121 Differential competitive resistance to methylating versus chloroethylating agents among five O6-alkylguanine DNA alkyltransferases in human hematopoietic cells Aparecida Maria Fontes,1 Brian M. Davis,1 G160S, L162V), MGMT-3 (C150Y, A154G, Y158F, Lance P. Encell,2 Karen Lingas,1 L162P, K165R), and MGMT-5 (N157T, Y158H, A170S; 3 3 M Dimas Tadeu Covas, Marco Antonio Zago, ED50 for benzylguanine, >1,000 mol/L)] were mixed, Lawrence A. Loeb,4 Anthony E. Pegg,5 and the virus produced from Phoenix cells was transduced and Stanton L. Gerson1 into K562 cells. Stringent selection used high doses of O6-benzylguanine (800 Mmol/L) and temozolomide (1,000 1Division of Hematology/Oncology and Comprehensive Cancer Mmol/L) or BCNU (20 Mmol/L) administered twice, and Center, Case Western Reserve University and University Hospitals following regrowth, surviving clones were isolated, and 2 of Cleveland, Cleveland, Ohio; Promega Corporation, Madison, the MGMT transgene was sequenced. None of the Wisconsin; 3Regional Blood Center and Faculdade de Medicina de Ribeira˜o Preto da Universidad de Sa˜o Paulo, Centro de Terapia mutants was lost during selection. Using temozolomide, Celular, Ribeira˜o Preto, Brazil; 4The Joseph Gottstein Memorial the enrichment factor was greatest for P140K-MGMT Cancer Research Laboratory, Department of Pathology, University (1.7-fold). Using BCNU selection, the greatest enrichment of Washington School of Medicine, Seattle, Washington; and was observed with MGMT-2 (1.5-fold). G156A-MGMT, 5 Department of Cellular and Molecular Physiology, Penn State which is the least O6-benzylguanine–resistant MGMT Medical Center, Hershey, Pennsylvania gene of the mutants tested, was not lost during selection but was selected against. The optimal mutant MGMT Abstract useful as a drug resistance gene may depend on whether P140K-MGMT and G156A-MGMT genes encode two a methylating or chloroethylating agent is used for drug O6-benzylguanine–resistant O6-alkylguanine DNA alkyl- selection. [Mol Cancer Ther 2006;5(1):121–8] transferase proteins that confer a high degree of O6- benzylguanine and 1,3-bis(2-chloroethyl)-1-nitrosourea Introduction 6 (BCNU) or O -benzylguanine and temozolomide resistance 6 O -benzylguanine, an inhibitor of the DNA repair protein, to primary hematopoietic cells. In this study, we directly 6 6 O -alkylguanine DNA alkyltransferase (AGT) plus 1,3-bis compared these and three other O -benzylguanine– 6 (2-chloroethyl)-1-nitrosourea (BCNU) or O -benzylguanine resistant MGMT genes for their ability to protect the plus temozolomide have increased therapeutic efficacy over human erythroleukemia cell line, K562, using a direct thechemotherapeuticagentsaloneinanimaltumorxenograft competitive selection strategy to identify the mutation models (1–8). Thus, these combinations are currently being that conferred the greatest degree of protection from O6- evaluated in clinical trials with cutaneous lymphomas, benzylguanine and either BCNU or temozolomide. MFG malignant melanoma, brain neoplasms, multiple myeloma, retroviral vector plasmids for each of these mutants and gastrointestinal carcinomas (9–12). However, the dam- [G156A-MGMT (ED for O6-benzylguanine, 60 Mmol/L); 50 age caused to the bone marrow by these alkylating agents and P140K-MGMT, MGMT-2 (S152H, A154G, Y158H, manifests in the short term as myelosuppression or pancyto- penia, the dose-limiting toxicity in clinical trials, and has the potential to induce therapy-related myelodysplasia or acute leukemia (13, 14). Received 7/11/05; revised 10/7/05; accepted 10/28/05. Mutations in human MGMT have been developed, which Grant support: USPHS grants RO1CA73062, RO1ES06288, retain AGT activity but are O6-benzylguanine resistant. UO1CA75525, and P30CA43703. Based on the crystal structure of the AGT active site pocket, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked it is apparent that these mutants affect the size, hydropho- advertisement in accordance with 18 U.S.C. Section 1734 solely to bicity, and flexion of the active site pocket, resulting in indicate this fact. exclusion of the benzyl moiety from the active site or a Note: A.M. Fontes is currently at the Regional Blood Center of Ribeira˜o Preto, Ribeira˜o Preto, SP, 14051-140 Brazil. B. Davis is currently at reduction in its ability to undergo an alkyl transfer reaction Virexis, Gaithersburg, Maryland. L. Encell is currently at Tanox, Inc., with the active site cysteine. G156A-MGMT, located within Department of Molecular Biology, 10301 Stella Link, Houston, TX 77025. the DNA-binding wing flanking the alkyl-binding pocket Requests for reprints: Stanton L. Gerson, Division of Hematology 6 (15), is moderately O -benzylguanine resistant (EC50 =60 Oncology, BEB-3 Case Western Reserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4955. Phone: 216-368-1177; Fax: 216-368-1166. Amol/L). It distorts the adjacent loop (residues 158–160) E-mail: [email protected] that constitutes one wall of the benzyl-binding pocket Copyright C 2006 American Association for Cancer Research. (16, 17). The P140K-MGMT mutant alters the floor of doi:10.1158/1535-7163.MCT-05-0236 the alkyl-binding pocket, excluding the benzyl group and Mol Cancer Ther 2006;5(1). January 2006 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. 