Cancer Therapy: Preclinical

Selective Inhibition of ADAM Metalloproteases as a Novel Approach for Modulating ErbB Pathways in Cancer Jordan S. Fridman,1Eian Caulder,1Michael Hansbury,1Xiangdong Liu,1Genjie Yang,1Qian Wang,1 Yvonne Lo,1Bin-Bing Zhou,1Maxwell Pan,1Sufi M. Thomas,2 Jennifer R. Grandis,2 Jincong Zhuo,1Wenqing Yao,1Robert C. Newton,1Steven M. Friedman,1 PeggyA. Scherle,1and KrisVaddi1

Abstract Purpose: ErbB receptor signaling pathways are important regulators of fate, and their dysregulation, through (epi)genetic alterations, plays an etiologic role in multiple cancers. ErbB ligands are synthesized as membrane-bound precursors that are cleaved by members of the ADAM family of zinc-dependent metalloproteases. This processing, termed ectodomain shedding, is essential for the functional activation of ErbB ligands. Recent studies suggest that elevated levels of ErbB ligands may circumvent the effectiveness of ErbB-targeted therapeutics. Here, we describe the discovery and preclinical development of potent, selective inhibitors of ErbB ligand shedding. Experimental Design: A series of biochemical and cell-based assays were established to identify selective inhibitors of ErbB ligand shedding. The therapeutic potential of these compounds was assessed in multiple in vivo models of cancer and matrix metalloprotease ^ related toxicity. Results: INCB3619 was identified as a representative selective, potent, orally bioavailable small-molecule inhibitor of a subset of ADAM that block shedding of ErbB ligands. Administration of INCB3619 to tumor-bearing mice reduced ErbB ligand shedding in vivo and inhibited ErbB pathway signaling (e.g., phosphorylation of Akt), tumor cell proliferation, and survival. Further, INCB3619 synergized with clinically relevant cancer therapeutics and showed no overt or compounding toxicities, including fibroplasia, the dose-limiting toxicity associated with broad-spectrum matrix metalloprotease inhibitors. Conclusions: Inhibition of ErbB ligand shedding offers a potentially novel and well-tolerated therapeutic strategy for the treatment of human cancers and is currently being evaluated in the clinic.

The ErbB family is composed of four members, epidermal activation (except for HER3 which lacks a functional kinase growth factor (EGF) receptor (EGFR; HER1 or ErbB1), HER2 ), receptor phosphorylation, and the activation of (neu or ErbB2), HER3 (ErbB3), and HER4 (ErbB4). Eleven multiple downstream signaling cascades with pleiotropic ligands have been reported to bind to EGFR, HER3, and HER4 oncogenic effects when dysregulated—a common event in and show some level of receptor selectivity (1). HER2has no cancer (3–5). Accordingly, the ErbB family represents a confirmed extracellular ligand and serves as the preferred validated therapeutic target for the treatment of cancer and dimerization partner for other ErbB receptors (2). Ligand has resulted in the development of multiple therapeutics, binding results in receptor dimerization, kinase domain including the antibodies trastuzumab (directed against HER2) and cetuximab (directed against EGFR) as well as low molecular weight, ATP-competitive tyrosine kinase inhibitors (TKI) targeting EGFR—gefitinib and erlotinib (reviewed in Authors’ Affiliations: 1Drug Discovery, Incyte Corporation,Wilmington, Delaware and 2Department of Otolaryngology, University of Pittsburgh Medical Center and ref. 5). Although such targeted therapeutics have shown dramatic University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania antitumor effects in a subset of patients, many do not respond Received 8/24/06; revised 11/17/06; accepted 11/30/06. and most eventually become resistant. The costs of publication of this article were defrayed in part by the payment of page Despite the fact that certain tumor traits have been identified charges. This article must therefore be hereby marked advertisement in accordance that may be associated with clinical responses to EGFR TKIs with 18 U.S.C. Section 1734 solely to indicate this fact. Conflict of interest statement. The authors (excluding S.M. Thomas and (e.g., EGFR amplification or a subset of somatic mutations J.R. Grandis) declare they all currently are, or have been, employed by Incyte in exons 18 to 21, or mesenchymal characteristics), they can Corporation. account for only a fraction of those patients achieving clinical Requests for reprints: Jordan S. Fridman, Drug Discovery, Experimental Station, benefit from treatment. Substantial preclinical and clinical Incyte Corporation, E400, Route 141and Henry Clay Road,Wilmington, DE19880. Phone: 302-498-6930; Fax: 302-425-2760; E-mail: [email protected]. evidence exist, suggesting that the different ErbB pathways may F 2007 American Association for Cancer Research. compensate for one another, thus providing a mechanism of doi:10.1158/1078-0432.CCR-06-2116 resistance against an agent targeting an individual pathway. For

