WO2012075484A2 - Novel drug targets to overcome de novo drug-resistance in multiple myeloma - Google Patents

Abstract

Topoisomerase II alpha (topo IIα) is exported from the cell nucleus in human myeloma cells by a chromosome-maintenance protein-1 (CRM1)-dependent mechanism, resulting in topo II inhibitor resistance. The nuclear export signal (NES) of topo IIα is unique, making it a potential target for small molecule inhibitors. Small molecules NES inhibitors were identified, which inhibited binding of topo IIα to the export receptor CRM1. Inhibition was specific to topo IIα as p53 trafficking was unaffected along with topo IIα protein expression and function (decatenation). These topo IIα-specific nuclear export inhibitors may potentially lead to a new approach in circumventing drug resistance in multiple myeloma. The compounds provide a protocol for treating multiple myeloma or an oncogenic disease. Further, the topoisomerase II nuclear export signal inhibitor may be combined with a topoisomerase II inhibitor.

Description

NOVEL DRUG TARGETS TO OVERCOME DE NOVO

DRUG-RESISTANCE IN MULTIPLE MYELOMA

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent Application No. 61/419,536, entitled "Novel Drug Targets to Overcome De Novo Drug-Resistance in Multiple Myeloma", filed on December 3, 2010, and U.S. Patent Application No. 13/159,016, entitled "Nuclear Export Inhibitors of Topoisomerase II Alpha", filed on June 13, 201 1 , which claims priority to International Application, Serial Number PCT/US2009/067922, entitled "Nuclear Export Inhibitors of Topoisomerase Ma" filed December 14, 2009, which claims priority to U.S. provisional patent application No. 61/122,098, entitled "Small Molecule Inhibitors of Nuclear Export of Topoisomerase II Alpha for the Treatment of Human Cancer", filed December 12, 2008, the contents of which are herein incorporated by reference.

FIELD OF INVENTION

This invention relates to cancer treatment. Specifically, the invention provides a method of treating multiple myeloma and other oncogenic diseases using novel compounds. BACKGROUND OF THE INVENTION

Multiple myeloma (MM) is a tumor of terminally-differentiated plasma cells. During development, genetic abnormalities in the forming plasma cells can result in malignant MM cells, which travel through the bloodstream and deposit in bone marrow and other organs, causing the variable symptoms of MM. Initial transformative are thought to occur post- germinal, as suggested by the hypermutation of IGV genes. Collections of abnormal cells accumulate in bones, where they cause bone lesions and elevated calcium levels from myeloma cell release of IL-6, and in the bone marrow where they interfere with the production of normal blood cells, resulting in anemia and impaired immune response. Most cases of myeloma also feature the production of a paraprotein, an abnormal antibody that can cause kidney problems and interferes with the production of normal antibodies leading to immunodeficiency. The disease develops in 1-4 per 100,000 people per year. It is more common in men, and is twice as common in African American populations than it is in Caucasians. With conventional treatment, the prognosis is 3-4 years, which may be extended to 5-7 years with advanced treatments. Multiple myeloma is the second most common hematological malignancy (13%) and constitutes 1% of all cancers. MM is diagnosed with blood tests, such as electrophoresis, peripheral blood smear, and microscopic examination of the bone marrow. Standard treatments include lenalidomide, bortezomit, thalidomite, doxirubicin, melphalan, cyclosphamide, prednisone, or dexamethasone. The disease is thought to progress due to dysregulation of the apoptotic mechanisms in plasma cells, which also is likely responsible for the resultant chemoresistance.

MM still remains an incurable disease despite improved treatment regimens that include bortezomib, lenalidomide and thalidomide. Other treatments that are thought to cause remission include steroids, chemotherapy, and stem cell transplants. Drug resistance, including resistance to topoisomerase II (topo II) inhibitors, is a major obstacle in the treatment of multiple myeloma. Cell adhesion-mediated drug resistance and stromal cell adherence are important parameters in the local bone marrow environment in patients with multiple myeloma and appear to be major determinants of drug resistance (Hazlehurst, et al., Reduction in drug-induced DNA double-strand breaks associated with betal integrin- mediated adhesion correlates with drug resistance in U937 cells. Blood 2001 ; 98:1897-903; Hazlehurst LA, et al., Cell adhesion to fibronectin (CAM-DR) influences acquired mitoxantrone resistance in U937 cells. Cancer Res 2006; 66:2338-45). Additionally, human multiple myeloma cell density is a determinant of sensitivity to topo II inhibitors (Valkov, et al., Cell density-dependent VP-16 sensitivity of leukaemic cells is accompanied by the translocation of topoisomerase llalpha from the nucleus to the cytoplasm. Br J Haematol 2000; 108:331-45; Turner JG, et al., Human topoisomerase llalpha nuclear export is mediated by two CRM-1- dependent nuclear export signals. J Cell Sci 2004; 1 17:3061-71 ). At increased cell densities, a considerable fraction of nuclear topo Ma (>90%) is exported to the cytoplasm, resulting in chemoresistance to VP-16 and doxorubicin. This appears to occur both in human myeloma cell lines and in CD138* cells isolated from patients with multiple myeloma. Intracellular mislocalization of tumor suppressor or nuclear drug target has been shown to decrease the effectiveness of antineoplastic agents, such as with the tumor suppressors and chemotherapeutic targets p53, APC/B-catenin, FOXO, p21^clp1, p27^KIP1, and topoisomerases I and II (Turner & Sullivan, CRM1-mediated nuclear export of proteins and drug resistance in cancer. Curr Med Chem 2008; 15: 2648-55). For proteins greater than 60 kDa to be exported to the cytoplasm, they must be transported through the nuclear-pore complex (Cook, et al., Structural biology of nucleocytoplasmic transport. Annu Rev Biochem 2007; 76: 647-71 ). This transport mechanism involves the binding of chromosome-maintenance protein-1 (CRM1 ) to a leucine-rich nuclear export signal (NES) on the target protein. This complex is then transported through the nuclear pore into the cytoplasm (Dong, et al., Structural basis for leucine-rich nuclear export signal recognition by CRM1. Nature 2009; 458: 1136-41).

