Combination Chemotherapy in Vitro Exploiting Glutamine Metabolism of Human Glioma and Medulloblastoma1

[CANCER RESEARCH 45, 4082-4086, September 1985]

Glenn Dranoff, Gertrude B. Elion, Henry S. Friedman,2 and Dareil D. Bigner3

Departments of Pathology (Neuropathology) [G. D., H. S. F., D. D. B.], Medicine [G. B. E.], Pediatrics [H. S. F.], and Pharmacology [G. B. E.], Duke University Medical Center, Durham, North Carolina 27710

ABSTRACT minai differentiation promote the emergence of permanent drug resistance (11). Considerable evidence indicates that primary The human glioma-derived cell line D-54 MG and the human central nervous system neoplasms develop extensive morpho medulloblastoma-derived cell line TE-671 have been shown to logical, karyotypic, biochemical, antigenic, and chemosensitive be sensitive in culture to the pharmacological interference with heterogeneity (2, 26, 34, 35). Therapeutic strategies targeted glutamine metabolism by acivicin, 6-diazo-5-oxo-u-norieucine, against this pleomorphism therefore should incorporate non- and methionine sulfoximine. Using as a guide the multiple con cross-resistant agents with complementary mechanisms of ac tributions of glutamine to the biosynthesis of proteins, purines, tion (3). Improved specificity may be accomplished by the bio and pyrimidines, we now have identified six additional antimetab- chemical potentiation of metabolically vulnerable sites and the olites active against these lines in vitro at clinically relevant selection of individually effective drugs which lack overlapping concentrations. The 50% growth-inhibitory levels of the drugs toxicities (5, 25). against D-54 MG in 6-day continuous exposure experiments We previously have demonstrated that primary central nervous were: L-asparaginase, 0.057 ID/ml; 5-fluorouracil, 0.5 fig/m\; 6- system tumors in culture are sensitive to the pharmacological mercaptopurine, 0.8 u.g/m\; actinomycin D, 0.0007 ng/m\; N- interference with the synthesis and utilization of glutamine (6). phosphonacetyl-L-aspartic acid, 2.3 nQÂ¡m\;and 5-azacytidine, We now have identified six additional antimetabolites active, at 0.2 fig/ml (3-day exposure). The corresponding 50% growth- clinically relevant concentrations, against the human glioma- inhibitory values in TE-671 were: L-asparaginase, 0.54 ID/ml; 5- derived cell line D-54 MG and the human medulloblastoma- fluorouracil, 1.5 ug/m\; 6-mercaptopurine, 4.7 /Â¿g/ml;actinomycin derived cell line TE-671 in vitro. The degree of potentiation D, 0.00044 jig/ml; W-phosphonacetyl-u-aspartic acid, 4.5 u.g/m\; achieved by several of these agents in biochemically designed and 5-azacytidine, 0.49 /Â¿g/ml.Dipyridamole up to 10 Â¿ig/mlwas two-drug combinations is presented. inactive against both lines. The isobologram method was used to evaluate the effectiveness of several two-drug combinations MATERIALS AND METHODS which were biochemically designed. The sums of the optimal fractional inhibitory concentrations for the pairs were: acivicin Tumor Lines. Cultured cell lines derived from a human glioma and plus L-asparaginase, 0.14; acivicin plus methionine sulfoximine, medulloblastoma were used. D-54 MG is the Duke University subline of 0.40; 