Delta MGMT-Transduced Bone Marrow Infusion Increases Tolerance to O6-Benzylguanine and 1,3-Bis(2-chloroethyl)1-nitrosourea and Allows Intensive Therapy of 1,3-Bis(2-chloroethyl)-1-nitrosourea-Resistant Human Colon Cancer Xenografts

Abstract

O6-Benzylguanine (BG) is a potent inhibitor of the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT), and sensitizes tumors to BCNU in vitro and in xenografts. The combination of BG and BCNU is now undergoing phase I clinical testing. The maximally tolerated dose of BC NU given after BG is expected to be lower then the doses tolerated as a single agent owing to BG sensitization of hematopoietic progenitors. We have previously shown that retroviral expression of G156A mutant MGMT ( Delta MGMT) in mouse and human marrow cells results in significant BG and BCNU resistance. In this study we evaluated the effect of Delta MGMT transduced marrow infusion on the therapeutic index of multiple BG and BCNU treatments in tumor-bearing nude (nu/nu athymic) mice. Prior to subcutaneous implantation of BCNU-resistant SW480 human colon cancer cells, cohorts of mice were given intraperitoneal injections of nonablative doses of BG (30 mg/kg) and BCNU (10 mg/kg, one-half of the LD10) and then infused with 1-2 x 10 6 isogeneic Delta MGMT (n = 29 mice) or lacZ-transduced (n = 20 mice) marrow cells. The xenograft-bearing mice were treated with multiple cycles of BG (30 mg/kg) and BCNU (10-25 mg/kg). After three cycles, Delta MGMT mouse bone marrow was repopulated with CFU containing the provirus, and demonstrated a 2.7-fold increase in AGT activity and a 5.5-fold increase in BCNU IC90 compared with LacZ mice. After five cycles, the BCNU IC90 of CFU cells increased nine-fold over control cells, indicating selective enrichment of CFU precursor cells expressing high levels of Delta MGMT. Starting with the third cycle of therapy, tolerance to BG and BCNU was significantly improved in Delta MGMT mice compared with LacZ mice, as evidenced by preserved peripheral blood counts, bone marrow cellularity, and CFU content 1 and 2 weeks posttreatment and a significantly higher survival rate. Xenograft growth was significantly delayed in mice tolerating multiple cycles and higher dose intensity of BG and BCNU as compared with mice receiving less intensive therapy. We conclude that Delta MGMT-transduced marrow cells can improve the therapeutic index of BG and BCNU by selectively repopulating the marrow and providing significant marrow tolerance to this combination, allowing intensive therapy of a BCNU-resistant tumor.