Targeted Modulation of MGMT: Clinical Implications

Although the exact physiologic function of O⁶-methylguanine DNA methyltransferase (MGMT) remains to be fully defined, MGMT is known to specifically remove the methyl/alkyl group from the O⁶-position of guanine and restore the guanine to its normal form without causing DNA strand breaks (Fig. 1). MGMT-mediated repair is unique compared with other DNA repair pathways: (a) it acts alone without relying on any other proteins or cofactors; (b) it transfers the alkyl group to an internal cysteine residue in the protein, acting as both a transferase and an acceptor of the alkyl-group; (c) it inactivates itself after receiving the alkyl-group from guanine, and thus, it is a suicidal protein; (d) it repairs in a stoichiometric fashion. Because one molecule of MGMT removes one alkyl molecule, an excess of DNA adducts at the O⁶-position could completely deplete MGMT. These properties make MGMT an important drug resistance factor and an ideal target for biochemical modulation of drug resistance (1, 2). A direct relationship between MGMT activity and resistance to alkylating therapeutic agents has been well documented (3, 4).

MGMT expression protects from alkylating agent cytotoxicities. The clinical effectiveness of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and related methylating compounds procarbazine, streptozotocin, or temozolomide is attributed, in part, to the potentially cytotoxic DNA lesions O⁶-chloroethylguanine, formed by BCNU, and O⁶-methylguanine (O⁶-meG) formed by the methylating agents. It has been known for several decades that O⁶-chloroethylguanine lesions induce cell death by forming G-C interstrand cross-links that are able to inhibit DNA replication and transcription, eventually leading to DNA double-strand breaks and apoptosis. O⁶-meG is a promutagenic and carcinogenic DNA lesion that becomes cytotoxic after persistent O⁶-meG adducts are recognized by the DNA mismatch repair (MMR) pathway (5). The mispairing of O⁶-meG with thymine during DNA replication initiates futile cycles of DNA MMR to generate DNA single- and double-strand breaks that activate apoptotic cell death (refs. 6, 7; Fig. 1B).

MGMT expression in tumors. MGMT is ubiquitously expressed in normal human tissues (8) but is overexpressed in all types of human tumors, including colon cancer, glioma, lung cancer, breast cancer, leukemia, lymphomas, and myeloma. Although tumor cells have many mechanisms of resistance to alkylating agents, the expression of MGMT in tumors has a protective effect against cell death induced by alkylating agents, showing a strong correlation between MGMT activity and tumor drug resistance. MGMT expressing tumor cells are 4- to 10-fold more resistant to BCNU, temozolomide, and related compounds (9, 10).

Silencing of MGMT. Tumor tissues have long been noted to be heterogenous with respect to MGMT expression with a broad range of expression between tumors, a lack of MGMT in patches of tumors, or a deficiency in MGMT in entire tumors. It is possible that epigenetic inactivation of the MGMT gene contributes to this variation. Several studies have reported transcriptional silencing of this gene in up to 30% of certain primary human tumors because of hypermethylation of the CpG island in its promoter region (11–14). Loss of MGMT has been shown to be associated with, and to precede the appearance of, G to A point mutations in the K-ras gene during lung, brain, and colorectal tumorigenesis. MGMT silencing is also often observed in tumors in which a number of other genes are silenced by methylation. The silenced gene lists are not consistent between tumors or tumor types (15, 16). Thus, the epigenetic alteration of MGMT behaves like a carcinogenic marker. MGMT silencing is observed in normal tissues, in premalignant lesions or in peritumor tissue, suggesting that it could be an early abnormality in premalignant processes (13, 17).

Recently, several clinical studies have shown that glioma patients with MGMT promoter methylation responded better to treatment with radiotherapy and either BCNU or temozolomide, and had improved survival compared with chemotherapy alone (18, 19). These observations suggest that the absence of MGMT expression is a positive survival predictive marker in patients with glioma. It would be appealing to consider a prospective clinical trial in which MGMT expression variables were used for personalized drug selection, electing alkylating agents for those tumors with low or absent MGMT expression, and other forms of treatment or inhibition of MGMT (vide infra) for those with MGMT expression. Tumor MGMT promoter methylation is being evaluated as a predictive marker. Because of the difficulty in obtaining these samples, however, measurement of circulating tumor or blood cell MGMT promoter methylation might be an effective surrogate for the status of tumor MGMT.

Depletion of MGMT. MGMT is an ideal target for modulation of its activity because it is a suicidal protein. O⁶-Benzylguanine (O⁶-BG) was developed based on its restricted mechanism of action (20). O⁶-BG reacts with MGMT by covalent transfer of the benzyl group to the active site-cysteine and causes an irreversible inactivation of the enzyme (Fig. 1A). At therapeutic levels, O⁶-BG is not toxic alone, but efficiently renders the tumor cells 2- to 14-fold more sensitive to alkylating agents in the in vitro and in vivo settings. This establishes the potential therapeutic effect of O⁶-BG as an enhancer of these drugs. Recent clinical trials established the maximal tolerated dose of O⁶-BG to achieve biochemical inhibition of MGMT to be 120 mg/m², and the maximal doses of BCNU and temozolomide were found to be 40 and 470 mg/m², respectively, given with this dose of O⁶-BG (21, 22). Phase 2 trials are currently under way. In addition to O⁶-BG, another alternative inactivator has been described [O(6)-(4-bromothenyl)guanine, PaTrin-2], which shows potential advantages over O⁶-BG in terms of higher reactivity against wild-type MGMT and oral formulation. A PaTrin-2-temozolomide combination is under clinical development in England (23, 24).