In multiple myeloma (MM), de novo drug resistance to topoisomerase (topo) II poisons occurs at high cell densities due to trafficking of topo lla from the nucleus to the cytoplasm where it is no longer in contact with the DNA and thus unable to induce cell death (Turner, et al., Human topoisomerase llalpha nuclear export is mediated by two CRM-1 -dependent nuclear export signals. J Cell Sci 2004; 117: 3061-71 ; Turner, et al. Human multiple myeloma cells are sensitized to topoisomerase II inhibitors by CRM1 inhibition. Cancer Res 2009; 69: 6899-905; Valkov, et al., Cell density-dependent VP-16 sensitivity of leukaemic cells is accompanied by the translocation of topoisomerase llalpha from the nucleus to the cytoplasm. Br J Haematol 2000; 108: 331 -45; Engel, et al., The cytoplasmic trafficking of DNA topoisomerase llalpha correlates with etoposide resistance in human myeloma cells. Exp Cell Res 2004; 295: 421- 31 ). Topo lla was previously demonstrated to be exported from the nucleus of human myeloma cells by a CRM 1 -dependent mechanism (Engel, et al., The cytoplasmic trafficking of DNA topoisomerase llalpha correlates with etoposide resistance in human myeloma cells. Exp Cell Res 2004; 295: 421-31 ), and the NES for topo lla was located to amino acids 1017- 28 (site A) and 1054-66 (site B) (Turner, et al., Human topoisomerase llalpha nuclear export is mediated by two CRM-1 -dependent nuclear export signals. J Cell Sci 2004; 1 17: 3061-71 ). Blocking nuclear export with a CRM1 inhibitor or by siRNA has been shown to sensitize drug- resistant MM cells to topo II poisons (Turner, et al., Human multiple myeloma cells are sensitized to topoisomerase II inhibitors by CRM1 inhibition. Cancer Res 2009; 69: 6899- 905).

However, use of CRM1 inhibition in cancer therapy has met with limited success. The first CRM1 inhibitor, leptomycin B, was found to efficiently inhibit nuclear export, but showed acute relative toxicities both in a human phase I trial (Newlands, et al., Phase I trial of elactocin. Br J Cancer 1996; 74:648-9) and in vitro. Leptomycin B in vitro studies found acute toxicity at concentrations <5 nmol/L for 1 hour. As such, new therapeutic targets are needed to further improve treatment outcomes of multiple myeolma.

SUMMARY OF THE INVENTION

Mislocalization of topo lla from the nucleus to the cytosol can render it ineffective as a chemotherapeutic target. High-density myeloma cells were shown to export topo lla into the cytoplasm, both in vivo and in vitro (Turner, et al., Human topoisomerase llalpha nuclear export is mediated by two CRM-1 -dependent nuclear export signals. J Cell Sci 2004; 1 17: 3061-71 ; Valkov, et al., Cell density-dependent VP-16 sensitivity of leukaemic cells is cytoplasm. Br J Haematol 2000; 108: 331-45; Engel, et al.Jhe cytoplasmic trafficking of DNA topoisomerase llalpha correlates with etoposide resistance in human myeloma cells. Exp Cell Res 2004; 295: 421-31 ). This nuclear export of topo lla contributes to drug resistance (Engel, et al., The cytoplasmic trafficking of DNA topoisomerase llalpha correlates with etoposide resistance in human myeloma cells. Exp Cell Res 2004; 295: 421-31), which is not due to differences in drug uptake, cell cycle, or total cellular topo lla protein levels. In addition, topo lla nuclear export has been shown to be CRM1 mediated, and topo lla protein has been found to contain two functional NES located at amino acids 1017-28 (site A) and 1054-66 (site B) (Turner, et al., Human topoisomerase llalpha nuclear export is mediated by two CRM-1 - dependent nuclear export signals. J Cell Sci 2004; 117: 3061-71 ). Export mediated by both signals was blocked by treatment of the cells with leptomycin B, indicating that a CRM1 - dependent pathway mediates export (Turner, et al., Human topoisomerase llalpha nuclear export is mediated by two CRM-1 -dependent nuclear export signals. J Cell Sci 2004; 1 17: 3061-71 ).

In MM, CRM1-mediated localization of topo lla to the cytoplasm prevents formation of lethal drug-induced topo II DNA strand breaks. Evidence suggests that agents blocking nuclear export of topo lla can reverse drug resistance to topo II poisons. A significant drawback of CRM1 inhibitors to prevent topo lla export is that they non-specifically block export of any nuclear protein exported by this pathway. To circumvent this shortcoming, computer- generated molecular modeling was used to screen the NCI database for compounds that selectively bind to the NES sites (1017-1028) of topo lla. Topo Ma's NES site A conforms to the hydrophobic amino acid motif for a NES (Engel, et al., The cytoplasmic trafficking of DNA topoisomerase llalpha correlates with etoposide resistance in human myeloma cells. Exp Cell Res 2004; 295: 421-31 ), but does not occur in any other human protein. In addition, this NES is in a pocket formed by the three-dimensional structure of the topo lla protein. These factors were used to identify drugs that exclusively block the NES of topo lla and not affect the CRM 1- dependent export of other nuclear proteins.

As such, a method is described herein for treating multiple myeloma or an oncogenic disease through administration of a therapeutically effective amount of a topoisomerase II nuclear export signal inhibitor to a patient. Exemplary topoisomerase II nuclear export signal inhibitors include NCI-36400, NCI-35847, NCI-80640, NCI-9138, NCI-155877, and NCI- 35024. In other variations of the invention, the topoisomerase II nuclear export signal inhibitors are

The topoisomerase II nuclear export signal inhibitor is optionally administered at a low micromolar concentration. Exemplary concentrations include 2 μΜ, 3 μ , 4 μΜ 5 μΜ, 10 μΜ, 15 μΜ, 20 μΜ, and 25 μΜ.

The identified topoisomerase II nuclear export signal inhibitors are expected to only interfere with Crm1 -mediated nuclear export of topo I la. Several of these small molecule inhibitors sensitized cells to the topo II poison doxorubicin, in a synergistic manner. In vitro cytotoxicity and apoptosis assays indicated that these drugs may be effective as single agents or in combination with currently used cancer drugs that target topo II. As such, the topoisomerase II nuclear export signal inhibitors are optionally combined with a topoisomerase II inhibitor, either concurrently or sequentially. Where the topoisomerase II nuclear export signal inhibitor and topoisomerase II inhibitor are administered sequentially, the topoisomerase II inhibitor is optionally administered about 4 hours after the topoisomerase II nuclear export signal inhibitor. For example, the topoisomerase II inhibitor may be administered 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, 3.5 hours, or 4 hours after the topoisomerase II nuclear export signal inhibitor. Non-limiting examples of the topoisomerase II inhibitor include amsacrine, etoposide, etoposide phosphate, teniposide, doxorubicin, VP-16, VM-26, mitoxantrone, epirubicin, daunomycin, mitoxantrone, and idarubicin. In specific variations, the tODOisomerase II inhibitor is administered in the range of about 0.01 to about 200 mg/m²/day for a human. For example, doxorubicin may be administered at 30-60 mg/m²/day, while daunomycin is administered at 45-90 mg/m²/day. The drug may also be administered at up to 500 mg/m²/day. Examples include 50 mg/m²/day, 75 mg/m²/day, 90 mg/m²/day, 93 mg/m²/day, 100 mg/m /day, 125 mg/m²/day, 150 mg/m²/day, 200 mg/m²/day, 250 mg/m²/day, 300 mg/m /day, 350 mg/m²/day, 400 mg/m²/day, or 450 mg/m²/day. Of particular note, the NES inhibitors may be used with doxol, doxorubicin, or eptosidoside, and synergistically act with some or all of these compounds.

