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Clinical Relevance of MGMT in the Treatment of Cancer

From the Division of Hematology/Oncology and Comprehensive Cancer Center, University Hospitals of Cleveland, Case Western Reserve University School of Medicine, and The University Hospitals Research Institute, Cleveland, OH.

Address reprint requests to Stanton L. Gerson, MD, Division of Hematology/Oncology, Comprehensive Cancer Center, Case Western Reserve University, 10900 Euclid Ave, BRB 3 West, Cleveland, OH 44106-4937; email: slg5{at}po.cwru.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION DNA ALKYLATION BY... REFERENCES

 
ABSTRACT: A number of DNA-damaging chemotherapeutic agents attack the O⁶ position on guanine, forming the most potent cytotoxic DNA adducts known. The DNA repair enzyme O⁶-alkylguanine DNA alkyltransferase (AGT), encoded by the gene MGMT, repairs alkylation at this site and is responsible for protecting both tumor and normal cells from these agents. Cells and tissues vary greatly in AGT expression, not only between tissues but also between individuals. AGT activity correlates inversely with sensitivity to agents that form O⁶-alkylguanine DNA adducts, such as carmustine (BCNU), temozolomide, streptozotocin, and dacarbazine. The one exception is those tumors lacking mismatch repair, which renders them resistant to methylating agents. A recent study in patients with gliomas confirmed the correlation between low-level expression of the MGMT gene and response and survival after BCNU. An inhibitor to AGT, O⁶-benzylguanine (BG), depletes AGT in human tumors without associated toxicity and is now in phase II clinical trials. Finally, mutations within the active site region of the MGMT gene render the AGT protein resistant to BG inactivation. As a result, mutant MGMT gene transfer into hematopoietic stem cells has been shown to selectively protect the marrow from the combination of an alkylating agent and BG, while at the same time sensitizing tumor cells. MGMT remains a paradigm for development of new agents that modulate known mechanisms of drug resistance in cancer cells and raise the spectra of combinatorial therapies that encompass known drug resistance mechanisms.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION DNA ALKYLATION BY... REFERENCES

 
A NUMBER OF chemotherapeutic agents attack DNA at the O⁶ position of guanine and mediate their cytotoxicity at this site. O⁶ alkylguanine DNA adducts are repaired by O⁶ alkylguanine DNA alkyltransferase (AGT) encoded by the gene MGMT. This protein acts as a "suicide" acceptor protein for the alkyl group, restoring DNA to normal but inactivating itself in the process. As such, it is a unique DNA repair protein. Tumor expression of AGT varies and correlates with therapeutic response to chemotherapy. An inhibitor, O⁶-benzylguanine (BG), is used in early phase II clinical studies to assess the ability of AGT inhibition to overcome chemotherapy resistance in a variety of tumors. Finally, MGMT gene transfer to selectively protect hematopoietic stem cells from the cytotoxic effects of chemotherapeutic alkylating agents has been evaluated extensively in preclinical models and is in early clinical trials. In this review, I will relate our understanding of the biochemistry of DNA alkylation and its repair by AGT to our understanding of the distribution of AGT activity in human tumors and recent advances in clinical trials with the AGT inhibitor BG.

	DNA ALKYLATION BY CHEMOTHERAPEUTIC AGENTS AND CELL DEATH

TOP ABSTRACT INTRODUCTION DNA ALKYLATION BY... REFERENCES

 
Chemotherapeutic agents that attack the O⁶ position of guanine fall into two major classes. Chloroethylating agents such as carmustine (BCNU) and lomustine (CCNU) attack initially at the O-6 position of guanine in an SN2 reaction followed by formation of a cyclic intermediate with attack at the N1 position of guanine giving rise to N¹O⁶ethanoguanine. Over the ensuing 10 to 18 hours, this unstable structure then rearranges from the oxygen to form a crosslink with the opposite strand cytosine yielding an interstrand crosslink.¹ The G-C crosslink induced by BCNU is poorly repaired and forces a halt to the DNA replication, formation of single- and double-strand breaks, and induction of p53 and p21, which leads to both caspase-3–mediated apoptotic cell death and necrotic cell death. However, cell death is not dependent on p53,² and even tumors with p53 mutations are sensitive to nitrosoureas. Repair of the crosslink requires lesion bypass but may also involve base excision repair and nucleotide excision repair (Table 1). Cell death caused by chloroethylating agents is quite potent and requires fewer then 10 lesions per cell to be toxic.³ Because the crosslink can linger in cells, cytotoxicity can be delayed for hours, days, or many weeks after treatment and seems to require DNA synthesis.

