MODULATION OF DNA REPAIR TO ENHANCE CHEMOTHERAPY

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Overwhelming evidence has demonstrated that the major mechanism of tumor cell resistance to the chloroethylnitrosoureas (CENU) results from the DNA repair activity of 06-methylguanine DNA methyltransferase (MGMT). This DNA repair protein is thought to protect cells from the cytotoxic DNA inter- strand crosslink (ISC) produced by the CENU by removing chloroethyl adducts from the 0-6 position of guanine before these adducts can rearrange to form a lethal crosslink. Studies conducts over the previous eight years have demonstrated that this DNA repair system can be temporarily inhibited by a variety of biochemical strategies including pre-incubation of tumor cells with DNA methylating agents such as streptozotocin (STZ) which product the natural substrate of MGMT, 06- methylguanine. Repair of this lesion depletes the tumor cell of MGMT due to MGMT's suicide repair activity. In addition, the free bases 06- methylguanine (MG) and 06-benzylguanine (6-BG) can also deplete cells of MGMT activity and subsequently sensitize tumor cells to treatment with BCNU. Recently, we have demonstrated that STZ combined with 6-BG and BCNU can produce a prolonged sensitization of resistant tumor cells in vitro and in xenograft tumors in vivo. Currently 6-BG plus BCNU is being tested in Phase I clinical trials at other institutions and 6-BG plus STZ plus BCNU will be tested at this institution. However, biochemical modulation strategies are not selective for tumor cells over normal cells. In this Project we propose to develop gene therapy strategies that will allow the protection for critical normal tissues while modulating tumor resistance to the CENU. The specific aims are: 1. To develop a in vitro and in vivo a 6-BG continuous exposure schedule using a bolus of 6-BG followed by a low dose continuous exposure to maximize the duration of MGMT depletion. 2. To determine the ability of transduced DNA repair genes to protect mouse and human cells from the cytotoxic killing by BCNU when combined with pretreatment regimens containing 6-BG or 6-BG plus STZ. 3. Using the NOD/SCID mouse model we will determine whether 6-BG (with and without STZ) and BCNU chemotherapy can be selectively administered to xenograft tumors in mice transplanted with human marrow. 4) To analyze MGMT activity and DNA cross linking in tumor cells from patients with relapsed B-cell malignancies from Phase 1 trials examining continuous infusion of 6-BG, or 6-BG in combination with STZ and BCNU.