Immunofluorescence microscopy and cell fractionation studies demonstrated that the NES inhibitors that target site A prevented nuclear export of topo I la. As such, therapeutically effective amount of a topoisomerase II nuclear export signal inhibitor is contacted on a cell to inhibit topoisomerase II export from a cell nucleus. The topoisomerase II nuclear export signal inhibitor is the NCI-36400, NCI-35847, NCI-80640, NCI-9138, NCI-155877, or NCI-35024. The topoisomerase II nuclear export signal inhibitor is optionally administered at 25 μΜ. Contacting cell with the topoisomerase II nuclear export signal inhibitor inhibited nuclear export in a topo ΙΙα-specific manner. Testing showed that the topoisomerase II nuclear export signal inhibitors did not prevent export of p53, a protein that is also exported by CR 1. Immunopreciptation data also indicated that the NES inhibitors prevented binding of topo lla to the nuclear export molecule CRM1. None of the compounds affected topo II decatenation activity or topo lla protein expression.

BRIEF DESCRIPTION OF THE DRAWINGS For a fuller understanding of the invention, reference should be made to the following detailed description, taken in connection with the accompanying drawings, in which:

Figure 1 is a graph showing CR 1 siRNA knockdown makes myeloma cells more sensitive to chemotherapy. Cell transfected with CRM1 siRNA, and treated with the topo II inhibitor doxorubicin (1 μΜ) more sensitive to topoisomerase inhibitors. Figure 2 is a computer image showing how the small molecule inhibitors interact with topo II via nuclear export signal (NES) sites. The structure of Saccharomyces cerevisiae topo II was used to create a homology model of human topo lla using the program PhyreA. Topo lla is displayed as a gray ribbon diagram, with the NES site A, NES site B, and the nuclear localization signal (NLS) indicated. Figure 3 is a computer image showing atomic homology model of human topoisomerase lla generated by the program Phyre. Molecular surfaces of the homology model of human topo lla is colored based on sequence similarity to S. cerevisiae topo II, calculated with clustalx and the Docker program (University of Florida, Gainesville, FL). Dark gray represents 100% sequence identity, and colors are graded from dark gray/ light gray/ medium gray which would represent no sequence identity. Nuclear export sites A(1) and B (2) were selected for high- throughput molecular docking to identify human topo I la-specific small molecules for functional assays. Figures 4(A)-(C) are illustrations of topo lla NES inhibitors (A) NCI-9138; (B) NCI-80640; and (C) NCI-155877.

Figure 5(A)-(D) are illustrations of topo lla NES inhibitors (A) NCI-35847; (B) NCI- 53040; (C) NCI-1 14057; and (D) NCI-82001.

Figure 6 is a graph showing nuclear localization of topo II sensitizes cells to doxorubicin. Human myeloma cells were grown for 16 hours at log or plateau densities, treated with 2 μΜ doxorubicin for 4 hours (n = 2). Apoptosis was determined by caspase 3 cleavage. Cells that maintained nuclear topo lla were more sensitive to topo I la-targeted chemotherapy.

Figure 7 is a graph overlain with compounds identified by NES docking scores. The compounds were obtained from NCI based on predicted docking scores for NES site A and site B. All compounds were further analyzed for chemical stability and structural integrity by both H-NMR and mass spec. After analysis of compounds, the following we were selected as the compounds that best prevented nuclear export of topo lla in high-density drug resistant myeloma cell (H929) and synergized with the topo II inhibitor doxorubicin (2 μΜ) (n=4), NCI- 35847, NCI-80640, NCI-9138 and NCI-15587 (25 μ ) (*P < 0.05). Figure 8 is a graph showing combination effects of topo II NES inhibitors and topo II inhibitors. NCI-9138 (25 μΜ) was combined with doxorubicin (2 μΜ), bortezomib (10 nM), dexamethasone (10 μΜ), lenalidomide (10 μΜ), melphalan (10 μ ), and topotecan (10 μΜ). Synergism was seen with doxorubicin.

Figures 9(A)-(E) are immunohistochemistry images of H929 cells treated with a topo II NES site A inhibitor. H929 cells were treated for 20 hours with (A) a 0.1 % DMSO vehicle, (B) NCI- 35847, (C) NCI-80640, (D) NCI-9138, or (E) NCI-155877 and stained with DAPI (light gray) and Alexa 594 (dark gray).

Figure 10(A)-(F) are images showing the present compounds prevent nuclear export of Topo lla. Human MM cells were treated with 25μΜ of NES inhibitor (A), (B) NCI-36400; (C) ,(D) NCI-35024; and (E), (F) NCI-35847 and placed at plateau densities (2 x 10⁶) for 20 hours. Cells were fixed and stained with DAPI (dark gray) and topo lla antibody Kis1 (light gray). Figure 11 is a molecular docking model of compound NCI-9138 to the NES of topo lla.

Figure 12 is a blot from co-immunoprecipitation of chromosome-maintenance protein-1 (CRM1 ) with topo lla. CRM-1 is blocked by the NES inhibitor NCI-9138 (50 μΜ for 20 hours).

Figures 13(A) and (B) are blots from nuclear (nuc)-cytoplasmic (cyto) fractionation of high- density MM cells (n = 2). Based on IC₅₀ and apoptosis data, 4 lead compounds were tested for anti-export activity of topo lla and compared to the anti-export activity of ratjadone C, a CRM1 -specific export inhibitor. Human myeloma RPMI-H929 cells were treated for 24 hours. Cells were then chemically separated into nuclear and cytoplasmic fractions. (A) Compounds NCI-35847 and NCI-9138 (25 μΜ) strongly blocked export of topo lla, while NCI-80640 and NCI-155877 had a smaller or little effect on topo II nucleus export inhibition. (B) Similar testing confirmed the inhibitory effect of NCI-9138 while NCI-32237, a control compound did not affect nuclear export of topo II. Ratjadone C, a CRM1-specific export inhibitor also prevented nuclear export.

Figure 14 is a blot of lead compounds tested to determine if nuclear export inhibition was specific to topo lla protein. p53 was cycled from the nucleus into the cytoplasm in MM cells cultured at log growth conditions. Cells grown at log-phase concentrations were treated with the lead NES inhibitors (25 μΜ) or a leptomycin control for 20 hours and chemically fractionated. Nuclei were assayed by SDS-PAGE Western blot analysis for p53 protein (anti- p53 (DO-1 ), Santa Cruz Biotech, Inc). NCI compounds 35847, 9138, and 155877 did not prevent export of p53. Lamin A C (anti-lamin A C, BD Pharmingen) is shown as a loading control.

