methylguanine-DNA Methyltransferase Activity Research Articles

Abstract 2035: MGMT inhibition leads to CDK4/6 inhibition and enhances palbociclib and abemaciclib activity in breast cancer

Abstract Background: MGMT (O6 methylguanine DNA methyltransferase), a DNA repair protein leading to chemotherapy resistance, increasingly studied for its cell cycle regulatory functions, also known to control ER expression and function, is overexpressed in a majority of cancers, including breast cancer. MGMT inhibition has been reported to restore ER function and sensitivity to hormonal therapy in tamoxifen resistant breast cancer. CDK4/6 is a cell cycle regulator targeted by a new class of drugs in the treatment of breast cancer in patients who had progressed during prior endocrine therapy. We investigated a potential correlative role between MGMT and CDK4/6 expression/activity. In this therapeutic context MGMT inhibition would have the dual role of increasing/restoring effect of endocrine therapy and facilitate activity of CDK4/6 inhibitors (Palbociclib and Abemaciclib). Methods: We have tested the effect of Antabuse (disulfiram, DSF), as an MGMT inhibitor, at nontoxic doses, on the expression of CDK4/6, or in combination with Palbociclib (PB) or Abemaciclib (LY2835219 - LY) on ER+ breast cancer cells. Results: DSF at very low doses (achievable in human serum with standard DSF clinical dosing) decreases ER+ breast cancer cell growth (MCF7, T47D and ZR75) in a dose-dependent manner. DSF further sensitizes breast cancer cells to PB or/and LY and significantly inhibits breast cancer growth without causing unwanted side effects on the normal breast epithelial cells. Dose effect and isobologram studies confirm synergistic activity of DSF + LY and moderate synergism for DSF + PB. DSF, alone or in combination with PB (DSF ± PB) and/or LY (DSF ± LY), significantly inhibits expression of MGMT, CDK4/6, ERα and aldehyde dehydrogenase activity - all involved in breast cancer cell cycle proliferation and tumorigenesis. Furthermore, PB and LY dose dependently decreased MGMT and CDK4 expression in breast cancer cells and significantly accumulated breast cancer cells in G1 phase of the cell cycle. DSF, alone or in combination with PB (DSF ± PB) and/or LY (DSF ± LY) caused significant apoptosis in breast cancer cells. DSF inhibited colony formation which was further enhanced by addition of PB/LY (DSF ± PB/LY). Similarly, DSF alone or in combination with PB (DSF ± PB) and/or LY (DSF ± LY) decreased the metastatic potential of breast cancer cells. Conclusions: Our findings suggest that DSF as an MGMT inhibitor significantly enhances the antitumor effect of CDK4/6 inhibitors (PB or LY) in ER+ breast cancer. Citation Format: George C. Bobustuc, Amin B. Kassam, Richard A. Rovin, Deborah L. Donohoe, Maxwell Albiero, Tarun Jella, Olivia Fukui, Cameron Piron, Santhi D. Konduri. MGMT inhibition leads to CDK4/6 inhibition and enhances palbociclib and abemaciclib activity in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2035. doi:10.1158/1538-7445.AM2017-2035

MGMT promoter methylation determined by HRM in comparison to MSP and pyrosequencing for predicting high-grade glioma response

BackgroundThe DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value.ResultsWe analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan–Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP.ConclusionsCompared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0204-7) contains supplementary material, which is available to authorized users.

Lipoic acid inhibits the DNA repair protein O 6-methylguanine-DNA methyltransferase (MGMT) and triggers its depletion in colorectal cancer cells with concomitant autophagy induction.

