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Clinical Cancer Research Vol. 12, 2583-2590, April 15, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

A Novel Methionine Aminopeptidase-2 Inhibitor, PPI-2458, Inhibits Non–Hodgkin's Lymphoma Cell Proliferation In vitro and In vivo

Andrew C. Cooper1, Russell M. Karp2, Edward J. Clark2, Nazbeh R. Taghizadeh2, Jennifer G. Hoyt2, Matthew T. Labenski2, Michael J. Murray3, Gerhard Hannig2, William F. Westlin2 and Charles D. Thompson4

Authors' Affiliations: 1 Department of Cell Biology, Repligen; 2 Department of Preclinical Research and Analytical Pharmacology, Praecis Pharmaceuticals Incorporated, Waltham, Massachusetts; 3 Department of Toxicology, Memory Pharmaceuticals, Montvale, New Jersey; and 4 Department of Drug Metabolism, Merck & Company Incorporated, West Point, Pennsylvania

Requests for reprints: William F. Westlin, Department of Preclinical Research, Praecis Pharmaceuticals Incorporated, 830 Winter Street, Waltham, MA 02451. Phone: 781-795-4395; E-mail: william.westlin{at}praecis.com.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Fumagillin and related compounds have potent antiproliferative activity through inhibition of methionine aminopeptidase-2 (MetAP-2). It has recently been reported that MetAP-2 is highly expressed in germinal center B cells and germinal center–derived non–Hodgkin's lymphomas (NHL), suggesting an important role for MetAP-2 in proliferating B cells. Therefore, we determined the importance of MetAP-2 in normal and transformed germinal center B cells by evaluating the effects of MetAP-2 inhibition on the form and function of germinal centers and germinal center–derived NHL cells.

Experimental Design: To examine the activity of PPI-2458 on germinal center morphology, spleen sections from cynomolgus monkeys treated with oral PPI-2458 were analyzed. Antiproliferative activity of PPI-2458 was assessed on germinal center–derived NHL lines in culture. A MetAP-2 pharmacodynamic assay was used to determine cellular MetAP-2 inhibition following PPI-2458 treatment. Finally, inhibition of MetAP-2 and proliferation by PPI-2458 was examined in the human SR NHL line in culture and in implanted xenografts.

Results: Oral PPI-2458 caused a reduction in germinal center size and number in lymphoid tissues from treated animals. PPI-2458 potently inhibited growth (GI50 = 0.2-1.9 nmol/L) of several NHL lines in a manner that correlated with MetAP-2 inhibition. Moreover, orally administered PPI-2458 significantly inhibited SR tumor growth, which correlated with inhibition of tumor MetAP-2 (>85% at 100 mg/kg) in mice.

Conclusions: These results show the potent antiproliferative activity of PPI-2458 on NHL lines in vitro and oral antitumor activity in vivo and suggest the therapeutic potential of PPI-2458 as a novel agent for treatment of NHL should be evaluated in the clinical setting.

Non–Hodgkin’s lymphoma (NHL) is the sixth leading cause of cancer death in the United States and has increased in incidence by >80% since 1973 (1, 2). NHL can be divided into at least 29 different subtypes of malignancy of either T- or B-lymphocyte origin. The vast majority of NHL malignancies are B cell derived. Moreover, a large number of these B-cell malignancies exhibit phenotypic characteristics consistent with a germinal center origin, the site where B cells undergo proliferation and somatic hypermutation.

Germinal center–derived B-cell lymphomas can be divided into at least three groups. Follicular lymphoma is responsible for 22% of all NHLs, and almost always exhibits aberrant expression of the antiapoptotic gene Bcl-2 (1, 3, 4). Follicular lymphoma typically follows an indolent course but often transforms into the more aggressive diffuse large B-cell lymphoma (DLBCL; ref. 5). Whereas at least half of all DLBCLs express germinal center markers (Bcl-6+, CD10+), the remainder may more closely resemble activated B lymphocytes and potentially deserve a distinct classification (6, 7). Under the current classification, DLBCLs represent 40% of all NHLs (8). A third type of germinal center–derived B-cell neoplasm, Burkitt's lymphoma, is highly aggressive but rare in the United States, representing <1% of NHL. In total, nearly half of all cases of NHL are malignancies of germinal center B lymphocytes.