Figure 15 is a graph showing synergy between the NES inhibitors (25 μΜ) with the topo II drug doxorubicin (2 μΜ) was measured by cleaved caspase 3 antibody staining and flow cytometry for high-density normal cells WI-38. Normal fibroblasts WI-38 cells were not sensitive to NES inhibitors and doxorubicin. Figure 16 is a graph showing synergy between the NES inhibitors (25 μΜ) with the topo II drug doxorubicin (2 μΜ) was measured by cleaved caspase 3 antibody staining and flow cytometry for high-density normal cells Flow 2000. Normal fibroblast Flow 2000 cells were not sensitive to NES inhibitors and doxorubicin.

Figure 17 is a graph showing synergy between the NES inhibitors (25 μΜ) with the topo II drug doxorubicin (2 μΜ) was measured by cleaved caspase 3 antibody staining and flow cytometry for high-density normal cells PBMCs. Normal PBMCs were not sensitive to NES inhibitors and doxorubicin. Figure 18 is a graph showing synergy between the NES inhibitors (25 μΜ) with the topo II drug doxorubicin (2 μΜ) was measured by cleaved caspase 3 antibody staining and flow cytometry for cancer cell line HL-60. HL-60 were not sensitive to NES inhibitors and doxorubicin.

Figure 19 is a graph showing synergy between the NES inhibitors (25 μΜ) with the topo II drug doxorubicin (2 μΜ) was measured by cleaved caspase 3 antibody staining and flow cytometry for cancer cell line KG1 a. KG1 a were not sensitive to NES inhibitors and doxorubicin.

Figure 20 is a graph showing synergy between the NES inhibitors (25 μΜ) with the topo II drug doxorubicin (2 μΜ) was measured by cleaved caspase 3 antibody staining and flow cytometry for cancer cell line MCF7. The breast cancer cell line MCF7 was sensitive to NES inhibitors and doxorubicin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

High-density myeloma cells export topo lla into the cytoplasm via a CRM1-mediated pathway, both in vivo and in vitro, contributing to drug resistance. Topo lla protein contains two functional NES located at amino acids 1017-28 (site A) and 1054-66 (site B). Targeting intracellular trafficking of proteins may sensitize cells to antitumor agents, and blocking topoisomerase II alpha (topo lla) trafficking from the nucleus to the cytoplasm in myeloma cells prevents drug resistance and enhances formation of lethal drug-induced topo II DNA strand breaks. Topoisomerase lla nuclear export inhibitors are described herein for treating multiple myeloma or an oncogenic disease, such as those listed below, through administration of the compounds to a patient, either alone or in combination with a topoisomerase inhibitor.

As used herein, "oncogenic disease" means a condition brought about by aberration of the cellular growth cycle and/or cellular differentiation. Oncogenic diseases include benign cancers, malignant cancers, and pre-cancerous lesions. The term cancer includes both solid tumors and non-solid cancers such as leukemias. Exemplary oncogenic diseases include multiple myeloma, breast cancer, ovarian cancer, testicular cancer, lung cancer, bronchogenic lung cancer, gastric cancer, soft tissue and osteogenic sarcomas, Ewing's sarcoma, malignant lymphoma (Hodgkin's and non-Hodgkin's), acute myeloblasts leukemia, acute lymphoblastic leukemia, and Kaposi's sarcoma.

Additionally, as used herein "patient", means members of the animal kingdom, including mammals, such as but not limited to, primates including humans, gorillas and monkeys; rodents, such as mice, fish, reptiles and birds. The patient may be any animal requiring therapy, treatment, or prophylaxis. The term treatment, as used in this definition only, is intended to mean that regiment described is continued until the underlying disease is resolved, whereas therapy requires that the regiment alleviate one or more symptoms of the underlying disease. Prophylaxis means that regiment is undertaken to prevent a possible occurrence, such as where a pre-cancerous lesion is identified.

The term "therapeutically effective amount" as used herein means that amount of active compound or pharmaceutical agent that elicits the biological or medicinal response in a tissue, system, animal or human that is being sought by a researcher, veterinarian, medical doctor or other clinician. In reference to cancers or other unwanted cell proliferation, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay occurrence and/or recurrence. An effective amount can be administered in one or more doses. In the case of cancer, the effective amount of the drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with the cancer.

The term "administration" and variants thereof (e.g., "administering" a compound) in reference to a compound of the invention means introducing the compound or a prodrug of the compound into the system of a patient in need of treatment. When a compound of the invention or prodrug thereof is provided in combination with one or more other active agents (e.g., a cytotoxic agent, etc.), "administration" and its variants are each understood to include concurrent and sequential introduction of the compound or prodrug thereof and other agents. A person of ordinary skill in the art may determine an appropriate dose of one of the instant compositions to administer to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most suitable for an individual patient and it will depend on a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy. The dosages disclosed herein are exemplary of the average case, though there will be instances where hiahpr or lower dosaae ranaes are merited, and such are within the scooe of this invention. In conducting the treatment method of the present invention, the anti-cancer agent or treatment can be administered in any effective manner known in the art, such as by oral, intravenous, intra-peritoneal, or subcutaneous, depending upon the type of cancer being treated, the type of anti-cancer agent or treatment being used, and the medical judgment of the prescribing physician as based, e.g., on the results of published clinical studies. When the anti-cancer agent or treatment is radiation or a radiochemical, the agent or treatment can be administered in any effective manner known in the art, as described briefly herein, above.

The anti-cancer agent or treatment can be administered with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, troches, hard candies, powders, sprays, creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, elixirs, syrups, and the like. Administration of such dosage forms can be carried out in single or multiple doses. Carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, etc. Oral pharmaceutical compositions can be suitably sweetened and/or flavored.

The anti-cancer agent or treatment can be combined together with various pharmaceutically acceptable inert carriers in the form of sprays, creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, and the like. Administration of such dosage forms can be carried out in single or multiple doses. A "pharmaceutically acceptable carrier" is a carrier, such as a solvent, suspending agent or vehicle that is suitable for use with humans and/or animals without undue adverse side effects (such as toxicity, irritation, and allergic response) commensurate with a reasonable benefit risk ratio. The carrier is provided for delivering the compound or compounds of the present invention to the animal or human, and may be liquid or solid based on the planned manner of administration, and may include liposomes or niosomes. As used herein, "carrier" includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.

Methods of preparing pharmaceutical compositions are also known in the art. In view of the teaching of the present invention, methods of preparing pharmaceutical compositions comprising both a anti-cancer agent or treatment and an NES inhibitor that sensitizes tumor cells to the pro-apoptotic effects of the anti-cancer agent or treatment will be apparent from the art, from other known standard references, such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18^th edition (1990). For oral administration of the anti-cancer agent or treatment or the NES inhibitor that sensitizes cells to the pro-apoptotic effects of the anti-cancer agent or treatment, tablets containing one or both of the active agents are combined with any of various excipients such as, for example, micro-crystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine, along with various disintegrants such as starch (and preferably corn, potato or tapioca starch), alginic acid and certain complex silicates, together with granulation binders like polyvinyl pyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tableting purposes. Solid compositions of a similar type may also be employed as fillers in gelatin capsules; preferred materials in this connection also include lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, active agents may be combined with various sweetening or flavoring agents, coloring matter or dyes, and, if so desired, emulsifying and/or suspending agents as well, together with such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.