Clin Cancer Res 2007;13(6) March 15, 2007 1892 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. Selective ADAM Inhibitors as Anticancer Agents instance, increased levels of the EGFR ligand transforming recommendations (R&D Systems). INCB3619 was also screened against growth factor-a (TGF-a) have been observed in trastuzumab- a panel of G- coupled receptors and ion channels for inhibitory treated breast cancer patients with progressive disease (6). activity (ExpresSProfile, Cerep, Inc., Paris, France). Similar results were obtained in a preclinical model of Cell culture and generation of stable cell lines. Cell lines were obtained from American Type Culture Collection (Manassas, VA) and trastuzumab resistance that showed a concordant increased were cultured as recommended by the supplier. Retroviral transduction sensitivity to an EGFR-selective TKI (7). Moreover, a nonbiased was used to generate derivatives of the NMuMG and Rat-1 cells as analysis of patients receiving gefitinib described significantly described previously (20). The coding region of the human cDNA for elevated expression of the ErbB ligands amphiregulin and TGF- TGF-a was cloned into pMSCVpuro (BD Biosciences, Mountain View, a in nonresponders, agreeing with in vitro data suggesting that CA) following amplification from a pool of human cDNA (BD QUICK- high ErbB ligand concentrations can circumvent the effective- Clone). Human umbilical vascular endothelial cells were obtained from ness of an EGFR-targeted agent (8, 9)3 or the less selective ErbB Cambrex Corp (East Rutherford, NJ) and treated following the kinase inhibitor GW572016 (10). These data are consistent manufacturer’s protocols. a with the preclinical observation that targeting the EGFR Analysis of ErbB ligand shedding and its effect. The TGF- cleavage A pathway at multiple points (ligand binding and ATP pocket) assay used SCC9 cells stimulated with 10 mol/L phorbol 12-myristate 13-acetate (Sigma-Aldrich, St. Louis, MO) for 30 min in serum- may be more efficacious than either method in isolation free medium following a 30-min pretreatment with DMSO or metal- (11, 12)—a hypothesis currently being evaluated clinically. loprotease inhibitor. Supernatants were collected and assayed for Finally, even in patients who do respond to EGFR-selective soluble TGF-a by a commercially available ELISA kit (R&D Systems). TKIs, the emergence of secondary mutations in the EGFR Alternatively, NMuMG or Rat-1 cells were grown for 24 h in the presence kinase domain affords resistance to the ATP-competitive TKIs of DMSO or inhibitor, and supernatants were analyzed in a (13–16). Although this is an evolving field, these and other similar manner. Amphiregulin release was measured in supernatants of data support the following concepts: (a) although ErbB- HCT116 stimulated with phorbol 12-myristate 13-acetate (1 Amol/L, 30 targeted agents have shown clinical promise, there is significant min) using an ELISA kit (R&D Systems). The heparin-binding EGF-like room for improvement; (b) a multifaceted approach to growth factor (HB-EGF) cleavage assay used Chinese hamster ovary cells inhibiting multiple ErbB pathways would be expected to stably transfected with human placental alkaline phosphatase–tagged improve therapeutic response; and (c) inhibiting the activation HB-EGF (In Vitro Biology Department of Incyte Corporation, Wilming- ton, DE). The transfected Chinese hamster ovary cells were grown in of multiple ErbB ligands represents a logical and perhaps 96-well plates and treated with DMSO or compound for 10 min clinically relevant point of intervention. followed by phorbol 12-myristate 13-acetate stimulation (2 Amol/L, Although certain broad spectrum matrix metalloprotease 45 min). Alkaline phosphatase activity was assessed using a luminescent (MMP) inhibitors (e.g., marimastat) can inhibit EGFR ligand substrate (Roche Molecular Biochemicals, Indianapolis, IN). Heregulin shedding in vitro (among other activities), this has not been cleavage was assessed using Chinese hamster ovary-K1 cells transiently investigated in vivo nor has the therapeutic hypothesis been transfected with the human heregulin-h1 cDNA in pCDNA3.2-DESTTM tested clinically due to the lesser potency of these drugs against (In Vitro Biology Department of Incyte Corporation) using Lipofect- key ADAM —relative to MMPs (see below)—and their AMINE 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s inability to be tolerated at higher doses (17). Here, we describe instructions. Following transfection, the cells were grown in serum- the discovery and preclinical development of selective inhib- free medium for 24 h. Cells were pretreated with DMSO or compound itors of ErbB ligand shedding and show that these compounds for 15 min and stimulated with phorbol 12-myristate 13-acetate (1 Amol/L, 60 min). Heregulin-h1 was quantified in the supernatants inhibit ErbB ligand shedding and tumor growth in vivo without using a commercially available human NRG1-h1/HRG1-h1 ELISA kit inducing joint fibroplasias, the dose-limiting toxicity of many (R&D Systems) according to the manufacturer’s instructions. MMP inhibitors. These compounds possess the pharmacologic Conditioned medium experiments used NMuMG-T cells grown in properties making them ideal candidates to test the therapeutic the presence of DMSO or INCB3619 for 24 h. Medium was collected potential of sheddase inhibition for the treatment of cancer in a and concentrated (Amicon Centriplus, Millipore Corp., Billerica, MA) clinical setting. and applied to serum-deprived (6 h) A431 cells for 30 min. Protein extracts were then subjected to immunoblotting using antibodies 1173 Materials and Methods against total EGFR and phosphorylated EGFR (Tyr ) antibodies (Cell Signaling, Beverly, MA). Primary head and neck cancer cells were obtained from a consenting In vitro metalloprotease and pharmacology assays. Sheddase inhib- patient with squamous cell carcinoma of the buccal mucosa (approved itors were synthesized at Incyte Corp as described (18).4 Human MMPs, by the University of Pittsburgh Institutional Review Board). The ADAM9, and ADAM10 were purchased from R&D Systems (Minneap- patient was not previously treated with standard therapies. Cells were olis, MN) and assayed as recommended by the manufacturer. The maintained in DMEM, 10% fetal bovine serum, and 2mmol/L peptide substrate (7-methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-Leu-(3- glutamine, and passed 1:10 weekly. Epithelial cells were separated [2,4-dinitrophenyl]-L-2, 3-diaminopropionyl)-Ala-Arg-NH2 was used from fibroblasts using selective trypsinization. For proliferation assays, for the MMP assays as recommended by the manufacturer (R&D cells were seeded at 103 per well in the in a 96-well plate and allowed to Systems). Partially purified porcine ADAM17 was obtained from spleen attach overnight. The medium was then replaced with 100 AL of low as described (19). The fluorogenic peptide substrate, (7-methoxycour- serum medium (2% fetal bovine serum) containing compound or marin-4-yl)-acetyl-Pro-Leu-Ala-Gln-Ala-Val-(3-[2,4-dinitrophenyl]-L-2, DMSO and maintained for 5 days. Viability was assessed following 3-diaminopropionyl)-Arg-Ser-Ser-Ser-Arg-NH , was used for the AD- 2 manufacturer’s protocols (Promega Cell-Titer Glo, Madison, WI) and AM9, ADAM10, and ADAM17 assays following the manufacturer’s percentage inhibition of growth was determined relative to the DMSO control. Amphiregulin shedding was determined from 4 105 cells 3 C. Arteaga, personal communication. per well in 12-well plates, which were allowed to attach overnight. 4 Yao W, Zhu J, Burns DM, et al. Discovery of a potent, selective, and orally active The medium was replaced with low serum medium (2% fetal human epidermal growth factor receptor-2 sheddase inhibitor for the treatment of bovine serum)–containing compound or DMSO and incubated cancer. JMed Chem. In press 2007. overnight. Centrifuged medium was collected and analyzed following