Because apoptosis is signaled during mismatch repair–mediated processing of O⁶-mG lesions, not surprisingly, cells and tumors with mismatch repair deficiency are remarkably resistant to methylating agents.^9-11 Mismatch repair–deficient cells with low AGT may be 10- to 50-fold more resistant to chemotherapeutic methylating agents than isogenic cells with intact mismatch repair.¹¹ Furthermore, not only are preexisting tumors and cancer cells resistant to methylating agents, as well as to 6-thioguanine and cisplatin, but these cells may preferentially survive and be selected for during chemotherapy.¹²

Mismatch repair–defective cells also have a higher mutation frequency and are more likely to develop other mechanisms of drug resistance by mutating key DNA repair genes. This has significant application in the clinic, where mismatch repair deficiency is observed in approximately 20% of colon cancers^13,14 and is associated with hereditary nonpolyposis colon cancer as well as with endometrial cancer, gastric carcinoma, and a small proportion of lymphoma and leukemias.^15,16 Although interruption of base excision repair is a potential mechanism for overcoming this resistance to methylating agents, clinical therapies have yet to be developed.¹¹

Methylating agents and chloroethylnitrosoureas are active agents in a number of malignancies. For instance, BCNU remains the mainstay for glioma therapy, and high-dose BCNU is used in a number of different preparative regiments for refractory non-Hodgkin’s lymphoma.¹⁷ Likewise, procarbazine and chloroethylnitrosoureas have been used for many years in first-line and relapse regimens for Hodgkin’s disease, and dacarbazine and temozolomide induce responses in melanoma.¹⁸ Temozolomide was approved for use in anaplastic astrocytomas and is used clinically in gliomas. BCNU is also active in myeloma although its use has been superseded by other active agents.

AGT
Definitive repair of lesions at the O⁶ position of guanine occurs almost entirely through the action of the AGT protein. This protein has been conserved through evolution, described in all species from bacteria to humans with a remarkably conserved active site (for review, see Pegg et al¹⁹). The exact physiologic function of AGT remains obscure: overexpression in mice is nontoxic²⁰ and mice carrying a disruption of the MGMT gene seem normal, although they are much more sensitive to both methylating and chloroethylating agents.²¹ Most of what we know about AGT function comes through methylating agent exposure in the setting of either acute toxicity or carcinogenesis or through the use of chemotherapeutic agents. Although two forms of the protein are present in bacteria, only one has survived into mammalian species and humans, and there does not seem to be a backup mechanism or any redundancy in the DNA repair process of O⁶-alkylguanine lesions.¹⁹ As noted, AGT specifically serves as the alkyl acceptor protein for O⁶-alkylguanine lesions and does not repair other sites. This reaction is efficient, and, in fact, one way to assess the level of DNA adduction at O⁶-guanine is to quantitatively measure depletion of endogenous or exogenous AGT protein through evidence of the alkyl-transfer reaction. As a suicide protein, it is unique among DNA repair proteins in two ways: first, it is the only protein that inactivates itself through the process of repair; second, it is the only DNA repair protein that acts alone, without relying on a sequential pathway of repair. To initiate repair, it binds DNA and scans individual bases for evidence of O⁶ alkylation.²² The alkyl group is taken up into the hydrophobic active site, where it hydrogen-bonds with side-chain residues adjacent to the acceptor cysteine at position 145 (Fig 1).²³ The alkylated protein then falls from the DNA and is ubiquinated and degraded much more rapidly than the active protein.²⁴ The degradation rate seems to vary between tumors and normal cells; for instance, it is much lower in normal lymphocytes than in most malignant cells.²⁵

View larger version (164K): [in this window] [in a new window]   Fig 1. Mechanism of action of AGT inhibition by O6-benzylguanine (BG). BG penetrates the active site pocket of AGT where it comes in contact with the sulfur of cysteine 145. A covalent transfer reaction inactivates the protein. Modified from Dolan ME, Pegg AE: O6-Benzylguanine and its role in chemotherapy. Clin Cancer Res 3:837-847, 1997.

