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. Despite ultimate plans to test the combination of depsipeptide and a resistance reversal agent, we have found that depsipeptide is effective in cutaneous and peripheral T cell lymphomas. We are conducting a multi-institutional Phase II trial in these diseases in order to determine precise response rates and duration of response. To date, both complete and partial responses are observed in roughly 50% of patients with cutaneous T cell lymphoma. The durability of these responses suggests that this agent will become a meaningful new approach to the therapy of this difficult disease. In addition, a Phase I trial with depsipeptide in a new, more dose intense schedule is now accruing patients. 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 tariquidar (XR9576) has been approved by the IRB and will be opening soon. This trial will combine XR9576 with taxotere in order to study to pharmacokinetic interaction of that combination. As a strategy for overcoming drug resistance, the development of P-glycoprotein inhibitors has been troubled by drug interactions and limited clinical success. Our goal is to develop a safe combination that can be tested in patients whose tumors overexpress the P-glycoprotein drug transporter. We have worked with the PET imaging department to develop an imaging agent, 94Tc-sestamibi that can detect tumors with Pgp overexpression. An imaging trial testing this agent with tariquidar (XR9576) has been IRB approved. We plan to study renal cell carcinomas with this imaging agent in order to determine the contribution of P-glycoprotein to drug resistance in that disease. 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, and have data suggesting that the Q141K SNP confers a reduced transport capability. In addition, we are studying the process by which this half-transporter dimerizes for its function. We have mutated multiple conserved residues seeking sites of potential dimerization. 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. The clinical/laboratory focus for the Molecular Therapeutics Section also includes laboratory support for our depsipeptide trials, as noted above. 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. This gene induction serves as a biomarker of drug effect, as well as indicating a potential mechanism of clinical 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 ABCG2. 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.
