Resistance to chemotherapeutic drugs is a major obstacle in the treatment of leukemia and multiple myeloma. We have previously found that myeloma and leukemic cells in transition from low-density log phase conditions to high-density plateau phase conditions export substantial amounts of endogenous topoisomerase II alpha from the nucleus to the cytoplasm. In order for topoisomerase-targeted chemotherapy to function, the topoisomerase target must have access to the nuclear DNA. Therefore, the nuclear export of topoisomerase II alpha may contribute to drug resistance, and defining this mechanism may lead to methods to preclude this avenue of resistance. We have identified nuclear export signals for topoisomerase II alpha at amino acids 1017-1028 and 1054-1066, using FITC-labeled BSA-export signal peptide conjugates microinjected into the nuclei of HeLa cells. Functional confirmation of both signals (1017-1028 and 1054-1066) was provided by transfection of human myeloma cells with plasmids containing the gene for a full-length human FLAG-topoisomerase fusion protein, mutated at hydrophobic amino acid residues in the export signals. Of the six putative export signals tested, the two sites above were found to induce export into the cytoplasm. Export by both signals was blocked by treatment of the cells with leptomycin B, indicating that a CRM-1-dependent pathway mediates export. Site-directed mutagenesis of two central hydrophobic residues in either export signal in full-length human topoisomerase blocked export of recombinant FLAG-topoisomerase II alpha, indicating that both signals may be required for export. Interestingly, this pair of nuclear export signals (1017-1028 and 1054-1066) also defines a dimerization domain of the topoisomerase II alpha molecule.

Purpose: A phase II trial of the novel camptothecin karenitecin (BNP1350) was conducted to determine its efficacy and tolerability in patients with metastatic melanoma. Patients were biopsied to determine topoisomerase expression at baseline and response to therapy. Patients and Methods: Eligible patients had metastatic melanoma with up to three prior chemotherapy and/or any number of immunotherapy regimens.Treatment consisted of an i.v. infusion of 1mg/m 2 karenitecin daily for 5 days with cycles repeated every 3 weeks. Fine-needle aspiration biopsies were done before treatment and on day 3 to determine topoisomerase expression from patients' tumors. Results: Forty-three patients were evaluable for response and toxicity. Most patients (72%) had stage M1C disease and were previously exposed to chemotherapy (56%). The investigational agent was well tolerated with limited gastrointestinal side effects or fatigue. The major toxicity seen was reversible noncumulative myelosuppression. One patient had a complete response after 11months of therapy. No partial responses were seen, but 33% of the patients had disease stabilization lasting z3 months. Topoisomerase I, IIA, and IIB expression and localization were determined in a subset of patients.Topoisomerase I expression was highest, followed by topoisomerase IIB and topoisomerase IIA. Conclusion: Karenitecin was a well-tolerated investigational agent in this phase II study; side effects were generally mild and mostly hematologic. Karenitecin has significant activity in metastatic melanoma. Melanoma metastases express high levels of topoisomerase I. We did not observe any compensatory increase in topoisomerase II upon treatment with karenitecin.

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