TUMOR EXPRESSION OF AGT AND ITS ROLE IN DRUG RESISTANCE
All types of human tumors express AGT, although expression levels vary between tumor isolates and some tumors may not express the protein at all. There also seems to be heterogeneity within individual tumors in AGT expression. In studies evaluating AGT expression in human tumors, high levels of expression have been noted in colon cancer,^28,29 melanoma,^30,31 pancreatic carcinoma,³² lung cancer,³³ and gliomas^34-36 (Table 2). In many instances, the AGT activity in these tumors is higher than in surrounding normal tissue. For instance, in immunohistochemical analysis of colon tumors and adjacent mucosa, statistically higher levels of AGT were found in tumors than in mucosa.²⁸ In this study, tumor grade was not correlated with AGT activity. In gliomas, a wide range of AGT activity was noted, with higher activity in higher-grade tumors and statistically higher activity than in adjacent brain tissue collected at surgery.³⁶ In a number of instances, the normal adjacent brain tissue did not express AGT, particularly in pediatric glioma patients.³⁶ In our own studies of myeloma, CD38⁺ plasma cells had higher AGT than normal CD34⁺ hematopoietic progenitors (unpublished data). The AGT levels in pancreatic tumors is also much higher than in normal pancreas.³² Numerous studies have evaluated AGT activity in primary tumors, as well as in established human tumor cell lines, and in large part, the predictions of the range of AGT activity and its impact on drug resistance have proven true when comparing tumor cell lines with primary tumors.^28-36

The most accurate assessment of AGT alkyl transfer activity relies on a measurement of the loss of a methyl group from substrate DNA measured by high-performance liquid chromatography (HPLC).^42,43 Alternative methods have included competitive assay using a sample with a known amount of AGT activity of either bacterial or human origin,⁴⁴ or the use of an oligonucleotide substrate containing an O⁶-mG that has altered restriction enzyme digestion characteristics when the alkyl group is removed.⁴⁵ These assays can give precise measurement of AGT activity down to the femtimolar range, which allows analysis of very small cell and tissue samples containing as few as 5 x 10⁴ cells. An alternative method for qualitative assessment of AGT activity is immunohistochemistry. The two most commonly used antibodies for this are MT 3.1 and MT 23.2.^28,46 The latter is better for immunohistochemistry analysis, whereas the former is better for Western blot and fluorescence-activated cell sorting analysis.⁴⁷ Immunohistochemistry assessment of heterogeneity within tumors has shown a marked range of AGT expression across the tumor, often with regions of intense staining and regions adjacent to cells lacking activity.^28,35 In other tumors, diffuse low-level staining can be observed. Immunohistochemistry also allows assessment of stromal, vascular, and infiltrative cells. To the extent that low-level AGT activity in tumor vasculature may contribute to overall tumor response, it is worthwhile to assess these regions as well as the primary tumor.

In addition to affecting the cellular response and resistance to nitrosoureas and methylating agents, AGT also seems to increase resistance to cyclophosphamide,⁴⁸ perhaps because acrolein, a metabolite of cyclophosphamide, becomes a DNA adduct recognized by AGT. This recent observation is being evaluated preclinically and may lead to additional clinical trials targeting AGT inhibition during therapy with cyclophosphamide.

MGMT PROMOTER METHYLATION AND LACK OF AGT ACTIVITY
The expression of many genes is regulated by methylation of their promoter, and MGMT is no exception. However, MGMT is a house-keeping gene and is expressed in all tissues, so for the most part, promoter methylation is limited to tumor cells. The methylation takes place on the cytosine of CpG islands and is mediated by 5'-methylcytosine methyltransferase, quite distinct from the O⁶ methylation that occurs after methylating agent exposure, and is repaired by AGT (Fig 2). Studies in human tumor cell lines identified that methylation of the MGMT promoter is associated with absence of AGT activity.⁴⁹ Furthermore, treatment of such cells with a demethylating agent, 5-azacytosine, which demethylates the promoter region, reactivates transcription and leads to restoration of AGT activity.⁵⁰ Human tumors were evaluated recently for evidence of promoter methylation. AGT promoter methylation occurs in up to one third of primary human tumors, often in conjunction with methylation of many other genes that promote tumorigenesis, and is associated with both regional and total loss of AGT protein within the tumor.⁵¹ Such promoter methylation was not observed in normal tissues. Although promoter methylation is associated with tumor progression and may be a hallmark of carcinogenesis, the specific genes that are shut off vary greatly among tumors. As yet, there is no clear correlation between genes with promoter methylation and specific tumors, stage, progression status, or genetic defect.

