. Unfortunately, however, conventional chemotherapy has largely reached a plateau of effectiveness in improving survival rates for lung cancer patients [
3,
4].
In recent years the advent of new molecularly-targeted agents and refinements to existing systemic therapies, such as the addition of the vascular endothelial growth factor (VEGF)-binding monoclonal antibody bevacizumab to platinum doublets, the epidermal growth factor receptor (EGFR)-binding monoclonal antibody cetuximab or the use of EGFR inhibitors erlotinib and gefitinib, as well as ELM4-ALK inhibitors such as crizotinib, have improved the therapeutic options for treating this disease [
5‐
7], resulting in modest improvements in overall survival and quality of life for certain patient populations. Despite this progress, treatment outcomes are still considered disappointing [
8]. Clearly, the development and use of novel therapeutic strategies to effectively combat NSCLC represents an urgent unmet medical need.
Heat shock protein 90 (Hsp90) is a molecular chaperone required for the post-translational stability and function of numerous key signal transduction proteins, termed ‘client’ proteins, many of which play critical roles in cell growth, differentiation and survival [
9,
10]. Importantly, it is now recognized that the chaperoning activity of Hsp90 can become subverted during tumorigenesis to help facilitate malignant progression [
9]. Since multiple signaling cascades are regulated by this molecule, the effects of pharmacological blockade of Hsp90 are transmitted to a variety of client proteins and biochemical pathways. Because of this unique characteristic, inhibition of Hsp90 can overcome signaling redundancies and mechanisms of drug resistance commonly observed in many cancers [
11‐
13]. In addition, because tumor cells contain elevated levels of the active form of the chaperone complex relative to normal cells, tumor cells have been shown to be selectively sensitive to Hsp90 inhibition [
14]. Thus, Hsp90 provides an attractive molecular target for the development of novel anticancer agents [
13,
15,
16].
Ganetespib (formerly STA-9090) is a potent and selective small molecule Hsp90 inhibitor [
17] currently being evaluated in multiple clinical trials in solid tumor and hematological malignancies. Recently, a Phase 2b/3 trial was initiated in which it is being combined with docetaxel to treat patients with advanced NSCLC. This indication is considered promising for the application of Hsp90 inhibitors [
18] and, importantly, has provided a compelling rationale for the feasibility of combining Hsp90 inhibitors with other therapeutic agents. For example, mutated EGFR, a known Hsp90 client protein, is an important oncogenic driver in a subset of NSCLC patients [
19]. Accordingly, Hsp90 inhibitors have demonstrated clinical efficacy when used in combination with EGFR tyrosine kinase inhibitors (TKIs), even in individuals who had progressed on TKI therapy [
20]. Of relevance here, Hsp90 inhibitors have also been shown to potentiate the cytotoxic effects of paclitaxel in multiple tumor models, including NSCLC [
21‐
24].
These considerations therefore prompted a more comprehensive evaluation of ganetespib activity in combination with taxanes in preclinical models of NSCLC. In the present study we show that combinatorial treatment results in synergistic antiproliferative and antitumor effects both in vitro and in vivo. Our findings support the potential therapeutic value of ganetespib, particularly in combination with docetaxel, for the treatment of patients with NSCLC.
Materials and methods
Cell lines, antibodies and reagents
All cell lines were obtained from the ATCC (Rockville, MD) and were maintained according to standard techniques at 37°C in 5% (v/v) CO2 using culture medium recommended by the supplier. All primary antibodies were purchased from Cell Signaling Technology (CST, Beverly, MA). Ganetespib [3-(2,4-dihydroxy-5-isopropylphenyl)-4-(1-methyl-1H-indol-5-yl)-1H-1,2,4-triazol-5(4H)-one] was synthesized by Synta Pharmaceuticals Corp. Paclitaxel, docetaxel and vincristine were purchased from LC Laboratories (Woburn, MA).
Cell viability assays
Twenty fours hours after plating in 96 well plates, H1975 cells were dosed with graded concentrations of the indicated compound or DMSO controls for 72 h. AlamarBlue (Invitrogen, Carlsbad, CA) was added (10% v/v) to the cells, and the plates incubated for 3 h and subjected to fluorescence detection in a SpectraMax Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA). Data were normalized to percent of control.
Western blotting
Following ganetespib treatment for 4 or 24 h, H1975 cells were disrupted in lysis buffer (CST) on ice for 10 min. Lysates were clarified by centrifugation and equal amounts of protein resolved by SDS-PAGE before transfer to nitrocellulose membranes (Invitrogen, Carlsbad CA). Membranes were blocked with 5% skim milk in TBS with 0.5% Tween and immunoblotted with the indicated antibodies. The antibody-antigen complex was visualized and quantitated using an Odyssey system (LI-COR, Lincoln, NE).
