Targeting KRAS-Mutant Non–Small Cell Lung Cancer with the Hsp90 Inhibitor Ganetespib

Published OnlineFirst September 25, 2012; DOI: 10.1158/1535-7163.MCT-12-0615

Molecular Cancer Preclinical Development Therapeutics

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 molecular 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. 2012 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 2012 American Association for Cancer Research. been linked to primary resistance to targeted EGFR-TKI

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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 (7.5 106) 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 American Type via the tail vein with either vehicle, ganetespib formu- Culture Collection (ATCC) and maintained according to lated in 10/18 DRD [10% dimethyl sulfoxide (DMSO), standard techniques. The cell lines were authenticated by 18% Cremophor RH 40, 3.6% dextrose, 68.4% water] or the routine ATCC Routine Cell Biology Program using per os dosed with BEZ235 formulated in PEG300/NMP short tandem repeat (STR) analysis (DNA profiling) and (90% PEG300, 10% N-methylpyrrolidone). Animals were used within 6 months of receipt for this study. All were treated with ganetespib at 50 mg/kg weekly or primary antibodies were purchased from Cell Signaling BEZ235 at 10 mg/kg 5 times a week, either alone or in Technology with the exception of the C-RAF and glycer- combination. Tumor growth inhibition was determined aldehyde-3-phosphate dehydrogenase (GAPDH) antibo- as described previously (24). dies which were obtained from Santa Cruz Biotechnology, Inc. Ganetespib [3-(2,4-dihydroxy-5-isopropylphenyl)-4- Multiple drug effect analysis (1-methyl-1H-indol-5-yl)-1H-1,2,4-triazol-5(4H)-one] was For combinatorial analysis, cells were seeded in 96-well synthesized by Synta Pharmaceuticals Corp. AZD6244, plates at a predetermined, optimum growth density for

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Ganetespib Activity in KRAS-Mutant NSCLCs

Figure 1. Chemical structures of ganetespib and all compounds used in the study.

24 hours before the addition of drug or vehicle to the the corresponding single-agent data for each cell line culture medium. Drug combinations were applied at a tested. nonconstant ratio over a range of concentrations for 72 or 96 hours. For each compound tested, a 7-point dose Reverse-phase protein array analysis range was generated on the basis of 1.5-fold serial dilu- For single-agent ganetespib proﬁling, H2009, A549, tions using IC50 values set as the midpoint. Cell viability H538, and Calu-1 cells were treated with DMSO (control) was assessed by either alamarBlue (Invitrogen) or Cell- or ganetespib (250 nmol/L) for 24 hours. In the combina- Titer-Glo assays and normalized to vehicle controls. For tion experiments, A549 cells were treated with ganetespib each combination study, the level of growth inhibition (25 nmol/L) or BEZ235 (20 nmol/L), either alone or in (fraction affected) is plotted relative to vehicle control. combination for 24 hours. Lysates were then prepared as Data are presented as one relevant combination point and recommended by the Reverse-Phase Protein Analysis