www.aacrjournals.org 1893 Clin Cancer Res 2007;13(6) March 15, 2007 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. Cancer Therapy: Preclinical manufacturer’s protocols by ELISA (R&D Systems-Human Amphiregulin to identify potent, selective, and well-tolerated inhibitors of a Duoset). Percentage inhibition was calculated relative to a DMSO control. subset of the ADAM family of metalloproteases, herein called studies. Animal studies were done under Animal Welfare . Sheddases are responsible for the cleavage and Regulation Guidelines in a facility accredited by the Association for the activation of all three types of ErbB ligands: (a) those that bind Assessment and Accreditation of Laboratory Animal Care. BALB/c or solely to EGFR (e.g., TGF-a), (b) those that bind both EGFR and CD-1 nu/nu mice (Charles River, Wilmington, MA) were injected s.c. with logarithmically growing cells suspended in either serum-free ErbB4 (e.g., HB-EGF), and (c) those that bind ErbB3 and/or culture medium, or a 1:1 mixture medium and Matrigel (BD ErbB4 (e.g., heregulin; reviewed in refs. 5, 22). Importantly, Biosciences). The MCF-7 tumor model required implantation of the ultimate goal was to discover molecules with drug-like estradiol pellets (17-h estradiol, 0.72mg/pellet, 60-day release; properties, including oral bioavailability and good pharmaco- Innovative Research of America, Sarasota, FL). NMuMG-T tumors were kinetics. To this end, we synthesized a number of novel implanted as s.c. tumor fragments (2 2mm). Tumor size was hydroxamic acid-derived metalloprotease inhibitors (18),3 followed using a caliper to measure the length (l) and width (w)of culminating in the identification of a series of molecules 2 tumors to calculate an approximate volume [(l w ) / 2]. In efficacy exemplified by INCB3619 (Fig. 1A). studies, treatment was usually initiated when mean tumor volumes The sheddases—ADAMs responsible for processing the were f150 mm3, whereas in some pharmacodynamic marker studies, membrane-bound ErbB proligands—have been reported to tumors were twice to thrice larger at the time of treatment. Tumor include ADAM17 (also known as -a growth delay represents the difference in time for the treated group (T) and a control group (C) to reach a predetermined mean tumor size and converting ) and ADAM10 (22–29). We thus estab- was calculated for long-term studies (TGD = T C). Alternatively, lished biochemical assays using recombinant and tumor growth inhibition (TGI) was calculated throughout the fluorogenic peptide substrates and screened proprietary experiment [TGI = (1 T / C) 100]. S.c. implantation of osmotic hydroxamic-based compounds (19) for inhibition of AD- pumps (Alzet, Cupertino, CA) was done under isofluorane anesthesia AM10/ADAM17 enzyme activity. Hydroxamic acids have following manufacturer’s protocol. These pumps were used to previously been shown to affect HB-EGF shedding through administer INCB3619 or marimastat in all animal studies. inhibition of an unknown enzyme (30). Compounds from our Determination of circulating TGF-a levels in mice was done in collection that showed >50% inhibition of ADAM10 and/or immunocompromised mice bearing s.c. well-established Rat-1-T tumors 3 ADAM17 at a concentration of 200 nmol/L were further (>200 mm ). Briefly, tumor-bearing were randomized into two characterized against a larger panel of metalloproteases and groups with similar mean tumor volumes. Plasma was then collected via ADAM proteins to assess selectivity. A structure-activity retro-orbital bleeding 2days after implantation of s.c. Alzet pumps delivering vehicle or INCB3619 (120 mg/kg/d). TGF-a levels were relationship was established and provided direction for the determined from plasma samples in similar fashion as described above. synthesis or more potent and/or selective compounds. Using The murine fibroplasia model was adapted from Renkiewicz et al. this strategy, INCB3619 was identified as a potent inhibitor (21). Synovial fibroplasias was scored on a scale of 0 (no change) to 3 of ADAM17 and ADAM10 activity with IC50 values of 14 and [severe deposition and fibroblast proliferation resulting in an 22 nmol/L, respectively. Moreover, INCB3619 shows excellent increase (>10-fold) in the ratio of synovium to subsynovial tissue mass]. selectivity both within the ADAM family (e.g., IC50 for ADAM9 Hematology and clinical chemistry analyses were done by DuPont is >5 Amol/L) and against a panel of MMPs (Table 1). Haskell Laboratories (Newark, DE). Functional cell-based assays confirmed the ability of INCB3619 Evaluation of compound levels in plasma was done using standard to inhibit the shedding of TGF-a, HB-EGF, amphiregulin, and liquid chromatography-tandem mass spectrometry techniques. heregulin with nanomolar potencies (Fig. 1B). Compounds Ex vivo analysis of tumor material. Tissues were fixed in 10% that possess MMP activity but lack ADAM10/17 activity did not neutral buffered formalin for at least 24 h at room temperature before block ErbB ligand shedding in these assays (see below). standard processing and treatment with 3% H2O2 for 15 min to quench any endogenous peroxidase activity. Preheated citrate buffer (Antigen In addition to metalloprotease selectivity, multiple structural Unmasking Solution, Vector Laboratories, Burlingame, CA) was used analogues were screened against a panel of >50 G-protein for antigen retrieval and background staining was reduced using the coupled receptors and ion channels (Cerep, Inc., ExpresSPro- /Biotin Blocking kit (Vector Laboratories) and CytoQ Back- file) and no significant binding or inhibition was observed at ground Buster (Innovex, Richmond, VA) following the manufacturer’s 1 Amol/L (data not shown). Pharmacokinetic analysis of protocols. Primary antibodies, rabbit anti-Ki67 (Novocastra Laborato- INCB3619 indicated that it was well-absorbed orally (10 mg/ ries, Newcastle upon Tyne, United Kingdom) or rabbit anti–phospho- kg but had a half-life of <2h in the mouse). 5 As such, for in vivo 473 j AKT:Ser (Cell Signaling), were incubated overnight at 4 Cina administration, we took advantage of the excellent solubility of humidified chamber. A biotinylated secondary antibody (goat anti- this compound and used implantable osmotic pumps. In larger rabbit, Vector Laboratories) was visualized using R.T.U. Vectastain Elite ABC Reagent and 3,3¶-diaminobenzidine (Vector Laboratories). Alter- species, such as dog, the half-life of INCB3619 was significantly natively, sections were examined for the presence of apoptotic cells by longer (6.1 h). Improved pharmacokinetics in canine and terminal deoxyribonucleotide –mediated nick-end labeling nonhuman primates is a common feature of hydroxamic acid– (TUNEL) staining (Apoptag, Chemicon International, Temecula, CA). based inhibitors. Therefore, continuous infusion in rodents is a