There are currently several approved treatment options for B-cell NHL. Aggressive B-cell lymphomas are treated with a broad spectrum of chemotherapeutics, frequently in combinations. The most commonly used regimen is cyclophosphamide-hydroxydoxorubicin-oncovin-prednisone; however, these drugs only produce a complete response in roughly half of DLBCL patients and are rarely curative for low grade follicular lymphoma (1, 6, 9). Rituxan, a humanized monoclonal antibody to the B-cell marker CD20, as well as recent Food and Drug Administration–approved radiolabeled anti-CD20 antibodies Zevalin (yttrium-90) and Bexxar (iodine-131), have shown promise in treating low-grade or refractory B-cell lymphoma, highlighting the utility of target-based approaches to treating B-cell NHL (10–12). Together, these therapies leave significant room for improvement, suggesting new molecularly targeted therapies may provide added benefit to current standard of care in NHL.

Methionine aminopeptidase-2 (MetAP-2) is the molecular target of the antiproliferative fumagillin class of compounds. MetAP-2 catalyzes the removal of N-terminal methionines from growing polypeptide chains, a precursor to subsequent posttranslational protein modifications, such as myristolyation, and is required for stability, activity, and intracellular localization of a number of proteins (13–15). MetAP-2 and a related isoform, MetAP-1, can use many of the same substrates but their relative activities differ based on the nature of flanking amino acids, primarily the amino acid residue adjacent to the N-terminal methionine. Fumagillin and structural analogues, such as TNP-470 (AGM-1470), selectively inhibit MetAP-2 enzymatic activity through irreversible covalent interactions with histidine-231 in the active site of the enzyme (16–19). This MetAP-2 blockade is thought to be responsible for the inhibition of endothelial cell proliferation observed with fumagillin analogues (20, 21). Fumagillin analogues also inhibit angiogenesis in vivo and TNP-470 has been evaluated in clinical trials (22).

There is increasing evidence implicating a role for MetAP-2, which is highly expressed in many germinal center–derived B-cell lymphomas, in tumor cell growth, survival, and metastasis (23). MetAP-2 is elevated in mesothelioma and may have a role in enhancing mesothelioma cell survival (24). Additionally, it was recently shown that MetAP-2 expression is elevated in colon cancer tissue, with highest MetAP-2 expression in metastatic cells (25). Although MetAP-2 is expressed in many normal human tissues, the highest level of expression is found in germinal center B cells as well as their neoplastic counterparts (23). Most follicular lymphoma and Burkitt's lymphoma express moderate to high levels of MetAP-2 and there is a statistically significant correlation between expression of Bcl-6 and MetAP-2 in DLBCL (23). Here, we show that a novel, orally active, fumagillin-class inhibitor of MetAP-2, PPI-2458, causes reversible depletion of germinal center B cells in animals, inhibits the growth of germinal center B-lymphoma cell lines in vitro, and potently inhibits the proliferation of SR NHL cells in culture and SR tumor growth in vivo in a severe combined immunodeficient mouse xenograft model (26, 27).


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Materials. PPI-2458 ([(1R)-1-Carbamoyl-2-methyl-propyl)]-carbamic acid-(3R, 4S, 5S, 6R)-5-methoxy-4-[(2R,3R)-2-methyl-3-(3-methyl-but-2-enyl)-oxiranyl]-1-oxa-spiro[2.5]oct-6-yl ester) and biotinylated PPI-2458 were synthesized at Praecis Pharmaceuticals Incorporated. The structure of PPI-2458 has been previously published (26).

Histopathology. Cynomolgus monkey studies were done at Sierra Biomedical (Sparks, NV). All studies were approved by the Sierra Biomedical Institutional Animal Care and Use Committee. Dose-range finding studies were done to identify doses that achieve maximal inhibition of MetAP-2 in tissue samples and that were well tolerated. These studies identified the cynomolgus monkey to be the most sensitive species requiring lower doses to achieve maximal effects compared with other species such as mouse. Forty-two experimentally naïve cynomolgus monkeys, 2 to 5 years of age, were administered vehicle (five males and five females) or PPI-2458 at 0.1 mg/kg (three males and three females), 0.3 mg/kg (three males and three females), 1.0 mg/kg (five males and five females), and 3.0 mg/kg (five males and five females) by nasogastric intubation every other day (QOD) for 13 days (total of seven treatments). Three animals per sex per group were sacrificed on day 15 (terminal sacrifice) and remaining animals were sacrificed on day 41 (recovery sacrifice), necropsied, and tissues were collected for immersion fixation in 10% neutral buffered formalin. Transverse sections of spleen were processed, paraffin-embedded, sectioned, and stained with H&E. Stained slides were examined microscopically by a board-certified veterinary pathologist and representative photomicrographs were acquired.