6-diazo-5-oxo-L-norieucine plus methionine sulfoximine, A-172 established by G. Todaro (10) from a human glioblastoma multi- 0.60; acivicin plus 6-mercaptopurine, 1.0, all in TE-671; and forme. Its morphological, karyotypic, and selected biochemical charac acivicin plus 5-fluorouracil, 0.79, in D-54 MG. Our findings sug teristics have been defined (2). TE-671 is a line initiated by McAllister (21) from a human medulloblastoma and further characterized by Fried gest that an antimetabolite regimen exploiting glutamine sensitiv man ef al. (9). ity might improve the chemotherapy of some human gliomas and The cell lines were grown in monolayer culture in Eagle's glutamine- medulloblastomas. and glutamic acid-free minimal essential medium (Grand Island Biological Co., Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum, 10 mw A/-2-hydroxyethylpiperazine-/v"-2-ethanesulfonic acid INTRODUCTION buffer, and 4 mw glutamine in a humidified 5% CO2 atmosphere at 37Â°C. At confluence, cells were mechanically harvested with a Pasteur pipet The limitations of current chemotherapy in modifying the nat following digestion with 0.125% trypsin:0.02% EDTA. All cell lines have ural history of primary central nervous system neoplasms are been tested to ensure the absence of HeLa cell, inter-, or intra-cell line well recognized (31). The addition of carmustine (bischloroethyl- contamination, and Mycoplasma infection (2). nitrosourea) to whole brain irradiation only slightly prolongs the Chemicals. Acivicin, DON,4 PALA, and 5-azacytidine were provided median and long-term survival of postsurgical patients with ma generously by the Developmental Therapeutics Program, National Can cer Institute. 6-Mercaptopurine was a gift from the Burroughs Wellcome lignant gliomas (12). Adjuvant chemotherapy is not consistently Co., Research Triangle Park, NC. i-Asparaginase and actinomycin D beneficial for patients with medulloblastoma, and although sev were obtained from Merck, Sharp, and Dohme, West Point, PA. 5- eral compounds produce temporary regressions in recurrent Fluorouracil was purchased from Roche Laboratories, Nutley, NJ. L- disease, cures are rarely effected (36). Methionine sulfoximine and dipyridamole were bought from Sigma Chem Stem cell heterogeneity is a critical factor underlying chemo- ical Co., St. Louis, MO. therapeutic failure (27), since tumor lineage, mutation, and ter- Efficacy Studies. Two x 10s cells were plated in 35-mm3 wells containing 2 ml of glutamine- and glutamic acid-free minimal essential ' This work was supported by NIH Grants P01 NSCA 200023-01 and CA 11898 medium supplemented with 10% heat-inactivated fetal calf serum, 10 and by Duke Comprehensive Cancer Center Developments Funds (CA 14236). 2 Recipient of an Association tor Brain Tumor Research Fellowship in memory ITIMA/-2-hydroxyethylpiperazine-A/'-2-ethanesulfonic acid buffer, 0.5 mw of Bryce Davis and an American Cancer Society Junior Clinical Faculty Fellowship. 3To whom requests for reprints should be addressed. 4 The abbreviations used are: DON, 6-diazo-5-oxo-L-norleucine; PALA, W-phos- Received 10/12/84; revised 4/3/85; accepted 5/22/85. phonacetyl-L-aspartic acid; FIC, fractional inhibitory concentration.