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Core Facility at MD Anderson Cancer Center (Houston, was accompanied by inactivation of downstream effectors TX). Serial diluted lysates were arrayed on nitrocellulose- (p-MEK, p-ERK, p-AKT) and induction of apoptotic mar- coated FAST slides (Whatman) and probed for a standard kers (cleaved PARP, increased BIM levels). AKT activa- list of antibodies as previously described (25, 26). tion involves the phosphorylation of 2 residues: serine 473 (Ser473) and threonine 308 (Thr308). Of note, phosphor- Results ylation of Thr308 has been correlated with activity in Loss of viability and client protein expression by NSCLCs (27), and p-AKT (Thr308) expression was acutely ganetespib in KRAS-mutant NSCLC cells sensitive to ganetespib treatment in H727 cells. Potent The cytotoxic activity of ganetespib was initially eval- destabilization of p-AKT (Ser473) was seen in both lines. uated in a panel of 20 NSCLC cell lines selected for Interestingly, mTOR signaling was affected by ganetespib expression of known KRAS mutations, including G12, in H441 cells, as evidenced by a reduction in levels of G13, and Q61 variants (Table 1). Ganetespib potently phosphorylated 4E-BP1 protein, a direct substrate of the reduced viability in all lines examined, with IC50 values mTOR-1 complex (mTORC1). Because AKT Ser473 is in the low nanomolar range. Representative viability regulated by mTOR complex-2 (mTORC2; ref. 28), these curves for 2 lines, H727 and H441, are shown in Fig. 2A data suggest that Hsp90 inhibition may coordinately (left). The IC50 values for these G12V-mutant KRAS– impact cross-talk between the mTOR pathway and other expressing cells were 28 and 14 nmol/L, respectively. KRAS-driven signaling cascades. KRAS itself is not a client protein of Hsp90 and was We have previously reported that even brief exposure therefore unaffected by ganetespib treatment (Supple- of NSCLC lines to ganetespib in vitro results in sustained mentary Table S1). However, as targeted degradation of activity and potent effects on cellular viability (23). There- client proteins is a feature of Hsp90 inhibition, we exam- fore, we exposed A549 and H2030 cells to graded con- ined expression changes in client and signaling pathway centrations of ganetespib for 1 hour and then investigated proteins associated with NSCLC progression. In H727 and the effects on client protein pathways at 24 hours. Doses H441 cells, ganetespib treatment resulted in a robust and were determined empirically for each line, using the dose-dependent decrease in EGFR and MET receptors, minimal concentrations required for client protein deple- both established Hsp90 clients (Fig. 2A, right panels). The tion. For comparison, we included a continuous 24-hour KRAS substrate C-RAF was potently destabilized in both exposure to ganetespib at the lowest relative dose (Fig. cell lines and, as expected, only a relatively modest reduc- 2B). Continuous low-dose ganetespib treatment (0.5 and tion in B-RAF protein levels was observed in H441 cells. 0.1 mmol/L for A549 and H2030 cells, respectively) Targeted degradation of these signaling intermediates resulted in targeted degradation of phosphorylated and total EGFR, C-RAF, p-MEK, p-AKT, and p-STAT3. How- ever, even a 1-hour exposure to ganetespib promoted a Table 1. In vitro cytotoxicity values of dose-dependent reduction in the levels of these same ganetespib in KRAS-mutant NSCLC lines proteins. Although complete abrogation of downstream STAT3 signaling was not achieved, 1-hour ganetespib KRAS Ganetespib treatment at 3 mmol/L for A549 cells or 0.5 mmol/L for Cell line mutation IC50, nmol/L H2030 cells was similarly effective as prolonged exposure H1355 G13C 5 at degrading p-EGFR, C-RAF, and p-MEK levels in both H157 G12R 7 cell lines (and p-AKT/AKT levels in H2030 cells) at the 24- H460 Q61H 8 hour time point. Taken together, these data show that IA-LM G12C 10 even brief exposure to the Hsp90 inhibitor was sufﬁcient HOP-62 G12C 11 to efﬁciently induce client protein destabilization in H23 G12C 11 KRAS-mutant NSCLC lines. H2030 G12C 12 H441 G12V 14 Multimodal suppression of oncogenic signaling by H2212 G12C 17 ganetespib confers superior cytotoxic activity over SK-LU-1 G12D 18 selective MEK and PI3K/mTOR inhibitors H2009 G12A 19 Recent preclinical studies have provided a convincing H1792 G12C 20 rationale for the combinatorial use of targeted MEK and COR-L23 G12V 22 PI3K/AKT inhibitors in NSCLCs, particularly in KRAS- H727 G12V 28 driven lung tumors (29, 30). Accordingly, here we focused H1734 G13C 28 on AZD6244, an allosteric inhibitor of the MEK1/2 H358 G12C 29 kinases, and BEZ235, a dual pan-PI3K and mTOR inhib- A549 G12S 43 itor, both of which are under clinical development. When H2122 G12C 53 the single-agent cell killing activity of these compounds Calu-1 G12C 58 was compared with that of ganetespib in a subset of Calu-6 Q61K 64 mutant KRAS NSCLC lines (Fig. 3A), ganetespib was typically more potent at reducing cell viability than either

