The Mechanism of Reactivation of Tumor Suppressor Genes by Inhibitors of DNA (Cytosine-5) Methyltransferase
The primary focus of my research program is the development of novel DNA (cytosine-5) methyltransferase (MT'ase) inhibitors for cancer therapy. An interest in both fluoropyrimidines and DNA methylation led to al collaboration with Dr. Steven Smith and Dr. Lawrence Sowers that demonstrated for the first time a covalent complex of human MT'ase with an oligonucleotide containing 5-fluoro-2'-deoxycytidine (FdCyd) and the methyl group from the cofactor AdoMet. We have continued to study the metabolism of FdCyd to FdCTP and the effects of its incorporation into cellular DNA on DNA methylation and gene expression.
Although FdCyd is rapidly degraded to FdUrd by cytidine/deoxycytidine deaminiase, this degradation can be inhibited by tetrahydrouridine (THU) without inhibiting its activation by deoxycytidine kinase. Thus, the effects of FdCyd on MT'ase and other potential intracellular targets can be examined in the presence of THU. Detailed molecular studies of the gene activation profile of FdCyd have continued in my laboratory, and preclinical toxicology studies of the FdCyd/THU combination were performed utilizing the Animal Resources and Pathology core facilities. The preclinical toxicology and other safety studies led to approval for the initial clinical studies of an Investigational New Drug (IND #54,223, 5-fluoro-2'-deoxycytidine) by the United States Food and Drug Administration (FDA).
The first Phase I clinical trial of FdCyd with THU in humans is currently being conducted at City of Hopeas described below. With funding from the NCI and the Department of Defense Breast Cancer Research Program, we have initiated studies to determine if FdCyd can alter DNA methylation and inhibit growth of human breast cancer xenografts in mice and to compare the effects of FdCyd with the effects of 5-Aza-2'-deoxycytidine (DAC). DAC is a drug with demonstrated activity in the treatment of acute myeloid leukemia and likely therapeutic utility in other leukemias and myelodysplasia. The rationale for using DAC as an antineoplastic agent is based on its ability to inhibit MT'ase and, thereby, to increase the expression of both tumor suppressors and tumor antigens silenced by DNA methylation. Thus, a detailed understanding of the mechanism by which FdCyd, DAC, and other inhibitors of MT'ase result in a decrease in the methylation of cytosines in DNA is critical for the future development of this class of agents.
Correlative Laboratory Studies Supporting the Clinical Development of FdCyd
In collaboration with Robert Morgan, Jr., M.D., and other clinical investigators we are currently conducting a Phase I trial of the combination of FdCyd, and THU, patients with advanced solid tumors. In addition to pharmacokinetic studies of FdCyd in plasma, we are conducting correlative studies in my laboratory in conjunction with the Phase I trial. The studies include the evaluation of the FdCyd-induced changes in DNA methylation in peripheral blood mononuclear cells and the induction of fetal hemoglobin expression during treatment as surrogate markers for the biological activity of FdCyd.