Cell cultures. D10, H2, and SU-DHL-16 B-cell lymphoma lines were a gift from Dr. Arnold Freedman (Dana-Farber Cancer Institute, Boston, MA). DB (CRL-2289), ST486 (CRL-1647), Ramos (CRL-1596), and SR (CRL-2262) cell lines were purchased from the American Type Culture Collection (Manassas, VA). All cells were maintained in RPMI (Life Technologies, Gaithersburg, MD) supplemented with 10% fetal bovine serum (Sigma, St. Louis, MO), 0.5 mg/mL gentamicin (Life Technologies), and 2 mmol/L glutamine (Mediatech, Herndon, VA) at 150,000 to 1,000,000 cells/mL in log-phase growth, unless otherwise indicated.

Proliferation assays. Cells in log phase growth were adjusted to 50,000 cells/mL, seeded in triplicate in T25 tissue culture flasks, and grown in the presence of 0.01 to 100 nmol/L concentrations of PPI-2458 prepared from a 10 mmol/L ethanol stock or vehicle (0.1% ethanol) for 5 to 6 days. Cell number was determined by hemocytometer and 50% growth inhibition (GI50) values were calculated from the averages of triplicate measurements using KaleidaGraph software.

MetAP-2 assay. Praecis Pharmaceuticals has developed an ELISA that measures the amount of MetAP-2 enzyme that is not covalently bound by PPI-2458 in cell culture samples. In this assay, 2.4 to 2,400 µg of cellular protein was incubated with a biotinylated analogue of PPI-2458. This analogue covalently binds to the catalytic site of MetAP-2 that has not already been derivatized by PPI-2458. After a 1-hour incubation period, the biotinylated MetAP-2–inhibitor complex was captured on a plate with immobilized streptavidin (Pierce, Rockford, IL). After 1 hour, the plates were washed and the immobilized biotinylated MetAP-2–inhibitor complex was then detected with an anti-MetAP-2 antibody (0.5 µg/mL). After 1-hour incubation, horseradish peroxidase–conjugated goat anti-rabbit IgG was added and incubated for 1 hour. After several washing steps, 100 µL TMB substrate [3,3',5,5'-tetramethylbenzidine and peroxidase solution (1:1), Kirkegaard and Perry Laboratories, Inc., Gaithersburg, MD] was added for 10 minutes. The reaction was stopped by adding 100 µL of 1 N H2SO4. Analysis was done by determining the absorption of each well at 450 nm using a Labsystems Multiskan plate spectrophotometer. Human recombinant MetAP-2 (Mediomics, St. Louis, MO), prebound to the biotinylated PPI-2458 analogue, was used to generate a standard curve for quantitation for each individual assay.

Western blots. To analyze protein expression in treated and untreated cells, whole cell lysates were prepared and 15 µg protein were subjected to SDS-PAGE and blotted to polyvinylidene difluoride membranes (Millipore, Billerica, MA). MetAP-2 polyclonal antibody CM33 (Zymed, San Francisco, CA) and Bcl-6 polyclonal antibody sc-858 (Santa Cruz Biotechnology, Santa Cruz, CA) were used for protein detection.

SR xenograft model. All animal studies were conducted according to a protocol approved by the Institutional Animal Care and Use Committee of Praecis Pharmaceuticals. SR lymphoma cells (5 x 106) were injected s.c. above the right hind leg of 60 female Fox Chase severe combined immunodeficient mice. Tumor growth was monitored twice to thrice a week using a CD-8'' CS digital caliper (Mitutoyo Corp., Aurora, IL) and tumor volume was calculated using the following equation: volume = (width x width) (length) / 2. On day 14 postimplantation, mice with tumor volumes between 50 and 200 mm3 were randomized into four groups (n = 8-9 animals per group). PPI-2458 was formulated at 1, 3, and 10 mg/mL in 11% hydroxypropyl cyclodextran and mice were dosed with vehicle (11% hydroxypropyl cyclodextran) or 10, 30, or 100 mg/kg PPI-2458 by oral gavage, QOD. Doses were chosen based on dose-range finding studies that identified doses that were well tolerated and achieved maximal inhibition of target MetAP-2 in tissues. Tumors were measured thrice a week until day 28 when the average tumor volume in the vehicle group reached 1,500 mm3 at which point all groups were euthanized. At necropsy, whole blood was collected into individual EDTA-coated Microtainer tubes (Becton Dickinson, Franklin Lakes, NJ) and later pooled by group. Whole blood samples were stored at 4°C. Tumors were removed and weighed and ~200 mg of each tumor was placed into an individual tissue cassette and snap frozen in liquid nitrogen. Tumor samples were stored at –70°C. Bone marrow was collected from both femurs using 0.9% saline to flush the marrow into individual microcentrifuge tubes. Bone marrow samples were stored at 4°C. Statistical analysis of tumor growth was done using Dunnett's multiple comparison test.

