Michael S. Bobola

O6-Methylguanine-DNA Methyltransferase, O6-Benzylguanine, and Resistance to Clinical Alkylators in Pediatric Primary Brain Tumor Cell Lines

Purpose: Primary brain tumors are the leading cause of cancer death in children. Our purpose is (a) to assess the contribution of the DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) to the resistance of pediatric brain tumor cell lines to clinical alkylating agents and (b) to evaluate variables for maximal potentiation of cell killing by the MGMT inhibitor O6-benzylguanine, currently in clinical trials. Few such data for pediatric glioma lines, particularly those from low-grade tumors, are currently available. Experimental design: We used clonogenic assays of proliferative survival to quantitate cytoxicity of the chloroethylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and the methylating agent temozolomide in 11 glioma and five medulloblastoma lines. Twelve lines are newly established and characterized here, nine of them from low-grade gliomas including pilocytic astrocytomas. Results: (a) MGMT is a major determinant of BCNU resistance and the predominant determinant of temozolomide resistance in both our glioma and medulloblastoma lines. On average, O6-benzylguanine reduced LD10 for BCNU and temozolomide, 2.6- and 26-fold, respectively, in 15 MGMT-expressing lines. (b) O6-Benzylguanine reduced DT (the threshold dose for killing) for BCNU and temozolomide, 3.3- and 138-fold, respectively. DT was decreased from levels higher than, to levels below, clinically achievable plasma doses for both alkylators. (c) Maximal potentiation by O6-benzylguanine required complete and prolonged suppression of MGMT. Conclusions: Our results support the use of O6-benzylguanine to achieve full benefit of alkylating agents, particularly temozolomide, in the chemotherapy of pediatric brain tumors.

Apurinic/apyrimidinic endonuclease activity is associated with response to radiation and chemotherapy in medulloblastoma and primitive neuroectodermal tumors

Purpose: Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair activity that confers resistance to radiation- and alkylator-induced cytotoxic abasic sites in human cells. We assayed apurinic/apyrimidinic endonuclease activity in medulloblastomas and primitive neuroectodermal tumors (PNET) to establish correlates with tumor and patient characteristics and with response to adjuvant radiation plus multiagent chemotherapy. Experimental Design: Ap endo activity was assayed in 52 medulloblastomas and 10 PNETs from patients 0.4 to 21 years old. Ape1/Ref-1, the predominant human Ap endo activity, was measured in 42 medulloblastomas by immunostaining. Cox proportional hazards regression models were used to analyze the association of activity with time to tumor progression (TTP). Results: Tumor Ap endo activity varied 180-fold and was significantly associated with age and gender. Tumor Ape1/Ref-1 was detected almost exclusively in nuclei. In a multivariate model, with Ap endo activity entered as a continuous variable, the hazard ratio for progression after adjuvant treatment in 46 medulloblastomas and four PNETs increased by a factor of 1.073 for every 0.01 unit increase in activity (P ≤ 0.001) and was independent of age and gender. Suppressing Ap endo activity in a human medulloblastoma cell line significantly increased sensitivity to 1,3-bis(2-chlororethyl)-1-nitrosourea and temozolomide, suggesting that the association of tumor activity with TTP reflected, at least in part, abasic site repair. Conclusions: Our data (a) suggest that Ap endo activity promotes resistance to radiation plus chemotherapy in medulloblastomas/PNETs, (b) provide a potential marker of treatment outcome, and (c) suggest clinical use of Ap endo inhibitors to overcome resistance.

