Alkylating Agent Resistance

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The most striking HSC selection results in vivo have been obtained using the O6-methylguanine-DNA methyltransferase (MGMT) gene. MGMT encodes O6-alkylgua-nine-DNA alkyltransferase (AGT), which repairs DNA damage induced by alkylating agents (see Fig. 4). Although most DNA repair pathways involve multiple protein constituents, AGT is singly responsible for the repair of O6-alkyl lesions. Repair is mediated by the covalent transfer of the O6-alkyl group from guanine to a cysteine thiol located in the AGT binding pocket (88). This irreversible reaction inactivates AGT. Thus, each AGT molecule is only capable of repairing one alkyl lesion, after which the protein is ubiquitinated and targeted for degradation (89).

Alkylation of the O6 position of guanine is the most cytotoxic lesion produced by methylating (e.g., temozolomide, streptozotocin, dacarbazine, and procarbazine), and chloroethylating (e.g., BCNU, and CCNU) agents used to treat a variety of cancers.

Normal AGT Repair

BG Inactivates

Unrepaired Lesions are Cytotoxic

AGT Point Mutants are Resistant to BG

Fig. 4. MGMT-mediated repair. MGMT repairs cytotoxic C^-alkylguanine lesions formed by methy-lating and chloroethylating agents. BG inactivates endogenous MGMT, thereby increasing the sensitivity of untransduced cells to alkylating agent treatment. Specific MGMT point mutants (MGMT*) are resistant to BG inactivation, but maintain the capacity for DNA repair. HSCs transduced with MGMT* are enriched in vivo following BG and alkylating agent treatment.

These agents are particularly myelosuppressive due to the low level of AGT activity in bone marrow cells (90,91). During DNA replication O6-methylguanine residues are mismatched with thymine (92). Therefore, DNA synthesis prior to AGT-mediated repair results in a G:T mispair that is corrected by the mismatch repair (MMR) pathway. Uncorrected methylguanine residues result in a futile MMR cycle in which thymine residues are continuously mispaired opposite O6-methylguanine, eventually leading to single strand breaks and cell death (93). The type of DNA adducts resulting from chloroethylating agents are particularly cytotoxic. Unrepaired O6-chloroethyl lesions rearrange to form both intra- and interstrand crosslinks to neighboring residues (94,95).

The significance of using drug resistance gene transfer to protect mammalian cells from DNA damage was first established after the bacterial MGMT homologue (ada) was cloned (96). Transfer and expression of ada in MGMT deficient cell lines was shown to dramatically reduce alkylating agent toxicity (97-99). These pivotal experiments set the stage for the last two decades of research aimed at using MGMT gene transfer to protect bone marrow cells from the myelosuppressive effects of alkylating agent chemotherapy, and as a mechanism for selecting transduced stem cells in vivo.

The first study to demonstrate MGMT-mediated protection of bone marrow (BM) cells was carried out using electroporation for ada gene delivery (100). Stable transfer of ada (101) or human MGMT (102,103) into murine BM cells with retroviral vectors was subsequently shown to reduce the myelosuppressive effects of choroethylating

Normal AGT Repair

BG Inactivates

Unrepaired Lesions are Cytotoxic

AGT Point Mutants are Resistant to BG

Fig. 4. MGMT-mediated repair. MGMT repairs cytotoxic C^-alkylguanine lesions formed by methy-lating and chloroethylating agents. BG inactivates endogenous MGMT, thereby increasing the sensitivity of untransduced cells to alkylating agent treatment. Specific MGMT point mutants (MGMT*) are resistant to BG inactivation, but maintain the capacity for DNA repair. HSCs transduced with MGMT* are enriched in vivo following BG and alkylating agent treatment.

agents in vivo. Increased resistance to multiple doses of BCNU correlated with increased percentages of MGMT-transduced murine progenitors in the bone morrow (104). Human CD34+ hematopoietic progenitor cells transduced with MGMT were also shown to tolerate higher doses of BCNU (105).