WBC lysate preparation. Four milliliters of the whole blood samples were transferred to sodium citrate vacutainer tubes (CPT, Becton Dickinson) and processed according to the instructions of the manufacturer. WBCs were isolated, combined with plasma, decanted into 15 mL conical tubes, and placed on ice. Cells were washed with cold PBS, resuspended in 600 µL modified radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH 7.4), 1% NP40, 0.25% deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 2 mmol/L Na3VO4, 1 mmol/L NaF, protease inhibitor complete (Roche, Nutley, NJ)], and lysed for 30 minutes at 2°C to 8°C. Lysates were clarified by centrifugation and supernatants were aliquoted and stored at –70°C until MetAP-2 analysis was done.

Bone marrow WBC preparation. Bone marrow samples were centrifuged and the supernatant was discarded. Pellets were incubated on ice in 1 mL erythrocyte lysis buffer (Qiagen, Valencia, CA) supplemented with protease inhibitors for 15 to 20 minutes. The samples were centrifuged and the supernatant was discarded. Once the pellets were resuspended in 200 µL modified radioimmunoprecipitation assay buffer, WBCs were lysed according to the protocol described above.

Tumor homogenization and lysis procedure. Tumor samples were weighed and homogenized in 5x (v/w) volume of PBS supplemented with protease inhibitors using disposable tissue grinders (Kendall, Mansfield, MA). PBS containing 10% NP40 was added, bringing the final protein concentration to 0.15 g/mL. The homogenized solutions were then incubated on ice for 30 minutes and vortexed intermittently. To collect the final lysate, the samples were centrifuged and the supernatant was collected, aliquoted, and stored at –70°C until MetAP-2 analysis was done.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
PPI-2458 reversibly depletes germinal center lymphocytes in nonhuman primates. The recent finding that MetAP-2 is highly expressed in germinal center B lymphocytes prompted an investigation of the effect of inhibiting MetAP-2 in germinal center B cells through treatment of animals with PPI-2458 (23). Lymph nodes and spleens from cynomolgus monkeys treated with vehicle or PPI-2458 for 13 days QOD with or without a 4-week recovery period were harvested, sectioned, and analyzed. In H&E-stained spleen sections from vehicle-treated animals (Fig. 1A ), a normal splenic architecture was observed, with multiple lymphoid follicles densely populated with germinal center B cells surrounded by resting mantle zone and marginal zone B lymphocytes. In contrast, lymphoid follicles from animals treated with 1 mg/kg PPI-2458 QOD exhibited a marked decrease in germinal center lymphocytes (Fig. 1B). In the 3 mg/kg QOD treatment group, there was a complete absence of germinal centers within lymphoid follicles with a collapse of the marginal zone into the center of the follicle (Fig. 1C). Importantly, germinal center depletion was reversible, as withdrawal from drug followed by a 3-week recovery period resulted in restoration of germinal centers (Fig. 1D) with histologic appearance of these sections similar to those from vehicle-treated animals (Fig. 1A). Similar observations were made in sections taken from mandibular, mesenteric, and inguinal lymph nodes (data not shown). Additionally, at lower doses (0.1 and 0.3 mg/kg) of PPI-2458, more modest decreases in germinal center lymphocytes were observed (data not shown). These findings suggest that PPI-2458 selectively and reversibly depletes proliferating B lymphocytes from lymphoid follicles, resulting in the loss of morphologically distinct germinal centers in lymphoid tissue.


Figure 1
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  		Fig. 1. Reversible depletion of germinal center lymphocytes in PPI-2458-treated animals. Transverse spleen sections from cynomolgus monkeys treated with vehicle or PPI-2458 for 13 days QOD were stained with H&E and photomicrographed at x10 magnification. A, lymphoid follicles from vehicle-treated animal with germinal centers (arrows) surrounded by a thin, darkly staining mantle zone (MZ) and broader marginal zone (MarZ). B, lymphoid follicles in 1 mg/kg PPI-2458–treated animal with lymphocyte depletion within the germinal center (arrows). C, lymphoid follicles from 3 mg/kg PPI-2458–treated animal with mantle zone collapse (arrows) and absence of germinal centers. D, restoration of lymphoid follicle and germinal center architecture in 3 mg/kg PPI-2458–treated animal following drug withdrawal and recovery for an additional 4 weeks. Histologic analysis was done on lymphoid organs from each animal in all groups. Representative data from spleen sections.