Alkylating agents are present in food and tobacco smoke, but are also used in cancer chemotherapy, inducing the DNA lesion O (6)-methylguanine. This critical adduct is repaired by O (6)-methylguanine-DNA methyltransferase (MGMT), resulting in MGMT inactivation and degradation. In the present study, we analyzed the effects of the natural disulfide compound lipoic acid (LA) on MGMT in vitro and in colorectal cancer cells. We show that LA, but not its reduced form dihydrolipoic acid, potently inhibits the activity of recombinant MGMT by interfering with its catalytic Cys-145 residue, which was partially reversible by N-acetyl cysteine. Incubation of HCT116 colorectal cancer cells with LA altered their glutathione pool and caused a decline in MGMT activity. This was mirrored by LA-induced depletion of MGMT protein, which was not attributable to changes in MGMT messenger RNA levels. Loss of MGMT protein coincided with LA-induced autophagy, a process resulting in lysosomal degradation of proteins, including presumably MGMT. LA-stimulated autophagy in a p53-independent manner as revealed by the response of isogenic HCT116 cell lines. Knockdown of the crucial autophagy component beclin-1 and chemical inhibitors blocked LA-induced autophagy, but did not abrogate LA-triggered MGMT degradation. Concomitant with MGMT depletion, LA pretreatment resulted in enhanced O (6)-methylguanine levels in DNA. It also increased the cytotoxicity of the alkylating anticancer drug temozolomide in temozolomide-resistant colorectal cancer cells. Taken together, our study showed that the natural compound LA inhibits MGMT and induces autophagy. Furthermore, LA enhanced the cytotoxic effects of temozolomide, which makes it a candidate for a supplement in cancer therapy.

Alkylation and Carbamylation Effects of Lomustine and Its Major Metabolites and MGMT Expression in Canine Cells.

DNA Alkylation is thought to be the reason for the efficacy of lomustine while carbamylation has been implicated as the cause for the side effects seen with lomustine treatment such as hepatotoxicity. In the alkylation study we show that lomustine and its metabolites form similar levels of the DNA adducts N7 hydroxyethylguanine and O6 hydroxyethyldeoxyguanosine. In terms of carbamylation, lomustine showed greater extent of carbamylation in the canine hepatocytes and lymphoma cell lines. The DNA repair enzyme O6 methylguanine DNA methyltransferase (MGMT) causes resistance of tumor cells to bifunctional nitrosourea, like lomustine. There is no data available regarding MGMT expression/activity in canine cells or tissues. Our study shows that there is low MGMT activity in the canine lymphoid cell line 17–71 while the GL-1 cells did not show any detectable enzyme activity or mRNA expression. The MGMT enzyme activity measured in canine hepatocytes is about 250–350 fmol/mg protein as compared to about 90 fmol/mg protein in 17–71 cells. We also show that MGMT mRNA expression in 17–71 cells and canine hepatocytes positively correlates with its enzyme activity in these cells.

Redox-responsive magnetic nanoparticle for targeted convection-enhanced delivery of O6-benzylguanine to brain tumors.

Resistance to temozolomide (TMZ) based chemotherapy in glioblastoma multiforme (GBM) has been attributed to the upregulation of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Inhibition of MGMT using O6-benzylguanine (BG) has shown promise in these patients, but its clinical use is hindered by poor pharmacokinetics that leads to unacceptable toxicity. To improve BG biodistribution and efficacy, we developed superparamagnetic iron oxide nanoparticles (NP) for targeted convection-enhanced delivery (CED) of BG to GBM. The nanoparticles (NPCP-BG-CTX) consist of a magnetic core coated with a redox-responsive, cross-linked, biocompatible chitosan-PEG copolymer surface coating (NPCP). NPCP was modified through covalent attachment of BG and tumor targeting peptide chlorotoxin (CTX). Controlled, localized BG release was achieved under reductive intracellular conditions and NPCP-BG-CTX demonstrated proper trafficking of BG in human GBM cells in vitro. NPCP-BG-CTX treated cells showed a significant reduction in MGMT activity and the potentiation of TMZ toxicity. In vivo, CED of NPCP-BG-CTX produced an excellent volume of distribution (Vd) within the brain of mice bearing orthotopic human primary GBM xenografts. Significantly, concurrent treatment with NPCP-BG-CTX and TMZ showed a 3-fold increase in median overall survival in comparison to NPCP-CTX/TMZ treated and untreated animals. Furthermore, NPCP-BG-CTX mitigated the myelosuppression observed with free BG in wild-type mice when administered concurrently with TMZ. The combination of favorable physicochemical properties, tumor cell specific BG delivery, controlled BG release, and improved in vivo efficacy demonstrates the great potential of these NPs as a treatment option that could lead to improved clinical outcomes.