122 O6-Alkylguanine DNA Alkyltransferases resulting in a very high degree of O6-benzylguanine anti-MGMT (monoclonal antibody clone MT3.1) was resistance (EC50 > 1.2 mmol/L; refs. 18, 19). Molecular purchased from Kamiya Biomedical Co. (Seattle, WA), evolution with selection allowed the identification of mul- and R-phycoerythrin–conjugated antibodies were pur- tiple MGMT mutants highly resistant to O6-benzylguanine, chased from Caltag Laboratories (Burlingame, CA). The which retain the AGT ability to remove alkyl adducts from mouse isotype IgG1 was purchased from Becton Dickinson the O6-guanine position in DNA (20). Three mutants (i.e., (San Jose, CA). MGMT-2, MGMT-3, and MGMT-5), each containing three Retroviral Vectors to five amino acid substitutions in the region of the active Retroviral vector pMFG-G156A was constructed to site, were the most resistant to O6-benzylguanine and express cDNA sequence for G156A-MGMT as previously BCNU (EC50 > 2 mmol/L; ref. 20). described (15). Retroviral vector pMFG-P140K was con- These mutants, O 6-benzylguanine–resistant MGMT structed to express cDNA sequence for P140K-MGMT genes, provide a potent selection strategy for drug resistance by Davis et al. (22) and was originally selected for gene transfer. We have used this for hematopoietic stem dual resistance to N-methyl-NV-nitro-N-nitrosoguanidine cell selection because two chemotherapeutic agents, BCNU, and O6-benzylguanine from a library of AGT mutants and methylating agents, such as temozolomide, are stem cell containing a random sequence at positions 138 to 140 (18). toxins, resulting in cumulative stem cell loss and delayed Retroviral vectors pMFG-MGMT-2, pMFG-MGMT-3, and myelosuppression. We first introduced the use of O6- pMFG-MGMT-5, containing the following mutations of benzylguanine–resistant G156A-MGMT to selectively pro- the MGMT gene,aspreviouslydescribed,MGMT-2 tect the human CD34 cells and suggested that this selective (S152H, A154G, Y158H, G160S, L162V), MGMT-3 (C150Y, protection could occur while sensitizing the tumor to A154G, Y158F, L162P, K165R), and MGMT-5 (N157T, chemotherapy using O6-benzylguanine (21). Selecting the Y158H, A170S), were subcloned into MFG retroviral vector best O6-benzylguanine–resistant MGMT for gene therapy (20). These three MGMT mutant genes were originally is an important therapeutic decision. These mutants selected for dual resistance to N-methyl-NV-nitro-N-nitro- vary in stability, level of O6-benzylguanine resistance, soguanidine and O6-benzylguanine from a library of MGMT and repair capacity for either O6-methylguanine or O6- mutant genes containing a random sequence at positions 150 chloroethylguanine. The aim of the present study was to to 172 (19) and conferred the greatest degree of resistance to compare the ability of five of the most potent MGMT O6-benzylguanine and BCNU in K562 cells (20). mutants to protect human hematopoietic cells against CellTransfections and Transductions toxicity from O6-benzylguanine + BCNU or O6-benzylgua- DNA from each retroviral vector plasmid was isolated nine + temozolomide treatment using a direct competitive using NucleoBond Plasmid Max kit from Clontech selection strategy. (Palo Alto, CA) and then pooled by mixing 2 Ag of each construct. A total of 10 Agofretroviralplasmidwas transfected into 5 Â 106 Phoenix amphotropic packaging Materials and Methods cells (a gift from G. Nolon, Stanford University, Stanford, Chemicals CA), using a calcium phosphate coprecipitation protocol.4 BCNU and temozolomide were obtained from the To transduce K562 cells, these amphotropic producer cells Developmental Therapeutics Branch, National Cancer were grown to confluence, and the virus was harvested in Institute (Bethesda, MD). The BCNU was solubilized DMEM supplemented with 10% heat-inactivated fetal in 100% ethanol before dilution in PBS and used within bovine serum. To remove contaminating producer cells, 10 minutes of reconstitution. Temozolomide was solubi- supernatant was filtered through a 0.45-Am filter (Millipore, lized in DMSO (Sigma, St. Louis, MO) before dilution in Bedford, MA). Polybrene was added at 5.5