O6-methylguanine-DNA-methyltransferase (MGMT) gene therapy targeting haematopoietic stem cells: Studies addressing safety issues

Sorg, Ursula R.; Kleff, Veronika; Fanaei, S A; Schumann, Alexandra; Moellmann, Michael; Opalka, Bertram; Thomale, Jürgen; Möritz, Thomas

doi:10.1016/j.dnarep.2007.03.021

Cited by 14 publications

(9 citation statements)

References 59 publications

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“…Immunocytologic assay for DNA alkylation products by adduct-specific antibodies was done as described (34) using either mouse anti–( O 6 -methylguanine) monoclonal antibody EM2-3 (35) or rabbit anti–imidazole-ring opened ( N 7 -methylguanine) serum (kindly provided by Dr. A Povey, University of Manchester, Manchester, United Kingdom). The alkaline comet assay was done on sorted lin – bone marrow cells as described (36).…”

Section: Methodsmentioning

confidence: 99%

Reciprocal Relationship between O6-Methylguanine-DNA Methyltransferase P140K Expression Level and Chemoprotection of Hematopoietic Stem Cells

et al. 2008

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Retroviral-mediated delivery of the P140K mutant O6-methylguanine-DNA methyltransferase (MGMTP140K) into hematopoietic stem cells (HSC) has been proposed as a means to protect against dose-limiting myelosuppressive toxicity ensuing from chemotherapy combining O6-alkylating agents (e.g., temozolomide) with pseudosubstrate inhibitors (such as O6-benzylguanine) of endogenous MGMT. Because detoxification of O6-alkylguanine adducts by MGMT is stoichiometric, it has been suggested that higher levels of MGMT will afford better protection to gene-modified HSC. However, accomplishing this goal would potentially be in conflict with current efforts in the gene therapy field, which aim to incorporate weaker enhancer elements to avoid insertional mutagenesis. Using a panel of self-inactivating gamma-retroviral vectors that express a range of MGMTP140K activity, we show that MGMTP140K expression by weaker cellular promoter/enhancers is sufficient for in vivo protection/selection following treatment with O6-benzylguanine/temozolomide. Conversely, the highest level of MGMTP140K activity did not promote efficient in vivo protection despite mediating detoxification of O6-alkylguanine adducts. Moreover, very high expression of MGMTP140K was associated with a competitive repopulation defect in HSC. Mechanistically, we show a defect in cellular proliferation associated with elevated expression of MGMTP140K, but not wild-type MGMT. This proliferation defect correlated with increased localization of MGMTP140K to the nucleus/chromatin. These data show that very high expression of MGMTP140K has a deleterious effect on cellular proliferation, engraftment, and chemoprotection. These studies have direct translational relevance to ongoing clinical gene therapy studies using MGMTP140K, whereas the novel mechanistic findings are relevant to the basic understanding of DNA repair by MGMT.

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Section: Methodsmentioning

confidence: 99%

Reciprocal Relationship between O6-Methylguanine-DNA Methyltransferase P140K Expression Level and Chemoprotection of Hematopoietic Stem Cells

et al. 2008

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“…It has also been shown that, in CD34 ? stem cells, which have low MGMT activity [24], very high level expression of a mutant form of MGMT that is inactivator-resistant inhibits stem cell proliferation, but the basis of this has not been established [25].…”

Section: The Dna Repair Protein Mgmtmentioning

confidence: 99%

Targeting O 6-methylguanine-DNA methyltransferase with specific inhibitors as a strategy in cancer therapy

Kaina

Margison

Christmann

2010

Cell. Mol. Life Sci.

127

116

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O (6)-methylguanine-DNA methyltransferase (MGMT) repairs the cancer chemotherapy-relevant DNA adducts, O (6)-methylguanine and O (6)-chloroethylguanine, induced by methylating and chloroethylating anticancer drugs, respectively. These adducts are cytotoxic, and given the overwhelming evidence that MGMT is a key factor in resistance, strategies for inactivating MGMT have been pursued. A number of drugs have been shown to inactivate MGMT in cells, human tumour models and cancer patients, and O (6)-benzylguanine and O (6)-[4-bromothenyl]guanine have been used in clinical trials. While these agents show no side effects per se, they also inactivate MGMT in normal tissues and hence exacerbate the toxic side effects of the alkylating drugs, requiring dose reduction. This might explain why, in any of the reported trials, the outcome has not been improved by their inclusion. It is, however, anticipated that, with the availability of tumour targeting strategies and hematopoetic stem cell protection, MGMT inactivators hold promise for enhancing the effectiveness of alkylating agent chemotherapy.

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“…Removal of antibody-binding sites after various repair times was determined after 10 J/m 2 UV-irradiation (3). Repair of N7-meG was measured in human lymphocytes after treatment with alkylating agents (7). …”

Section: Introductionmentioning

confidence: 99%

DNA double-strand-break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice

Schipler

Iliakis

2013

Nucleic Acids Research

245

232

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Although the DNA double-strand break (DSB) is defined as a rupture in the double-stranded DNA molecule that can occur without chemical modification in any of the constituent building blocks, it is recognized that this form is restricted to enzyme-induced DSBs. DSBs generated by physical or chemical agents can include at the break site a spectrum of base alterations (lesions). The nature and number of such chemical alterations define the complexity of the DSB and are considered putative determinants for repair pathway choice and the probability that errors will occur during this processing. As the pathways engaged in DSB processing show distinct and frequently inherent propensities for errors, pathway choice also defines the error-levels cells opt to accept. Here, we present a classification of DSBs on the basis of increasing complexity and discuss how complexity may affect processing, as well as how it may cause lethal or carcinogenic processing errors. By critically analyzing the characteristics of DSB repair pathways, we suggest that all repair pathways can in principle remove lesions clustering at the DSB but are likely to fail when they encounter clusters of DSBs that cause a local form of chromothripsis. In the same framework, we also analyze the rational of DSB repair pathway choice.

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O6-methylguanine-DNA-methyltransferase (MGMT) gene therapy targeting haematopoietic stem cells: Studies addressing safety issues

Cited by 14 publications

References 59 publications

Reciprocal Relationship between O6-Methylguanine-DNA Methyltransferase P140K Expression Level and Chemoprotection of Hematopoietic Stem Cells

Reciprocal Relationship between O6-Methylguanine-DNA Methyltransferase P140K Expression Level and Chemoprotection of Hematopoietic Stem Cells

Targeting O 6-methylguanine-DNA methyltransferase with specific inhibitors as a strategy in cancer therapy

DNA double-strand-break complexity levels and their possible contributions to the probability for error-prone processing and repair pathway choice

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