View larger version (118K): [in this window] [in a new window]   Fig 2. 5'Methylcytosine methylation of the MGMT promoter shuts off expression of the gene. A region of the promoter of MGMT is preferentially methylated, resulting in loss of MGMT expression both in cell lines and human primary tumors but not in normal tissues.

CORRELATION BETWEEN MGMT EXPRESSION AND RESPONSE TO NITROSOUREAS AND METHYLATING AGENTS
A number of preclinical studies describe a strong correlation between AGT activity and resistance to chloroethylating agents and methylating agents.^61-66 Interventional studies showed that AGT depletion, using either methylating agents or O⁶-mG, sensitized colon cancer, melanoma, and rhabdomyosarcoma tumor cell lines^61-66 to nitrosoureas. In xenografts, tumors with high AGT are typically resistant to BCNU and other chloroethylating agents⁶⁵ and methylating agents such as temozolomide (Fig 3).^67,68 However, in vivo treatment with AGT inhibitors sensitized these tumors to nitrosoureas to a considerable extent. In many instances, tumors completely resistant to these agents could be induced to respond after AGT depletion. From these studies it can be generally concluded that AGT plays a very important role mediating tumor drug resistance to chloroethylating agents and, in the context of intact mismatch repair, to methylating agents. Although numerous other drug resistance mechanisms undoubtedly exist, including mutated p53 or p21, increased bcl-2, and other antiapoptotic mediators, AGT seems to play a central role in many tumors and thus is an appropriate target for therapeutic inhibition. Therefore, it is not surprising that some glioma cell lines lacking AGT were found to be BCNU-resistant,⁶⁹ although the exact mechanism remains to be elucidated.

View larger version (88K): [in this window] [in a new window]   Fig 3. O6 alkylation by temozolomide and carmustine (BCNU). The methylating agent temozolomide forms O6-methylguanine DNA adducts that induce cell death by invoking mismatch repair. The chloroethylating agent BCNU initially forms O6-chloroethylguanine DNA adducts that then rearrange to a 1,6-ethanoguanine cyclic intermediate followed by a crosslink with the cytosine directly on the opposite strand.

DRUG SENSITIZATION STUDIES
Given that there is a strong statistical correlation between AGT activity and drug resistance, a number of therapeutic approaches have been initiated to modulate AGT and increase drug responses in the clinical setting. Initial efforts combined in a sequential fashion a methylating agent to deplete AGT with BCNU. A number of clinical trials were performed with streptozotocin as the methylating agent followed by BCNU. In one trial significant depletion of AGT occurred in both tumor and peripheral-blood lymphocytes following streptozotocin at doses of 2 g/m².^72,73 Toxicity from BCNU was observed at lower-than-expected doses, suggesting synergy had been achieved as a result of targeted AGT depletion. These studies were superseded by the discovery of O⁶ benzylguanine as a potent inhibitor of AGT.