Following staining, sections were washed with H2O, counterstained better reflection of the predicted human pharmacokinetics with hematoxylin, dehydrated, and mounted. Observations were made and also serves to specifically test the therapeutic hypothesis on a Nikon Eclipse E800 system and images captured with ACT-1 by avoiding maximal drug exposures sufficient to potentially software (Nikon Corporation, Melville, NY). inhibit an array of MMPs nonspecifically. In vitro and in vivo sequelae of selective ADAM inhibi- Results tion. To begin to assess the biological effect of inhibiting ErbB Identification of potent selective inhibitors of ErbB ligand ligand shedding, we first engineered a cellular model reliant on shedding. With the goal of discovering a single agent capable of inhibiting multiple ErbB pathways concomitantly, we set out 5 J.S. Fridman and M. Pan, unpublished data.

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Fig. 1. INCB3619 exemplifies a series of potent and selective sheddase inhibitors that prevent the proteolytic processing of multiple ErbB proligands. A, the chemical structure of INCB3619. B, INCB3619 inhibits the shedding of TGF-a,amphiregulin (AR), HB-EGF, and heregulin (HRG)in cell-based assays.

TGF-a expression for its transformed phenotype (Fig. 2A). consenting patient with squamous cell carcinoma of the buccal NMuMG cells (immortalized mammary epithelial cells) were mucosa. This tumor type was chosen for investigation based on transduced with either an empty expression vector (NMuMG-V) the clinical efficacy observed with antibodies that inhibit EGFR or a vector encoding TGF-a (NMuMG-T). After confirming ligand binding. Using these cells, we show that INCB3619 can the ability of transfected cells to produce TGF-a, we tested the inhibit the release of amphiregulin (Fig. 2D) in primary tumor ability of INCB3619 to inhibit the shedding of TGF-a from the cells in addition to ErbB ligands in established cell lines. cell surface by analyzing tissue culture supernatants from cells Treatment with INCB3619 resulted in a significant decrease in grown in the presence or absence of compound (Fig. 2B). In proliferation of these primary tumor cells (Fig. 2E). The EC50 contrast to INCB3619, which inhibits ADAM10 and ADAM17, for proliferation was estimated to be near the EC90 for amphi- neither INCB8765 (ref. 9; an ADAM10 selective inhibitor; regulin shedding, suggesting that the ADAM sheddases may Fig. 2B, E) nor an inhibitor of MMPs that is both ADAM10 and require marked inhibition for efficacy in this model system. ADAM17 sparing (Fig. 2B, 4; ref. 9) inhibited TGF-a shedding. Although the results in these primary cells are consistent with This agrees with the knockout data implicating ADAM17 as the our cell line data, we cannot conclude that similar effects will enzyme responsible for TGF-a shedding (24, 31). The ability be observed in all cancer cells. In contrast, in normal cells to potently inhibit ErbB ligand cleavage was not specific to (human umbilical vascular endothelial cells or primary INCB3619 as additional compounds having similar enzymatic fibroblasts), there was no effect on proliferation at concen- activities as INCB3619 were also able to do so (data not trations >10 times those effective on the tumor cells (data not shown). Moreover, this pharmacology was not limited to cells shown). of epithelial origin as nearly identical results were obtained Although these experiments show that selective sheddase using immortalized fibroblasts (Fig. 2C). To extend these inhibition can diminish the release of EGFR ligands from the findings beyond contrived cell lines, we investigated the effect cell surface, they do not show that this can affect EGFR signaling. of sheddase inhibition on EGFR ligand release and cell To illustrate that inhibition of the release of TGF-a can reduce proliferation in primary tumor cells surgically isolated from a EGFR pathway activation, we treated the EGF-responsive A431