MGMT gene silencing by promoter hypermethylation in gastric cancer in a high incidence area.

Inactivation of tumor suppressor and DNA repair genes by promoter hypermethylation does commonly occur in human cancers. O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that removes methyl groups as well as larger adducts at the O(6) position of guanine. In the absence of MGMT activity, O(6)-methylguanine mispairs with thymine during DNA replication, resulting in G:C to A:T transitions. Promoter hypermethylation of the MGMT gene has been observed in various cancers, including gastric cancer. Here, we aimed at assessing the promoter hypermethylation, mutation and expression status of the MGMT gene in patients from a geographic region with a high incidence of gastric cancer (Kashmir, North India) and to investigate their association with various clinicopathological characteristics. In this study 82 gastric cancer samples and adjacent normal tissues were included. Mutations in the MGMT gene were detected by single stranded conformational polymorphism (SSCP) analysis and direct sequencing. Methylation-specific polymerase chain reaction (MS-PCR) and Western blot analyses were performed to detect promoter hypermethylation and concomitant (loss of) expression of the MGMT gene. Promoter hypermethylation of the MGMT gene was found in 52.44% (43 of 82) of the tumor samples and loss of MGMT protein expression was detected in 45.12% (37 of 82) of the tumor samples. Hypermethylation and loss of expression were significantly associated with higher tumor grades (moderately/poorly differentiated) (P < 0.05) and higher tumor stages (III/IV) (P < 0.05). In addition, MGMT hypermethylation and loss of expression were found to be significantly associated with high salt tea consumption (P < 0.05). Our results indicate that MGMT promoter hypermethylation and concomitant loss of MGMT protein expression may play an important role in the development of gastric cancer in the Kashmiri population. High salt tea consumption may be a risk factor.

Disulfiram is a direct and potent inhibitor of human O6-methylguanine-DNA methyltransferase (MGMT) in brain tumor cells and mouse brain and markedly increases the alkylating DNA damage

The alcohol aversion drug disulfiram (DSF) reacts and conjugates with the protein-bound nucleophilic cysteines and is known to elicit anticancer effects alone or improve the efficacy of many cancer drugs. We investigated the effects of DSF on human O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein and chemotherapy target that removes the mutagenic O(6)-akyl groups from guanines, and thus confers resistance to alkylating agents in brain tumors. We used DSF, copper-chelated DSF or CuCl2-DSF combination and found that all treatments inhibited the MGMT activity in two brain tumor cell lines in a rapid and dose-dependent manner. The drug treatments resulted in the loss of MGMT protein from tumor cells through the ubiquitin-proteasome pathway. Evidence showed that Cys145, a reactive cysteine, critical for DNA repair was the sole site of DSF modification in the MGMT protein. DSF was a weaker inhibitor of MGMT, compared with the established O(6)-benzylguanine; nevertheless, the 24-36h suppression of MGMT activity in cell cultures vastly increased the alkylation-induced DNA interstrand cross-linking, G2/M cell cycle blockade, cytotoxicity and the levels of apoptotic markers. Normal mice treated with DSF showed significantly attenuated levels of MGMT activity and protein in the liver and brain tissues. In nude mice bearing T98 glioblastoma xenografts, there was a preferential inhibition of tumor MGMT. Our studies demonstrate a strong and direct inhibition of MGMT by DSF and support the repurposing of this brain penetrating drug for glioma therapy. The findings also imply an increased risk for alkylation damage in alcoholic patients taking DSF.

