Tumor Suppression by a Rationally Designed Reversible Inhibitor of Methionine Aminopeptidase-2

[CANCER RESEARCH 63, 7861–7869, November 15, 2003] Tumor Suppression by a Rationally Designed Reversible Inhibitor of Methionine Aminopeptidase-2

Jieyi Wang,1 George S. Sheppard,1 Pingping Lou,1 Megumi Kawai,1 Nwe BaMaung,1 Scott A. Erickson,1 Lora Tucker-Garcia,1 Chang Park,2 Jennifer Bouska,1 Yi-Chun Wang,1 David Frost,1 Paul Tapang,1 Daniel H. Albert,1 Sherry J. Morgan,3 Michael Morowitz,4 Suzanne Shusterman,5 John M. Maris,5 Rick Lesniewski,1 and Jack Henkin,1 1Cancer Research, 2Advanced Technology, and 3Preclinical Safety, Global PharmaceuticalR&D,Abbott Laboratories, Abbott Park, Illinois, and 4Departments of Surgery and 5Pediatric Oncology, The University of Pennsylvania School of Medicine and The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania

ABSTRACT cancer were observed with TNP-470 (20–22). Stable disease was also reported in TNP-470-treated patients with sarcoma, melanoma, and Methionine aminopeptidase (MetAP)-2 has been suggested as a novel adenocarcinoma (23, 24). However, dose-limiting neurotoxicity asso- target for cancer therapy because the anticancer agent TNP-470 irrevers- ciated with TNP-470 was reported in these clinical trials. ibly inactivates the catalytic activity of this enzyme. However, the impor- tance of MetAP2 in cell growth and tumor progression was uncertain MetAP2 is one of the two MetAPs known in eukaryotes (25). because previous data were based on the chemically reactive TNP-470. MetAPs are intracellular metalloenzymes responsible for removing Here we show that a rationally designed reversible MetAP2 inhibitor, the NH2-terminal initiator methionine residue from nascent proteins. A-357300, suppresses tumor growth preclinically without the toxicities They are required for protein cotranslational and/or posttranslational observed with TNP-470. We have synthesized this bestatin-type MetAP2 modifications, such as NH2-terminal myristoylation, and for protein inhibitor with the aid of crystal structures of the enzyme-inhibitor com- stability (26, 27). Inhibition of MetAP activity could therefore affect plexes and parallel synthesis. A-357300 induces cytostasis by cell cycle protein biological activity, proper subcellular localization, and degra- arrest at the G1 phase selectively in endothelial cells and in a subset of dation and result in interference of normal cell signal transduction and tumor cells, but not in most primary cells of nonendothelial type. cell cycle progression. Both MetAP1 and MetAP2 are expressed in all A-357300 inhibits angiogenesis both in vitro and in vivo and shows potent mammalian tissues and cells examined (7, 28), but only MetAP2 is antitumor efficacy in carcinoma, sarcoma, and neuroblastoma murine models. These data affirm that MetAP2 plays a pivotal role in cell growth up-regulated during cell proliferation (7). MetAP2 is also found at and establish that reversible MetAP2 inhibitors are promising novel higher concentrations in tumors as compared with normal cells (29, cancer therapeutic agents. 30). Antisense of MetAP2 induces apoptosis in human mesothelioma cells (31) and in rat hepatoma cells (32). These studies suggest that MetAP2 may play a critical role in cell proliferation and tumor INTRODUCTION growth. MetAP2 is a protein with two functions: processing initiator

6 methionine from nascent proteins and stabilizing eukaryotic initiation Selective inhibition of tumor cells and tumor ECs by targeting a factor 2 (32). These dual functions of MetAP2 are mediated by common molecule required for signal transduction of multiple growth different domains of the protein (32). The TNP-470-inactivated stimulators could provide an effective approach to treat cancer. The MetAP2 retains its capacity to stabilize eukaryotic initiation factor 2 intracellular enzyme MetAP2 became such a candidate molecule (2), consistent with the hypothesis that MetAP2 catalytic activity is when it was identified as a target for the widely investigated antican- required for cell growth. cer agent TNP-470 (1, 2). TNP-470, as well as its precursor, fumagil- TNP-470 inhibits MetAP2 by forming a covalent adduct through lin, irreversibly inhibited MetAP2 catalytic activity by alkylating a epoxide alkylation of the His231 residue in the enzyme active site (3, histidine residue in the enzyme active center (3, 4) and selectively 4). The short in vivo half-life (2–6 min in human; Ref. 24), the inhibited the proliferation of ECs and a subset of tumor cells, with presence of multiple metabolites, and concerns that TNP-470 may little effect on most primary cells of nonendothelial type (5–8). alkylate additional proteins complicate the linking of MetAP2 enzyme TNP-470 was postulated to inhibit cell proliferation by inactivating inhibition to the observed in vivo therapeutic and toxic effects by this cellular MetAP2 catalytic activity because the dose-response curves agent. To examine the effects of targeted MetAP2 inhibition in vitro for cell growth inhibition and cellular MetAP2 inactivation were and in vivo, we have synthesized highly specific and reversible superimposable (9). The antitumor efficacy of TNP-470 was demon- MetAP2 inhibitors using structure-based design. Here we show that a strated extensively in animal models including a transgenic mouse reversible MetAP2 enzyme inhibitor induces cytostasis in tumor cells pancreatic cancer (10), chemical or viral induced tumors (11, 12), a and ECs in vitro and inhibits tumor growth in murine models without wide variety of syngeneic tumors and xenografts of human tumors the toxicities observed with TNP-470. The data show that reversible (13–16), and metastases in the lung, liver, bone, and mesenchymal MetAP2 inhibitors are promising novel cancer therapeutics. tissues (17–19). In clinical trials, anecdotal cases of complete remission of metastatic cervical cancer and regression of metastatic breast MATERIALS AND METHODS MetAP1 and MetAP2 Enzymes and Activity Assay Received 5/15/03; revised 7/11/03; accepted 9/2/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with Recombinant human MetAP1 and MetAP2 were prepared as described 18 U.S.C. Section 1734 solely to indicate this fact. previously, and a coupled-enzyme chromogenic assay was used to measure Requests for reprints: Jieyi Wang, R48R, AP9, Abbott Laboratories, Abbott Park, MetAP activity by monitoring the production of free methionine with L-amino Illinois 60064. Phone: (847) 938-0434; Fax: (847) 937-4150; E-mail: Jieyi.Wang@ acid oxidase and horseradish peroxidase (33). abbott.com. 6 The abbreviations used are: EC, endothelial cell; MetAP, methionine aminopepti- MetAP2 Inhibitor A-357300 dase; HMVEC, human microvascular endothelial cell; CEC, circulating endothelial cell; T/C, the ratio of tumor volume of drug-treated versus vehicle control groups; VEGF, Detailed chemistry of preparation of bestatin-type MetAP2 inhibitors has vascular endothelial growth factor; bFGF, basic fibroblast growth factor; FBS, fetal bovine serum; HPMC, hydroxypropyl methylcellulose; SCID, severe combined immuno- been described previously (34). A scheme used to synthesize A-357300 is deficient; Rb, retinoblastoma; HUVEC, human umbilical vein endothelial cell. shown below (Scheme 1). 7861