Cell cycle analysis
Docetaxel- and ganetespib-treated H1975 cells were incubated with the respective compounds (0–30 nM) for 24 h prior to harvest. Cells were fixed, washed and stained with propidium iodide before being analyzed by flow cytometry. The percentage of cells in each phase of the cell cycle (sub-G1, G1, S and G2/M) was determined from the FL2-A histogram.
H1975 cells were seeded into the viability assay and combination treatments of ganetespib with paclitaxel, docetaxel or vincristine were performed at fixed, non-constant ratios of the compounds. Drugs were added to cell cultures for 72 h and viability measured by alamarBlue assay. The nature of the combinatorial interactions were evaluated using the combination index (CI) method [
25] and values generated using Median Effect analysis (Calcusyn Software; Biosoft, Cambridge, UK).
In vivo NSCLC tumor models
Female immunodeficient CB-17/Icr-
Prkdc
scid
/Crl (SCID) mice (Charles River Laboratories, Wilmington, MA) were maintained in a pathogen-free environment, and all in vivo procedures were approved by the Synta Pharmaceuticals Corp. Institutional Animal Care and Use Committee in accordance with the Guide for Care and Use of Laboratory Animals. NSCLC cell lines were subcutaneously implanted into mice. Mice bearing established tumors (~150 mm
3) were randomized into treatment groups (5–8 animals per group) and i.v. dosed via the tail vein with ganetespib, paclitaxel or docetaxel formulated in 10/18 DRD (10% DMSO, 18% Cremophore RH 40, 3.8% dextrose) either as single agents or concurrently on a 1X/week schedule. Tumor volume measurements were made twice weekly and tumor growth inhibition determined as described previously [
26].
Statistical analysis
A mixed-model, repeated measures analysis of variance was used to analyze the tumor growth inhibition data. The model included the tumor measurement as dependent variable and treatment (fixed effect), days after tumor implantation (fixed effect), the interaction between treatment and days after tumor implantation (fixed effect) and mice (random effect) as independent variables. P-values are obtained from pair-wise comparisons of each drug to the vehicle. SAS Version 9.1 was used for analysis.
Histological analysis and apoptosis assessment
Mice bearing established H1437 xenografts were administered a single dose of ganetespib (50 mg/kg), docetaxel (4 mg/kg), ganetespib plus docetaxel, or vehicle (10/18 DRD) for 24 h. Tumors were excised and formalin fixed. Paraffin embedded sections were subject to immunohistochemical staining for TUNEL expression using the ApopTag Peroxidase ISOL Apoptosis Detection kit (Millipore, Billerica, MA) according to manufacturers instructions. Images were acquired using a Nikon E800 microscope and Leica DC camera linked to Image-Pro plus software (Media Cybernetics, Inc., Bethesda, MA). Image analysis was performed using a total of 6 sections per slide at 20X magnification. The percentage of apoptotic area was determined by the Average apoptotic area/Average total area. Statistical significance was determined using one-way ANOVA.
Discussion
Providing improved treatment options for NSCLC remains a daunting challenge for clinicians and oncologists worldwide. Systemic combination chemotherapy is considered the standard of care for patients with advanced NSCLC despite intense efforts at modifying treatments toward improving survival outcomes [
3]. The use of standard chemotherapy is restricted to a defined number of cycles, as continuation results in added toxicity without meaningful improvements in progression-free or overall survival [
27]. The addition of a third cytotoxic agent to the two-drug combination has been evaluated, however no differences in efficacy were seen and the toxicity profile worsens [
28]. More recently, the use of maintenance therapy has evolved as a promising treatment option for NSCLC, based on emerging clinical data using novel molecularly targeted agents or chemotherapeutic drugs that exhibit a more favorable therapeutic index [
6,
27].
One strategy to improve objective response rates in patients is through the use of molecularly targeted agents in combination with front line chemotherapeutics, such as bevacizumab and cetuximab. Indeed, combining complimentary agents that possess different presumed mechanisms of action and non-overlapping toxicities has proven important for the control of many human malignancies [
29]. This biologically rational approach in NSCLC has revealed that benefit from targeted therapy combinations [
5] is typically observed within subsets of patients and correlates with specific tumor histology and/or molecular phenotypes [
8]. The coordinate impact of Hsp90 blockade on multiple oncogenic pathways and processes, as well as substantial evidence of the clinical promise of using Hsp90 inhibitors in combination with other therapeutic agents [
30], provide a compelling rationale for investigating novel combinatorial strategies. Ganetespib is a next generation Hsp90 inhibitor, structurally unrelated to the prototypic ansamycin class, which exhibits superior pharmacologic and biological properties in terms of potency and safety [
17]. Accordingly, an important finding of this study is that ganetespib greatly enhances the efficacy of chemotherapeutics commonly used in the treatment of advanced NSCLC.
