Targeting KRAS-Mutant Non–Small Cell Lung Cancer with the Hsp90 Inhibitor Ganetespib
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Published OnlineFirst September 25, 2012; DOI: 10.1158/1535-7163.MCT-12-0615 Molecular Cancer Preclinical Development Therapeutics Targeting KRAS-Mutant Non–Small Cell Lung Cancer with the Hsp90 Inhibitor Ganetespib Jaime Acquaviva, Donald L. Smith, Jim Sang, Julie C. Friedland, Suqin He, Manuel Sequeira, Chaohua Zhang, Yumiko Wada, and David A. Proia Abstract Mutant KRAS is a feature of more than 25% of non–small cell lung cancers (NSCLC) and represents one of the most prevalent oncogenic drivers in this disease. NSCLC tumors with oncogenic KRAS respond poorly to current therapies, necessitating the pursuit of new treatment strategies. Targeted inhibition of the molec- ular chaperone Hsp90 results in the coordinated blockade of multiple oncogenic signaling pathways in tumor cells and has thus emerged as an attractive avenue for therapeutic intervention in human malignancies. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90 currently in clinical trials for NSCLCs in a panel of lung cancer cell lines harboring a diverse spectrum of KRAS mutations. In vitro, ganetespib was potently cytotoxic in all lines, with concomitant destabilization of KRAS signaling effectors. Combinations of low-dose ganetespib with MEK or PI3K/mTOR inhibitors resulted in superior cytotoxic activity than single agents alone in a subset of mutant KRAS cells, and the antitumor efficacy of ganetespib was potentiated by cotreatment with the PI3K/mTOR inhibitor BEZ235 in A549 xenografts in vivo. At the molecular level, ganetespib suppressed activating feedback signaling loops that occurred in response to MEK and PI3K/mTOR inhibition, although this activity was not the sole determinant of combinatorial benefit. In addition, ganetespib sensitized mutant KRAS NSCLC cells to standard-of-care chemotherapeutics of the antimitotic, topoisomerase inhibitor, and alkylating agent classes. Taken together, these data underscore the promise of ganetespib as a single-agent or combination treatment in KRAS-driven lung tumors. Mol Cancer Ther; 11(12); 2633–43. Ó2012 AACR. Introduction for NSCLCs has been revolutionized by agents that inhibit Lung cancer is the leading cause of cancer death world- the EGF receptor (EGFR) in tumors that harbor activating wide (1), and non–small cell lung cancer (NSCLC) mutations in this receptor tyrosine kinase, including the accounts for 85% of all cases. Platinum-based combination tyrosine kinase inhibitors (TKI) gefitinib and erlotinib (6). chemotherapy represents the standard-of-care for indivi- More recently, discovery of the anaplastic lymphoma duals with advanced NSCLCs (2), although this approach kinase (ALK) gene rearrangement in a subset of patients has largely reached a plateau of effectiveness in improv- with NSCLCs led to the rapid approval of the multi- ing overall survival (3, 4). Intensive research into the targeted TKI crizotinib as another genotype-driven ther- underlying biologic and molecular basis of NSCLCs has apy (7). Despite this progress, however, treatment out- provided critical insights into essential oncogenic path- comes for NSCLCs are still considered disappointing (8). ways that become dysregulated during tumorigenesis KRAS is a member of the RAS family of oncogenes, a and, in turn, has identified important targets for drug collection of small guanosine triphosphate (GTP)-binding development. These findings have subsequently translat- proteins that integrate extracellular cues and activate ed into significant clinical advances due to the inclusion of intracellular signaling pathways to regulate cell prolifer- molecularly targeted agents in novel therapeutic strate- ation, differentiation, and survival (9). Gain-of-function gies for this disease (5). As an example, first-line therapy mutations that confer transforming capacity are frequent- ly observed in KRAS, predominantly arising as single amino acid substitutions at residues G12, G13, or Q61 Authors' Affiliation: Synta Pharmaceuticals Corp., Lexington, Massa- (9). Constitutive activation of KRAS leads to the persistent chusetts stimulation of downstream signaling pathways that pro- Note: Supplementary data for this article are available at Molecular Cancer mote tumorigenesis, including the RAF/MEK/ERK and Therapeutics Online (http://mct.aacrjournals.org/). PI3K/AKT/mTOR cascades (10). In NSCLCs, KRAS Corresponding Author: David A. Proia, Synta Pharmaceuticals, Corp., mutations are highly prevalent (20%–30%) and are asso- 125 Hartwell Avenue, Lexington, MA 02421. Phone: 781-541-7236; Fax: ciated with unfavorable clinical outcomes (11, 12). Muta- 781-274-8228; E-mail: [email protected] tions in KRAS appear mutually exclusive with those in doi: 10.1158/1535-7163.MCT-12-0615 