CANCER RESEARCH VOL. 45 SEPTEMBER 1985 4082

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1985 American Association for Cancer Research. CHEMOTHERAPY OF HUMAN GLIOMA AND MEDULLOBLASTOMA glutamine, and varying concentrations of inhibitor. The fetal calf serum Table 1 was stored at 4Â°Cfor 2 wk prior to use and contributed less than 0.06 Concentrations of antimetabolites producing 50% growth inhibition in human HIM glutamine (determined with the Beckman Model 6300 high-perform medulloblastoma line TE-671 and human glioma line D-54 MG ance amino acid analyzer; Beckman Instruments, Inc., Berkeley, CA) to 50% growth-inhibitory /ml)* the total level in the experimental solutions. This value is not included in the reported concentration of this substrate. The asparagine content Drug"L-Asparaginase5-Fluorouracil6-MercaptopurineActinomycinMG0.0570.500.800.000700.202.30.0150.04 was less than 0.03 rriM, all derived from the fetal calf serum. The osmolality in the dishes ranged between 277 and 296 mOsmol (deter mined with an Advanced osmometer; Advanced Instruments, Newton Highlands, MA). Medium was replaced on alternate days. Viable cell D5-AzacytidinePALAAcivicinDONMethionme counts, as assessed by trypan blue exclusion, were determined on Day 6. One or two drops of 5% trypan blue were added to 1 or 2 ml of medium, and 20 to 200 cells were counted, depending upon growth. Each point was run in triplicate. Mean values are expressed. 6-Mercap- sulfoximineTE-6710.541.54.70.000440.494.50.0550.0253.6D-54 topurine was first dissolved in dimethyl sulfoxide. The final concentration " The 50% growth-inhibitory level was determined in a 6-day continuous expo sure experiment following plating 2 x 104 cells in 0.5 mu glutamine. of dimethyl sulfoxide was less than 0.003% (v/v) and had no effect on " Units for L-asparaginase are ID/ml. cell viability. For the 5-azacytidine experiments, medium was furnished on Day 0, and viable cell counts were measured on Day 3. Combination Studies. The isobologram method of Elion (7) was used â€¢ OlU/ml Asporog.nase to evaluate the effectiveness of two antimetabolites in combination. Â»00632lU/ml Asparagiaie Dose-response curves of each agent alone in 0.5 HIM glutamine were 100 â€¢020IU/ml Asporaginose defined in a 6-day continuous exposure experiment, as described above. 0.632 lU/ml Asparagmase 80 Three concentrations of the second inhibitor were run concurrently with three concentrations of the first inhibitor, generating three additional dose-response curves of the combinations. A percentage of control 60 growth (no drugs) allowing analysis of the greatest number of curves 40 simultaneously was chosen. The concentrations of drugs, alone and in combination, corresponding to this level of growth were determined. FICs were calculated by dividing the concentration of the drug in the 20 combination by the concentration of the drug alone required to produce the selected percentage of control growth. 000316 001 00316 010 00632 020 0632 20 ACIVICIN yu-a/ml ASPARAGINASE IlMnl Chart 2. Combination studies of acivicin and L-asparaginase in TE-671. Control RESULTS growth on Day 6 was 2.99 Â±0.10 x 10* viable cells.

Efficacy Studies. The effects of several antimetabolites op inactive. Increasing the concentration of actinomycin D to 0.01 the growth in culture of the human medulloblastoma-derived line Mg/ml resulted in the killing of virtually all 2 x 105 tumor cells TE-671 were investigated (Chart 1). The concentrations of the plated per dish. 5-Azacytidine at 10 Â¿ig/mlwas weakly cytocidal, various drugs producing 50% growth inhibition in 0.5 mw gluta achieving a cell kill of approximately 50%. mine, the level in cerebrospinal fluid (23), were determined (Table The 50% growth-inhibitory values for these compounds 1). The duration of drug exposure was 6 days, since previous against the human glioma-derived line D-54 MG were defined in experiments established that, by this time, there was a dose- similar experiments (Table 1). D-54 MG was more sensitive than response relationship between glutamine concentration and TE-671 to all of the active inhibitors except actinomycin D. growth of TE-671 in vitro (6). The short half-life of 5-azacytidine Dipyridamole had no effect up to 10 *ig/ml. Actinomycin D at in solution, 65 h at 25Â°C(22), however, required abbreviating its 0.01 i/g/ml destroyed almost all the glioma cells. 5-Fluorouracil exposure to only 3 days. Dipyridamole up to 10 ^g/ml was and 5-azacytidine at 3.16 Â¿/g/mlandL-asparaginase at 0.632 IU/ ml were slightly cytocidal, producing 25 to 50% kill. 100 Combination Studies. Since the antimetabolites were se lected within a strategy to exploit glutamine sensitivity, it was of interest to examine the degree of potentiation achieved by coupling these agents with a glutamine antagonist. Dose-re sponse curves in TE-671 of acivicin and L-asparaginase, individ ually and in combination, were established (Chart 2). The con 6-MP centrations of the drugs at which growth was inhibited to 33% of control were identified, and fractional inhibitory concentrations were derived (Table 2). This required extrapolating the dose- response curve of acivicin from 36 to 33% of control growth. The FICs for each pair of inhibitors were plotted, and intersecting lines were drawn (Chart 3). Since the resulting curve was left of 00001 10.0 the line connecting unity on both axes, the combination of acivicin and L-asparaginase was synergistic. The FICs for the most Chart 1. Influence of antimetabolites on the growth of TE-671. Control growth on Day 6 ranged from 1.46 to 4.93 x 10* viable cells. 5-FU, 5-fluorouracil; 6-MP. effective balance of the drugs, indicated by the point of intersec 6-mercaptopurine. tion, were 0.03 for acivicin and 0.11 for L-asparaginase. In terms