In Vivo Studies

Drug Formulation. A-357300 was dissolved in 0.2% HPMC to yield a final working concentration of 5–10 mg/ml. This solution was sonicated for 30 min at 37°C and adjusted to pH 8.3. A-357300, dissolved in water, was used for mini-osmotic pump delivery (Alzet; Durect Corp., Cupertino, CA). Mouse Cornea Angiogenesis. On study day 0, twenty-four 30-g CF1 mice (Charles River Laboratories, Wilmington, MA) were anesthetized with ket- amine and Rompum before surgery. After anesthesia, eyes were checked to insure that there was no evidence of active or recent ophthalmic infection/ inflammation and that there were no residual capillary vessels within the corneal stroma. All whiskers and fur around the eyes were clipped, and the area was flushed with sterile saline. A corneal pocket was made by surgical incision approximately 0.7 mm from the limbus. Then one hydron-sucralfate pellet, Scheme 1. Reagents and conditions: a, Na, i-PrBr, NH3; b, BOC2O, i-PrOH, H2O; c, containing either 30 ng of bFGF (right cornea) or 150 ng of VEGF (left cornea) HN(OCH )CH HCl, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl, 1-hydroxy- 3 3 was inserted into the top of the pocket, and Neosporin ointment was applied to benzotriazole, 1-methylmorpholine, CH2Cl2; d, LiAlH4,Et2O; e, TMSCN, ClCH2CH2Cl 90°C; f, HCl, dioxane, H2O, 100°C; g, BOC2O, 1-methylmorpholine, dioxane, H2O; h, each eye to prevent infection. Drug treatments were started on day 0. 3-chlorobenzhydrazide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl, 1-hydroxy- A-357300 was given by twice daily s.c. injections at 25, 75, and 150 mg/kg/ benzotriazole, 1-methylmorpholine, CH2Cl2; i, diastereomer separation by high-perfor- day. On day 5 (right cornea with bFGF) and again on day 7 (left cornea with mance liquid chromatography (Silica, 20% acetone/hexanes); j, HCl, dioxane. VEGF pellet), the eyes were vasodilated by injecting 0.1 ml of 100 nM Nitropress i.p. 60 min before imaging. The mice were anesthetized immedi- Cell Culture and Proliferation Assay ately before imaging, and their eyes were moistened with saline to reduce reflections and prevent drying. A magnified corneal image was obtained by All normal human primary cells and their recommended culture media were using a digital camera attached to a slit lamp biomicroscope. The neovascular purchased from Clonetics (San Diego, CA) as described previously (7). Human measurement field was the area between the pellet and the limbus where CECs were isolated as described by Lin et al. (35). Briefly, buffy coat neovascularization had occurred. Data acquisition and storage were achieved mononuclear cells from 50 ml of blood freshly obtained from healthy donors with Leica imaging software, and the data computation was done with Mi- were suspended in 5 ml of EGM2 medium (Clonetics) and seeded into 1 well crosoft Excel. The statistical significance was evaluated with a two-tailed t test. of a 6-well plate coated with type I collagen (Becton Dickinson, Sparks, MD). CHP-134 Neuroblastoma Xenograft. The CHP-134 cell line was derived