Transfer of MGMT gene into bone marrow cells. Alkylating agents represent effective antitumor drugs. Unfortunately, these compounds are highly toxic to both malignant and normal cells. Bone marrow cells are especially sensitive to damage by alkylating agents because of reduced MGMT activity compared with tumor tissues. Thus, toxicity to the bone marrow is dose-limiting in cancer patients. MGMT has emerged as a lead target gene for the protection of bone marrow cells against the cytotoxic effects of many chemotherapeutic alkylating agents. Most gene transfer studies have evaluated two mutant forms of MGMT (G156A and P140K) that are resistant to O⁶-BG-induced inactivation up to 60-fold to >500-fold, respectively (25, 26). Retroviral and lentiviral gene transfer of mutant MGMT into hematopoietic stem cells followed by transplantation has shown a very high degree of stem cell selection and reconstitution under both ablative and myeloablative conditions in mouse and dog models. Importantly, gene transfer of mutant MGMT into hematopoietic stem cells confers significant levels of drug resistance to the combination of O⁶-BG and either temozolomide or BCNU. By this approach, tumor-bearing mice tolerated dose escalation of O⁶-BG and BCNU with considerable moderation of myelosuppression, resulting in improved tumor response (27). Furthermore, because MGMT overexpression protects from leukemogenesis, it is possible that this approach would lessen the incidence of secondary leukemias after alkylating agent therapy (28). Clinical studies with gene transfer of mutant MGMT in CD34 cells collected from patients are ongoing to determine the gene transfer modeling and clinical applications (29). Potential applications include stem cell selection and protection in patients undergoing dose escalation of temozolomide for gliomas or other tumors and, at lower doses, as a selection strategy for genetically altered stem cells in other nonmalignant diseases.

DNA repair through MGMT and MMR pathways influences cell sensitivity to methylating agents. The relationship between MGMT activity and methylating agent resistance is complex, because O⁶-meG lesions not repaired by MGMT are processed by MMR (Fig. 1B). O⁶-meG preferentially pairs with thymine during DNA replicative synthesis and this mispair is bound by the mut S complex of MMR and the newly synthesized strand is excised by the mut L heterodimer of MMR. Unfortunately, the opposing strand O⁶-meG is then paired with thymine again, reinitiating a futile repair process. In the process, extended single-strand patches are present, overlapping double-strand breaks could occur, triggering apoptotic cell death as well as G to A point mutations. Thus, O⁶-meG is a potent mutagenic and cytotoxic DNA adduct.

MMR-deficient tumors are very resistant to methylating agents. Furthermore, inactivation of MGMT by O⁶-BG fails to potentiate temozolomide-toxicity in MMR-deficient tumor cells. These results indicate that even large numbers of O⁶-meG DNA adducts are not cytotoxic in the absence of the MMR recognition, although they may be very mutagenic (30, 31). In addition, MGMT mutants acquired in MMR-deficient cell lines selected for resistance to O⁶-BG and BCNU are resistant to inactivation and sensitization of alkylating agents by O⁶-BG, indicating that MGMT mutations might emerge in human tumors after drug exposure leading to drug resistance (32, 33). These studies also indicate that the function of MMR is to prevent mutations and to sensitize genetically unstable cells to certain agents. When MMR is inactivated, however, cells exhibit a high mutation rate, and the relationship of drug resistance to methylating agents with MGMT becomes even more complicated. Thus, MMR-proficient tumors are resistant to methylating agents on the basis of MGMT activity, whereas MMR-deficient tumors are drug-resistant regardless of MGMT activity (34).

Inactivation of MGMT coupled with interruption of base excision repair enhances therapeutic efficacy of alkylating agents. Therapeutic methylating therapeutic agents, such as temozolomide, from O⁶-meG (∼6% of adducts formed), as well as 7-methylguanine (N⁷-meG; 70% of adducts formed), and 3-methyladenine (N³-mA; 9% of adducts formed). The latter adducts, N⁷-mG and N³-mA, are removed by the methylpurine glycosylase followed by AP endonuclease, the first two enzymes in the base excision repair (BER) pathway (Fig. 1C). Efficient BER repair minimizes the effect of these lesions in normal and tumor cells. Thus, these abundant N-methylated DNA adducts become highly cytotoxic only when BER is disrupted (35, 36). Most importantly, BER disruption is able to bypass other drug-resistant factors such as MMR defects and high MGMT activity.

Several previous studies have shown considerable efforts toward improving the therapeutic efficacy of methylating agents (37–40) by blocking BER using methoxyamine, which binds the aldehyde group at the AP site and prevents AP endonuclease from clipping the backbone and thereby blocking the BER pathway. When combined with temozolomide or BCNU, methoxyamine potentiated therapeutic efficacy in colon cancer and malignant glioma xenografts. Importantly, no additive systemic toxicity was noted in mice after receiving treatment with temozolomide or BCNU and methoxyamine. Combinations of O⁶-BG, methoxyamine, and a lower dose temozolomide or BCNU (1/3 of effective doses of agent used alone) showed significant antitumor effects without toxic death. Thus, modulation of MGMT, but not BER, requires a MMR-proficient background. The simultaneous targeting of MGMT and BER is a promising strategy to improve the therapeutic efficacy of alkylating agents in both MMR-deficient and MMR-proficient tumors.

It has recently become clear that MGMT may have a more complex function in the cell. The protein is phosphorylated, ubiquitinated, and may function in transcription regulation, cell cycle control, and apoptotic signaling. Whether MGMT inhibition will enhance drug efficacy, be of predictive value in patient chemotherapy selection, and become a target for other anticancer agents remain fruitful areas of discovery.