CANCER RESEARCH VOL. 45 SEPTEMBER 1985 4083 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1985 American Association for Cancer Research. CHEMOTHERAPY OF HUMAN GLIOMA AND MEDULLOBLASTOMA of concentrations, the equivalent values were 0.0038 ^g/ml and tions of methionine sulfoximine, as its optimal FIG with acivicin 0.066 lU/ml, respectively. The sum of the optimal FICs, a quan was only 0.03 in comparison to 0.20 with DON. Mildly synergistic titative measure of drug potentiation with a total of 1 denoting was the combination of acivicin and 5-fluorouracil, their optimal addition and lower values synergy, was 0.14. FICs totalling 0.79. The balance between the two compounds The isobologram method was applied in evaluating the effec was skewed, with the respective FICs being 0.65 and 0.14. tiveness of several other antimetabolite combinations (Table 3). Acivicin and 6-mercaptopurine together were strictly additive. The percentages of control growth selected for analysis ranged from 40 to 56, depending upon the shapes of the dose-response DISCUSSION curves. The glutamine antagonist DON and glutamine syntnetase inhibitor methionine sulfoximine, whose individual dose-response The susceptibility of a tumor cell to the pharmacological inter curves previously have been reported (6), were moderately syn- ference with glutamine metabolism furnishes considerable insight ergistic in TE-671, the sum of their optimal FICs being 0.60. into targeting chemotherapy more effectively. The several func Acivicin and methionine sulfoximine in the medulloblastoma were tions of glutamine in protein, purine, and pyrimidine biosynthesis more synergistic at 0.40. The difference in potentiation between suggest that these critical pathways for cellular growth may be the two pairs of drugs was due largely to the relative contribu- exploitable with additional antimetabolites. We previously estab lished that the human glioma-derived line D-54 MG and the Table 2 human medulloblastoma-derived line TE-671 are sensitive to Concentrationsof combinationsoÃacivicinand L-asparaginaseproducing 33% of control growth in human medulloblastomaline TE-671 glutamine antagonists in culture (6). We therefore used this conceptual framework as a basis for identifying the activity of L- asparaginase, 5-fluorouracil, 6-mercaptopurine, actinomycin D, inhibitory inhibitory 5-azacytidine, and PALA. Comparison of the 50% growth-inhib ng/rrit0.125 concentration61 concentration0 itory values of the compounds in a physiological level of gluta 0.018 0.144 0.10 mine (Table 1) with their achievable plasma concentrations in 0.00316 0.025 0.20 0.33 patients (Table 4) indicates that the experiments potentially are 0Acivicin*Fractional 0L-AsparaginaselU/ml00.06320.60Fractional 1 a Control growth was 2.99 Â±0.10 x 10eviable cells on Day 6 after plating 2 x clinically relevant. 105cells in 0.5 mw glutamine on Day 0. An advantage of selecting drugs through a biochemical strat " Calculated by dividing the concentration of the inhibitor in the combination by egy is the possible potentiation accomplished by using the the concentration of the inhibitor alone required to produce 33% of control growth. inhibitors in combination. The synergism we observed between the glutamine synthetase inhibitor methionine sulfoximine and the glutamine antagonists DON and acivicin illustrates the prin ciple of sequential blockade. The very low concentration of methionine sulfoximine required with acivicin is particularly en couraging in view of the former's dose-dependent central nerv