The plate was incubated at 37°C with 5% CO2. The medium was changed from the diagnostic primary tumor biopsy from of a neuroblastoma patient twice weekly, and cell colonies appeared within 3 weeks. These cells were whose tumor showed MYCN amplification, deletion of the distal short arm of expanded into a T-75 flask as passage 1. After two more expansions, sufficient chromosome 1, and unbalanced gain of chromosome 17q material, and its cells were obtained and stored in liquid nitrogen. These CECs stained positive xenografting characteristics in athymic mice were as described previously (15). for CD31, CD34, and CD146 by flow cytometry. Primary BAECs and their Tumor growth was observed within 14 days after inoculation in 90% of growth medium were purchased from Clonetics. A transformed mouse EC line animals. Fourteen mice were randomized to receive either A-357300 or HPMC derived from brain microvasculature (bEND3) was kindly provided W. Risau vehicle when tumor volumes reached 200 mm3 (ϳ3 weeks after inoculation). (36). Human HT-1080 fibrosarcoma cell line and other tumor lines were A dose of 50 mg/kg A-357300 was administered s.c. twice daily, and an purchased from American Type Culture Collection (Manassas, VA). They equivalent volume of HPMC was given to control mice. Tumor measurements were grown in DMEM supplemented with 10% FBS (Invitrogen, San Diego, were made by Vernier calipers twice weekly, and tumor volumes were calcu- CA). Proliferation assays were performed in a fashion similar to that described lated using the ellipsoid formula: length ϫ width ϫ height ϫ 0.52 (39). previously (7). Treatment was continued for 30 days or until tumor volume exceeded 3.0 cm. Animals were sacrificed at this time and necropsied. These studies were Overexpression of MetAP2 in HT-1080 Cells approved by the Animal Care and Use Committee of the Children’s Hospital of Philadelphia. MetAP2 cDNA was cloned by reverse transcription-PCR using HMVEC HT-1080 Fibrosarcoma Xenograft. Eight-week-old SCID-beige mice total RNA as the template, as described previously (33). The primers used were (Charles River Laboratories) were inoculated with HT-1080 human fibrosar- 5Ј-att-aat-gct-agc-ccacc-atg-gcg-ggc-gtg-gag-gag-gta-gcg-gcct-3Ј and 5Ј-att- coma cells. The cells were reconstituted at 2 ϫ 106 cells/ml in 20% saline and aat-ctc-gag-tct-aga-cggtccg-tta-ata-gtc-atc-tcc-tct-gct-gac-aact-3Ј. The ampli- 80% phenol red-free Matrigel (Becton Dickinson Labware, Palo Alto, CA). fied MetAP2 cDNA was cut with NheI and XbaI and cloned into pcDNA3.1 Cells (0.25 ml; 0.5 million cells) were injected s.c. in the left upper abdominal (Invitrogen). The DNA sequence of the final construct, pcDNA-MetAP2, was quadrant. Dosing and tumor measurements were started on day 8 (after 7 days), confirmed by standard sequencing techniques. Transient expression was car- when the tumor was established. Tumor measurements were made by Vernier ried out in HMVEC primary culture. The cells were grown in T-75 flasks to calipers every other day. reach ϳ50% confluence. Four ␮g of DNA were incubated with 30 ␮lof MDA-435 Breast Carcinoma Xenograft. MDA-435-LM is a metastatic LipofectAMINE (Invitrogen) and transfected into the HMVECs in a T-75 flask isolate derived from MDA-MB-435 (American Type Culture Collection). using the conditions recommended by the manufacturer. The cells were then Approximately 7-week-old female SCID-C.B17 mice (C.B-17/IcrCrl-scid-BR; incubated with EGM2 medium for 24 h, trypsinized, and used in the prolif- Charles River Laboratories) were inoculated s.c. in the right flank with 0.2 ml eration assay. Stable transfection was carried out in HT-1080 cells by electro- of 1 ϫ 106 MDA-435 LM cells (1:1 Matrigel) on study day 0. All mice were poration. Briefly, 5 ϫ 106 cells were mixed with 15 ␮g of DNA, and the ear tagged. Treatments were started at day 4. The tumors were measured by a mixture was subjected to 250 V and 950 ␮F electroporation. Transfected cells pair of Vernier calipers twice a week after tumors were palpable (day 10), and were selected by 300 ␮g/ml G418 (Invitrogen), and single-cell clones were the tumor volumes were calculated according to the formula V ϭ L ϫ W2/2 (V, obtained by limited dilution of the transfected cells. volume; L, length; W, width). Perfusion fixation under halothane anesthesia Cellular MetAP2 Activity Assay was performed at the end of the study. Histology was performed on the major tissues/organs (bone marrow, brain, liver, lymphoid tissues, kidney, lung, and Inhibition of cellular MetAP2 activity was determined using the recently heart) as well as the mass. published method (33).

EC Tube Formation in Fibrin Matrix RESULTS

This protocol was adapted from a previously published method (37) as Discovery of the Reversible MetAP2 Inhibitor A-357300. To described in detail (38). determine the requirement of MetAP2 catalytic activity for cell 7862

Table 1 Enzyme inhibition assay results for compounds 1–9

Examples of bestatin-type inhibitors were listed to show their activity on MetAP2 and MetAP1 enzyme inhibition. The IC50 numbers were an average of two or more separate measurements.