O⁶ BENZYLGUANINE: A POTENT INHIBITOR OF AGT ACTIVITY
In 1991, Pegg, Moschel, and Dolan observed that O⁶ benzylguanine (BG) inhibited AGT and potentiated the cytotoxicity of both chloroethylating agents and methylating agents.^74-76 In a series of important observations, they fully characterized the interaction between BG and AGT and its therapeutic impact. They showed that BG binds AGT, transferring the benzyl moiety to the active-site cysteine.⁷⁷ The reaction is very rapid and more potent than any other previously known AGT inhibitor. BG is not incorporated into DNA in living cells and reacts directly with both cytoplasmic and nuclear AGT. The median effective dose of BG toward human AGT is approximately 0.2 µmol/L, whereas for mouse AGT, it is approximately 10-fold higher.⁷⁸ Bacterial AGT is much more resistant to BG inactivation because of amino acid differences near the active site.⁷⁹ BG concentrations of 1 to 5 µmol/L completely inhibit AGT in tumor cell culture and potentiate BCNU cytotoxicity three- to five-fold and temozolomide cytotoxicity up to 10-fold.^40,52,53 In vivo, BG inhibits human tumor xenograft AGT in the mouse when given at concentrations of 10 to 30 mg/kg.^45,80 BG readily crosses the blood-brain barrier, reaching concentrations that equal that of serum after a single dose.⁸¹ Human tumor xenograft AGT is depleted within 30 minutes, and depletion is maintained for 6 to 8 hours, after which endogenous regeneration of AGT occurs through synthesis of new protein.^45,80 Repeated dosing of BG in the mouse can maintain depletion of AGT activity in tumors and will potentiate the tumor growth delay of a single dose of BCNU or a methylating agent such as temozolomide. In the former instance, repeated BG dosing prevents minimal regeneration of AGT. This is important because even small amounts of newly synthesized AGT can remove the precrosslink adduct, whereas with methylating agents, repeated dosing ensures maximal persistence of O⁶-mG adducts. Preclinical toxicology indicated a therapeutic window of approximately 30-fold between doses that induced toxicity in animals and those which depleted normal tissue AGT.⁸² Because no toxicity to BG has been observed clinically, we will not further explore the preclinical toxicology of BG alone.

These studies established a number of points that have driven clinical trial development. First, therapeutic doses of BG seemed to be nontoxic. Second, BG was a specific inhibitor of AGT, and depletion, rather than toxicity, could be used as an effective end point in the clinical development of the combination. Third, complete depletion was required, because residual AGT would remove the DNA adducts formed and reduce the efficacy of the nitrosourea. Fourth, the preclinical studies predicted that the maximum-tolerated doses of nitrosoureas with BG inhibition of AGT would be approximately 25% of those used without BG.^80,83 Furthermore, the duration of AGT depletion would depend on both dose schedule and the type of compound. Because it takes approximately 12 to 16 hours for the O⁶-chloroethylguanine adduct to crosslink the DNA, then after a single dose of BCNU,⁶⁵ AGT depletion for 16 to 18 hours was predicted to be optimal. On the other hand, with the daily administration of a methylating agents, prolonged depletion of AGT would be optimal because regeneration of AGT activity would allow the O⁶-mG DNA adducts to be removed, thus limiting their toxicity.

In addition to BG, another compound, O⁶-(4-bromothenyl)guanine, has been developed in England for clinical use.⁸⁴ Like BG, this compound results in a covalent transfer reaction on incubation with AGT, causing its irreversible inactivation. It has similar efficacy in preclinical cytotoxicity experiments and in xenograft models. Favorable pharmacokinetics and animal toxicology have enabled its introduction into phase I clinical trials in England. There have been no reports, as of yet, of its therapeutic outcome, but it will likely also be an alternative to BG in the clinical setting as a potentiator of nitrosourea toxicity.

CLINICAL TRIALS WITH BG TO POTENTIATE NITROSOUREA ANTITUMOR EFFICACY
Initial clinical trials with BG began in 1995, a remarkably short time after its discovery and initial preclinical development. The first trial to begin, at University Hospitals of Cleveland (UHC) and Case Western Reserve University (CWRU) School of Medicine, was designed to dose escalate BG to the end point of complete depletion of tumor AGT activity.⁴² Sequential biopsies were performed to compare the effect of BG on tumor AGT. In the first cohort of patients, pretreatment and 2-hour posttreatment biopsy specimens were obtained to determine that tumor AGT depletion had taken place. In all subsequent groups, pretreatment and 18-hour posttreatment biopsy specimens were analyzed to bracket the pharmacodynamic end point of maintenance of depletion over the 12- to 18-hour period required for completion of the BCNU crosslink, as determined in vitro.²⁷ The starting dose of BG was 10 mg/m² with a fixed dose of BCNU at 13 mg/m², each over 11 hours. Persistent tumor AGT activity was observed 18 hours after BG up to doses of 80 mg/m², and depletion was observed in all patients receiving 120 mg/m². This dose was well tolerated when given alone or in combination with BCNU 13 mg/m².⁴² In this trial, patients tolerated sequential tumor biopsies using a 14-gauge cutting needle. Most patients underwent two computed tomography–guided biopsies of liver metastases.⁸⁵ In all cases the tumor biopsy was sectioned to provide histologic confirmation of tumor adjacent to sections analyzed for biochemical AGT activity. As expected, a wide range in tumor AGT was observed in baseline samples more than eight-fold, but complete depletion was observed at the 120-mg/m² dose. One of 24 patients experienced a minor complication to the computed tomography–guided biopsy with a small hematoma not requiring transfusion. All remaining patients tolerated the procedure quite well. No correlation was found between baseline AGT in tumor and peripheral-blood mononuclear cells, and the latter was not predictive of the dose required for complete depletion of AGT in tumor. On the basis of the tumor biopsy data, the National Cancer Institute’s Cancer Treatment Evaluation Program (CTEP) proposed dose escalation of BCNU followed by phase II development of BG at doses of 120 mg/m².