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Figure 2. Ganetespib effects on cell viability and Hsp90-dependent signaling in mutant KRAS NSCLC cell lines. A, H727 and H441 NSCLC cells were treated with ganetespib over a broad range (0.1–1000 nmol/L) and cell viability assessed after 72 hours (left). For signaling (right), H727 (top) and H441 (bottom) cells were exposed to graded concentrations of ganetespib (0–100 nmol/L) for 24 hours and cell lysates immunoblotted with the indicated antibodies. GAPDH was included as a loading control. B, A549 cells were exposed to 0, 0.5, 1, 2, or 3 mmol/L ganetespib and H2030 cells to 0, 0.1, 0.25, or 0.5 mmol/L ganetespib for 1 hour. A549 and H2030 cells were also treated with ganetespib at 0.5 and 0.1 mmol/L, respectively, continuously for 24 hours. At the 24-hour time point, cell lysates were immunoblotted with the indicated antibodies. agent. AZD6244, in particular, was largely ineffective as a their molecular targets, both kinase inhibitors triggered monotherapy, with IC50 values 1,000 nmol/L in the 4 compensatory signaling activity within the tumor cells. lines examined. In contrast, ganetespib treatment resulted in loss of C- We next examined modulation of the cellular signaling RAF, p-MEK, p-ERK, total/p-AKT (and partial suppres- cascades targeted by these agents using Western blot sion of 4E-BP1 activity) across all lines and without con- analysis (Fig. 3B). As expected, MEK blockade by comitant upregulation of feedback signaling (Fig. 3B). In AZD6244 resulted in consequent loss of downstream ERK agreement with the cytotoxicity profiles shown in Fig. 3A, activity with no observed effects on upstream (B-RAF or ganetespib treatment additionally manifested the highest C-RAF) AKT or mTOR signaling. However, in all of the pro-apoptotic activity, as shown by consistent elevations lines, AZD6244 induced a significant accumulation of in cleaved PARP expression. These findings were sup- phosphorylated MEK protein, indicating that treatment ported by a more extensive reverse-phase protein array with this inhibitor resulted in the activation of a feedback analysis of ganetespib exposure in the same cell panel signaling loop. Induction of feedback signaling was also (Supplementary Table S1). Besides leading to the expected observed in each of the 4 lines following exposure to the downregulation of client protein receptors (including PI3K/mTOR inhibitor BEZ235 (Fig. 3B). Consistent with EGFR, MET, and HER2) and signaling intermediates of its targeted activity, BEZ235 significantly suppressed pathways regulated by these oncogenic drivers (Src, p-AKT levels and completely abrogated mTOR activity, STAT3, GSK3), ganetespib treatment also selectively including complete loss of phosphorylated 4E-BP1 pro- altered the expression of a number of proteins involved tein. Unexpectedly, BEZ235 treatment also increased in mTOR signaling (S6, 4E-BP1, PDK1), cell-cycle regula- p-MEK levels and this was associated with a robust tion, and apoptosis. Taken together, these coordinate upregulation of activated (i.e., phosphorylated) ERK impacts on multiple signaling cascades conferred by expression (Fig. 3B). Therefore, despite direct effects on Hsp90 chaperone inhibition underlies the superior

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Figure 3. Cellular viability and pathway modulation in mutant KRAS cell lines following Hsp90, MEK, and PI3K/mTOR inhibition. A, A549, H2009, Calu-1, and H358 cells were treated with increasing concentrations of ganetespib, AZD6244, or BEZ235, and cell viability was assessed after 72 hours. B, A549, H2009, Calu-1, and H358 NSCLC cells were exposed to 500 nmol/L ganetespib, 1,000 nmol/L AZD6244, or 500 nmol/L BEZ235 for 24 hours and cell lysates immunoblotted with the indicated antibodies.