Randomized trial of chemoradiotherapy and adjuvant chemotherapy with nimustine (ACNU) versus nimustine plus procarbazine for newly diagnosed anaplastic astrocytoma and glioblastoma (JCOG0305)

Glioblastoma (GBM) is one of the worst cancers in terms of prognosis. Standard therapy consists of resection with concomitant chemoradiotherapy. Resistance to nimustine hydrochloride (ACNU), an alkylating agent, has been linked to methylguanine DNA methyltransferase (MGMT). Daily administration of procarbazine (PCZ) has been reported to decrease MGMT activity. This study investigated the efficacy of ACNU + PCZ compared to ACNU alone for GBM and anaplastic astrocytoma (AA). Patients (20-69 years) who had newly diagnosed AA and GBM were randomly assigned to receive radiotherapy with ACNU alone or with ACNU + PCZ. The primary endpoint was overall survival (OS). This was designed as a phase II/III trial with a total sample size of 310 patients and was registered as UMIN-CTR C000000108. After 111 patients from 19 centers in Japan were enrolled, this study was terminated early because temozolomide was newly approved in Japan. The median OS and median progression-free survival (PFS) with ACNU alone (n = 55) or ACNU + PCZ (n = 56) in the intention-to-treat population were 27.4 and 22.4 months (p = 0.75), and 8.6 and 6.9 months, respectively. The median OS and median PFS of the GBM subgroup treated with ACNU alone (n = 40) or ACNU + PCZ (n = 41) were 19.0 and 19.5 months, and 6.2 and 6.3 months, respectively. Grade 3/4 hematologic adverse events occurred in more than 40 % of patients in both arms, and 27 % of patients discontinued treatment because of adverse events. The addition of PCZ to ACNU was not beneficial, in comparison with ACNU alone, for patients with newly diagnosed AA and GBM.

Transfection of a human glioblastoma cell line with liver‐type glutaminase (LGA) down‐regulates the expression of DNA‐repair gene MGMT and sensitizes the cells to alkylating agents

O(6)-methylguanine-DNA methyltransferase (MGMT) is a DNA-repair protein promoting resistance of tumor cells to alkylating chemotherapeutic agents. Glioma cells are particularly resistant to this class of drugs which include temozolomide (TMZ) and carmustine (BCNU). A previous study using the RNA microarray technique showed that decrease of MGMT mRNA stands out among the alterations in gene expression caused by the cell growth-depressing transfection of a T98G glioma cell line with liver-type glutaminase (LGA) [Szeliga et al. (2009) Glia, 57, 1014]. Here, we show that stably LGA-transfected cells (TLGA) exhibit decreased MGMT protein expression and activity as compared with non-transfected or mock transfected cells (controls). However, the decrease of expression occurs in the absence of changes in the methylation of the promoter region, indicating that LGA circumvents, by an as yet unknown route, the most common mechanism of MGMT silencing. TLGA turned out to be significantly more sensitive to treatment with 100-1000 μM of TMZ and BCNU in the acute cell growth inhibition assay (MTT). In the clonogenic survival assay, TLGA cells displayed increased sensitivity even to 10 μM TMZ and BCNU. Our results indicate that enrichment with LGA, in addition to inhibiting glioma growth, may facilitate chemotherapeutic intervention.

5-day dosing schedule of temozolomide in relapsed sensitive or refractory small cell lung cancer (SCLC) and methyl-guanine-DNA methyltransferase (MGMT) analysis in a phase II trial.

7052 Background: MGMT promoter methylation and loss of MGMT activity are associated with improved sensitivity to alkylating agents. We recently reported that the oral alkylating agent temozolomide is active in 2nd- and 3rd-line treatment of relapsed SCLC at 75mg/m2/day for 21 out of 28 days (Pietanza et al, Clin Can Res 2012). Here we evaluate the 5-day dosing schedule of temozolomide in a second cohort of patients and analyze MGMT activity in both cohorts of patients on the same study. Methods: Patients with disease progression after 1 or 2 prior chemotherapy regimens received temozolomide at 200mg/m2/day for 5 out of 28 days (n=25). Those with sensitive (S-SCLC, n=16) and refractory (R-SCLC, n=9) disease were enrolled to assess safety. Available tumor tissue from patients treated according to either schedule was assessed for MGMT promoter methylation status by PCR and MGMT expression by immunohistochemistry (IHC). Results: An overall response rate of 12% was noted (3 partial responses: 1 S-SCLC, 2 R-SCLC). 4 patients had stable disease for at least 3 cycles. Median progression-free survival and overall survival for all patients were 1.3 months and 7.9 months, respectively. Grade ≥3 thrombocytopenia and neutropenia was observed in 20% with a shorter mean duration of myelosuppression compared to the 21-day schedule. Results from MGMT evaluation of tumor samples from both dosing schedules were combined. Promoter methylation of MGMT was not significantly associated with response (p=0.23). However, patients that lacked MGMT expression by IHC had a higher response rate compared to those with MGMT-positive tumors (43% vs. 13%, p = 0.052; see table). Conclusions: The 5-day dosing schedule of temozolomide is both active and safe in patients with relapsed SCLC. A strong trend toward increased response was demonstrated in patients with MGMT-negative tumors compared to patients with MGMT-positive tumors by IHC. A trial combining temozolomide with the PARP inhibitor, ABT888, is planned. [Table: see text]