MetAP2 MetAP1 1 2 ␮ ␮ Compounds R NHR IC50 ( M) IC50 ( M)

1CH3SCH2CH2 Ethyl (2S)-2-aminopropionate 6.0 63 2CH3CH2SCH2CH2 Ethyl (2S)-2-aminopropionate 1.3 69 3 (CH3)2CHCH2SCH2 Ethyl (2S)-2-aminopropionate 1.1 66 4CH3CH2SCH2CH2 (S) 1-(1-Naphthyl) ethylamine 0.022 0.37 5 (CH3)2CHCH2SCH2 (S) 1-(1-Naphthyl) ethylamine 0.048 2.2 6 (CH3)2CHSCH2CH2 (S) 1-(1-Naphthyl) ethylamine 0.19 12 7CH3CH2SCH2CH2 3-Chloro-benzhydrazide 0.11 20 8 (CH3)2CHCH2SCH2 3-Chloro-benzhydrazide 0.13 8.1 9 (CH3)2CHSCH2CH2 3-Chloro-benzhydrazide 0.12 57 (A-357300) growth, we sought to design substrate-like MetAP2 inhibitors based improved activity and greater selectivity for MetAP2 versus MetAP1 2 on the known leucine aminopeptidase inhibitor bestatin (40). With the (Table 1). We observed that large hydrophobic amides at the R site aid of crystal structures of the enzyme-inhibitor complexes and par- provided potent inhibitors of MetAP2, despite the preference of allel synthesis, we undertook iterative optimization of this 2-hydroxy- MetAP2 for protein or peptide substrates with small amino acids 1 3-aminoamide series. Examination of the R groups indicated that adjacent to the NH2-terminal methionine residue. Modification of the extension of the methionine-like hydrophobic side chain provided link to the aromatic group from amide to diacyl hydrazine provided

Fig. 1. Structure of the reversible MetAP2 inhibitor A-357300. A, the chemical structure of A-357300. B, crystal structure of MetAP2 active site with A-357300 (shown in green). The 2- hydroxy-3-aminoamide grouping of A-357300 interacted with the two manganese ions (solid spheres) with the oxygen substituent bridging between them. The thioether-containing side chain largely filled the adjacent hydrophobic site (Tyr444 shown as an example), whereas the 3-chlorophenyl aromatic group lies face to face with the His339 imidazole and occupies the adjacent opening of the active site. C, the chemical structure of TNP-470. D, an overlay of crystal structures of MetAP2 active site with TNP-470 (shown in magenta) and A-357300. The covalent inhibitor TNP-470 through its ring epoxide alkylated His231.

7863

Table 2 Antiproliferative activity of MetAP2 inhibitors species was not unexpected because there is Ͼ95% amino acid Cells grown in 96-well plates were treated with A-357300 or fumagillin at 0.1 nM to identity between human and mouse MetAP2. This high degree of ␮ 100 M for 3 days. IC50 for proliferation inhibition was the average of two or more separate measurements. similarity provided an advantage for generalizing from results of MetAP2 inhibitors in mouse models. Most of the nonendothelial IC (␮M) 50 human primary cells we examined were not inhibited by A-357300 Cell type A-357300 Fumagillin (Table 2). The human tumor cell lines we tested showed distinct ECs sensitivity to growth inhibition by A-357300 (Table 2). HT-1080 HMVECs/HUVECs 0.1 0.001 fibrosarcoma and CHP-134 neuroblastoma were among the most Human CECs 0.1 0.001 bEND3 (murine) 0.2 0.002 sensitive cells and were inhibited by A-357300 with IC50 values of BAEC (bovine) 0.1 0.001 0.1–0.2 ␮M and displayed sensitivity similar to that of ECs. Other Human primary cells Mammary epithelial Ͼ100 Ͼ1 tumor cell lines such as PC3 prostate carcinoma and MCF7 breast Prostate epithelial Ͼ100 Ͼ1 carcinoma were not significantly inhibited by A-357300. The sensi- Astrocytes 53.6 Ͼ1 tivity of certain tumor cells to MetAP2 inhibition may augment the Fibroblasts 2.0 Ͼ1 Tumor cells efficacy of MetAP2 inhibitors as anticancer agents because of the PC3 Ͼ100 Ͼ1 combination of an antiangiogenic effect with direct tumor cytostasis. Ͼ Ͼ MCF7 100 1 Similar ranking of cell sensitivities to A-357300 and fumagillin (Ta- MDA-435-LM 2.0 Ͼ1 MiaPaCa2 1.0 Ͼ1 ble 2) implies their common mechanism of action, i.e., inhibition of A549 0.2 0.002 the MetAP2 enzyme. DLD-1 0.2 0.002 HCT-15 0.2 0.003 A-357300 Inhibits Cell Proliferation by Inhibiting Intracellular NCI-H460 0.2 0.003 MetAP2. We further demonstrated that the cytostasis by A-357300 HT-1080 0.1 0.001 was a result of cellular MetAP2 inhibition by examining the effects of CHP-134 0.1 0.001 MetAP2 overexpression and the NH2-terminal methionine status of cellular proteins. Human HT-1080 fibrosarcoma cells were sensitive to MetAP2 inhibitors as shown above (Table 2). Whereas the growth potent MetAP2 inhibitors with improved selectivity, particularly when of HT-1080 cells was not affected by transfection of pcDNA-MetAP2 combined with larger R1 groups. A-357300 was identified as the plasmid or vector control, the cells overexpressing MetAP2 (ϳ5-fold optimized compound (Fig. 1A). It selectively inhibited MetAP2 cat- of control determined by Western blot; data not shown) showed there ␮ alytic activity with an IC50 of 0.12 M (Table 1) and did not inhibit was an approximately 4-fold decrease in the potency of A-357300 or MetAP1 or leucine aminopeptidases at concentrations below 10 ␮M. fumagillin, but not that of cytotoxic agents such as paclitaxel and The X-ray crystal structure of the enzyme-inhibitor complex of 5-flurouracil (Table 3), supporting the hypothesis that A-357300 A-357300 (Fig. 1B) indicates the mode of binding. The 2-hydroxy- inhibits cell growth by inhibiting the cellular MetAP2 enzyme. 3-aminoamide grouping interacts with the two manganese metal ions To demonstrate that A-357300 inhibits cellular MetAP2 enzyme