The next phase of the UHC/CWRU study was to determine the maximum-tolerated dose of the combination. While maintaining the BG constant at 120 mg/m², dose escalation of BCNU to 40 mg/m² intravenously resulted in grade 4 myelosuppression with thrombocytopenia and neutropenia 4 to 6 weeks after treatment. Otherwise, treatment was well tolerated with no evidence of pulmonary or renal toxicity, which is observed with high doses of BCNU alone. One patient continues on treatment on this phase I trial with stable disease for more than 4 years, suggesting that cumulative BG exposure is not associated with toxicity.

A second phase I study at the University of Chicago analyzed toxicity of BG plus BCNU and performed AGT depletion studies in peripheral-blood mononuclear cells.⁸⁶ In this study a staggered dose escalation was performed of both BG and BCNU. Depletion of blood-cell AGT was observed at BG doses of 40 and 80 mg/m² and a maximum-tolerated dose for the combination of BG and BCNU was reached at 40 mg/m²; the dose-limiting toxicity was reached at BCNU 50 mg/m².

Pharmacokinetic studies in these two trials show a remarkably short half-life of the compound BG, with initial half-life of 25 to 30 minutes and rapid conversion to 8-oxobenzylguanine suggesting that BG may act as a prodrug. 8-Oxobenzylguanine has a terminal half-life of 4 hours and a dose-dependent maximum concentration. At BG 120 mg/m², the maximum concentration of BG is approximately 1.5 µg/mL, and for 8-oxobenzylguanine, it is 5 µg/mL. Thus, it seems that the initial depletion of tumor AGT is due to the immediate action of BG, but that sustained depletion of AGT for 18 to 24 hours is due to 8-oxo BG.^42,86 Metabolism of BG by the 1A2 cytochrome P450,⁸⁷ variably expressed in humans, may account for the different metabolic degradation rates of BG and 8-oxobenzylguanine.

A third phase I trial in brain tumors was performed at Duke University Medical Center.⁸⁸ In this study, patients had escalating doses of BG administered over 1 hour, followed approximately 18 hours later by surgical resection of the brain malignancy and assay for AGT. Although no baseline studies were performed, uniform depletion of AGT was observed at BG 100 mg/m², suggesting that BG readily crosses the blood-brain barrier in patients with malignancy and that dose escalation above that required for systemic AGT depletion would not be required.

Two additional phase I studies are ongoing at UHC/CWRU; one is evaluating the pharmacokinetics of continuous-infusion BG over 24 hours, with or without a 1-hour bolus; and the other, in patients with T-cell lymphomas, administers systemic BG at 120 mg/m² over 1 hour, followed by cutaneous administration of BCNU to areas of affected plaque-like disease. An initial report suggests that these patients tolerate the combination quite well with very good objective responses observed and no chronic sequela or systemic toxicity (G. Woods and K. Cooper et al, personal communication, July 2001). In all instances, the cutaneous lymphoma AGT activity was completely depleted by the BG infusion (L. Liu, personal communication). Another phase I trial underway evaluates systemic BG with Gliadel wafer placement intraoperatively for patients undergoing primary resection of brain tumors. In these and other studies, repeated administration of BG and/or the use of continuous infusion to maintain tissue depletion of AGT will be important in the assessment of synergy and therapeutic benefit. A number of phase I trials are also planned using temozolomide and BG (Table 3). These trials will address the question of prolonged AGT depletion by either repeated or continuous dosing with BG.