 
PPI-2458 inhibits proliferation of germinal center–derived B-lymphoma cell lines. To further examine the activity of PPI-2458 on germinal center B cells, we investigated the effect of PPI-2458 on the proliferation of SU-DHL-16, a DLBCL cell line derived from a transformed germinal center–derived follicular lymphoma (5). PPI-2458 inhibited SU-DHL-16 proliferation in a dose-dependent fashion with maximum inhibition of ~60% achieved at the highest concentration (100 nmol/L) and GI50 at 1.9 nmol/L (Fig. 2A ). No increase in cell death, as determined by trypan blue exclusion, was observed, suggesting a cytostatic activity for PPI-2458 growth inhibition (data not shown). Although proliferation was not completely inhibited even at the highest concentration of PPI-2458, the degree of inhibition was substantial at low-nanomolar concentrations of PPI-2458 provided as single doses at the start of the experiment. Further, growth inhibition was reversible as removal of drug from cultures resulted in resumption of exponential growth similar to that of untreated cells (data not shown); data are consistent with prior observations made with human fibroblast-like synoviocytes from RA patients (26).


Figure 2
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  		Fig. 2. PPI-2458 inhibits proliferation and MetAP-2 in SU-DHL-16 cells. SU-DHL-16 cells were cultured at 50,000 cells/mL and treated with PPI-2458 for 6 days. A, concentration-response curve of cell counts at day 6 demonstrating a PPI-2458 concentration-dependent decrease in proliferation with GI50 at 1.9 nmol/L, as determined mathematically from averaged data using KaleidaGraph software. Points, average; bars, SD. Multiple experiments were done. Representative experiment done in triplicate. B, representative Western blot for total MetAP-2 protein. C, MetAP-2 pharmacodynamic assay measuring uninhibited MetAP-2 in PPI-2458- or vehicle-treated lysates harvested on day 5.

 
Fumagillin class MetAP-2 inhibitors irreversibly inhibit MetAP-2 through covalent interactions in the active site of the enzyme and likely inhibit cellular proliferation by interfering with downstream events that are dependent on MetAP-2 enzymatic activity (16, 17). MetAP-2 protein levels in lysates from SU-DHL-16 cells treated with vehicle or PPI-2458 were analyzed by Western blot. Total MetAP-2 expression was relatively unchanged over the entire concentration range of PPI-2458, although a modest increase was noted at 10 and 100 nmol/L, suggesting induced or stabilized expression of MetAP-2 at high drug concentrations (Fig. 2B). In cells treated with ≤1 nmol/L PPI-2458, the ELISA-based MetAP-2 assay showed that uninhibited MetAP-2 was similar to that of vehicle-treated cells. However, virtually all MetAP-2 was occupied by PPI-2458 at concentrations ≥10 nmol/L, suggesting near-complete enzymatic inhibition within a PPI-2458 concentration range where cell growth was maximally inhibited.

A panel of additional DLBCL, follicular lymphoma, and Burkitt's lymphoma cell lines were assayed for sensitivity to PPI-2458. With the exception of the Burkitt's lymphoma cell line Ramos, PPI-2458 inhibited the growth of all cell lines tested by 42% to 60% (Table 1 ). Although 50% inhibition was not achieved in DB cells, maximum inhibition occurred at 10 nmol/L PPI-2458 (data not shown). All remaining PPI-2458-sensitive cell lines had low-nanomolar to subnanomolar GI50 values (Table 1).


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  		Table 1. Summary of PPI-2458 inhibitory activity on selected B-cell NHL cell lines

 
Expression of Bcl-6 and MetAP-2 was examined in NHL cell lines. SU-DHL-16, DB, H2, D10, ST486, and Ramos cell lines all expressed Bcl-6 protein, consistent with a germinal center origin for these cell lines (Fig. 3A ). Furthermore, expression of MetAP-2 protein was observed in all vehicle-treated cells and at modestly increased levels in 100 nmol/L PPI-2458–treated cells (Fig. 3B). To examine the extent of MetAP-2 occupation by PPI-2458, uninhibited MetAP-2 present in cell lysates was quantitated by the MetAP-2 ELISA. Uninhibited MetAP-2 was reduced to undetectable levels by 100 nmol/L PPI-2458 treatment in each of the six cell lines tested (Fig. 3C). PPI-2458 treatment had no effect on the level of Bcl-6 expression in any of the cell lines tested (data not shown). These data show that all cell lines in this study are likely derived from germinal center B cells and express similar levels of MetAP-2 that is nearly completely occupied at 100 nmol/L PPI-2458.


Figure 3
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  		Fig. 3. Bcl-6 and MetAP-2 expression and free MetAP-2 depletion in B-cell NHL cell lines. A, Western blot demonstrating expression of the germinal center B-cell marker Bcl-6 in all cell lines. Lysates from cells treated for 5 to 6 days with vehicle or 100 nmol/L PPI-2458 were analyzed by (B) Western blot for total MetAP-2 expression or (C) MetAP-2 assay. Uninhibited MetAP-2 in all cells treated with 100 nmol/L PPI-2458 was below the limit of detection in this assay. Experiments were done at least twice.