O6-methylguanine-DNA methyltransferase in glioma therapy: Promise and problems

Gliomas are the most frequent adult primary brain tumor, and are invariably fatal. The most common diagnosis glioblastoma multiforme (GBM) afflicts 12,500 new patients in the U.S. annually, and has a median survival of approximately one year when treated with the current standard of care. Alkylating agents have long been central in the chemotherapy of GBM and other gliomas. The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), the principal human activity that removes cytotoxic O(6)-alkylguanine adducts from DNA, promotes resistance to anti-glioma alkylators, including temozolomide and BCNU, in GBM cell lines and xenografts. Moreover, MGMT expression assessed by immunohistochemistry, biochemical activity or promoter CpG methylation status is associated with the response of GBM to alkylator-based therapies, providing evidence that MGMT promotes clinical resistance to alkylating agents. These observations suggest a role for MGMT in directing adjuvant therapy of GBM and other gliomas. Promoter methylation status is the most clinically tractable measure of MGMT, and there is considerable enthusiasm for exploring its utility as a marker to assign therapy to individual patients. Here, we provide an overview of the biochemical, genetic and biological characteristics of MGMT as they relate to glioma therapy. We consider current methods to assess MGMT expression and discuss their utility as predictors of treatment response. Particular emphasis is given to promoter methylation status and the methodological and conceptual impediments that limit its use to direct treatment. We conclude by considering approaches that may improve the utility of MGMT methylation status in planning optimal therapies tailored to individual patients.

Apurinic endonuclease activity in adult gliomas and time to tumor progression after alkylating agent-based chemotherapy and after radiotherapy

Purpose: Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair enzyme that cleaves DNA at cytotoxic abasic sites caused by alkylating agents and radiation. We have observed that human glioma cells deficient in Ap endo activity are hypersensitive to clinically used alkylators (Silber et al., Clin Cancer Res 2002;8:3008.). Here we examine the association of glioma Ap endo activity with clinical response after alkylating agent-based chemotherapy or after radiotherapy. Experimental Design: Cox proportional hazards regression models were used to analyze the relationship of Ap endo activity with time to tumor progression (TTP). Results: In a univariate model with Ap endo activity entered as a continuous variable, the hazard ratio (HR) for progression after alkylator therapy in 30 grade III gliomas increased by a factor of 1.061 for every 0.01 increase in activity (P = 0.013). Adjusting for age, gender, extent of resection, and prior treatment strengthened slightly the association (HR = 1.094; P = 0.003). Similarly, the HR for progression after radiotherapy in 44 grade II and III tumors increased by a factor of 1.069 (P = 0.008). Adjusting for the aforementioned variables had little effect on the association. In contrast, we observed no association between activity and TTP in grade IV gliomas after either alkylator therapy in 34 tumors or radiotherapy in 26 tumors. Conclusions: Our data suggest that Ap endo activity mediates resistance to alkylating agents and radiation and may be a useful predictor of progression after adjuvant therapy in a subset of gliomas.

Characterization of paclitaxel (Taxol) sensitivity in human glioma- and medulloblastoma-derived cell lines.

Paclitaxel (Taxol), a cytotoxic natural product that disrupts microtubule integrity, is being clinically evaluated for use against gliomas. We examined paclitaxel-induced killing in seven cell lines derived from human malignant astrocytic gliomas and medulloblastomas with the goal of characterizing range of sensitivity, contribution of P-glycoprotein 170-mediated drug efflux to resistance, and cross-resistance with alkylating agents. Exposure to paclitaxel for 8 h or less produced biphasic survival curves for all lines, with 40-75% of cells comprising a subpopulation that was 9-26 times more resistant to paclitaxel than the more sensitive fraction. Increasing exposure to 24 h eliminated the resistant subpopulation, increasing sensitivity 50- to 400-fold. The dose producing one log of kill (LD10) after a 24-h exposure ranged from 4 to 18 nM, comparable to concentrations in the cerebrospinal fluid of brain tumor patients given a 3-h infusion of paclitaxel. Concurrent exposure to paclitaxel and either nimodipine or verapamil, inhibitors of P-glycoprotein activity, did not increase sensitivity, demonstrating that the fivefold range in sensitivity was not due to P-glycoprotein-mediated drug efflux. Importantly, there was no correlation between LD10 for paclitaxel and LD10 for 1,3-bis(2-chloroethyl)-1-nitrosourea, streptozotocin, and temozolomide, indicating no expression of cross-resistance to these different classes of tumoricidal agents. Our results suggest that greater clinical efficacy of paclitaxel against malignant brain tumors may be obtained by infusion for 24 h or longer and support the use of paclitaxel in combination with alkylating agents.

