Reciprocal relationship between O6-methylguanine-DNA methyltransferase P140K expression level and chemoprotection of hematopoletic stem cells

Retroviral-mediated delivery of the P140K mutant 06 -methylguanine-DNA methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSC) has been proposed as a means to protect against dose-limiting myelosuppressive toxicity ensuing from chemotherapy combining O(6)-alkylating agents (e.g., temozolomide) with pseudosubstrate inhibitors (such as O(6) -benzylguanine) of endogenous MGMT. Because detoxification of O(6)-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 garnma-retroviral vectors that express a range of MGMTP 140K activity, we show that MGMTP40K expression by weaker cellular promoter/enhancers is sufficient for in vivo protection/ selection following treatment with O(6)-benzylguanine/temozolomide. Conversely, the highest level of MGMTP140K activity did not promote efficient in vivo protection despite mediating detoxification of O(6)-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 MGMT P140K, but not wild-type MGMT. This proliferation defect correlated with increased localization of MGMT P 140K to the nucleus/chromatin. These data show that very high expression of MGMTP 140K has a deleterious effect on cellular proliferation, engraftment, and chernoprotection. These studies have direct translational relevance to ongoing clinical gene therapy studies using P140K MGMT, whereas the novel mechanistic findings are relevant to the basic understanding of DNA repair by MGMT.