 
PPI-2458 inhibits SR cell proliferation in vitro and tumor growth in vivo in an SR xenograft model. The activity of PPI-2458 on SR, a human NHL cell line selected due to its expression of MetAP-2 and well-established utility as a human lymphoma xenograft model, was next examined. SR cells were highly sensitive to PPI-2458, with a GI50 of 0.5 nmol/L and maximum growth inhibition of ~80% (Fig. 4A ). Total MetAP-2 expression was examined by Western blot in vehicle- and PPI-2458-treated SR cells (Fig. 4B). At low concentrations (0.01-0.1 nmol/L) of PPI-2458, MetAP-2 expression was relatively unchanged when compared with vehicle-treated cells. However, at concentrations (1-100 nmol/L) that substantially inhibited growth, MetAP-2 expression increased in a dose-dependent fashion in response to PPI-2458, suggesting MetAP-2 enzyme induction or stabilization (Fig. 4B). Despite increased MetAP-2 expression, little or no uninhibited MetAP-2 remained at PPI-2458 concentrations ≥10 nmol/L (Fig. 4C). Thus, 50% of the total MetAP-2 was bound by PPI-2458 at a concentration between 0.1 and 1 nmol/L, which correlates directly with the GI50 of 0.5 nmol/L calculated from the SR growth curve (Fig. 4A).


Figure 4
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  		Fig. 4. PPI-2458 inhibits SR lymphoma cell proliferation and depletes uninhibited MetAP-2 at low nanomolar concentrations. SR cells were cultured at 50,000 cells/mL and treated with PPI-2458 for 6 days. A, PPI-2458 concentration-response curve with ~80% maximum inhibition of SR proliferation as determined by averaged cell counts and GI50 of 0.5 nmol/L determined mathematically from average values using KaleidaGraph software. Points, average; bars, SD. B, Western blot for total MetAP-2. C, MetAP-2 assay for uninhibited MetAP-2 in lysates from SR cells treated with vehicle or PPI-2458. Experiments were repeated at least thrice. Representative data from an experiment done in triplicate.

 
To establish PPI-2458 as an inhibitor of NHL tumor growth, we investigated the effect of PPI-2458 on growth of SR tumor xenografts in severe combined immunodeficient mice. Fourteen days following tumor implantation, PPI-2458 at 10, 30, or 100 mg/kg or vehicle was administered orally to mice QOD. Oral PPI-2458 administration significantly suppressed tumor growth in a dose-dependent manner (Fig. 5A ). Tumor growth inhibition was apparent in all PPI-2458 treatment groups as early as 2 days following the initial dose. Inhibition in the 10 and 30 mg/kg PPI-2458 treatment groups was similar over the course of the study and at day 28 postimplantation was inhibited by 41% and 35% (P < 0.05), respectively (Fig. 5A). PPI-2458 administered at 100 mg/kg produced the greatest degree of tumor growth inhibition, which was 57% (P < 0.001) at the end of the study. PPI-2458 was generally well tolerated, with no weight loss observed in any of the treatment groups (data not shown).


Figure 5
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  		Fig. 5. PPI-2458 inhibits SR lymphoma tumor growth and depletes free MetAP-2 in a mouse xenograft model. A, tumor measurements were made in SR tumor-bearing mice 3 d/wk beginning on day 14 postimplantation in animals treated orally QOD with vehicle (bullet), 10 mg/kg PPI-2458 ({blacksquare}), 30 mg/kg PPI-2458 ({blacktriangleup}), or 100 mg/kg PPI-2458 ({blacklozenge}). B, percentage of MetAP-2 inhibited in circulating (black columns) and bone marrow (white columns) WBCs, and in SR tumors (stippled columns) from PPI-2458-treated animals compared with vehicle-treated animals as determined by the MetAP-2 pharmacodynamic assay. Columns, mean of nine animals per group in the drug-treated group or eight animals per group in the vehicle-treated group; bars, SE. Statistical analysis of tumor growth was done using Dunnett's multiple comparison test. This experiment has been repeated with similar results.