Minimally cytotoxic doses of temozolomide produce radiosensitization in human glioblastoma cells regardless of MGMT expression

Concurrent treatment with the methylating agent temozolomide during radiotherapy has yielded the first significant improvement in the survival of adult glioblastomas (GBM) in the last three decades. However, improved survival is observed in a minority of patients, most frequently those whose tumors display CpG methylation of the O6-methylguanine (O6-meG)-DNA methyltransferase (MGMT) promoter, and adult GBMs remain invariably fatal. Some, although not all, preclinical studies have shown that temozolomide can increase radiosensitivity in GBM cells that lack MGMT, the sole activity in human cells that removes O6-meG from DNA. Here, we systematically examined the temozolomide dose dependence of radiation killing in established GBM cell lines that differ in ability to remove O6-meG or tolerate its lethality. Our results show that minimally cytotoxic doses of temozolomide can produce dose-dependent radiosensitization in MGMT-deficient cells, MGMT-proficient cells, and MGMT-deficient cells that lack mismatch repair, a process that renders cells tolerant of the lethality of O6-meG. In cells that either possess or lack MGMT activity, radiosensitization requires exposure to temozolomide before but not after radiation and is accompanied by formation of double-strand breaks within 45 minutes of radiation. Moreover, suppressing alkyladenine-DNA glycosylase, the only activity in human cells that excises 3-methyladenine from DNA, reduces the temozolomide dose dependence of radiosensitization, indicating that radiosensitization is mediated by 3-methyladenine as well as by O6-meG. These results provide novel information on which to base further mechanistic study of radiosensitization by temozolomide in human GBM cells and to develop strategies to improve the outcome of concurrent temozolomide radiotherapy. Mol Cancer Ther; 9(5); 1208–18. ©2010 AACR.

Human Glioma Cell Sensitivity to the Sequence-Specific Alkylating Agent Methyl-Lexitropsin

Purpose: Defining the cytotoxicity of individual adducts in DNA is necessary for mechanistic understanding of human brain tumor resistance to therapeutic alkylating agents and for design of DNA repair-related antiresistance strategies. Our purpose is to characterize the sensitivity of human glioma cells to methyl-lexitropsin (Me-lex), a sequence-specific alkylator that produces 3-methyladenine (3-meA) as the predominant (>90%) DNA lesion. Experimental Design: We quantitated the Me-lex cytotoxicity of 10 human glioma cell lines that differ in O6-methylguanine (O6-meG)-DNA methyltransferase (MGMT) and mismatch repair activity. We used antisense suppression of alkyladenine DNA glycosylase (AAG) and Ape1 to assess the contribution of 3-meA and abasic sites to lethality and measured abasic sites. Results: (a) The LD10 for Me-lex varied widely among the cell lines. (b) MGMT-proficient lines were more resistant than MGMT-deficient lines, an unexpected finding because Me-lex produces very little O6-meG. (c) Suppression of AAG increased Me-lex killing and reduced abasic site content. (d) Suppression of Ape1 increased Me-lex killing and increased abasic site content. (e) Ablation of MGMT had no effect on Me-lex cytotoxicity. Conclusions: (a) Me-lex is cytotoxic in human glioma cells and AAG promotes resistance, indicating that 3-meA is a lethal lesion in these cells. (b) Abasic sites resulting from 3-meA repair are cytotoxic and Ape1 promotes resistance to these derivative lesions. (c) A factor(s) associated with MGMT expression, other than repair of O6-meG, contributes to Me-lex resistance. (d) Me-lex may have clinical utility in the adjuvant therapy of gliomas. (e) AAG and Ape1 inhibitors may be useful in targeting alkylating agent resistance.

Repair of 3-methyladenine and abasic sites by base excision repair mediates glioblastoma resistance to temozolomide