in the active site (33), with the oxygen substituent bridging between activity, we measured the NH2-terminal initiator methionine status of them. The thioether-containing side chain largely fills the adjacent cellular proteins. HT-1080 cells were incubated with MetAP2 inhib- hydrophobic site, whereas the 3-chlorophenyl aromatic group lies face itors for 4 h before [35S]methionine was added to the culture. After an to face with a histidine imidazole (His339) and occupies the adjacent additional2hofincubation, cellular proteins were isolated and 1 2 opening of the active site. Both the R and R hydrophobic groups applied to a Reactive Red dye-agarose column. The NH2-terminal interact with MetAP2 near the site of an insertion of 60 amino acids initiator [35S]methionine of these proteins, if unprocessed inside cells, (Tyr444 shown as an example), which is absent in MetAP1 (25). A was then released by the exposure to recombinant MetAP2 enzyme. modeled structure of MetAP1 indicates that the active site of MetAP1, Increases in this initiator [35S]methionine (i.e., the unprocessed initi- lacking the insertion, has less room in this region, providing a basis ator [35S]methionine released by exogenously added MetAP2) re- for the selectivity observed. The chemical structure of TNP-470 (Fig. flected the inhibition of cellular MetAP2 enzyme activity by the 1C) and a crystal structure overlay of TNP-470 with A-357300 in compounds tested. A-357300 blocked cellular MetAP2 enzyme activ- MetAP2 active site (Fig. 1D) are shown as a comparison. ity, as indicated by the increase in unprocessed initiator [35S]methi- ␮ A-357300 Selectively Inhibits EC and Tumor Cell Growth. onine (Fig. 2A), with an IC50 of 0.15 M. The similar IC50 of A-357300 induced cytostasis in ECs and a subset of tumor cells A-357300 for inhibiting both cell proliferation and cellular MetAP2 without cytotoxicity in the in vitro studies. A-357300 inhibited the enzyme activity further supports the postulated mechanism of action proliferation of ECs, such as HMVECs and HUVECs, grown in of this compound.

complete growth medium containing a combination of growth factors A-357300 Induces Cytostasis by Cell Cycle Arrest at the G1 (VEGF, bFGF, epidermal growth factor, and insulin-like growth fac- Phase. To understand the mechanism of cytostasis by A-357300, we ␮ tor) and FBS in a 3-day proliferation assay with an IC50 of 0.1 M analyzed effects of this agent on cell cycle proteins and cell cycle (Table 2). Continuous exposure of A-357300 was required for its maximal potency against proliferation, and cells exposed to 10 ␮M A-357300 for only 4 h were not inhibited. The inhibition was cyto- Table 3 Selective sensitivity to A-357300 in MetAP2-overexpressing cells static rather than cytotoxic because cell numbers did not fall below the HT-1080 cell clones transfected with pcDNA3.1 (vector) or pcDNA-MetAP2 (MetAP2) were used in a 3-day proliferation assay. IC50 for proliferation inhibition was initial values, and no cytotoxicity was observed with concentrations of the average of three separate measurements. Paclitaxel and 5-fluorouracil were used to A-357300 below 100 ␮M as measured by cellular lactate dehydrogen- show that there was no nonspecific shift of sensitivity of these cells. ␮ ase release (data not shown). Furthermore, no apoptosis was detected Proliferation IC50 ( M) by caspase-3 measurements in drug-treated cells. A-357300 also in- Compound HT-1080 (vector) HT-1080 (MetAP2) hibited the proliferation of CECs (Table 2) isolated from human A-357300 0.11 Ϯ 0.06 0.47 Ϯ 0.22 peripheral blood (35) that may contribute to angiogenesis (41). Fumagillin 0.002 Ϯ 0.001 0.01 Ϯ 0.006 Ϯ Ϯ A-357300 inhibited murine and bovine ECs with IC50 values similar Paclitaxel 0.024 0.015 0.031 0.011 to those for HMVECs and HUVECs. The potency of A-357300 across 5-FU 0.014 Ϯ 0.06 0.010 Ϯ 0.004 7864

Fig. 2. A-357300 inhibits cellular MetAP2 and induces cell cycle arrest at the G1 phase. A, cellular MetAP2 activity. Inhibition of cellular MetAP2 enzyme activity was measured by determining the unprocessed initiator methionine of newly synthesized cellular proteins labeled with [35S]methionine in HT-1080 cells treated with inhibitors at various concentrations. The unprocessed initiator [35S]methionine released by exog- enous MetAP2 and quantified in a scintillation counter reflects the inhibition of cellular MetAP2. B, Western blots. Cell lysates of HMVECs treated with A-357300 for 24 h were analyzed for Rb, cyclin A, and cyclin D1 protein (all antibodies from Santa Cruz Biotechnology). 6% SDS-PAGE gel was used to separate Rb forms of hyper- or hypophosphorylation. C, flow cytometry. HM- VECs or HT-1080 cells treated with A-357300 (10 ␮M) for 3 days were stained with propidium iodine and analyzed for cell cycle distributions.