 
To further correlate inhibition of tumor growth with MetAP-2 enzyme inhibition, the levels of uninhibited MetAP-2 were examined in WBC and SR tumor extracts taken at the conclusion of the study. PPI-2458 produced a near-complete depletion of uninhibited MetAP-2 in circulating WBC in all PPI-2458-treated animals relative to the vehicle group. In animals treated with 10 mg/kg PPI-2458, MetAP-2 was inhibited ~75% relative to the vehicle group levels in bone marrow WBCs and was further inhibited in the 30 and 100 mg/kg treatment in a dose-dependent manner. Active MetAP-2 was also potently and dose-dependently depleted in SR tumor cells from all PPI-2458-treated animals. In tumors from 100 mg/kg PPI-2458–treated animals, PPI-2458 inhibited MetAP-2 by >85% relative to tumors from vehicle-treated animals. These results show that inhibition of SR tumor growth in mice by PPI-2458 is accompanied by a substantial reduction in uninhibited MetAP-2, providing further support for the notion that MetAP-2 enzyme inhibition is responsible for the antitumor activity of PPI-2458.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
PPI-2458 and the closely related molecules fumagillin and TNP-470 (AGM-1470) inhibit proliferation of activated endothelial cells and specific tumor cell lines through the selective inhibition of MetAP-2, an enzyme critically involved in the processing of proteins (16–21, 26, 27). In cell systems, PPI-2458 was shown to deplete free, active MetAP-2 and inhibit endothelial cell proliferation in a coordinated, concentration-dependent manner, thereby suggesting a causal relationship between MetAP-2 enzyme inhibition and endothelial growth inhibition (26, 27). Furthermore, the dependence of PPI-2458 activity on the expression of MetAP-2 was confirmed as depletion of MetAP-2 by small interfering RNA rendered cells refractory to further growth inhibition by PPI-2458 (27). Germinal center B lymphocytes express high levels of MetAP-2 relative to other primary tissues, suggesting that MetAP-2 plays a prominent role in the cellular function of germinal center B lymphocytes (23). Here, we report that PPI-2458, a novel, selective, and orally active inhibitor of MetAP-2, depletes germinal center B cells in cynomolgus monkeys, inhibits germinal center–derived NHL line proliferation, and inhibits NHL tumor growth in xenograft models.

PPI-2458 caused a dose-dependent and reversible depletion of germinal center B cells in cynomolgus monkeys. Within lymphoid tissues, B-lymphocyte depletion was limited to the germinal center and was not observed in the surrounding areas (marginal or mantle zones) of resting B lymphocytes in the lymphoid follicle. Although fumagillin analogues have been shown to possess both positive and negative effects on T cell–dependent B-cell proliferation (28–30), our results show for the first time that an inhibitor of MetAP-2 can selectively deplete germinal center lymphocytes and suggest inhibition of B-lymphocyte proliferation as a likely mechanism.

The depletion of germinal centers in nonhuman primates and high-level MetAP-2 expression in many germinal center–derived tumors provided the rationale to examine the effect of PPI-2458 on the proliferation of B lymphocyte–derived NHL cell lines in vitro. The results described here show that PPI-2458 is a potent inhibitor of B-lymphoma cell line proliferation, with responses in most cell lines at subnanomolar concentrations and maximum inhibition ranging from 42% to 60%. Growth inhibition could not be explained by decreased expression of MetAP-2 in response to PPI-2458; rather, modest increases in expression were observed, suggesting induction of protein expression and/or stabilization of PPI-2458-inhibited MetAP-2 at higher drug concentrations. In addition, variable MetAP-2 protein expression between cell lines did not predict the degree of sensitivity to PPI-2458. To determine the functional significance of MetAP-2 inhibition in cell growth in the presence of PPI-2458, we developed a MetAP-2 pharmacodynamic assay that measures the amount of residual uninhibited cellular MetAP-2 following exposure to PPI-2458. Complete MetAP-2 enzyme inhibition was achieved with PPI-2458 in vitro in B-lymphoma cell lines within the dose range studied. Although proliferation was not completely inhibited at any concentration of PPI-2458, there was a direct concentration-dependent correlation of MetAP-2 inhibition and cell growth inhibition in sensitive lines. The cytostatic nature of the growth inhibition observed in response to treatment with PPI-2458 may suggest that therapeutic benefit in the clinical setting may be further enhanced when used in combination with cytotoxic chemotherapeutic agents.

All B-cell lymphoma cell lines in this study exhibited expression of Bcl-6, a transcriptional repressor expressed in germinal center B lymphocytes and germinal center–derived B-cell lymphomas. Bcl-6 is essential for germinal center formation and is down-regulated as B cells undergo terminal differentiation (31–34). Chromosomal lesions resulting in deregulated Bcl-6 expression are frequent in germinal center B lymphoma and it is likely that sustained Bcl-6 expression prevents terminal differentiation and promotes lymphomagenesis. However, PPI-2458 exposure did not decrease Bcl-6 expression. It is, therefore, unlikely that growth inhibition was a result of decreased Bcl-6 activity (data not shown). Additionally, loss of Bcl-6 function would be expected to cause terminal B-cell differentiation. This was not observed in PPI-2458 growth-inhibited B-lymphoma lines, as suggested by the finding that normal cell growth resumed following removal of PPI-2458 from cultures (data not shown). Moreover, the initial level of Bcl-6 expression did not correlate with the degree of PPI-2458 sensitivity in the cell lines studied. Therefore, these data do not support a role of MetAP-2 enzymatic activity in Bcl-6 expression or function.