therapeutic utility of ganetespib in KRAS-mutant lung abrogated in these cells (Fig. 4D). However, cellular expo- cancer lines. sure to both mTOR inhibitors induced feedback pathway activation, again evidenced by an accumulation of p-MEK Ganetespib suppresses feedback pathway activation and p-ERK protein levels. Concurrent administration of and enhances the activity of MEK and PI3K/mTOR ganetespib effectively suppressed this response in both inhibitors in vitro lines (Fig. 4D). Of note, ganetespib treatment also sup- To extend these observations, we subsequently tested pressed feedback signaling induced by both AZD6244 ganetespib in combination with inhibitors of MEK and and BEZ235 in Calu-1 and H358 cells (Supplementary PI3K/mTOR. Concurrent administration of low (IC50 or Fig. S1). Calu-1 cells showed enhanced antiproliferative þ IC20) doses of ganetespib with AZD6244 substantially activity for the ganetespib BEZ235 combination but not increased cell death in A549 and H2009 cells (Fig. 4A). for ganetespib þ AZD6244, and H358 cells were a line in This effect was not universal, however, as no additional which no benefit was observed for ganetespib with either benefit was seen in the Calu-1 or H358 lines when the inhibitor. Thus, while targeted Hsp90 inhibition could drugs were combined at similar ratios (Fig. 4A). At the overcome compensatory signaling loops in mutant KRAS molecular level, single-agent ganetespib robustly inhib- NSCLC lines, this effect was not the sole determinant of ited the C-RAF/MEK/ERK signaling axis in A549 and the improved therapeutic activity conferred by ganete- H2009 cells (Fig. 4B). AZD6244 treatment alone reduced spib treatment. downstream p-ERK expression yet induced p-MEK accu- To examine potential mechanisms underlying the mulation in both cell types, consistent with the data enhanced cytotoxicity seen with ganetespib þ BEZ235 shown in Fig. 3B. Importantly, co-treatment with ganete- treatment, we conducted reverse-phase protein array spib blocked this effect, suggesting that the feedback loop analysis using A549 cells treated with IC30 concentrations (s) responsible for promoting signaling redundancy in of each agent either alone or in combination (Table 2). these NSCLC cell lines were sensitive to Hsp90 inhibition. Consistent with its mTOR inhibitory activity, BEZ235 In agreement with the cytotoxicity data (Fig. 4A), the exposure caused a 6.8-fold reduction in phosphorylated increased PARP cleavage observed in H2009 cells high- S6 protein levels and robust decreases in p70S6K and lights the more potent pro-apoptotic activity seen with the phospho-4E-BP1. Single-agent effects of ganetespib on ganetespib–AZD6244 combination. these mTOR signaling intermediates were comparatively Combinatorial benefit was also observed in 5 mutant moderate. Notably, combination treatment promoted KRAS NSCLC lines when ganetespib was dosed in com- greater cellular loss of these proteins, suggesting that bination with BEZ235 (Fig. 4C). Selecting 2 of these lines, mTOR signaling represents a point of convergence we found that single-agent treatment of A549 and H2030 between the 2 agents. Unexpectedly, this assay also iden- cells with either BEZ235 or another mTOR inhibitor, tified Y-box protein 1 (YB-1), a translational repressor and AZD8055, inhibited phosphorylation of 4E-BP1 as well AKT substrate, as a potential molecular effector linked to as AKT (Ser473); thus, mTOR activation was effectively the pro-apoptotic response. YB-1 protein levels were

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Figure 4. Combinations of ganetespib with MEK and mTOR inhibitors in mutant KRAS NSCLC cells. A, A549, H2009, Calu-1, and H358 cells were treated with the indicated concentrations of ganetespib or the MEK inhibitor AZD6244 either alone or in combination. Cell viability was assessed at 72 hours. B, A549 and H2009 cells were treated with 100 nmol/L ganetespib or 1 mmol/L AZD6244 either alone or in combination for 24 hours. The levels of C-RAF, p-MEK, p-ERK, cleaved PARP, and GAPDH were determined by immunoblotting. C, A549, H2030, Calu-1, H2122, and H441 cells were treated with ganetespib or the PI3K/mTOR inhibitor BEZ235 at the indicated concentrations either alone or in combination. Viability was assessed at 72 hours. D, A549 and H2030 cells were treated with 100 nmol/L ganetespib either alone or in combination with the BEZ235 (500 nmol/L) or the mTOR inhibitor AZD8055 (500 nmol/L) for 24 hours. The levels of p-MEK, p-ERK, p-AKT p-4E-BP1, cleaved PARP, and BIM proteins were determined by immunoblotting. unaffected by either compound, and only a minor reduc- observed. Increased BIM levels confirmed apoptotic tion in phosphorylated YB-1 was seen following ganete- induction by the combination (Table 2). spib exposure. However, when the 2 drugs were combined, a greater than 1.4-fold loss of YB-1 expression was The antitumor efficacy of ganetespib is potentiated by PI3K/mTOR inhibition in a KRAS-mutant in vivo Table 2. Fold changes in protein expression xenograft model following single-agent and combination To evaluate whether the combinatorial benefits on cell viability observed in vitro translated to improved efficacy treatment in A549 NSCLC cells using reverse- in vivo, mice bearing A549 xenografts were treated with phase protein array analysis ganetespib and BEZ235, both as single agents and in combination. We have previously determined that the Ganetespib BEZ235 highest nonseverely toxic dose (HNSTD) of ganetespib (IC30) (IC30) Combination on a weekly dosing regimen is 150 mg/kg (23). As shown S6 (pS240/S244) 1.28 6.80 9.07 in Fig. 5, weekly administration of a suboptimal dose of S6 (pS235/S236) 1.70 6.86 7.66 ganetespib (50 mg/kg) and 5/wk dosing with BEZ235 p70S6K 1.38 1.54 1.72 (10 mg/kg) each reduced tumor growth by 44% (T/C 4E-BP1 (pS65) 1.13 1.54 1.66 value 56%). Consistent with the in vitro findings above, YB-1 (pS102) 1.25 1.03 1.46 concurrent treatment with both drugs at the same dose YB-1 1.02 1.03 1.41 levels resulted in a significant enhancement of antitumor BIM 1.14 1.00 þ1.31 activity, inhibiting tumor growth by 79%. Combination treatment was well tolerated, with no significant loss of