Alkylating agents have long played a central role in the adjuvant therapy of glioblastoma (GBM). More recently, inclusion of temozolomide (TMZ), an orally administered methylating agent with low systemic toxicity, during and after radiotherapy has markedly improved survival. Extensive in vitro and in vivo evidence has shown that TMZ-induced O6-methylguanine (O6-meG) mediates GBM cell killing. Moreover, low or absent expression of O6-methylguanine-DNA methyltransferase (MGMT), the sole human repair protein that removes O6-meG from DNA, is frequently associated with longer survival in GBMs treated with TMZ, promoting interest in developing inhibitors of MGMT to counter resistance. However, the clinical efficacy of TMZ is unlikely to be due solely to O6-meG, as the agent produces approximately a dozen additional DNA adducts, including cytotoxic N3-methyladenine (3-meA) and abasic sites. Repair of 3-meA and abasic sites, both of which are produced in greater abundance than O6-meG, is mediated by the base excision repair (BER) pathway, and occurs independently of removal of O6-meG. These observations indicate that BER activities are also potential targets for strategies to potentiate TMZ cytotoxicity. Here we review the evidence that 3-meA and abasic sites mediate killing of GBM cells. We also present in vitro and in vivo evidence that alkyladenine-DNA glycosylase, the sole repair activity that excises 3-meA from DNA, and Ape1, the major human abasic site endonuclease, mediate TMZ resistance in GBMs and represent potential anti-resistance targets.

Apurinic/Apyrimidinic Endonuclease Is Inversely Associated with Response to Radiotherapy in Pediatric Ependymoma

Apurinic/apyrimidinic endonuclease (Ap endo) is a key DNA repair activity that confers radiation resistance in human cells. Here we examined the association between Ap endo activity and response to radiotherapy in pediatric ependymomas, tumors for which treatment options are limited and survival rates are only about 50%. We assayed Ap endo activity in 36 ependymomas and expression of Ape1/Ref‐1, the predominant Ap endo activity in humans, in 44 tumors by immunostaining. Cox proportional hazards regression models were used to analyze the association of activity or expression with progression‐free survival or with overall survival. Activity varied 13‐fold and was not associated with tumor or patient characteristics. In univariate models with Ap endo activity entered as a continuous variable, the hazard ratio for progression increased by a factor of 2.18 for every 0.01 unit increase in activity (p ≤ 0.003) in 24 grade II ependymomas. Risk for death increased by a factor of 1.89 (p ≤ 0.02) in the same population. The fraction of Ape1/Ref‐1 immunopositive cells varied widely within individual tumors and was not associated with either progression‐free or with overall survival. Suppressing Ap endo activity in pediatric ependymoma cells significantly increased radiation sensitivity, suggesting that the association of activity with radiation response reflected, at least in part, repair of radiation‐induced DNA lesions. Our data indicate that Ap endo activity is predictive of outcome following radiotherapy, and suggest that Ape1/Ref‐1 promotes radiation resistance in pediatric ependymomas. Our findings support the use of inhibitors of Ap endo activity to overcome resistance.

Synthesis and Characterization of DNA Minor Groove Binding Alkylating Agents

Derivatives of methyl 3-(1-methyl-5-(1-methyl-5-(propylcarbamoyl)-1H-pyrrol-3-ylcarbamoyl)-1H-pyrrol-3-ylamino)-3-oxopropane-1-sulfonate (1), a peptide-based DNA minor groove binding methylating agent, were synthesized and characterized. In all cases, the N-terminus was appended with an O-methyl sulfonate ester, while the C-terminus group was varied with nonpolar and polar side chains. In addition, the number of pyrrole rings was varied from 2 (dipeptide) to 3 (tripeptide). The ability of the different analogues to efficiently generate N3-methyladenine was demonstrated as was their selectivity for minor groove (N3-methyladenine) versus major groove (N7-methylguanine) methylation. Induced circular dichroism studies were used to measure the DNA equilibrium binding properties of the stable sulfone analogues; the tripeptide binds with affinity that is >10-fold higher than that of the dipeptide. The toxicities of the compounds were evaluated in alkA/tag glycosylase mutant E. coli and in human WT glioma cells and in cells overexpressing and under-expressing N-methylpurine-DNA glycosylase, which excises N3-methyladenine from DNA. The results show that equilibrium binding correlates with the levels of N3-methyladenine produced and cellular toxicity. The toxicity of 1 was inversely related to the expression of MPG in both the bacterial and mammalian cell lines. The enhanced toxicity parallels the reduced activation of PARP and the diminished rate of formation of aldehyde reactive sites observed in the MPG knockdown cells. It is proposed that unrepaired N3-methyladenine is toxic due to its ability to directly block DNA polymerization.