For parenteral administration of either or both of the active agents, solutions in either sesame or peanut oil or in aqueous propylene glycol may be employed, as well as sterile aqueous solutions comprising the active agent or a corresponding water-soluble salt thereof. Such sterile aqueous solutions are preferably suitably buffered, and are also preferably rendered isotonic, e.g., with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes. The oily solutions are suitable for intra-articular, intramuscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

The term "treating", "treating cancer", "treatment", or "treatment of cancer" refers to obtaining beneficial or desired clinical results via administration of the present compounds to a mammal afflicted with a cancerous condition. Beneficial or desired clinical results refer to an effect that alleviates the cancerous condition by inhibiting of growth and/or metastasis of the cancer or killing the cancerous cells. This includes, but is not limited to, any one or more of: alleviation of one or more symptoms (such as tumor growth or metastasis), diminishment of extent of cancer, stabilized (i.e., not worsening) state of cancer, preventing or delaying spread (e.g., metastasis) of the cancer, preventing or delaying occurrence or recurrence of cancer, delay or slowing of cancer progression, amelioration of the cancer state, and remission (whether partial or total). The methods of the invention contemplate any one or more of these aspects of treatment. The term "about" or "approximately" as used herein means within ±5% of a given value or range. Other than in the operating examples, or unless otherwise expressly specified, all of the numerical ranges, amounts, values and percentages such as those for amounts of materials, times and temperatures of reaction, ratios of amounts, values for molecular weight (whether number average molecular weight ("M_n") or weight average molecular weight ("M_w"), and others in the following portion of the specification may be read as if prefaced by the word "about" even though the term "about" may not expressly appear with the value, amount or range. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present disclosure. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements. Furthermore, when numerical ranges of varying scope are set forth herein, it is contemplated that any combination of these values inclusive of the recited values may be used.

Kits for practicing the methods of the invention are further provided. By "kit" is intended any manufacture (e.g., a package or a container) comprising at least one reagent, e.g., an NES inhibitor of the invention. The kit may be promoted, distributed, or sold as a unit for performing the methods of the present invention. Additionally, the kits may contain a package insert describing the kit and methods for its use. Any or all of the kit reagents may be provided within containers that protect them from the external environment, such as in sealed containers or pouches.

Topoisomerase inhibitors are agents designed to interfere with the action of topoisomerase (topoisomerase I and II), which control changes in DNA structure by catalyzing the breaking and rejoining of the phosphodiester backbone of DNA strands during the normal cell cycle. Inhibitors are often divided according to which type of enzyme it inhibits. Topoisomerase inhibitors may be administered at any dose known effective in the art, such as in the range of about 0.01 to about 200 mg/m²/day for a human. The range used for topoisomerase II inhibitors may be altered based on the drug, patient, and disease, such as using 0.01 to about 10 mg/m²/day for a human. For example, doxorubicin may be administered at 30-60 mg/m²/day, while daunomycin is administered at 45-90 mg/m²/day. The treatment may be repeated on a daily, bi-weekly, semi-weekly, weekly, or monthly basis. In some embodiments, a treatment period may be followed by a rest period of from one day to several days, or from one to several weeks. In combination with an NES inhibitor, the NES inhibitor and the topoisomerase inhibitor may be dosed on the same day or may be dosed on separate days. Other cytotoxic, chemotherapeutic or anti-cancer agents, or compounds that enhance the effects of the topo inhibitors may be used without departing from this invention. Such compounds include, for example: alkylating agents or agents with an alkylating action, such as cyclophosphamide (CTX; e.g. CYTOXANtp), chlorambucil (CHL; e.g. LEUKERAN®), cisplatin (Cis P; e.g. PLATINOL®) busulfan (e.g. MYLERAN®), melphalan, carmustine (BCNU), streptozotocin, triethylenemelamine (TEM), mitomycin C, and the like; antimetabolites, such as methotrexate (MTX), etoposide (VP16; e.g. VEPESID®), 6- mercaptopurine (6MP), 6-thiocguanine (6TG), cytarabine (Ara-C), 5-fluorouracil (5-FU), capecitabine (e.g. XELODA®), dacarbazine (DTIC), and the like; antibiotics, such as actinomycin D, doxorubicin (DXR; e.g. ADRIAMYCIN®), daunorubicin (daunomycin), bleomycin, mithramycin and the like; alkaloids, such as vinca alkaloids such as vincristine (VCR), vinblastine, and the like; and other antitumor agents, such as paclitaxel (e.g. TAXOL®) and pactitaxel derivatives, the cytostatic agents, glucocorticoids such as dexamethasone (DEX; e.g. DECADRON®) and corticosteroids such as prednisone, nucleoside enzyme inhibitors such as hydroxyurea, amino acid depleting enzymes such as asparaginase, leucovorin and other folic acid derivatives, and similar, diverse antitumor agents. The following agents may also be used as additional agents: arnifostine (e.g. ETHYOL®), dactinomycin, mechlorethamine (nitrogen mustard), streptozocin, cyclophosphamide, lomustine (CCNU), doxorubicin lipo (e.g. DOXIL®), gemcitabine (e.g. GEMZAR®), daunorubicin lipo (e.g. DAUNOXOME®), procarbazine, mitomycin, docetaxel (e.g. TAXOTERE®), aldesleukin, carboplatin, oxaliplatin, cladribine, camptothecin, CPT 11 (irinotecan), 10-hydroxy 7-ethyl-camptothecin (SN38), floxuridine, fludarabine, ifosfamide, idarubicin, mesna, interferon beta, interferon alpha, mitoxantrone, topotecan, leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracil mustard, vinorelbine, chlorambucil.

Example 1

To date, there are no known agents that target the NES of a specific protein that are being developed to treat cancer. Because drug resistance to topo II inhibitors occurs when topo lla is trafficked from the nucleus to the cytoplasm where it is no longer in contact with the DNA and unable to induce cell death (Valkov, et al., Cell density-dependent VP-16 sensitivity of leukaemic cells is accompanied by the translocation of topoisomerase llalpha from the nucleus to the cytoplasm. Br J Haematol 2000; 108: 331-45, Engel, et al., The cytoplasmic trafficking of DNA topoisomerase llalpha correlates with etoposide resistance in human myeloma cells. Exp Cell Res 2004; 295: 421-31 ), a specific NES in topo lla was targeted as an innovative treatment approach in MM. This provides a very focused and potent combination with topo II inhibitors, which may also overcome de novo drug resistance in MM. The nuclear export signals (NES) for topo lla have also identified at amino acids 1017-28 (site A) and 1054-66 (site B) (Turner et al., Human topoisomerase llalpha nuclear export is mediated by two CRM-1 -dependent nuclear export signals. J Cell Sci 2004; 117: 3061-71). Blocking nuclear export of topo lla with a CRM1 inhibitor or by siRNA has been shown to sensitize MM cells to topo II poisons, as seen in Figure 1. Analysis of potential drugs against topoisomerase II was performed based on the unique topo II NES site at 1017-1028. Though this site conforms to the hydrophobic amino acid motif for an NES, the amino acid sequence does not occur in any other human protein. In addition, this NES is in a pocket formed by the three-dimensional structure of the topo lla protein. These factors were used to development drugs that exclusively block the NES of topo lla without affecting the export of other nuclear proteins, as occurs with other known CRM1 inhibitors.