Temozolomide for Pediatric High-Grade Gliomas

Introduction: Pediatric high-grade gliomas, excluding those arising in the brain stem which is frequently not histologically confirmed, comprise approximately 5% to 10% of all childhood brain tumors. Within the subgrouping are anaplastic astrocytomas and glioblastoma multiforme [1, 2]. Other subtypes may occur such as malignant oligodendrogliomas or oligoastrocytomas, but are much less common in pediatrics, compared with tumors arising in adults [1, 2]. Overall prognosis for pediatric high-grade gliomas is poor, although some studies have suggested somewhat higher survival rates than those seen in patients with similar tumors in adults [3–5]. The validity of early pediatric clinical studies has been questioned, as on central pathologic reviews, one study of pediatric high-grade astrocytomas reporting apparently better overall survival rates was contaminated by the inclusion of patients with low-grade neoplasms [4]. In general, 2-year survival rates have ranged from 10% to 30% for patients with supratentorial pediatric high-grade gliomas, and the single most independent prognostic factor across pediatric-age groups has been the degree of surgical resection; with patients undergoing a total resection or near-total resection having a better prognosis [3–5]. Recent studies have demonstrated a survival advantage for patients greater than 21 years of age with highgrade gliomas treated with temozolomide during and after tumor site radiotherapy [6, 7]. In adults with glioblastoma multiforme, the addition of temozolomide to radiation resulted in a 26% 2-year overall survival rate compared with 10% for those treated with radiation alone [6]. Temozolomide is an alkylating agent whose primary mechanism of cytotoxicity is alkylation at the 06 position of guanine. In the adult studies, the survival benefit was most apparent in patients whose tumors had methylation of the methylguanine DNA methyltransferase (MGMT) promoter, which is thought to result in diminished MGMT activity [8]. The MGMT gene encodes a DNArepair enzyme, which reduces the efficacy of alkylating chemotherapies. To determine if a similar survival rate could be seen in children, a study was undertaken by the Children’s Oncology Group (COG).

The strategy for enhancing temozolomide against malignant glioma

A combined therapy of the alkylating agent temozolomide (TMZ) and radiotherapy is standard treatment, and it improves the survival of patients with newly diagnosed glioblastoma (GBM). The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) removes the most cytotoxic lesions generated by TMZ, O6-methylguanine, establishing MGMT as one of the most important DNA repair mechanisms of TMZ-induced DNA damage. Thus, the expression of MGMT, its activity, and its promoter methylation status are associated with the response of GBM to TMZ, confirming that MGMT promotes clinical resistance to TMZ. Previous studies have shown that a variety of drugs such as interferon-β (IFN-β), levetiracetam (LEV), resveratrol, and valproic acid (VAP) increased the sensitivity of TMZ through MGMT-dependent or MGMT-independent mechanisms. In this review, we describe drugs and promising molecules that influence the responsiveness of GBM to TMZ and discuss their putative mechanism of action. In MGMT-positive GBMs, drugs that modulate MGMT activity could enhance the therapeutic activity of TMZ. Thus, administration of these drugs as an adjunct to TMZ chemotherapy may have clinical applications in patients with malignant gliomas to improve the outcome.