www.aacrjournals.org 1895 Clin Cancer Res 2007;13(6) March 15, 2007 Downloaded from clincancerres.aacrjournals.org on October 2, 2021. © 2007 American Association for Cancer Research. Cancer Therapy: Preclinical cells with conditioned medium harvested from INCB3619- As we and others have hypothesized that the antitumor treated NMuMG-T cells. Treatment of NMuMG-T cells with effects obtained by inhibiting a single node of an ErbB pathway INCB3619 markedly inhibited activation of EGFR in the A431 in isolation using one treatment modality may not be optimal, cells, confirming that NMuMG-T cells release a soluble factor we investigated the potential of improving responses to an capable of activating EGFR and that this is prevented with a EGFR TKI by concomitantly treating with INCB3619. Mice with selective sheddase inhibitor (Fig. 2F). Similar experiments in s.c. human non–small cell lung carcinoma xenografts were which A431 cells were first incubated with an EGFR-neutralizing treated with gefitinib, INCB3619, or the combination and antibody showed no EGFR activation after addition of tumor growth was followed over a 2-week period (Fig. 4C). As conditioned medium from NMuMG-T cells grown in the single agents, neither compound was efficacious; however, in presence or absence of INCB3619 (data not shown), consistent combination, INCB3619 and gefitinib markedly inhibited with the conclusion that selective sheddase inhibition prevents tumor growth. Therapeutic benefit was obtained without activation of an autocrine TGF-a loop. Likewise, we have notation of compounding toxicity. Similar results have been previously shown in multiple autonomous cell-based systems obtained using erlotinib in place of gefitinib (data not shown). that pharmacologic or genetic inhibition of ADAM10/17 can These data are consistent with that recently published inhibit ErbB receptor phosphorylation and downstream signal- exemplifying the ability of INCB3619 to potentiate the effects ing (9). However, as none of the cell lines studied here of gefitinib in vitro (9). Collectively, these data support that a overexpress HER2, we did not detect significant changes to the sheddase inhibitor may be an efficacious and well-tolerated amount of HER2receptor phosphorylation (data not shown). treatment modality for affecting ErbB pathways in cancer as a The ability of sheddase inhibition to reduce TGF-a shedding single agent or in combination with other targeted agents. in vivo (Fig. 3A) as well as EGFR-driven tumor growth was Because particular cancer types have been linked to elevated investigated in both tumors of fibroblast (Fig. 3B; 42% TGI) ErbB ligand expression, and hence increased EGFR activity and epithelial origin (Fig. 3C; 38% TGI). In agreement with (reviewed in ref. 38), we evaluated the activity of INCB3619 in data that has been generated using less selective compounds, a xenograft model of one such tumor type, head and neck the direct effect of sheddase inhibition addition on ex vivo squamous cell carcinoma. Subcutaneous tumors were estab- cellular proliferation of established cell lines was not dramatic lished using the SCC4 cell line. As platinum-based therapeutic (refs. 32–35; data not shown)—similar to what was observed regimens are the standard of care for inoperable tumors of this by others using the C225 anti-EGFR antibody. This was not type, we compared the efficacy of INCB3619 to the cytotoxic unexpected as any of a number of signaling pathways may be agent cisplatin, alone or in combination with INCB3619. sufficient to drive proliferation in in vitro established ell culture INCB3619, as a single agent, nearly completely inhibited tumor systems and/or to activate the ErbB receptors via crosstalk growth during the treatment period (30% TGI) and subsequent- among signaling pathways. However, three-dimensional tumor ly resulted in a 13-day TGD (Fig. 5A). In contrast, cisplatin was growth in vivo is different than growth in culture and ErbB mostly ineffective (0% TGI and a 2-day TGD) although it was signaling may play a more crucial role. Supporting this administered near its maximally tolerated dose (Fig. 5A and data hypothesis, it has been shown that xenografts arising from not shown). Although cisplatin was lackluster as a single agent, cultured cells significantly up-regulate key players in ErbB the addition of INCB3619 was synergistic (66% TGI and a signaling (36). Therefore, evaluating the effect of sheddase 24-day TGD), producing sporadic tumor regressions (in three inhibition in the more clinically relevant in vivo setting was of seven mice). Importantly, INCB3619 did not exacerbate imperative. the toxic effects of cisplatin, as assessed by animal weights To determine if sheddase inhibition could be as efficacious as and clinical observations (data not shown). To understand the direct kinase inhibition using a low molecular weight TKI, such as gefitinib (Iressa), we treated mice bearing well established MCF-7 human breast cancer xenografts with either INCB3619 Table 1. INCB3619, as opposed to the broad- (60 mg/kg/d) or a maximally tolerated regimen of gefitinib spectrum MMP inhibitor marimastat, potently and selectively inhibits ADAM10 and ADAM17, (100 mg/kg/d) and concluded that both treatments resulted in compared with a panel of other zinc-dependent equivalent tumor inhibition (50% and 48% TGI, respectively, metalloproteases and 19-day TGD for both; Fig. 4A). Further, in support of the targeted nature of INCB3619 and gefitinib, neither agent Enzyme assays INCB3619 Marimastat showed efficacy against MDA-MB-231 xenografts, in which IC50 (nmol/L) IC50 (nmol/L) proliferation is driven, not by EGFR, but by mutated k-Ras ADAM8 1,000 ND (ref. 37; Fig. 4B and data not shown). In contrast, the ADAM9 >5,000 ND nonselective MMP inhibitor marimastat, which inhibits both ADAM10 22 78 ADAM17 14 12 MMPs and sheddases (Fig. 1B), was efficacious (60% TGI; ADAM33 1,036 ND Fig. 4B), implying that the antitumor effect of marimastat is due MMP1 >5,000 4 to MMP inhibition and that INCB3619 is a selective compound MMP2 35 3 and that its antitumor activity is not a result of its somewhat MMP3 3,500 30 weak potency against MMP2. Indeed, a selective ADAM 10/17 MMP7 >5,000 20 MMP9 304 <5 inhibitor with 10-fold greater selectivity against MMP2 MMP12 17 <5 (INCB9725) retained its ability to inhibit tumor growth MMP14 772 <5 similarly to INCB3619 in the MDA-MB-435 tumor model (INCB3619: TGI, 35%; TGD, 10 days; INCB9725: TGI, 44%; Abbreviation: ND, not determined. TGD, 10 days; not shown).