Although the crystal structure of human topo lla has not been solved to date, the homologous S. cerevisiae topo II is sufficiently similar to permit generation of a high confidence atomic model of human topo lla. Thus, the structure of S. cerevisiae topo II was used to create a computer generated hybrid molecule using the known three dimensional structure of yeast topo II and an atomic homology model of human topo lla isozyme (Homo sapiens) (gi19913406/NP_001058.2) was generated using the Phyre Protein Fold Recognition Server (Kelley & Sternberg, Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 2009; 4: 363-71), as seen in Figure 2. The nuclear export sequences were mapped, as seen in Figure 3, and these coordinates provided the basis for molecular docking. To prepare the site for docking, all water molecules were removed. Protonation of topo lla residues was done with SYBYL (Tripos, St. Louis, MO) (Kelley & Sternberg, Protein structure prediction on the Web: a case study using the Phyre server. Nat Protoc 2009; 4: 363-71). Molecules were docked in silico using atomic coordinates for each small molecule in a National Cancer Institute (NCI) small molecule database of the NCI Developmental Therapeutics Program (Driscoll, The preclinical new drug research program of the National Cancer Institute. Cancer Treat Rep 1984; 68: 63-76). 139,735 small molecules (MW<500) were positioned in each of the two selected structural pockets (hinge region outside active site* in 1.000 different orientations, with the best orientation and scores (contact and electrostatic) calculated. The scores were ranked based on predicted polar (H-bond) and nonpolar (van der Waals) interactions. The 20 highest scoring compounds for each structural pocket were obtained for in vitro assays and functional testing.

The molecular surface of the structure was explored using sets of spheres to describe potential binding pockets. The sites selected for molecular docking were defined using the SPHGEN program and filtered through the CLUSTER program (Gschwend , et al., Molecular docking towards drug discovery. J Mol Recognit 1996; 9: 175-86). Intermolecular AMBER energy scoring (van der Waals + coulombic), contact scoring, and bump filtering were implemented in DOCK v5.1.0 (Gschwend, et al., Molecular docking towards drug discovery. J Mol Recognit 1996; 9: 175-86; Ewing, et al., DOCK 4.0: search strategies for automated molecular docking of flexible molecule databases. J Comput Aided Mol Des 2001 ; 15: 41 1- 28). The UCSF Chimera software package (Pertersen, et al., UCSF Chimera-a visualization system for exploratory research and analysis. J Comput Chem 2004; 25: 1605-12) was used to generate molecular graphic images. The University of Florida High-Performance Computing Center Linux cluster was used to run the docking jobs. Four compounds were identified from the NCI database that bind to the NES of topo lla and improve the effectiveness of topo II directed therapeutics in myeloma cells. These additional compounds were obtained from NCI based on predicted docking scores for the topo lla NES site A.

Example 2 Compounds identified in Example 1 as the top scoring potential inhibitors for each of the two NES (20 each) were obtained from NCI and structures of these molecules confirmed using NMR and mass spectroscopy analysis, seen in Figures 4(A)-(C) and 5(A)-(D). The potential inhibitors were followed up using in vitro model cell cultures. MM cell lines NCI-H929 (H929) and RPMI-8226 (8226) were obtained from and tested for authenticity by the American Type Culture Collection (Manassas, VA). All cell lines were grown as described previously (Turner, et al., Human topoisomerase llalpha nuclear export is mediated by two CRM-1 -dependent nuclear export signals. J Cell Sci 2004; 117: 3061 -71 ).

The molecules with the highest docking scores were assayed for IC50 values. Cultures were tested for cytotoxicity using the CellTiter-Glo assay (CT-Glo cytotoxicity assay; Promega, Madison, Wl), a luminescence-based homogeneous method for determining the number of viable cells in culture based on quantitation of the ATP present, which signals the presence of metabolically active cells (Corsino, et al., A novel class of cyclin-dependent kinase inhibitors iH^pntifiprl hw molpn ilar rlnrkinn ar.t thrniinh a i inini ip mpr.hanism .1 Riol Ohflm 9009· ?84· 29945-55; Homsi, et al., Src activation in melanoma and Src inhibitors as therapeutic agents in melanoma. Melanoma Res 2009; 19: 167-75). All cells were assayed at log-phase growth conditions. Luminescence was measured with a Synergy 4 microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). IC50 values were determined using a sigmoidal equilibrium model regression using XLfit version 5.2 (ID Business Solutions Ltd., Surrey, UK) (Corsino, et al., A novel class of cyclin-dependent kinase inhibitors identified by molecular docking act through a unique mechanism. J Biol Chem 2009; 284: 29945-55).

The IC50 values obtained from single drug cell viability assays in low-density 8226 cells revealed four lead compounds with IC50 values of 7.2 ± 1.4 μΜ (NCI-35847), 12.0 ± 4.2 μΜ (NCI-80640), 12.7 ± 4.5 μΜ (NCI-9138), and 19.5 ± 5.4 μΜ (NCI-155877), as seen in Table 1. Only NCI-9138 affected the viability of high-density cells (IC50 of 9.4 μΜ) as a single agent (data not shown).

Table 1. ICso data.

IC50 results (in μΜ) are shown as average ± SD. Results were obtained from 72-h CT-Glo cytotoxicity assays. ND, not determined.

Data from apoptosis assays indicate that three of the inhibitors (NCI-36400, NCI-35847, NCI- 35024) that dock to NES site A significantly (p<0.05) sensitize high density MM cells to doxorubicin. CT-Glo assays determined that several of the site A inhibitors had antiproliferative activity. CT blue (Promega Corp., Madison, Wl) robotic cell viability assays confirmed that several of the NES site A inhibitors had anti-proliferative activity. However, none of the NES site B molecules exhibited any anti-neoplastic activity with or without a topo II inhibitor (data not shown).

Example 3 The compounds identified in Example 1 were tested for induction of apoptosis (cleaved caspase 3) as previously described (Turner, et al., Human multiple myeloma cells are sensitized to topoisomerase II inhibitors by CRM1 inhibition. Cancer Res 2009; 69: 6899- 905). Inhibition of topo lla export enhances DNA damaging agents, like intercalation agents such as doxorubicin. Human myeloma cells that maintained nuclear topo lla were more sensitive to topo I la-targeted chemotherapy in a synergistic manner, as seen in Figure 6. As such, cells were treated with 25 μΜ of each identified NES compound for 20 hours followed by 4 hours with either doxorubicin (2 μΜ), bortezomib (10 nM), dexamethasone (10 μΜ), lenalidomide (10 μΜ), melphalan (10 μΜ), and topotecan (10 μΜ).

