The Molecular Therapeutics Section conducts both clinical and laboratory studies in drug resistance. Our clinical trials this year focused on the effects of depsipeptide in the treatment of cutaneous and peripheral T cell lymphoma. While we plan to study the ability of a Pgp antagonist to overcome resistance to depsipeptide in solid tumors, we have found that depsipeptide is effective in cutaneous and peripheral T cell lymphomas. We are conducting a Phase II trial in these diseases in order to determine precise response rates and duration of response. In addition, a Phase I trial with depsipeptide in a new, more dose intense schedule has undergone both IRB and CTEP review and is in the final stages of approval. One goal of this study will be to evaluate gene induction. Patients with thyroid cancer will be identified for potential enrollment on the study, since we have found in laboratory studies that depsipeptide induces expression of NIS, the sodium iodide symporter responsible for radio-iodine uptake in thyroid cells. Finally a new trial with the Pgp antagonist XR9576 is being written in order to continue our studies with that agent. Our ultimate goal is to combine depsipeptide with XR9576. However, we plan an intermediate study with XR9576 with taxotere in order to study to pharmacokinetic interaction of that combination. Our laboratory continues to be interested in increasing our understanding of non-P-glycoprotein mechanisms of drug resistance; the mechanism of action of depsipeptide FR901228, and the intrinsic mechanisms of resistance in renal cell carcinomas. Finally, our laboratory is dedicated to providing translational support for the clinical trials run in our section. Laboratory studies previously identified overexpression of a new non-P-glycoprotein ATP-dependent transporter, termed MXR1 or ABCG2 for mitoxantrone resistance gene in a doxorubicin-resistant breast cancer subline and a mitoxantrone-resistant colon cancer subline. This gene was cloned and characterized. MXR has particular activity for mitoxantrone, topotecan and CPT 11. The gene is localized to chromosome 4, is amplified in the breast cancer cells and rearranged in the colon cancer cells. The gene encodes a predicted half transporter molecule. Using a polyclonal antibody raised against a peptide fragment predicted from the sequence, immunoblot analysis has demonstrated overexpression of a 72 kDa protein in the two sublines. Immuno-localization studies suggest that MXR is localized at the plasma membrane. Efflux cannot be reversed by standard P-glycoprotein antagonists; however, two agents able to inhibit MXR-mediated resistance are under study with an aim to develop them for clinical trials. Evaluation of the substrate specificity of MXR revealed that in two selected cell lines, mutation at amino acid 482 markedly altered the substrate profile. Both R482G and R482T mutations resulted in the addition of doxorubicin, daunorubicin, and rhodamine as substrates. We examined 20 other resistant cell lines for the presence of this mutation and found it overexpressed without mutation in those cell lines. In addition, we sequenced 90 DNAs from the Coriell Repository and identified 3 nonsynonmyous SNPs. We are currently evaluating functional differences due to those SNPs. In addition, we are studying the process by which this half-transporter dimerizes for its function. We have expanded laboratory studies with the depsipeptide FR901228. The compound is a histone deacetylase inhibitor and induces a mitotic arrest in susceptible cells. Both a G1 and a G2 arrest are identified; we have determined that the G1 arrest is p21-dependent. The G2 arrest occurs in prometaphase following chromosomal condensation, and chromosomes do not attach properly to the mitotic spindle. Currently, the molecular basis of these abnormalities is being sought. Translational studies in conjunction with a Phase II clinical trial of depsipeptide have confirmed increased histone acetylation in normal and malignant circulating mononuclear cells. The increased histone acetylation is accompanied by induction of specific gene expression, also confirmed in malignant circulating cells. The clinical/laboratory focus for the Molecular Therapeutics Section includes laboratory support for our depsipeptide trials, as noted above. The Phase I study of depsipeptide identified the dose limiting toxicities as fatigue and thrombocytopenia. EKG changes and a cardiac arrythmia were also noted. Depsipeptide is transported very efficiently by Pgp, making it likely that the drug will be ineffective in patients whose tumors express Pgp. Thus, studies are planned combining a Pgp antagonist with depsipeptide. In addition, we have developed an assay to determine the effect of depsipeptide on tumor tissue, measuring the change in histone acetylation in tumor and in peripheral T cells, following treatment with depsipeptide. Both malignant and normal circulating mononuclear cells show increased histone acetylation following treatment with depsipeptide. In addition, induction of gene expression has been measured following depsipeptide in patient samples. The Pgp/MDR1 gene is consistently induced, indicating that depsipeptide induces its own mechanism of drug resistance. In summary, the focus of the Molecular Therapeutics Section has been the identification of mechanisms of drug resistance, and approaches to overcome them. As long as chemotherapy remains in the anticancer armamentarium, drug resistance will be a problem. Studies have shown that many of the agents developed for new molecular targets are susceptible to the same drug efflux mechanisms identified for the old agents. For example, the newly approved topoisomerase I inhibitors, Topotecan and CPT 11 are substrates for MXR, the transporter encoded by the new mitoxantrone resistance gene. Depsipeptide, so effective in cutaneous and peripheral T cell lymphoma induces MDR1 in normal and malignant cells, thereby inducing its own mechanism of resistance. Thus, resistance modulation continues to be a critical area for cancer treatment.