progression. Western blot showed that Rb protein was predominantly against VEGF (Fig. 3, B and C), and against bFGF (Fig. 3D). hypophosphorylated in HMVECs treated with A-357300 (Fig. 2B), A-357300 at 75 and 150 mg/kg/day inhibited VEGF-induced vessel displaying a dose response similar to that for cellular MetAP2 inhi- area by 34% (P Ͻ 0.005) and 55% (P Ͻ 0.001), respectively, but bition and proliferation inhibition. A-357300 also caused a reduction showed no significant inhibition at the lowest dose of 25 mg/kg/day. of cyclin A but had no effect on cyclin D1 concentrations (Fig. 2B). It also inhibited bFGF-stimulated vessel area by 43%, 52%, and 60% Similar results were seen with fumagillin (data not shown). Both (all P Ͻ 0.001) for the three doses, respectively. Plasma A-357300 HMVECs and HT-1080 tumor cells treated with A-357300 showed concentrations measured at the 6 h time point after the terminal dose ␮ arrest at the G1 phase of the cell cycle as shown by flow cytometry were 0.24, 0.38, and 2.2 M for 25, 75, and 150 mg/kg/day groups,

(Fig. 2C). No accumulation of sub-G1 cell population was detected, respectively, and correlated to efficacy in a dose-dependent manner. consistent with the cytostatic effect of A-357300 seen in the prolif- As a comparison, TNP-470 given near the maximal tolerated dose (30 eration assay. TNP-470 and fumagillin were reported to arrest cells at mg/kg by s.c. injections every other day) resulted in a 54% inhibition

the G1 phase of cell cycle (5, 6). Taken together, these observations of vessel area with bFGF induction. with MetAP2 inhibitors support that MetAP2 inhibition leads to A-357300 Inhibits Tumor Growth in Vivo. To evaluate its anti-

cytostasis by G1 arrest in susceptible cells. tumor efficacy, we then tested A-357300 in three human xenograft A-357300 Inhibits Angiogenesis in Vitro and in Vivo. We next tumor models in mice. These were CHP-134 neuroblastoma, HT-1080 evaluated A-357300 for its antiangiogenic activity both in vitro and in fibrosarcoma, and MDA-435-LM breast carcinoma. Of these, the vivo. Sprout and tube formation in three-dimensional fibrin matrix is CHP-134 human neuroblastoma xenograft in nude mice was reported a feature of activated ECs, which provides a useful angiogenesis previously to be inhibited by TNP-470 (15). A-357300 at 100 mg/kg/ model in vitro (37). HMVECs attached to microcarrier beads embed- day by twice daily s.c. injections, starting 3 weeks after tumor inoc- ded in fibrin gel were stimulated to grow, migrate, sprout, and form ulation, significantly suppressed growth of this established tumor tubule structures in the presence of angiogenesis inducers VEGF and xenograft with a T/C of 0.185 (P Ͻ 0.001) on day 24 after the bFGF (Fig. 3A). A-357300 at 0.4 ␮M completely blocked this self- initiation of treatment (Fig. 4A). A-357300-treated mice continued to organization of HMVECs. In addition, A-357300 showed inhibition gain weight, and no signs of adverse effects were observed. of mouse cornea angiogenesis in vivo. s.c. injections of A-357300 A-357300 inhibited HT-1080 human fibrosarcoma, grown in the twice daily at 25, 75, and 150 mg/kg/day inhibited growth factor- s.c. flank of SCID-beige mice, in a dose-dependent manner when induced cornea neovascularization in a dose-dependent manner administered by twice daily s.c. injections starting on day 7 after 7865

Fig. 3. Inhibition of angiogenesis by A-357300 both in vitro and in vivo. A, HMVECs grown in microcarriers sprout and form tubule structures when embedded in fibrin gel in the presence of VEGF, bFGF, and serum. A-357300 at final concentrations of 0, 0.08, 0.4, and 2 ␮M was added into the culture medium. Images were taken after a 3-day incubation. B, these photographs show the representative mouse cornea neovessels from the VEGF experimental group. The numbers are the daily dose of A-357300 in mg/kg/day by twice daily s.c. injections. C, quantitative vessel area of the cornea images was determined with Leica imaging software (n ϭ 6). VEGF was the inducer of neovascularization. D, quantitative vessel area of the cornea images was achieved with Leica imaging software (n ϭ 6). bFGF was the inducer of neovascularization.