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heterogeneity of the disease and the development of new molecularly targeted agents. Indeed the clinical successes of EGFR and ALK inhibitors have ushered in a new era of drug development for NSCLCs. Of relevance, this particular malignancy is considered promising for the applica- tion of Hsp90 inhibitors (19), and evidence of clinical efficacy by small-molecule Hsp90 inhibition has recently been reported (32). At present, however, there are no effective therapies for KRAS-driven lung cancer and mutations in KRAS are associated with poor prognosis and resistance to both adjuvant therapy and targeted EGFR inhibitors (12). New preclinical data suggest that KRAS mutations may confer sensitivity to Hsp90 inhibition in lung adenocarcinoma (33), thereby suggesting a putative role for highly potent inhibitory agents targeting In vivo Figure 5. activity of ganetespib in combination with BEZ235 in a Hsp90 as novel treatment strategies for this subset of mutant KRAS NSCLC xenograft model. Mice bearing established A549 xenografts (n ¼ 8 mice/group) were i.v. dosed with ganetespib (50 mg/kg) patients. In support of this premise, we observed tumor once weekly and BEZ235 (10 mg/kg) administered orally 5/wk, either shrinkage in 47% of patients (8 of 17) who harbored KRAS alone or in combination as indicated (arrowheads). % T/C values are mutations in a recent phase II monotherapy trial of gane- indicated to the right of each growth curve and the error bars are the SEM. tespib in advanced NSCLCs, although no objective The combination of ganetespib and BEZ235 displayed significantly greater efficacy than either agent alone (, P < 0.05). p.o., orally. responses were seen (34). Here, we have presented additional compelling evidence for the potential use of ganetespib in KRAS-dependent NSCLCs. body weight seen after 3 weeks of dosing (data not Targeted knockdown of KRAS in NSCLC cell lines has shown). We also investigated the combination of ganete- provided critical preclinical confirmation of the role of this spib with AZD6244 (3 mg/kg, 5 /wk) in the same model; driver in tumorigenesis, resulting in both the suppression however, no significant improvement in efficacy over of tumor growth and sensitization to inhibitors of other ganetespib alone was observed on this dosing regimen signaling pathways (35). However, in practical terms, (Supplementary Fig. S2). KRAS itself has proven to be an intractable target for the development of therapeutics (16), and considerable effort Ganetespib sensitizes mutant KRAS NSCLC cells to has thus focused on inhibition of downstream effectors to standard-of-care chemotherapeutics perturb persistent activation of oncogenic signaling path- Finally, we examined the potential for improved ther- ways. The canonical RAF/MEK/ERK kinase cascade is apeutic benefit by combining ganetespib with standard- the primary mitogenic pathway stimulated by KRAS of-care agents used in the clinical treatment of lung cancer. under both physiologic and pathologic conditions (9, These included chemotherapeutics of the antimitotic, 10). RAF serine/threonine kinases are direct substrates antimetabolite, topoisomerase inhibitor, and alkylating of the RAS family of proteins and, importantly, are also agent classes (Fig. 6). Combinatorial benefit was cell line known Hsp90 clients. Here, we showed that C-RAF was specific; for example, in some lines, ganetespib potenti- more sensitive to destabilization by ganetespib in KRAS- ated the cytotoxic effects of docetaxel (Fig. 6A), in agree- mutant NSCLC lines than B-RAF, consistent with the ment with an earlier study (31). When ganetespib was observation that wild-type B-RAF is not dependent on combined with either pemetrexed or gemcitabine, Hsp90 for stability (36). While this result reflects differing enhanced cell killing was seen in H2030 and H2009 cells sensitivities of individual client proteins to Hsp90 inhibi- (Fig. 6B and C). Synergistic effects, most notably in the tion (37), it may additionally have important implications H2030 line, were also seen when the topoisomerase inhi- for lung cancer. Two elegant studies have recently pro- bitors camptothecin and SN-38 were used as co-treatment vided convincing evidence that C-RAF, but not B-RAF, is with ganetespib (Fig. 6D and E). Combinatorial benefit essential for the initiation and development of KRAS- was also seen in Calu-1 cells when ganetespib was used in driven NSCLC using transgenic mouse models (38, 39). combination with either oxaliplatin or cisplatin (Fig. 6F). Therefore, the potent degradation and loss of C-RAF These findings indicate that, depending on the cellular protein afforded by ganetespib treatment provides an context, ganetespib may enhance the anticancer activity of effective means to uncouple aberrant KRAS signaling conventional chemotherapies. from downstream MEK and ERK activation in NSCLC cells and likely contributes to the antiproliferative activity Discussion of the compound. Individualized therapy is an emerging clinical reality With low nanomolar potency, ganetespib was cytotoxic for patients with advanced NSCLCs. In recent years, an to all mutant KRAS-bearing cell lines and even a brief increasing array of treatment options has become avail- exposure (1 hour) was sufficient to negatively impact able, based on an improved understanding of the biologic Hsp90 activity, highlighting the durable response