EGFR in NSCLC tumors (13); more importantly, they have Ó2012 American Association for Cancer Research. been linked to primary resistance to targeted EGFR-TKI www.aacrjournals.org 2633 Downloaded from mct.aacrjournals.org on September 27, 2021. © 2012 American Association for Cancer Research. Published OnlineFirst September 25, 2012; DOI: 10.1158/1535-7163.MCT-12-0615 Acquaviva et al. therapies (14). As a therapeutic target, oncogenic KRAS AZD8055, gemcitabine, camptothecin, and pemetrexed has proven to be "undruggable," and extensive efforts at were purchased from Selleck Chemicals; cisplatin from developing specific anti-RAS agents have failed clinically EMD Chemicals; docetaxel and BEZ235 from LC Labora- (15, 16). Alternative strategies, such as blockade of RAS tories; SN-38 from TCI America; and oxaliplatin from effectors are being evaluated; yet there remains an urgent Sigma-Aldrich. The chemical structures of all compounds unmet need for more effective therapies. are shown in Fig. 1. In this regard, targeting the molecular chaperone Hsp90 represents a promising avenue for therapeutic interven- Cell viability assays tion. Hsp90 is required for the stability and maturation of Cellular viability was assessed using the CellTiter-Glo numerous key signal transduction proteins, termed "cli- Luminescent Cell Viability Assay (Promega) according to ent" proteins (17, 18). Many of these clients play indis- the manufacturer’s protocol. KRAS-mutant NSCLC cell pensable roles in cell growth and survival and include lines were seeded into 96-well plates on the basis of such proteins as EGFR, RAF, and AKT. Of note, cancer optimal growth rates determined empirically for each cells exploit the Hsp90 chaperone machinery as a bio- line. Twenty-four hours after plating, cells were dosed chemical buffer to protect a variety of mutated and/or with graded concentrations of ganetespib for 72 hours. overexpressed oncoproteins from targeted degradation, CellTiter-Glo was added (50% v/v) to the cells, and the thereby facilitating aberrant cell survival and oncogene plates incubated for 10 minutes before luminescent detec- addiction (18, 19). Importantly, inhibition of Hsp90 activ- tion in a SpectraMax Plus 384 microplate reader (Molec- ity targets its clients for proteasomal destruction. Because ular Devices). Data were normalized to percentage of of its coordinate and simultaneous impact on multiple control, and IC50 values used to determine the sensitivity signaling cascades, pharmacologic blockade of Hsp90 can of each line. For the comparative analysis study with MEK therefore overcome signaling redundancies and drug and PI3K/mTOR inhibitors, A549, H2009, Calu-1, and resistance mechanisms commonly seen in many cancers H358 cells were treated with graded concentrations of (20–22). As such, Hsp90 has become an attractive molec- ganetespib, AZD6244, or BEZ235 for 72 hours, and cell ular target for the development of novel anticancer viability measured as above. agents. Ganetespib (formerly STA-9090) is a small-molecule Western blotting inhibitor of Hsp90 with pharmacologic and biologic prop- Following treatment, tumor cells were disrupted in erties that distinguish it from other first- and second- lysis buffer (Cell Signaling Technology) on ice for 10 generation inhibitors in terms of superior antitumor activ- minutes. Lysates were clarified by centrifugation and ity, potency, and safety (23). In addition, ganetespib is equal amounts of proteins resolved by SDS-PAGE before presently undergoing evaluation in multiple human clin- transfer to nitrocellulose membranes (Invitrogen). Mem- ical trials, including patients with advanced NSCLCs. branes were immunoblotted with the indicated antibodies Here, we evaluated ganetespib activity in a panel of and antibody–antigen complexes visualized using an KRAS-mutant NSCLC lines to determine the effects on Odyssey system (LI-COR). signaling cascades responsible for KRAS-driven NSCLC cell survival. Furthermore, we examined the potential In vivo tumor growth inhibition study combinatorial activity of ganetespib with clinical agents Female immunodeficient CD-1 (nude) mice (Charles targeting key nodal components of these pathways as well River Laboratories) were maintained in a pathogen-free as standard-of-care therapeutics for NSCLCs. Taken environment, and all in vivo procedures were approved together, the findings support a potential clinical use for by the Synta Pharmaceuticals Corp. Institutional Ani- ganetespib in patients with KRAS-mutant NSCLCs. mal Care and Use Committee. A549 NSCLC cells (7.5 Â 106) were subcutaneously implanted into the animals. Materials and Methods Mice bearing established tumors (100–200 mm3)were Cell lines, antibodies, and reagents randomized into treatment groups of 8 and i.v. dosed All cell lines were obtained from the