< ous system toxicity (19). The coupling of acivicin and L-asparaginase exemplifies the 1 concept of complementary inhibition. Since some tumor cells become resistant to L-asparaginase through increased asparagine synthetase activity, combination therapy with a glutamine antagonist may help circumvent treatment failure (13, 14, 24). Antitumor effects have also been improved by administering L- asparaginase concurrently with actinomycin D or 6-mercapto purine (16). The sensitivity of D-54 MG and TE-671 in culture to L-asparaginase is intriguing in the context of the drug's reported

Chart 3. Isobologram of acivicin and L-asparaginasein TE-671 as determined activity against meningea! leukemia and notable central nervous at 33% of control growth. system toxicity (4).

Tables antimetabolitesCombination8DON Effectivenessof combinations of of optimal fractional fractional inhib inhibitory con itory concen concen of control centrations60.40, trations00.60 trations(M9/ml)c0.0084, growth"55

+ methionine sulfoximine 0.20 0.72 Acivicin + methionine sulfoximine 0.37, 0.03 0.40 0.013,1.026 40 Acivicin + 5-fluorouracN 0.65, 0.14 0.79 0.016, 0.074 47 Acivicin + 6-mercaptopurine 0.50, 0.50 1.00 0.009, 2.25 56 Acivicin + L-asparaginaseOptimal 0.03,0.11Sum 0.14Optimal 0.0038, 0.066% 33 " Drugs were tested in a 6-day continuous exposure experiment after plating 2x10Â° cells in 0.5 mw glutamine. All combinations were run in TE-671 except for acivicin plus 5-fluorouracilwhich was evaluated in D-54 MG. 6 Calculated by dividing the concentration of the inhibitor in the combination by the concentration of the inhibitor alone required to produce a selected percentageof control growth. c Units for L-asparaginaseare lU/ml. " This value varied depending on the shapes of the dose-responsecurves.