Just as high BM AGT expression reduces alkylating agent-induced myelosuppres-sion, tumor cells with upregulated AGT are also tolerant to these treatments (106-108). Therefore, the modest levels of protection achieved by MGMT gene transfer experiments are unlikely to have a dramatic therapeutic impact. However, two major advancements brought MGMT-mediated chemoprotection to the forefront. First was the discovery of the potent MGMT inactivator, O6-benzylguanine (BG). BG provided a mechanism for depleting AGT activity, sensitizing tumors to drug treatment (109-111). However, BG-mediated inactivation of AGT is not specific, and thus sensitizes both tumor and bone marrow cells to alkylating agents (112). The second major advancement came from the identification of specific point mutations in MGMT that conferred significant resistance to BG inactivation without altering the O6-alkyltransferase activity (113). Additional BG-resistant MGMT mutants were identified from randomized MGMT libraries using BG and O6-alkylating agent selection schemes (114,115). Specific MGMT mutants were then shown to efficiently protect transduced human bone marrow progenitors from BG-mediated sensitization to chloroethylating (116) and methylating (117) agent toxicity (see Fig. 4). Davis et al. demonstrated that murine bone marrow progenitors transduced with the BG-resistant MGMT-G156A point mutant could be enriched in vivo with combined doses of BG and BCNU, and this enrichment protected transplant recipients from doses that were lethal to animals transplanted with control bone marrow cells (118). Other MGMT point mutants were also shown to protect mice from combined BG and temozolomide (119), or BG and BCNU treatments (120). These studies also demonstrated that selective enrichment occurred at the stem cell level.

The true potential for using MGMT mutants for stem cell selection was demonstrated by Davis et al. using nonmyeloablated transplant recipients (121). Transduced bone marrow CFU were enriched up to 47% in mice infused with as few as 5 x 104 transduced cells and selected with three rounds of BG and BCNU treatments. Enrichment to 97% was obtained when 1 x 105 cells were infused prior to drug treatments. In vivo enrichment for MGMT-transduced human CD34+ cord blood progenitors has also been achieved in NOD/SCID recipients preconditioned with irradiation (122) or a mild dose of BG and BCNU (123). The potency of MGMT-mediated stem cell selection has recently been demonstrated in a large animal canine model (124,125). Transgene-expressing granulocytes were enriched to over 98% in both animals studied (from 3 and 16% initial cell expression percentages), following incremental dosing with BG and temozolomide. Remarkably, polyclonal marking and long-term expression was achieved with an average of only one integration event per cell.

The use of MGMT point mutants to differentially protect the hematopoietic compartment while sensitizing tumors has also been reported in animal xenograft models (126-128). Clinical trials using gene transfer of MGMT have been proposed by several investigators, and one phase I trial in patients with advanced malignancies such as melanoma, sarcoma and other solid tumors is in progress (129). The objective of this trial is to protect bone marrow stem cells from the toxic effects of chemotherapy and select for MGMT-G156A transduced cells during treatment. This strategy is expected to result in less toxicity to bone marrow and blood cells while enriching for the number of genetically altered drug resistant stem cells over time, perhaps even from undetectable levels. The hypothesis of this study is based on preclinical data that shows this gene can provide HSCs with more than 500-fold survival advantage compared with HSCs not carrying the gene. In this clinical protocol, peripheral blood stem cells are collected from patients, exposed to a MMLRV containing the G156A MGMT gene in the laboratory and immediately reinfused into the patient. Starting 2 d prior to cell infusion and every 6 wk thereafter, patients are treated with BG and BCNU to inhibit tumor growth and provide selective resistance to the stem cells carrying the gene. To date, 5 patients have been enrolled and the level of gene transfer into the stem cells before infusion ranged from 11 to 36%. No complications related to cell infusion or chemotherapy administration have been observed. In one patient, evidence of genetically altered cells was observed by molecular analysis in the bone marrow 5 wk after the infusion and prior to the chemotherapy treatment. Although preliminary, these results indicate that infusion of HSCs transduced with retroviral mutant MGMT is feasible and safe. This is an important trial because stem cell selection with MGMT may be useful in other planned clinical applications including the use of MGMT in combination with therapeutic genes to correct for genetic disorders and the use of MGMT stem cell protection during allogeneic transplantation as a selection strategy to encourage donor engraftment. Because the theoretical risks of oncogenesis associated with oncoretroviral vector integration has now been observed in a successful human gene therapy trial for SCID XI (130), the next generation of gene therapy trials will likely incorporate lentiviral vectors. These vectors are thought to have a decreased risk of insertional oncogenesis, and their increased stem cell transduction efficiencies indicate that lower multiplicities of infection (MOIs) may be used to achieve the same endpoint.

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