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mechanism behind the observed synergy, we analyzed similarly (Fig. 5B, bottom). This latter effect likely accounts for the treated tumor samples ex vivo. Immunohistochemical analyses increased responsiveness to the cytotoxic insult induced by indicated that INCB3619 alone was sufficient to modestly treatment with cisplatin (Fig. 5A and C). However, even as a decrease tumor cell proliferation nearly by nearly 10% (Fig. 5B, single agent, INCB3619 was sufficient to induce tumor cell top) and activation of the Akt/PKB survival signaling pathway apoptosis (Fig. 5C, top right). Similar synergism was observed in

Fig. 2. Selective sheddase inhibition prevents proteolytic processing of ErbB ligands and activation of an EGFR receptor ligand autocrine/paracrine loop. A, transduction of the immortalized murine mammary epithelial cell line NMuMG withTGF-a results in an aggressively tumorigenic cells. NMuMG cells were transduced with an empty retroviral expression vector (n) or one expressingTGF-a (y). Pooled populations of transduced cells were injected s.c. into immunocompromised mice, and tumor growth was followed over time. B, ErbB ligand shedding was evaluated in NMuMG cells expressingTGF-a in the presence of increasing concentrations of INCB3619 (5), the ADAM10 selective compound INCB8765 (E), or the ADAM10/17-sparing MMP inhibitor INCB8278 (4). C, TGF-a shedding was also inhibited in rat fibroblasts (Rat-1-T) grown for 24 h in the presence of DMSO (control) or INCB3619, thus indicating that this effect is not cell type specific. D, INCB3619 also potently inhibited amphiregulin shedding from primary tumor cell cultures of a treatment naive head and neck cancer patient. E, INCB3619 inhibits the proliferation of primary head and neck carcinoma cells. Cells were grown in low serum (2%) for 5 d in the presence of increasing concentrations of INCB3619 or DMSO, and proliferation was assessed using a luciferase-based assay (see Materials and Methods for details). No effect on proliferation was observed in normal cells (human umbilical vascular endothelial cells or BJ fibroblasts) at similar concentrations of INCB3619 (not shown). F, inhibition of ligand shedding resulted in inhibition of EGFR activation in serum-deprived (6 h) A431cells treated with conditioned medium fromTGF-a ^ transduced cells grown in the presence or absence of INCB3619 (2 Amol/L) for 30 min as determined by immunoblot analysis. An EGFR blocking antibody similarly inhibited EGFR activation (not shown).

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Fig. 3. Sheddase inhibition reduces the levels of circulatingTGF-a and retards tumor growth in vivo. A, TGF-a levels were determined in plasma from mice bearing Rat-1-T tumors before treatment and 2 d after treatment with INCB3619 (120 mg/kg/d; n =8).B, Rat-1-T tumors were treated with vehicle (gray)or INCB3619 (black ; 60 mg/kg/d) for 7 d, and tumor size was determined (n =7). C, carcinoma growth was also reduced by sheddase inhibition as exemplified by the epithelial NMuMG-T tumors treated with INCB3619 (black columns ; 60 mg/kg/d) or vehicle (gray columns)for7d(n =13).

a breast cancer xenograft model (MDA-MB-435) when INCB3619 was used in combination with paclitaxel, thus indicating that these effects were not particular to a specific tumor type or chemotherapeutic combination (paclitaxel, 10- day TGD; INCB3619, 20-day TGD; combination, 50-day TGD; Fig. 5D and E). INCB3619 also improved the therapeutic responses to other cytotoxic agents in models of pancreatic, non–small cell lung, and colon cancer (data not shown). Although INCB3619 and related molecules are potent and highly selective, they are not completely devoid of MMP Fig. 4. Selective sheddase inhibition is equally as efficacious as an EGFR TKI, activity. For example, selectivity against MMP2has proven and their combination results in improved tumor growth inhibition. A, s.c. human breast cancer xenografts (MCF-7) were treated with INCB3619 (60 mg/kg/d) or difficult due to the similarity of its active site to those of gefitinib (100 mg/kg/d) for 2 wk and their tumor volumes were measured (n =8). ADAM10 and ADAM17.6 As discussed above, although the B, MDA-MB-231xenografts, which have a mutated K-ras and which are gefitinib insensitive, were treated with INCB3619 (y) or marimastat (4) (60 mg/kg/d) for efficacy of selective sheddase inhibitors do not require MMP2 14 d, and tumor size was followed over time (n =9).C, sheddase inhibition activity, it is possible that inhibition of MMP2has potential improves the therapeutic response to EGFR kinase inhibition.Well-established A549 non ^ small cell lung carcinoma tumor xenografts were treated for 14 d with vehicle (n), INCB3619 (4; 60 mg/kg/d), gefitinib (y; 100 mg/kg, bid), or a combination of the two agents (o).Tumors were measured twice weekly using calipers. 6 W.Yao, unpublished observations. Points, mean tumor volumes (n = 7/group); bars, SE.