Here we showed that ganetespib synergistically potentiated the cytotoxic and antitumor activity of paclitaxel or docetaxel in a panel of NSCLC cell lines both in vitro and in vivo. As a single agent, ganetespib treatment resulted in loss of EGFR client protein expression and blockade of AKT signaling in the H1975 cell line, and significantly suppressed H1975 tumor growth in vivo when used in combination with paclitaxel or docetaxel. These findings are in agreement with a previous report showing that degradation of mutant EGFR and inhibition of AKT activity by the ansamycin Hsp90 inhibitor 17-allyamino-17-demethoxygeldanamycin (17-AAG) could sensitize NSCLC tumors to paclitaxel [
24]. In addition, the Hsp90 inhibitor CUDC-305 was recently shown to potentiate paclitaxel activity in H1975 xenografts [
31] where it was proposed that this occurred, at least in part, via suppression of AKT. Other studies in breast and ovarian cancer cell lines have also suggested that suppression of AKT activity by 17-AAG resulted in an enhanced sensitivity to the proapoptotic effects of paclitaxel [
23,
32]. In this regard, we have additionally found that ganetespib treatment promotes synergistic improvements in docetaxel activity in breast, colon and prostate cell lines (D. Proia, unpublished observations). Taken together, these data imply that the modulation of Hsp90 function and consequent loss of pro-survival pathways may render cancer cells more susceptible to taxane treatment, and that this mechanism is conserved across tumor types.
However, the mechanism(s) by which ganetespib achieves improved therapeutic indices in combination with taxanes is likely to be multifactorial. Other, not mutually exclusive, molecular interactions likely exist - including complementary effects of ganetespib on the cell cycle machinery that enhance taxane activity. Paclitaxel and docetaxel each cause microtubule stabilization, mitotic arrest of cycling tumor cells, and subsequent apoptosis. Ganetespib treatment itself exerts profound effects on cell cycle regulatory proteins, in addition to oncogenic signaling pathways, that contribute to its potent antitumor activity [
26]. Further, it is known that mitotic catastrophe can be exacerbated by Hsp90 inhibition in cell lines with defects in the function of the cell checkpoint regulators BRCA1 [
33] and RB [
22], an effect presumably linked to interference with Hsp90’s role in centrosome organization [
34,
35]. The Hsp90 client protein CRAF has also now been shown to localize to the mitotic spindle of proliferating tumor cells to promote progression in a MEK-independent manner [
36]. Inhibition of this process results in prometaphase arrest thus providing another avenue for targeted mitotic interference by Hsp90 inhibitors. It is reasonable to suggest, therefore, that modulation of the cell division machinery may represent an important component of the cytotoxic sensitizing property of ganetespib. This premise is supported by our findings that additive and synergistic benefit was also seen for ganetespib in combination with vincristine, another microtubule-targeted agent that induces mitotic arrest prior to cell death.
Notably, we observed that concurrent exposure to ganetespib and docetaxel was significantly more efficacious than either agent alone in 5 of 6 xenograft models in mice bearing established NSCLC tumors. In the one exception, H2228 cells, it appeared that the concentrations tested masked any potential benefit as each drug alone caused pronounced antiproliferative effects. These data have important clinical considerations. Docetaxel is the only agent that is approved for both first- and second-line therapy in advanced NSCLC [
4]. Meta-analyses of the current treatment regimens for these patients have shown that docetaxel is associated with better disease control than paclitaxel combinations [
3]. In addition, tumor histology does not exert any influence over the activity or efficacy of docetaxel, as opposed other third-generation cytotoxics [
3]. Thus, docetaxel represents an optimal front line candidate for combination with a targeted agent such as ganetespib. In light of these observations, we have recently initiated a Phase 2b/3 trial evaluating this combination in patients with refractory NSCLC.
In summary, the capacity of ganetespib to potentiate the cytotoxic effects of taxanes, in particular docetaxel, provides a molecular rationale for combining these agents as a clinical strategy for NSCLC. Overall, the data presented here provide strong preclinical support for the exploration of this combination as a novel therapeutic approach in patients suffering from this disease.