MGMT activity, promoter methylation and immunohistochemistry of pretreatment and recurrent malignant gliomas: a comparative study on astrocytoma and glioblastoma

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a key player in tumor cell resistance. Promoter methylation, MGMT activity and immunohistochemistry are used for determining the MGMT status. However, it is unclear whether MGMT promoter methylation correlates with MGMT activity and whether MGMT promoter methylation of the pretreatment tumor predicts the MGMT status of recurrences. To address these questions, we determined MGMT activity promoter methylation and immunoreactivity in pretreatment and recurrent glioblastomas (GB, WHO Grade IV), and in astrocytomas (WHO Grade III). We show that GB that were promoter methylated display a range of 0-62 fmol/mg MGMT and tumors that were nonmethylated 0-423 fmol/mg protein. For astrocytomas, promoter-methylated samples displayed 0-28 fmol/mg and, nonmethylated samples, 23-107 fmol/mg. No correlation was found between the intensity of promoter methylation and MGMT activity. Given a threshold level of 30 fmol/mg of protein, we found a correlation between promoter methylation and no/low MGMT activity in 82.4% of the tumors. This high correlation level was only observed when tumors were excluded showing a hemimethylated promoter (20%). Therefore, classification of hemimethylated tumors remains questionable. Further, we show that 39.1% of pretreatment GB and 5.3% of recurrences were promoter methylated, which is in line with the observed increase of MGMT activity in recurrences. Although individual exceptions were found, the data show an overall correlation between promoter methylation and lack/low MGMT activity in GB and astrocytomas. We also show that promoter methylation assay is superior over immunohistochemistry in determining the MGMT status defined by a given MGMT activity level.

Effect of alternative temozolomide schedules on glioblastoma O6-methylguanine-DNA methyltransferase activity and survival

Background:O6-methylguanine-DNA methyltransferase (MGMT) expression in glioblastoma correlates with temozolomide resistance. Dose-intense temozolomide schedules deplete MGMT activity in peripheral blood mononuclear cells; however, no published data exist evaluating the effect of temozolomide schedules on intracranial tumour MGMT activity.Methods:Human glioblastoma cells (GBM43) with an unmethylated MGMT promoter were implanted intracranially in immunodeficient rodents. Three weeks later, animals received temozolomide 200 mg m−2 for 5 days (schedule A, standard dose) or 100 mg m−2 for 21 days (schedule B, dose intense).Results:Tumour MGMT activity was depleted by day 6 in both treatment groups compared with baseline. O6-methylguanine-DNA methyltransferase activity returned to baseline by day 22 in the schedule A group, but remained suppressed in the schedule B group. By day 29, MGMT activity had returned to baseline in both groups. Mean tumour volume was significantly decreased compared with untreated controls with either schedule (P<0.01), although neither schedule was superior (P=0.60). Median survival was 64, 42, and 28 days for schedule A, schedule B, and no drug, respectively (P<0.001 A or B vs control, P=NS A vs B).Conclusions:Dose-intense temozolomide prolongs tumour MGMT activity depletion compared with standard dosing, however, survival was not improved in this model.

Glucocorticoid-Dependent Expression ofO6-Methylguanine-DNA Methyltransferase Gene Modulates Dacarbazine-Induced Hepatotoxicity in Mice

O(6)-methylguanine-DNA methyltransferase (MGMT) plays a crucial role in the defense against the alkylating agent-induced cytotoxic lesion O(6)-alkylguanine in DNA. Although a significant circadian variation in MGMT activity has been found in the liver of mice, the exact mechanism of the variation remains poorly understood. In this study, we present evidence that glucocorticoids were required for the 24-h oscillation of MGMT expression in mouse liver. The exposure of mouse hepatic cells (Hepa1-6) to dexamethasone (DEX) significantly increased the mRNA levels of MGMT in a dose-dependent manner. The DEX-induced increase in MGMT expression was reversed by concomitant treatment with RU486 [11beta-[p-(dimethylamino) phenyl]-17beta-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one], a glucocorticoid receptor antagonist. The mRNA levels of MGMT and its enzymatic activity in the liver of mice showed significant 24-h oscillations, which were not observed in adrenalectomized mice. A single administration of DEX to adrenalectomized mice significantly increased the mRNA levels of MGMT in the liver. These findings suggest that the 24-h oscillation in the hepatic expression of MGMT is caused by the endogenous rhythm of glucocorticoid secretion. Dacarbazine (DTIC), a potent O(6)-guanine-alkylating agent, causes serious hepatotoxicity accompanied by hepatocellular necrosis and hepatic vein thrombosis. DTIC-induced hepatotoxicity in mice was attenuated by administering the drug at the time of day when MGMT expression was abundant. The present findings suggest that glucocorticoid-regulated oscillation in the hepatic MGMT expression is the underlying cause of dosing time-dependent changes in DTIC-induced hepatotoxicity.