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Ganetespib Activity in KRAS-Mutant NSCLCs

Figure 6. Combinatorial activity of ganetespib with standard-of-care chemotherapeutics in vitro. NSCLC cell lines were treated with the indicated concentrations of ganetespib either alone or in combination with docetaxel (A), pemetrexed (B), gemcitabine (C), camptothecin (D), SN-38 (E), or the platins (oxaliplatin and cisplatin; F). Cell viability was assessed after 72 hours (96 hours for pemetrexed and gemcitabine). property of the drug. At the molecular level, Hsp90 together in the A549 and H2009 cell lines. In another inhibition by ganetespib resulted in blockade of the report, significant synergy in shrinking KRAS-driven PI3K/AKT/mTOR signaling axis, which mediates pro- lung tumors was observed when BEZ235 was combined survival signaling downstream of mutant KRAS. Impor- with the MEK inhibitor ARRY-142886 (30), further sup- tantly, a feature of Hsp90 inhibition is the capacity to porting the potential benefit of coordinate blockade of the simultaneously block multiple growth and survival path- mitogenic and survival pathways activated by KRAS ways, and concomitant inhibition of the PI3K/AKT/ mutation. When ganetespib was used in combination with mTOR and RAF/MEK/ERK effector pathways repre- BEZ235, we observed superior cytotoxic activity in a sents an intrinsically intuitive strategy to counteract onco- greater number of KRAS-mutated cell lines than seen genic KRAS activity. Indeed this approach has previously with the MEK inhibitor, as well as a significant enhance- been explored using drug combinations. For example, ment of antitumor efficacy using the A549 xenograft Meng and colleagues (29) have shown that dual-agent model. These data are in agreement with an earlier study combination therapy using AZD6244 and the AKT inhib- showing synergistic benefit by combining the mTOR itor MK2206 resulted in synergistic effects on cell viability inhibitor rapamycin with the Hsp90 inhibitor IPI-504 in vitro as well as tumor growth in KRAS-driven NSCLC (40) and suggest that Hsp90 inhibition alongside mTOR tumors in vivo. Our data revealed that ganetespib was blockade may represent an effective drug combination for more potent than AZD6244 in reducing viability when the treatment of KRAS-mutant lung cancer. each agent was used as a monotherapy and combinatorial The mTOR pathway was also shown to be a critical benefit was seen when the 2 compounds were used point of convergence for combined ganetespib–BEZ235

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Acquaviva et al.