Data from caspase 3 apoptosis assays showed that the four lead compounds that dock to NES site A significantly (P < 0.05) increased sensitivity of high-density MM cells to doxorubicin, as seen in Figure 7. NES site A inhibitors (25 μΜ) alone did not induce a significant level of apoptosis versus DMSO (dimethyl sulfoxide) controls. Additionally, the most active lead compound (NCI-9138) was assayed at 25 μΜ with other drug combinations, including bortezomib, dexamethasone, lenalidomide, melphalan, and topotecan, as seen in Figure 8. Tests in cells modeling AML, breast cancer and fibroblastoma show the NES site A inhibitor NCI-9138 synergized exclusively with the topo II inhibitor doxorubicin.

Lead compounds from cytotoxicity and apoptosis assays were evaluated by immunofluorescence microscopy for their effect on the intracellular location of topo lla in treated MM cells as previous described (Turner, et al., Human multiple myeloma cells are sensitized to topoisomerase II inhibitors by CRM1 inhibition. Cancer Res 2009; 69: 6899- 905). Slides were viewed with a fully automated, upright Zeiss Axio-lmagerZ.1 microscope (Carl Zeiss AG, Oberkochen, Germany). Briefly, the DAPI image was used as a mask to segment the nuclear region from the Alexa 594 image. The remaining portion of the Alexa 594 image represents the cytosol, and individual nucleus and cytosol images were analyzed. Immunofluorescence microscopy revealed a 2- to 3-fold increase in topo lla in the cell nucleus of high-density MM cells treated for 20 hours with the four lead site A inhibitors, as ^' seen in Figures 9(A)-(E). H929 cells were mock treated with 0.1 % DMSO in media or with 25 μΜ of NES inhibitor. The high density control cells, seen in Figure 9(A) show high levels of topo II staining outside the nucleus, evidencing export of topo II from the nucleus. However, treatment with NCI-35847, NCI-80640, NCI-9138, and NCI-155877 show overlapping staining of topo II with DAPI, as seen in Figures 9(B)-(E), respectively. This indicates that these NES site inhibitors prevented export of the topo II enzyme from the nucleus, consistent with the hypothesis that the NES site A docking molecules prevented nuclear export of topo lla. Further, the NES site A inhibitors, when administered with doxorubicin, showed drug synergism, as seen in Table 2.

Table 2. NES inhibitors effect on topo Ha nuclear export and synergism with doxorubicin.

* Immunofluorescence microscopy, H929 human myeloma cells. Change in fluorescence intensity of topo lla was compared in the nucleus v cytoplasm of drug-treated cells (25 μΜ NES inhibitor) and compared to untreated high-density controls. Nuclear and cytoplasmic fluorescence was averaged from 100-500 MM cells per drug treatment group.

** Apoptosis as measured by cleaved caspase 3 in cells treated with dox/drug (2 μΜ) was divided by apoptosis in cells treated with dox alone (N/A, not available).

Myeloma cells (RPMI-8226) were tested to determine if the observed effect of nuclear export inhibition of topo lla at high-density growth conditions was due to a decrease in topo lla protein expression. Equal numbers of cells were incubated with the lead NES (25 μΜ for 20 hours), and total protein was assayed by Western blot using anti-topo lla Kis1 antibody (Millipore). Fifty micrograms of protein was loaded into each lane. GAPDH was co-assayed with topo lla as a protein loading control (anti-GAPDH). Nuclear-cytoplasmic fractionation revealed that topo lla NES site A docking molecules prevent nuclear export of topo lla, as seen in Figures 10(A)-(F).

Lead compounds were tested for the inhibition of topo II decatenation activity with a kit purchased from TopoGen, Inc (Port Orange, FL) and using the protocols established by Muller (Muller, et al., Single-strand DNA cleavages by eukaryotic topoisomerase II. Biochemistry 1988; 27: 8369-79). Each drug was tested at concentrations of 25 and 50 μΜ, with mitoxantrone used as a control (1-10 μΜ). None of the NES compounds (up to 50 μΜ) inhibited the decatenation activity of topo II. In addition, NES inhibitors did not significantly alter total cellular levels of topo lla in MM cells treated for 20 hours with 25 μΜ of each inhibitor Cdata not shownV Example 4

A molecular docking model of compound NCI-9138 to the NES of topo lla, seen in Figure 1 1 , indicated that inhibitors may function by preventing binding of CR -1 signal export to leucine- rich nuclear export signals. Lead compounds identified in Example 1 were evaluated by cellular fractionation to confirm whether they prevented nuclear export of topo lla. Human myeloma cells were placed at plateau concentrations (4 x 10⁶ cells/ml), and NES inhibitors were added at 25 μ for 20 hours. Cells were collected, washed in PBS, and fractionated by a method adapted from Li et al. (Li Y, Bor YC, Misawa Y, Xue Y, Rekosh D, Hammarskjold ML. An intron with a constitutive transport element is retained in a Tap messenger RNA. Nature 2006; 443: 234-7). Separated fractions containing nuclei or precipitated cytosol were dissolved in 2% SDS buffer and assayed by SDS-PAGE Western blot analysis. Blots were probed with an anti-tppo lla Kis1 antibody (Millipore Corp.). Lead compounds were also tested to determine if nuclear export inhibition was specific to topo lla protein. Cells grown at log-phase concentrations were also fractionated. After MM cells were treated with NCI-9138 at 25 μΜ for 20 hours, CRM1 -topo lla complexes were isolated from cells by immunoprecipitation using a combination of both protein A and protein G magnetic beads according to the manufacturer's protocol (Miltenyi Biotec, Cologne, Germany). Co- immunoprecipitation was performed with 2 g of anti-CRM1 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Immunoprecipitation samples were separated by SDS- PAGE, transferred to nitrocellulose membranes, and probed with anti-topo lla Kis1 antibody (Millipore, Corp., Billerica, MA) and anti-mouse horseradish peroxidase secondary antibody.

Co-immunoprecipitation of topo lla with a CRM1 antibody and subsequent Western blot analysis showed that topo lla was prevented from binding to CRM1 by the NES inhibitor NCI- 9138, as seen in Figure 12. Western blot analysis of nuclear and cytoplasmic fractions confirmed that the lead NES compounds increased nuclear localization of topo lla. In human myeloma RPMI-H929 cells, the addition of compounds NCI-35847, NCI-80640, and NCI-9138 (100 μΜ, 24 hours) reduced cytoplasmic levels of topo lla equivalent to that produced by the CRM-1 inhibitor ratjadone C, seen in Figures 13(A) and (B). However, compound NCI-155877 did not significantly reduce the level of cytoplasmic topo lla relative to untreated controls. NCI- 32237, a control compound did not affect nuclear export of topo II, confirming that the inhibitors likely function through NES inhibition.