Poly(ADP-ribose) polymerase inhibitor ABT-888 potentiates the cytotoxic activity of temozolomide in leukemia cells: influence of mismatch repair status and O6-methylguanine-DNA methyltransferase activity.

The poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 potentiates the antitumor activity of temozolomide (TMZ). TMZ resistance results from increased O(6)-methylguanine-DNA methyltransferase (MGMT) activity and from mismatch repair (MMR) system mutations. We evaluated the relative importance of MGMT activity, MMR deficiency, nonhomologous end joining (NHEJ), and PARP activity in ABT-888 potentiation of TMZ. MMR-proficient and MMR-deficient leukemia cells with varying MGMT activity, as well as primary leukemia samples, were used to determine TMZ IC(50) alone and with ABT-888. ABT-888 effectively inhibited PARP activity and enhanced TMZ growth inhibition in most leukemia cells. ABT-888 potentiation was most effective in MMR-deficient cells with low MGMT activity [potentiation factor (PF) = 21]. ABT-888 also potentiated TMZ activity in MMR-deficient cells with elevated MGMT activity. Unexpectedly, ABT-888 also enhanced TMZ activity in MMR-proficient cells (PF = 3-7). ABT-888 potentiation was unrelated to NHEJ activity. ABT-888 potentiated TMZ (PF = 2-5) in two of four acute myeloid leukemia patient samples but showed little potentiation in primary acute lymphoblastic leukemia. In conclusion, although ABT-888 potentiation of TMZ was most pronounced in MMR-deficient cells with low MGMT activity, neither MMR proficiency nor MGMT overexpression completely abrogated ABT-888 potentiation of TMZ.

New (alternative) temozolomide regimens for the treatment of glioma.

One barrier to successful treatment of malignant glioma is resistance to alkylating agents such as temozolomide. The cytotoxic activity of temozolomide and other alkylating agents is believed to manifest largely by the formation of O(6)-methylguanine DNA adducts. Consequently, the primary mechanism of resistance to temozolomide is a function of the activity of the DNA repair enzyme O(6)-methylguanine DNA methyltransferase (MGMT). Fortuitously, MGMT is inactivated after each reaction (i.e., suicide enzyme). Therefore, if the rate of DNA alkylation were to outpace the rate of MGMT protein synthesis, the enzyme could, in theory, be depleted. Several studies have shown that prolonged exposure to temozolomide can deplete MGMT activity in blood cells, a process that could potentially increase the antitumor activity of the drug. To date, however, there are limited data demonstrating the depletion of MGMT activity in tumor tissue exposed to temozolomide. A variety of dosing schedules that increase the duration of exposure and the cumulative dose of temozolomide are currently being investigated for the treatment of glioma, with the goal of improving antitumor activity and overcoming resistance. These alternative dosing regimens have been shown to deplete MGMT activity in peripheral blood mononuclear cells, but the regimen that provides the best balance between enhanced antitumor activity and acceptable hematologic toxicity has yet to be determined.