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Fig. 5. INCB3619 inhibits tumor cell proliferation and survival signaling resulting in synergism with cytotoxic chemotherapy. A, SCC4 xenografts were treated with vehicle (n), INCB3619 (y;60mg/kg/d14 d, horizontal arrow), cisplatin (4;10 mg/kg, 2/wk 2, vertical arrows), or a combination of the two agents (o), and tumor growth was followed over time (n =7).B, tumors were harvested from similarly treated mice 8 h after the first dose of cisplatin and inhibition of cellular proliferation (Ki67, top)andsurvival signaling (p-Akt, bottom) was examined by immunohistochemistry (n =4).C, apoptosis was also assessed usingTUNEL on these same samples (n = 4).Arrows, examples of apoptotic cells. D, INCB3619also synergizes with taxanes in ahumanbreast cancer xenograft model. S.c. well-established MDA-MB-435 tumors were treatedwith vehicle (n), INCB3619 (y;60mg/kg/d14 d, horizontal arrow), paclitaxel (4;10mg/kg,2/wk 2, vertical arrows), or a combination of the two agents (o), and tumor growth was followed over time (n = 6/group). Points, mean tumor volumes; bars, SE. E, apoptosis was also assessed usingTUNEL on tumor samples collected 24 h after the first dose of taxane (n = 3). Arrows, examples of apoptotic cells.

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Fig. 6. Selective sheddase inhibition does not cause the musculoskeletal side effects associated with broad-spectrum MMP inhibition. A, mice were treated with INCB3619 or the broad-spectrum MMPinhibitor marimastat (60 mg/kg/d) for14d. Knee joints were isolated and examined for histopathology. Arrow, fibrosis; arrowhead, angiogenesis; s, synovium; m, meniscus; c, cartilage. B, the extent of knee joint fibrosis was quantified by blinded scoring on a scale of 0 (no fibrosis) to 4 (severe fibrosis; n =4).

safety liabilities. For example, the clinical utility of broad- endothelial cells or primary fibroblasts) were resistant to the spectrum MMP inhibitors has been hampered by unexpected growth-inhibitory effects of INCB3619 at concentrations 10 musculoskeletal side effects. Because others have hypothesized times higher than those effective in transformed cells.7 These that inhibition of sheddases may be responsible for this findings suggest selective ADAM10/17 inhibitors, such as unwanted activity, we determined whether INCB3619 would INCB3619, have the potential to effect tumor growth and induce these undesirable effects. To do so, we established a survival while being better tolerated than the broad-spectrum murine model of the MMP-induced musculoskeletal side effects MMP inhibitors. similar to that described previously in the rat (21). Mice were treated with INCB3619 or the broad-spectrum MMP inhibitor Discussion marimastat at doses significantly higher than those required for efficacy in xenograft models. After 14 days of continuous Targeted therapies for the treatment of cancer have advanced treatment, mice were sacrificed and their knee joints were significantly in recent years and continue to show great analyzed. Marimastat treatment clearly induced joint fibropla- promise. The challenges that the medical and scientific sia, whereas administration of a selective sheddase inhibitor did communities now face involve the identification of populations not (Fig. 6A and B). Of note, de novo angiogenesis was also that will derive clinical benefit from these agents and the observed in the marimastat-treated mice in the area of fibrosis. delineation of an optimal strategy for combining these agents Similar steady-state drug exposures were achieved for both with current cytotoxic regimens to improve response rates while compounds (INCB3619, 691 F 228 nmol/L; marimastat, decreasing exposure to unnecessary side effects. Nonetheless, 448 F 74 nmol/L). Thus, sheddase inhibition can be shown significant advances are rapidly being made. For example, the to be both efficacious and devoid of the toxicity that prevented identification of the mechanism(s) of resistance to some of the exploration of this mechanism in earlier clinical trials with these new therapies, such as imatinib (41–43) and the EGFR- drugs such as marimastat. In addition, INCB3619 is somewhat selective TKIs (14, 15), has occurred within a short time after active against MMP12, and studies in knockout mice have these agents were introduced as approved therapies. As such, suggested a potential role for MMP12in the vascularization of complementary or alternative therapies are actively being murine lung tumor via the reduced formation of pursued. Our identification of potent, selective inhibitors of angiostatin—a reported endogenous angiogenesis inhibitor the ErbB ligand sheddases that are efficacious, well tolerated, and substrate of MMP12(39). The role of such molecules in and have suitable drug-like properties represents a new the human condition, however, is a point of contention. opportunity to affect clearly validated oncology targets. These Indeed, recent clinical data supports a beneficial role of MMP12 molecules prevent the release of ligands for multiple ErbB inhibition in non–small cell lung carcinoma, in which MMP12 family members representing a new class of pan-ErbB expression correlates with metastasis while inversely correlating inhibitors. with relapse-free survival (40). Taken together, these data There is strong evidence that ligand shedding is critical for suggest that the selective sheddase inhibitors, such as the transforming potential of EGFR (reviewed in ref. 44) and INCB3619, are capable of preventing the functional activation increased ErbB ligand expression is a common event in human of ErbB ligands and have therapeutic potential both as single cancers (38). This latter observation my be explained, in part, agents and when used in combination with additional by observations demonstrating metalloprotease-dependent therapeutics. Furthermore, these compounds are extremely well ErbB ligand shedding is increased upon activation of a number tolerated in xenograft experiments and do not exacerbate the of diverse signaling pathways linked to human cancers toxicities of cytotoxic therapies. In addition, we evaluated (e.g., WNT, prostaglandin E2, and LPA; refs. 3, 5, 22, 23). toxicologic variables indicative of hematologic, renal, and Further, correlative clinical and preclinical data suggest that hepatic dysfunction and found no significant effect in rodents ErbB pathways may compensate for one another, thus after continuous administration of INCB3619 at a dose significantly greater than that required for efficacy (Table 2). Moreover, untransformed cells (e.g., human umbilical vascular 7 J.S. Fridman and M. Hansbury, unpublished observations.