CANCER RESEARCH VOL. 45 SEPTEMBER 1985 4084

Table 4 Ruoslahti, E., Herschman,H., Eng, L. F., and Wikstrand, C. J. Heterogeneity Achievable plasma concentrations in patients ol antimetabolites active against of genotypic and phenotypic characteristics of fifteen permanent cell lines human glioma line D-54 MG and human medulloblastoma line TE-671 derivedfrom humangliomas.J. Neuropathol.Exp. Neural.,40:201 -229,1981. 3. Bigner, D. D., Pedersen, H. B., Bigner. S. H., and McComb, R. A proposed level basis for the therapeutic resistance of gliomas. Semin. Neural., 1: 169-179, DrugL-Asparaginase infusion)1000IU/kga(continuous Â«ml)8-20* 1981. (4) 4. Capizzi, R. L, Berlino, J. R., and Handschumacher, R. E. L-Asparaginase. 5-Fluorouracil(8) 1100 mg/m2for 4 days 0.20-0.49Â° Annu. Rev. Med., 27: 433-444,1970. 6-Mercaptopurine(37) 50mg/m2/hfor 12-24 h 0.66-1.20" 5. DeVita, V. T., and Schein, P. S. The use of drugs in combination for the 10 Mg/kg'or 5 days" 0.001e Actinomycin D (29) treatment of cancer. N. Engl. J. Med., 288: 998-1006, 1973. 5-Azacytidine(15) 75 mg/m2for 6 days 1.33' 6. Dranoff, G., Elion, G. B., Friedman, H. S., Campbell, G. L., and Bigner, D. D. 5.05 PALA(1)Dosage 940 mg/m2for 5 days9Plasma Influenceof glutamine on the growth of human glioma and medulloblastoma in culture. Cancer Res., 45: 4077-4081, 1985. " i.v. bolus. 6 Twenty-four h postinjection; units are Ill/ml. 7. Elion, G. B., Singer, S., and Hitchings, G. H. Antagonists of nucleic acid derivatives. VIII. Synergism in combinations of biochemicallyrelated antime c Ninety-six h after initiating infusion. tabolites. J. Biol. Chem., 208: 477-488, 1954. " Steady state. 8. Fraile, R. J., Baker, L. H., Buroker, T. R., Horwitz, J., and Vaitkevicius, V. K. 8 Ninety-six h after last injection. Level in lymphocytes at this time was 0.01 Â¡Â¡gl Pharmacokineticsof 5-fluorouracil administered orally, by rapid infusion and ml. by slow infusion. Cancer Res., 40: 2223-2228,1980. ' Twelve h into infusion. 9. Friedman, H. S., Bigner, S. H., McComb, R. D., Schold, S. C., Pasternak, J. 9 In combination with 5-day continuous infusion 5-fluorouracil (180 to 325 mg/ F., Groothuis, D. R., and Bigner, D. D. A model for human medulloblastomaâ€” m2/day). growth, morphology, and chromosomal analysis in vitro and in athymic mice. J. Neuropathol.Exp. Neural., 42. 485-503,1983. The susceptibility of our human anaplastic glioma and medul- 10. Giard, D. J., Aaronson,S. A., Todaro, G. J., Amstein, P., Kersey,J. H., Dosile, H., and Parks, W. P. In vitro cultivation of human tumors: establishmentof cell loblastoma-derived lines to a number of pyrimidine antagonists lines derived from a series of solid tumors. J. Nati. Cancer Inst., 57: 1417- provides important opportunities to assess the capabilities of 1423,1973. 11. Goldie,J. H., and Coldman,A. J. Quantitative model for multiple levelsof drug combination therapy. The interaction of acivicin and 5-fluorouracil resistancein clinicaltumors. Cancer Treat. Rep., 67: 923-931,1983. is synergistic, with the optimal FIC of 5-fluorouracil equal to 0.14. 12. Green, S. B., Byar, D. P., Walker, M. D., Pistenmaa, D. A., Alexander, E., A Phase I study of azotomycin, a glutamine antagonist, and 5- Batzdorf, V., Brooks, W. H., Hunt, W. E., Meatey, J., Odom, G. L., Paoletti, P., Ransohoff, J., Robertson, J. T., Selker, R. G., Shapiro, W. R., Smith, K. fluorouracil against colon carcinoma yielded encouraging results R., Wilson, C. B., and Strike, T. A. Comparisonsof carmustine, procarbazine, (33). The growth inhibition induced by PALA in D-54 MG and TE- and high-dose methylprednisoloneas additions to surgery and radiotherapy 671 is in accordance with the drug's prior demonstrated activity for the treatment of malignantglioma.Cancer Treat. Rep., 67: 121-132,1983. 13. Haskell, C. M., and Caneltos, G. P. Asparagine biosynthesis in human KB against the glioma 26 model (17). The interaction of PALA and tumor cells: inhibitor studies with asparagine and glutamine antagonists. acivicin is synergistic in vitro as well (20) and capable of coun Cancer Res., 30: 1081-1083,1970. 14. Holcenberg,J. S. Enhanced effect of an i-glutamine antagonist, L-(as-5s)-a- teracting in vivo the resistance to PALA in a variant of the Lewis amino-3-chloro-4,5-dihydro-5-isoxazoleaceticacid,by Acinetobacter L-glutam- lung carcinoma (18). inase-L-asparaginase.CancerTreat. Rep., 63:1109-1114,1979. Other agents that have important relationships to glutamine 15. Israili,Z. H., Vogler, W. R., Mingioli, E. S., Pirkle, J. L., Smithwick, R. W., and antagonists include 6-mercaptopurine and actinomycin D. Our Goldstein,J. H. The disposition and pharmacokineticsin humans of 5-azacytidine administeredintravenouslyas a bolus or by continuous infusion. 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Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 1985 American Association for Cancer Research. Combination Chemotherapy in Vitro Exploiting Glutamine Metabolism of Human Glioma and Medulloblastoma

Glenn Dranoff, Gertrude B. Elion, Henry S. Friedman, et al.

Cancer Res 1985;45:4082-4086.

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