Promoter Methylation and Polymorphisms of the MGMT Gene in Glioblastomas: A Population-Based Study

O⁶-methylguanine-DNA methyltransferase (MGMT) is a repair enzyme that removes promutagenic O⁶-methylguanine adducts in DNA, to protect cells from acquisition of G:C→ A:T mutations. MGMT promoter methylation and polymorphisms may affect MGMT expression and activity. In the present study, we assessed MGMT promoter methylation and polymorphisms (Leu84Phe, Ile143Val, c.–56C>T) in 371 glioblastomas diagnosed at the population level. MGMT methylation was observed in 165 (44%) glioblastomas, with a higher frequency in females than males (53 vs. 39%; p = 0.0106) and in secondary than primary glioblastomas (73 vs. 43%; p = 0.0074). The frequency of TP53 G:C→A:T mutations in glioblastomas with MGMT methylation was 25%, which was significantly higher than that in glioblastomas with MGMT methylation (16%; Fisher exact test; p = 0.0385). MGMT 143 Val allele in glioblastomas was significantly less frequent than in a healthy European Caucasian population, and was associated with longer survival than those with the MGMT 143 Ile allele (hazard ratio 0.70; 95% CI 0.48–1.01). These results suggest that MGMT methylation may be associated with susceptibility to acquire TP53 G:C→A:T mutations, and that MGMT polymorphisms may affect the risk and prognosis of glioblastomas.

Mechanisms of Disease: temozolomide and glioblastoma—look to the future

Glioblastoma is both the most common and most aggressive primary brain tumor. Until recently, the standard of care involved maximal safe surgical resection followed by radiation therapy with or without nitrosourea-based chemotherapy. In 2005, the results of a large clinical trial examining the role of adjuvant chemotherapy in management of newly diagnosed glioblastoma were published. This study created a new standard of adjuvant treatment, using concurrent and sequential temozolomide in the initial therapy of glioblastoma. A companion tumor biology study identified the prognostic role of O(6)-methylguanine-DNA methyltransferase (MGMT) status in patients with newly diagnosed glioblastoma. Several preliminary studies have been initiated to address the issue of resistance and suppression of MGMT activity, and have used alternative temozolomide dosing schedules and O(6)-guanine mimetic agents as substrates for MGMT. In addition, recent studies have attempted to define mechanisms responsible for the apparent synergy between temozolomide and radiotherapy. Lastly, an increased understanding of the molecular biology of glioblastoma has provided new leads for the adjuvant treatment of this disease. This Review summarizes new developments in treatment of glioblastoma and speculates on possible future treatment strategies for managing this aggressive cancer.

Increased expression of the MGMT repair protein mediated by cysteine prodrugs and chemopreventative natural products in human lymphocytes and tumor cell lines

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein which protects the cellular genome and critical oncogenic genes from the mutagenic action of endogenous and exogenous alkylating agents. An expedited elimination of O6-alkylguanines by increasing MGMT activity levels is likely to be a successful chemoprevention strategy. Here, we report for the first time that cysteine/glutathione enhancing drugs and certain plant antioxidants possess the ability to increase human MGMT expression beyond its steady-state levels that may afford protection. The non-toxic cysteine prodrugs, 2-oxothiazolidine-4-carboxylic acid (OTC) and N-acetyl-L-cysteine (NAC), metabolized, respectively by 5-oxoprolinase and acylases, increased the MGMT protein and its repair activity levels in a dose- and time-dependent manner in several cancer cell lines and peripheral blood lymphocytes with a maximum of 3-fold increase by 72 h. The natural antioxidants, namely, curcumin, silymarin, sulforaphane and resveratrol were also effective in raising the MGMT levels to different extents. Among the synthetic agents, oltipraz and N-(4-hydroxyphenyl) retinamide (4-HPR) also increased MGMT expression, albeit to a lesser extent. Augmented mRNA levels accounted at least, in part, for the increased activity of MGMT in this setting. However, evidence from cysteine/methionine deprivation, acivicin treatment, and protein synthesis measurements in OTC-treated cells suggested that an increased cysteine flux also contributed significantly to enhanced MGMT expression. Many of these treatments increased the glutathione S-transferase-P1 (GSTP1) levels as well. These findings raise the possibility of MGMT-targeted chemoprevention strategies through dietary supplementation of OTC and herbal antioxidants. Further, the studies reveal the antioxidant responsiveness of the human MGMT gene.