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Table 2. Hematologic and clinical chemistry variables are not altered upon treatment with INCB3619

RBC RET WBC ALB TP GLOB ALT BILI (106/mL) (103/mL) (103/mL) (g/dL) (g/dL) (%) (units/L) (mg/dL) Vehicle 10.23 F 0.43 425.4 F 60.47 8.23 F 1.86 3.22 F 0.08 5.62 F 0.11 2.40 F 0.04 47.00 F 4.03 0.21 F 0.02 INCB3619 10.88 F 0.14 388.5 F 14.53 7.05 F 0.83 2.88 F 0.16 5.12 F 0.23 2.23 F 0.08 58.83 F 11.57 0.26 F 0.03 Statistically No No No No No No No No significant

NOTE: Immunocompetent mice were treated with vehicle or INCB3619 (45 mg/kg/d) by continuous infusion for 7 d (n = 6/group). Whole blood was evaluated by looking at RBC counts, reticulocytes, and WBC. Plasma was used to quantify the levels of albumin, total protein, globulin, alanine aminotransferase, and bilirubin, changes in which may indicate altered hepatic and/or renal function. Abbreviations: RET, reticulocytes; ALB, albumin; TP, total protein; GLOB, globulin; ALT, alanine aminotransferase; BILI, bilirubin.

providing a potential mechanism of ‘‘resistance’’ to selective MMP inhibitors. It remains unclear why this latter class of ErbB-targeted therapies. Although this observation has yet to agents failed in the clinic but it is intriguing to find that some be conclusively proven, it suggests that sheddase inhibitors MMP activities have been linked to the generation of should synergize with TKIs (or other targeted therapies) antiangiogenic molecules (e.g., endostatin and tumstatin; because they inhibit unique aspects of a common pathway. ref. 50). It is therefore tempting to speculate that the effect Our studies demonstrating that INCB3619 enhances gefitinib of these antiangiogenic products of MMP activity may be sensitivity (Fig. 4C; ref. 9) support this concept and may greater in human cancer than in the mouse or that in the represent a well-tolerated alternative to combining EGFR TKIs preclinical mouse models, the concentration of MMP inhib- with antibodies specific solely for the EGFR and its ligands. itors was high enough to affect the sheddases, thus partly Clearly, the understanding of intrinsic sensitivity to ErbB explaining their efficacy against some tumor xenografts. inhibition in the clinic remains an evolving field. Although In this report, we have shown that selective sheddase EGFR amplification and/or mutation may affect response rates, inhibitors can prevent the release of ErbB ligands from cell multiple mechanisms that affect sensitivity may exist either in lines and primary patient tumor cells and that this can result parallel or in series. in inhibition of ErbB signaling pathways in vitro and in vivo. Selective sheddase inhibition seems to be well tolerated Although we cannot conclude that inhibition of sheddases in and can be equally efficacious as a TKI administered at a addition to ADAM10 and ADAM17 can be important for this maximally tolerated dose. The mechanism of this apparent activity (e.g., ADAM12or ADAM15), we have clearly shown differential tolerability is an active area of study; however, that MMP sparing protease inhibition is sufficient for clearly, inhibiting ligand-stimulated receptor activation is efficacy. Moreover, our previous in vitro studies have shown different than inhibiting basal kinase activity through direct that selective genetic (RNA interference) inhibition of kinase inhibition. For example, ligand-independent activation ADAM17 and not ADAM9 or ADAM15 is sufficient to affect of ErbB receptors, as has been observed upon - ErbB signaling pathways (9). These molecules thus have mediated binding to , is unlikely to be antitumor activity in tumor models reported to be sensitive perturbed by sheddase inhibition (45). Alternatively, a number to perturbations in ErbB pathways, but not elsewhere (e.g., of reports have shown that expression of tissue inhibitor of MDA-MB-231 tumors). Although sheddase inhibitors do -3 (TIMP-3), a gene encoding for an endoge- possess significant single-agent antitumor activity, their nous sheddase inhibitor, is often silenced in solid tumors, via ability to inhibit survival pathways such as Akt and our methylation (46–48). TIMP-3, as opposed to TIMP- preclinical findings demonstrating additive or synergistic 1, TIMP-2, and TIMP-4, is the only endogenous inhibitor effects support the use of combination therapy in the clinic. known to inhibit both ADAM10 and ADAM17 (ref. 49 and This is further substantiated by their excellent tolerability references therein) and may, therefore, regulate basal levels of and their lack of exacerbating the toxicity associated with ErbB ligand production. Perhaps, selective sheddase inhibitors standard chemotherapeutics or targeted agents. Further, have been so well tolerated because they merely complement combining sheddase inhibitors with other ErbB inhibitors or compensate for a loss of TIMP-3 activity in a pathologic may thwart multiple mechanisms of resistance, such as setting. kinase (gatekeeper) mutations or increased ligand expres- Although INCB3619 and related molecules are zinc-depen- sion. As such, selective sheddase inhibitors may complement dent metalloprotease inhibitors, they are highly selective the existing arsenal of anticancer agents; hence, clinical against a broad panel of MMPs and thus do not share their investigation of sheddase inhibition is undoubtedly war- musculoskeletal side effects. In addition, unlike broad- ranted. spectrum MMP inhibitors, the sheddase-selective compounds do not show efficacy in a xenograft model, which has a mutated K-ras, and do not show efficacy in in vitro Acknowledgments angiogenesis systems (i.e., inhibition of human umbilical We thank Kamna Katiyar and Yanlong Li for the screening of all compounds in the vascular endothelial cell proliferation, migration, invasion, biochemical metalloprotease assays; Xiaomei Gu for assistance with compound and tube formation; data not shown), further distinguishing pharmacokinetics; and Frederic Baribaud, Nancy Contel, and Phil Czerniak for their mechanism of tumor growth inhibition from that of technical and intellectual input.

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