tumor inoculation (Fig. 4B). Highest tumor growth inhibition (T/C of DISCUSSION 0.34 at day 20; P Ͻ 0.001) was achieved with a 100 mg/kg/day dose, without overt signs of toxicity. When given via osmotic minipumps at We have presented data showing an essential role for MetAP2 in 15 mg/kg/day, A-357300 also significantly inhibited HT-1080 tumor cell growth and tumor progression. Selective inhibition of MetAP2 growth (Fig. 4C), giving a T/C value of 0.38 (P Ͻ 0.001) at day 23, catalytic activity by a rationally designed reversible inhibitor resulted although twice daily s.c. injections of double this daily dose had failed in the G1 cell cycle arrest of ECs and many tumor cells. MetAP2 to produce a significant antitumor effect (Fig. 4B). These data suggest inhibitors arrest ECs stimulated by a combination of growth factors (bFGF, VEGF, insulin-like growth factor, and epidermal growth fac- that in vivo activity of A-357300 was not driven by high Cmax values but was related to a continuous minimum drug exposure over a period tor) and serum, suggesting that MetAP2 enzyme activity is required of time. These observations were in agreement with our in vitro for signal transduction by multiple growth signals. MetAP2 enzyme finding that continuous exposure to MetAP2 inhibitors was required activity is also required for the growth of many tumor lines. These for optimal inhibition of cell proliferation and consistent with the observations strongly support the hypothesis that MetAP2 is essential mode of action of MetAP2 inhibitors (namely, reversible cytostasis). for proliferation of tumor cells and is an appropriate target for anti- We further examined both the efficacy and toxicity of A-357300 in cancer therapy. MDA-435-LM human breast carcinoma s.c. xenograft model in SCID The molecular mechanism of MetAP2 inhibition leading to the mice, and we compared it with TNP-470. A-357300 or TNP-470 was activation of p53 pathway (8, 42) and subsequent G1 cell cycle arrest administered starting on day 4 after tumor inoculation. A-357300 has not been established. MetAP2 is one of the two known enzymes inhibited tumor growth in a dose-dependent manner, with T/C values responsible for the removal of the NH2-terminal initiator methionine on day 32 of 0.59 (P Ͻ 0.01) and 0.32 (P Ͻ 0.01), respectively, for from nascent proteins. Whereas MetAP1 and MetAP2 share an en- 50 and 100 mg/kg/day doses by twice daily s.c. injections (Fig. 5A). zyme activity and might compensate for each other, MetAP2 is The efficacy of A-357300 at 100 mg/kg/day (T/C value of 0.32 on day Ͼ1000-fold more efficient at catalyzing methionine removal from 32; P Ͻ 0.01) was superior to that of TNP-470 (T/C value of 0.5 on peptide sequences of glyceraldehyde-3-phosphate dehydrogenase (9). day 32; P Ͻ 0.01; Fig. 5B), which was given at 16 or 20 mg/kg by s.c. Other MetAP2-specific cellular substrate proteins, such as cyclophilin injections every other day in the SCID mice. No overt signs of toxicity A and 14-3-3␥, have been identified (9, 43). Therefore, MetAP2 were observed during the 28 days of drug treatment with A-357300. inhibition may result in unprocessed initiator methionine in only a In contrast, TNP-470-treated mice exhibited severe skin irritation at small subset of cellular proteins, which are very poor substrates for the injection site, dehydration, rough coat, and diminished weight MetAP1. Altered NH2 termini of these proteins may interfere with gain. Histological evaluation of these chronically treated mice on subsequent modification(s) and affect their function, subcellular lo- termination revealed that TNP-470, at doses of 16 and 20 mg/kg given calization, or turnover. For example, removal of initiator methionine by s.c. injection every other day, caused pleural (Fig. 5C) and epicar- is required for protein N-myristoylation. Proteins destined to become dial fibrosis, as well as suppurative vasculitis with medial hypertrophy myristoylated begin with the NH2-terminal sequence Met-Gly. The at nontumor sites. None of these lesions were seen in animals treated initiator Met is removed by MetAPs before myristate is linked to the with A-357300 at any doses, suggesting that these toxic effects of Gly via an amide bond catalyzed by N-myristoyl transferase. Protein TNP-470 are not related to MetAP2 inhibition but rather are effects N-myristoylation is required for membrane binding of many important related to the chemical nature of TNP-470. signal transduction proteins including Src family tyrosine kinases, Abl 7866

angiogenesis that is targeted by the N-end rule pathway through its

NH2-terminal Cys residue (46). Therefore, we postulate that removal of the initiator methionine in specific cellular proteins by MetAP2 cannot be compensated by MetAP1 and that this first step of protein posttranslational modification may be required for the activity, stability, or subcellular localization of these MetAP2-specific substrate proteins essential for the cell cycle progression in susceptible cells. Application of proteomics technology should help delineate the critical proteins in the MetAP2 pathway and may uncover novel cell cycle regulators or controlling mechanisms. We have demonstrated that a reversible MetAP2-specific inhibitor showed potent single agent antitumor efficacy in xenograft models of carcinoma, sarcoma, and neuroblastoma. These data support the view that reversible MetAP2 inhibitors are promising therapeutics for cancer treatment. MetAP2 inhibitors as novel anticancer agents may have several potential advantages over the chemotherapeutics in the clinic and other agents in development. First, MetAP2 inhibition blocks multiple growth signals. MetAP2 inhibitors arrest EC growth in the presence of FBS and many other added growth factors. Tumor cells secrete multiple angiogenesis inducers, and it is important that effective angiogenesis inhibitors block ECs in response to multiple stimulators. Next, MetAP2 inhibition also results in an antiproliferative effect in many tumor cells (Table 2). The sensitivity of these tumor cells to MetAP2 inhibitors does not seem to correlate strictly with their p53 status. MCF7 cells have wild-type p53 but are not inhibited by A-357300 or fumagillin, whereas p53-null cells (HCT-15 and DLD1) are inhibited by these agents. p53 pathway has been shown to be required for inhibition of primary mouse ECs by TNP-470 (8, 42). However, MetAP2 inhibitors may use alternative mechanisms to block proliferation in tumor cells without functional p53. This hypothesis is also supported by a previously published observation that the p53-null prostate cancer PC3 cells in monolayer culture were insensitive to TNP-470 but were inhibited when grown in soft agar (47). The combined antiangiogenic and direct antitumor properties of a MetAP2 inhibitor could prove to be a significant advantage. MDA- 435-LM cells are less sensitive to A-357300 in vitro, whereas this agent significantly inhibited the growth of MDA-435-LM tumor in vivo, which could be attributed to the antiangiogenic effect of A-357300. Conventional cytotoxic drugs may also have this dual effect, but they are less attractive because they kill other normal cells in addition to ECs. MetAP2 activity seems less critical for the growth of most nonendothelial primary cells. Finally, MetAP2 inhibition is