NEW DIRECTIONS IN MODULATING RESISTANCE TO NITROSOUREAS
The most important aspect of the development of AGT as a target and BG as its inhibitor is that AGT depletion can safely be achieved clinically in human tumors. Nonetheless, it is unlikely that AGT depletion will result in increased responses to nitrosoureas in the majority of tumors both because of the increased toxicity of the combination and because of multiple mechanisms of drug resistance. As noted in the next section, the myelosuppression of BG combinations may be prevented by protection of the marrow. The next question will be whether AGT depletion can enhance the role of other agents and whether it can be used to build regimens that target a variety of drug resistance mechanisms. For instance, agents that target other forms of DNA repair (including topoisomerase I and II inhibitors),⁸⁹ strand break repair, and base excision repair¹¹ are all promising. Combination therapy of BG, a nitrosourea, and agents that increase apoptosis after DNA damage without exacerbating marrow suppression, such as bcl-2 antisense oligonucleotides, proteosome inhibitors, and cdk inhibitors, is a new direction that has yet to be developed in either preclinical or clinical settings.

DRUG RESISTANCE GENE THERAPY USING BG-RESISTANT MUTANT MGMT
Because myelosuppression is dose limiting with BCNU and methylating agents, especially in combination with BG, there has been interest in evaluating the use of MGMT gene transduction into hematopoietic progenitor cells as a means of selective drug protection. The use of a series of mutant MGMTs, with altered amino acids in the active site rendering them resistant to BG, has resulted in preclinical in vitro studies of hematopoietic progenitor protection from the combination of BG and BCNU or temozolomide.^90,91 The two MGMT mutants most commonly used, P140K and G156A, originally described by Pegg et al,^79,92 have retained repair activity and drug selection capacity in hematopoietic cells.⁹³ A new series of mutants, identified by random mutagenesis and screening for resistance in hematopoietic cells, is even more BG- and BCNU-resistant and may have more favorable reaction kinetics with the lesions in DNA than the previously described mutants.⁹⁴

Animal studies show that hematopoietic cells transduced with G156A or P140K MGMT have increased tolerance to BG and BCNU^59,95,96 or temozolomide.⁹⁷ In mice transplanted without myeloablation, selection in favor of the transduced cells up to 1,000-fold was observed,⁹⁶ suggesting that selection at the stem-cell level rather than simply at the progenitor level is taking place and that myeloablation would not be required for successful clinical applications of stem-cell selection. Expression and selection of the transduced hematopoietic cells in secondary transplant recipients has been observed, confirming that the repopulating stem cells of the mouse are transduced, express the mutant MGMT, and can be selected for by BCNU and BG treatment.⁹⁵ Furthermore, human tumor xenograft-bearing mouse recipients of mutant MGMT-transduced marrow before treatment tolerated more cycles of BG and BCNU and had less myelosuppression than mice infused with lacZ-transduced marrow.⁹⁸ A number of clinical trials are now proposed to exploit this strategy with the aim of determining the extent to which human hematopoietic progenitors can (1) be transduced with a retrovirus containing a mutant MGMT, (2) be safely infused back into the patient, (3) be selected for by BG and BCNU treatment, resulting in an increased proportion of transduced cells recovered in the marrow, and (4) result in less myelosuppression. This drug-resistance approach might also be used to select for stem cells corrected for a genetic disorder utilizing a vector expressing MGMT and an additional therapeutic gene.

Thus, the MGMT gene product AGT is an important determinant of clinical drug resistance to methylating and chloroethylating agents. A potent AGT inhibitor, BG is now in phase II testing to determine whether this compound will improve patient response rates to this class of alkylating agents. Using a mutant MGMT, a series of gene transfer studies is underway to determine whether it is possible to select for human hematopoietic progenitor cells during drug treatment with BG and BCNU. This mechanism-based approach has led to exciting new therapeutic strategies that may improve the utility of alkylating agents.

	ACKNOWLEDGMENTS

 
Supported in part by National Public Health Service grant nos. RO1CA63193, RO1CA73062, RO1CA86357, and UO1CA75525.

The author thanks Drs L. Liu, A. Pegg, E. Dolan, H. Friedman, R. Moschel, J. Willson, C. Hoppel, T. Spiro, L. Erickson, C. Schold, D. Kokkinakus, and D. Yarosh for years of collaboration under the auspices of the National Cooperative Drug Discovery Group grant no. NCI UO1CA75525, without which much of the work reviewed here would not have been completed.

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