Direct reversal of DNA damage by mutant methyltransferase protein protectsmice against dose-intensified chemotherapy and leads to in vivo selection of hematopoietic stem cells

Direct reversal of O-6 adducts caused by chemotherapy agents is accomplishe d in mammalian cells by the protein O-6-methylguanine DNA methyltransferase (MGMT). Some tumors overexpress MGMT and are resistant to alkylator therap y, One future approach to treatment of these tumors may rely on concurrent pharmacological depletion of tumor MGMT with O-6-benzylguanine (6-BG) and p rotection of sensitive tissues, such as hematopoietic stem and progenitor c ells, using genetic modification with 6-BG-resistant MGMT mutants. We have used retroviral-mediated gene transfer to transduce murine hematopoietic bo ne marrow cells with MGMT point mutants showing resistance to 6-BG depletio n irt vitro. These mutants include proline to alanine and proline to lysine substitutions at the 140 position (P140A and P140K, respectively), which s how 40- and 1000-fold resistance to 6-BG compared with wild-type (WT) MGMT, Lethally irradiated mice were reconstituted with murine stem cells transdu ced with murine stem cell virus retrovirus expressing each mutant, WT MGMT, or mock-infected cells and then treated with a combination of 30 mg/kg 6-B G and 10 mg/kg 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or with 40 mg/kg BCNU alone, Compared with mice treated with BCNU alone, significant myeloi d toxicity and death occurred in mice reconstituted with mock-infected or W T MGMT (<0.1 probability of survival) or the P140A mutant (0.13 probability of survival) MGMT cDNAs, In contrast, after an initial period of mild cyto penia, mice reconstituted with the P140K mutant (0.83 probability of surviv al) recovered nearly normal blood counts, even during continued treatment. Comparison of peripheral blood neutrophils after completion of 5 weekly tre atments in these animals showed a direct correlation between the treatment and in vivo selection for progeny of transduced cells (pretreatment, simila r to 8-12% transduced cells; no treatment, similar to 6% transduced cells; BCNU only, 51% transduced cells; 6-BG/BCNU, 93% transduced cells), To deter mine whether this selection occurred at the stem cell level, bone marrow fr om each treatment group was infused into secondary recipients, Whereas anim als that received bone marrow from untreated animals reconstituted with 2% transduced cells, animals receiving marrow from 6-BG/BCNU-treated animals r econstituted with 94% transduced cells, demonstrating nearly complete selec tion for stem cells in the primary animals. Mice reconstituted with marrow from animals treated with BCNU only demonstrated 23% transduced cells, cons istent with partial selection of stem cells in the primary mice. The levels of transduced cells also correlated with survival during a second round of intensive combination chemotherapy (probability of survival: 6-BG/BCNU, 1. 0; BCNU alone, >0.70; no treatment, <0.1). These data demonstrate that muta nt MGMT expressed in the bone marrow can protect mice from time- and dose-i ntensive chemotherapy and that the combination of 6-BG and BCNU leads to un iform selection of transduced stem cells in vivo in mice.