treatment. Interestingly, reverse-phase protein analysis present within the cell lines. Overall, however, the ability also implicated a role for the translational and transcrip- of ganetespib to potentiate the cytotoxic effects of chemo- tional factor YB-1 in the enhanced cytotoxic activity of the therapeutic agents in the KRAS-mutant setting provides a combination. Notably, YB-1 has been identified as an strong rationale for combinatorial approaches as a poten- essential regulator of proliferation and gene expression in tially useful therapeutic strategy. In this regard, we and KRAS oncogene–transformed cancer cells (41) and also others have previously shown that Hsp90 inhibition plays a role in mediating drug resistance (42). Phosphor- potentiates the activity of taxanes in preclinical NSCLC ylation at serine 102 by AKT attenuates the translational models (31, 47, 48). Perhaps, more importantly, from our repressor activity of the molecule (43) and also weakens ongoing phase IIb/III GALAXY trial, comparing stan- its mRNA cap–binding capability, in turn, facilitating acti- dard-of-care docetaxel with the combination of ganete- vation of silenced mRNA species (44). We found that total spib plus docetaxel in the second-line advanced NSCLC and phosphorylated YB-1 protein levels were unaffected by treatment setting, we have observed encouraging activity single-agent exposure but were robustly reduced following with ganetespib in the mutant KRAS population. Taken combination treatment. In this context, downregulation of together, the data we are presenting here suggest that YB-1 would result in the loss of oncogenic and prosurvival ganetespib may offer considerable promise for patients signals, in turn, promoting apoptosis, as has been reported suffering from these generally untreatable cancers. in breast carcinoma (45). Studies confirming the role of YB-1 in the enhanced therapeutic benefit conferred by ganete- Disclosure of Potential Conflicts of Interest spib–BEZ235 co-treatment are underway. No potential conflicts of interest were disclosed. One interesting observation to arise from this study involved the drug-induced activation of compensatory Authors' Contributions feedback signaling pathways in mutant KRAS NSCLC Conception and design: J. Acquaviva, Y. Wada, D.A. Proia Development of methodology: D.L. Smith, J. Sang, S. He, D.A. Proia lines following exposure to either MEK or mTOR inhi- Acquisition of data (provided animals, acquired and managed patients, bitors. This response typically arises due to relief of provided facilities, etc.): J. Acquaviva, D.L. Smith, J. Sang, S. He, M. Sequeira, C. Zhang, D.A. Proia negative feedback pathways that maintain cellular Analysis and interpretation of data (e.g., statistical analysis, biostatis- homeostasis, and this mechanism has recently been tics, computational analysis): J. Acquaviva, J. Sang, S. He, Y. Wada, D.A. proposed to contribute to adaptive resistance to thera- Proia Writing, review, and/or revision of the manuscript: J. Acquaviva, C. peutic agents (46). Ganetespib treatment potently sup- Zhang, Y. Wada, D.A. Proia pressed these signaling loops, even in NSCLC lines that Administrative, technical, or material support (i.e., reporting or orga- did not show combinatorial benefit. Therefore, while nizing data, constructing databases): D.L. Smith, J. Sang, J.C. Friedland, S. He, D.A. Proia attenuation of this feedback signaling activation did not Study supervision: J. Sang, D.A. Proia appear to account for the improved therapeutic index of ganetespib in combination with MEK or mTOR inhibi- Acknowledgments tion, the more universal response suggests that Hsp90 The authors thank Dr. Richard Bates who provided drafts and editorial inhibition may provide a means to overcome adaptive assistance during preparation of the manuscript and the RPPA Facility at MD Anderson Cancer Center for their assistance in the reverse-phase resistance to targeted agents, and this possibility is protein array analysis. currently under investigation. Finally, we showed that ganetespib sensitized mutant Grant Support KRAS cell lines to standard-of-care therapeutics currently All work was funded by Synta Pharmaceuticals Corp. and no authors received any grant support. in clinical use for NSCLC treatment. Again, benefit was The costs of publication of this article were defrayed in part by the not observed in all lines, further highlighting the com- payment of page charges. This article must therefore be hereby marked plexity of treating this particular subset of patients with advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cancer. It is reasonable to suggest that the heterogeneous response may simply reflect the cellular context, addi- Received June 20, 2012; revised August 17, 2012; accepted September 18, tional mutational status, and/or signaling redundancies 2012; published OnlineFirst September 25, 2012.

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Targeting KRAS-Mutant Non−Small Cell Lung Cancer with the Hsp90 Inhibitor Ganetespib

Jaime Acquaviva, Donald L. Smith, Jim Sang, et al.

Mol Cancer Ther 2012;11:2633-2643. Published OnlineFirst September 25, 2012.

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