The P140K mutant of human O6-methylguanine-DNA-methyltransferase (MGMT) confers resistance in vitro and in vivo to temozolomide in combination with the novel MGMT inactivator O6-(4-bromothenyl)guanine

The O6‐methylguanine‐DNA‐methyltransferase (MGMT) inactivator O6‐benzylguanine (O6‐beG) is currently under clinical investigation as a potential tumour‐sensitising agent. In clinical trials its use has been associated with increased myelotoxicity and a reduced maximum tolerated dose (MTD) for BCNU. Thus the concept of myeloprotection by gene therapy with an O6‐beG‐insensitive mutant of MGMT is soon to be tested. Recently, an alternative inactivator has been described (O6‐(4‐bromothenyl)guanine, PaTrin‐2), which shows potential advantages over O6‐beG in terms of higher activity against wild‐type MGMT and oral formulation. The use of PaTrin‐2 has also been associated with increased myelotoxicity in clinical trials and thus PaTrin‐2 may also be a candidate for use in conjunction with mutant MGMT gene transfer in genetic chemoprotective strategies. However, its activity against mutant MGMTs has not been reported. We show here that the P140K mutant of MGMT is highly resistant to inactivation by PaTrin‐2. Furthermore, we show that a human haemopoietic cell line (K562) transduced with a retroviral vector encoding MGMTP140K is highly resistant to the cytotoxic effects of PaTrin‐2 in combination with the methylating agent temozolomide, and that cells expressing MGMTP140K can be effectively enriched in vitro following challenge with this drug combination. Finally, we show that animals reconstituted with bone marrow expressing MGMTP140K exhibit haemopoietic resistance to PaTrin‐2/temozolomide, which results in in vivo selection of gene‐modified cells. All of these effects were comparable to those also achieved using O6‐beG/temozolomide. Thus our data show that MGMTP140K is a suitable candidate for chemoprotective gene therapy where PaTrin‐2 is being used in conjunction with temozolomide. Copyright © 2005 John Wiley & Sons, Ltd.