exclusively cytostatic. MetAP2 inhibitors induce cell arrest at the G1 phase of cell cycle without apoptosis. They do not cause cytotoxicity

at concentrations up to 1000-fold of the IC50 for inhibition of cellular MetAP2 and proliferation. A-357300 could be dosed for 100 days continuously in the murine models without overt signs of toxicity in Fig. 4. Inhibition of tumor growth by A-357300. A, CHP-134 human neuroblastoma the treated mice (data not shown). Thus, MetAP2 inhibitors may be cells were s.c. implanted in athymic mice, and mice were allowed to form tumors of ϳ200 tolerated for chronic use in the clinic because of the selective cyto- 3 mm in size and then were treated with A-357300 (100 mg/kg/day) by twice daily s.c. static mechanism of action. These biological features of MetAP2 injections. B and C, SCID-beige mice inoculated with HT-1080 human fibrosarcoma in the flank were allowed to form tumors of ϳ450 mm3 in size and then treated with distinguish it from other known cell growth controlling targets. A-357300 at either 30, 60, or 100 mg/kg/day doses by twice daily s.c. injections (B)orat MetAP2 inhibitors therefore are a promising new generation of anti- 15 mg/kg/day by s.c. osmotic minipumps (C). cancer therapeutics. The potential utility of MetAP2 inhibitors in the treatment of cancer tyrosine kinases, Ser/Thr kinases such as cAMP-dependent protein is underscored by clinical observations with TNP-470. Anticancer kinase, phosphatases such as calcineurin B, guanine nucleotide-bind- activity of TNP-470 was observed in four patients in a Phase I study ing proteins, myristoylated alanine-rich C kinase substrate protein, of 18 patients with inoperable recurring metastatic squamous cell and others (44). Protein turnover in a given cell follows the N-end rule cancer of the cervix (20, 21). Patients with lung metastasis appeared that relates the half-life of a protein to the identity of its NH2-terminal to benefit most frequently from TNP-470 treatment. Four of seven residue (45). MetAP2 inhibitors could affect protein turnover due to patients with lung metastasis demonstrated clinical benefit (three the uncleaved NH2-terminal methionine. It has been speculated that patients experienced disease stabilization, and one patient obtained the effect of MetAP2 inhibitors may stem from inhibition of the complete resolution of all her pulmonary metastases). In another

NH2-terminal Met-Cys cleavage in a normally short-lived regulator of study, regression of metastatic breast cancer in multiple organs in a 7867

Fig. 5. Comparison of the antitumor activity and toxicity of A-357300 and TNP-470. SCID mice inoculated with MDA-435-LM human breast carcinoma cells were allowed to form tumors for 3 days and then treated with A-357300 at 50 and 100 mg/kg/day doses by twice daily s.c. injections (A) or with TNP-470 at 8, 16, and 20 mg/kg doses by s.c. injections every other day (B). The lungs (H&E staining) of representative mice in the vehicle-, TNP-470-, and A-357300-treated groups are shown in C. SCID mice inoculated with MDA-435-LM human breast carcinoma cells in the flank were allowed to form tumors for 3 days and then treated as described in A and B. On day 32, the mouse tissues were fixed by perfusion and subjected to histological evaluation.

patient treated with TNP-470 was observed (22). In addition, three appeared to be reversible. TNP-470 caused pleural and epicardial patients experienced disease stabilization in a Phase I trial of TNP-470 fibrosis, as well as suppurative vasculitis with medial hypertrophy at in patients who had solid tumors refractory to the best available nontumor sites in mice (Fig. 5C). None of these lesions were seen in treatment or with a high risk of recurrence (23, 24). In a Phase I study animals treated with A-357300, which produced superior antitumor published in 2002, the combination of TNP-470 and paclitaxel in 16 efficacy (Fig. 5). We postulate that the chemical properties of TNP- NSCLC patients produced a favorable effect on patient survival when 470 cause it to be difficult to deliver, metabolized quickly, and exhibit compared with published reports for paclitaxel alone (48). However, toxicities, thus limiting its utility in the clinic. Rationally designed TNP-470 has significant liabilities that limit its usefulness in the reversible MetAP2 inhibitors should overcome these liabilities and be clinic. The mean plasma half-lives of TNP-470 and its principal amenable to chronic use in the clinic to suppress tumor growth and metabolite AGM-1883 were extremely short (harmonic mean t1/2 of 2 improve patient survival. and 6 min, respectively). Although in vitro data suggest that TNP-470 In summary, we have discovered a potent, selective, and reversible is cytostatic at low concentrations, this drug resembles a cytotoxic MetAP2 inhibitor that suppresses tumor growth in murine models. It does agent in vivo. Neurotoxicity (weakness, nystagmus, diplopia, and not cause the adverse pathological changes observed with TNP-470, ataxia) observed with TNP-470 was sudden and dose-limiting but supporting the hypothesis that the toxicity of TNP-470 is unrelated to 7868

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