A major obstacle to effective treatment with alkylating agents in cancer is the presence of elaborate mechanisms of DNA repair. For instance, methylating agents, such as temozolomide (TMZ) form O6-methylguanine (O6/mG), 7-methylguanine (N7mG), and 3-methyladenine (N3mA) DNA adducts, repaired by at least two mechanisms and a third mechanism that mediates cell death. The O6/mG DNA adduct is cyotoxic when mismatch repair (MMR) triggers apoptosis by aberrant repair but is successfully repaired by O6-methylguanine DNA-methyltransferase (MGMT). On the other hand, N7mG, the dominant lesion formed by methylating agents, and N3mA, are removed by the base excision repair (BER) pathway. Efficient BER repair minimizes the impact of these lesions in normal and tumor cells, perhaps explaining why this pathway has received little investigation as a therapeutic target. The investigators recent work found that when BER is disrupted, these N-methylated DNA adducts become highly cytotoxic. BER was blocked by methoxyamine binding of the basic [AP] site sugar aldehyde, preventing DNA backbone cleavage by Ap-endonuclease (APE) in t he BER pathway. When combined with TMZ, MX sensitized colon cancer and glioma cell lines and xenografts regardless of MMR and MGMT activity. These successful studies enabled the investigators to submit and receive approval for MX to be developed by the NCI-RAID program as a """"""""first in its class"""""""" BER inhibitor with potential therapeutic value. The investigators will now address the following Aims: First, the exact mechanism of killing by persistent MX-bound AP sites in glioma and colon cancer. Recent data suggest that MX-bound AP sites may poison topoiosomerase II (topo II). The hypothesis to be tested is that MX-AP sites, when located within a topo I/II DNA cleavage site, act as topoisomerase poisons and stimulate enzyme-mediated DNA scission. Second, since susceptibility to BER disruption may be modulated by differential tumor cell reliance on BER short patch (single nucleotide) rather than BER long patch (2 to 15 nucleotides) repair, these sub-pathways will be analyzed and disrupted in tumor cell lines. Third, the role of mitochondrial DNA damage and BER repair inhibition in apoptotic signaling will be analyzed since recent data indicate that this organelle may mediate DNA damage-induced cell death. Finally, studies will be performed that optimize dosing of MX and TMZ in gliomas and colon cancers to define the therapeutic efficacy of BER disruption with correlative studies of the markers identified in the earlier aims. These studies will provide evidence that interruption of BER with molecules that bind to the basic site synergistically improve the therapeutic efficacy of DNA damaging agents, promoting BER as a new cancer therapeutic target.