Fumagillin and related MetAP-2 inhibitors arrest human umbilical vein endothelial cell growth in the G1 phase of the cell cycle (20, 21). However, we were unable to show a G1 arrest in any B-cell lymphoma lines by propidium iodide staining and flow cytometry (data not shown), suggesting different or only partially overlapping mechanisms (downstream of MetAP-2 inhibition) of growth inhibition in human umbilical vein endothelial cells and NHL lines. These findings suggest that the mechanism of growth inhibition is complex and likely involves multiple downstream MetAP-2 effector proteins. The critical downstream substrates of MetAP-2 in B lymphocytes and the mechanism(s) of growth inhibition of NHL cells by MetAP-2 inhibition with PPI-2458 exposure remain areas of intensive investigation.

It was of particular interest that the MetAP-2-positive Ramos Burkitt's lymphoma cell line was refractory to growth inhibition by PPI-2458. Resistance could not be explained by insufficient concentration of PPI-2458 or lack of MetAP-2 binding. In addition, MetAP-1 was expressed similarly in Ramos and ST486 Burkitt's lymphoma cells in the presence or absence of PPI-2458 (data not shown) and, therefore, it is unlikely that MetAP-1 was compensating for MetAP-2 inactivation, consistent with previously published findings (35). It is possible that proliferation of Ramos is less dependent than ST486 on yet unidentified MetAP-2 substrates or downstream effectors. Indeed, these cell lines respond differently to other stimuli, such as tumor necrosis factor-{alpha}, which inhibits IgM-induced apoptosis in Ramos but not ST486 cells (36). A comparison of the transcriptional and protein profiles of Ramos and ST486 with and without PPI-2458 treatment may help to elucidate critical components, and perhaps downstream effector proteins, in PPI-2458-mediated growth inhibition.

PPI-2458 is an orally available inhibitor of MetAP-2 and tumor growth in the SR human NHL xenograft model. SR tumor growth in mice was inhibited in a dose-dependent fashion with a maximum inhibition observed of 57% at 100 mg/kg. As these cells are sensitive to PPI-2458-mediated growth inhibition in vitro, inhibition of SR tumor growth in mice is likely due, at least in part, to the direct antitumor activity of PPI-2458. Previous studies have shown that in vivo effects of PPI-2458 on tumor growth are greater in cell lines that are growth inhibited in response to MetAP-2 inhibition in vitro compared with those that are not (37). Nonetheless, we cannot rule out the possibility that inhibition was due, in part, to antiangiogenic activity because PPI-2458 is a potent inhibitor of human umbilical vein endothelial cell proliferation in vitro and angiogenesis in vivo (26, 38). Tumor growth inhibition was accompanied by decreases in levels of active MetAP-2 by 70% to 85% in SR tumors, further demonstrating the oral bioavailability of PPI-2458 and supporting a correlation between inhibition of MetAP-2 enzyme and growth inhibition in vivo. In addition, PPI-2458 has been shown to inhibit MetAP-2 and proliferation of several nonhematologic tumor cell lines in vitro and inhibits growth of these tumor cell lines grown as implanted xenografts (37, 38).

Because the SR lymphoma model was run in immunocompromised mice, the effect of PPI-2458 on immunomediated effects on tumor growth cannot be established in this model. Preclinical studies have shown that PPI-2458 does not have a significant effect on circulating B-cell levels, suggesting that the effects of PPI-2458 to reduce germinal center B cells is a local lymphoid tissue effect and not a systemic effect (data not shown). The effect of PPI-2458 on immune surveillance of human cancers will need to be determined more conclusively in the clinic.

In conclusion, the data presented here show that PPI-2458 selectively depletes germinal center B lymphocytes, inhibits the growth of several germinal center–derived lymphoma cell lines in vitro, and inhibits tumor growth in a human NHL xenograft model. Therefore, PPI-2458, currently under investigation in phase I clinical trials, and other inhibitors of the molecular target MetAP-2 have the potential as novel therapies for the treatment of NHL.


   Acknowledgments
 
We thank Dr. Pat Lappin for microscopic examination of H&E slides and image acquisition.


   Footnotes
 
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 18 U.S.C. Section 1734 solely to indicate this fact.

Received 4/20/05; revised 11/29/05; accepted 12/21/05.


   References
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 Abstract
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 References
 

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