Nuclear fractions of low-density myeloma cells (H929) were assayed for p53 to determine if nuclear export inhibition was specific to topo lla protein, as seen in Figure 14. p53 is known to undergo nuclear to cytoplasmic shuttling in logarithmically growing myeloma cells and therefore was used to test the whether nuclear export was topo lla specific. p53 was exported from the nucleus to the cytoplasm in myeloma cells cultured at low concentrations (2 x 10⁵/mL). Cells grown at low concentrations were treated with the lead NES inhibitors (25 μΜ) or a leptomycin B (5 n ) control for 20 hours and chemically fractionated. Nuclei were assayed by SDS-PAGE Western blot analysis for p53 protein. NCI compounds 35847, 9138, and 155877 did not prevent export of p53, indicating that these compounds may specifically inhibit topo lla export, as seen in Figure 14. Interestingly, NCI-80640 was found to significantly inhibit nuclear export of p53, indicating that this drug can affect transport of other nuclear proteins into the cytoplasm.

Example 5

Additional cancer and normal cell lines were assayed to determine whether inhibition of topo lla nuclear export sensitized these cell lines to the topo II inhibitor, doxorubicin. Cell cultures were continuously treated with NES inhibitors (25 μΜ) for 24 hours. Doxorubicin was added to a final concentration of 2 μΜ during the final 4h of treatment and then apoptosis was measured by cleaved caspase 3 antibody staining (Cell Signaling Technology, Beverly, MA) and flow cytometry. Apoptosis was measured by cleaved caspase 3 antibody staining (Cell Signaling Technology) and flow cytometry. Cells lines assayed included normal fibroblasts WI-38 (American Type Culture Collection, Manassas, VA) and Flow 2000 (Flow Laboratories, Inc., Rockville, MD) peripheral blood mononuclear cells (PBMCs) from normal donors (Florida Blood Services, St. Petersburg, FL), MCF7 breast cancer cells (American Type Culture Collection), and acute myeloid leukemia (AML) cell lines HL-60 and KG1a (American Type Culture Collection). Volunteer donors to Florida Blood Services provided informed consent to allow the use of their cells in accordance with the Declaration of Helsinki.

High-density cultures of normal fibroblasts WI-38 and Flow 2000, seen in Figures 15 and 16, and normal PBMCs and AML cells HL-60 and KG1a, seen in Figures 17-19, were not sensitized to doxorubicin. However, the breast cancer cell line MCF7, seen in Figure 20, was sensitive to NES inhibitors and doxorubicin. In contrast to MM cells, topo lla does not preferentially localize to the cytoplasm of MCF7 cells under high-density growth conditions (unpublished data). The present combination shows high specificity to MM, as promyelocytic leukemia, myeloblasts leukemia, and normal fibroblast models do not sensitize to topo II NES inhibitors or combinations of to topo II NES inhibitors and topo II inhibitors.

Table 3. Additional compounds identified as topoisomerase II nuclear export signal inhibitors.

NCI-281703

NCI-203305 1 -bromo-15,3,5,7- N-isobutyl-1 ,3,5-

CID: 430738

CID: 4003104 tetraazatricyclo[3.3.1.1 -3,7- triazatricyclo[3.3.1.1 -3,7-]

ripran-7.aminp ] decane (ACD/Name 4.0) (ACD/Name 4.0)

Docking score -42.3161 Docking -42.1261

score

VDW -7.03116 VDW -13.8052

ES -35.285 ES -28.3209

MW 220.0916 MW 210.3216

Formula C₆H₁₂BrN₄ Formula C11H22N

HDonors 0 HDonors 1

HAcceptors 3 HAcceptors 4

Active pass Bisphosphonate: 0.029 Active pass Carbonic anhydrase inhibitor: 0.043 screen (p < Ribonucleoside diphosphate screen (p <

0.05) reductase inhibitor: 0.041 0.05)

Active pass Anesthetic inhalation: Active pass Antiviral: 0.0080

0.0080

screen (p < screen (p < Carbonic anhydrase

Mutagenic: 0.0060

0.05) 0.05) inhibitor: 0.0080

Table 4. A list of identified topoisomerase II nuclear export signal inhibitors.

1-(2,3-dibromc-2-propenyl)-15,3,5,7- -44.03179932 tetraazatricyclo[3.3.1.1 ~3,7~]decane

(ACD/Name 4.0)

C9H15Br2N4

339.0523

NCI-168615

N1 ,N4-bis(4-(4,5-dihydro-1 H-imidazol-2- -44.02162552 yl)phenyl)-2-(methylthio)terephthalamide

(ACD/Name 4.0)

C27H26N602S

498.6014

NCI-72382

4-((4-((2 ,4-dihydroxy-3- -43.9335556 quinolinyl)diazenyl)phenyl)diazenyl)benz

enesulfonic acid (ACD/Name 4.0)

C21 H15N505S

449.44

NCI-45588

2,4,6-triphenyl-1 ,3,5- -43.88329315 triazabicyclo[3.1.OJhexane (ACD/Name

4.0)

C21 H 19N3

313.4012

NCI-634537

1-allyl-15,3,5,7- -43.86736679 tetraazatricyclo[3.3.1.1 ~3,7~]decane

(ACD/Name 4.0)

C9H17N4

181.2601

NCI-5062 ^A

1-(7-methyl-8-(3,4,5-trimethoxyphenyl)- -42.81098938

7,8-dihydro-6H-[1 ,3]dioxolo[4,5- g]chromen-6-yl)hydrazine (ACD/Name

4.0)

C20H24N2O6

388.4194

NCI-667918

1 -(2-oxiranylmet yl)-15,3,5,7- -42.72693634 tetraazatricyclo[3.3.1.1 ~3,7~]decane

(ACD/Name 4.0)

C9H17N40

197.2595

NCI-177979

1-benzyl-15,3,5,7- -42.64362335 tetraazatricyclo[3.3.1.1 -3, 7~]decane

(ACD/Name 4.0)

C13H19N4

231.3199

NCI-10408

N-(2-((2- -42.60683441 pyrrolidinylcarbonyl)amino)ethyl)-2- pyrrolidinecarboxamide (ACD/Name 4.0)

C12H22N402

254.3314

NCI-621603

1-(15,3,5,7- -42.55291367 tetraazatricyclo[3.3.1.1 ~3,7-]dec-1 - yl)acetone (ACD/Name 4.0)

C9H17N40

197.2595

NCI-177977

In the preceding specification, all documents, acts, or information disclosed do not constitute an admission that the document, act, or information of any combination thereof was publicly available, known to the public, part of the general knowledge in the art, or was known to be relevant to solve any problem at the time of priority.

The disclosures of all publications cited above are expressly incorporated herein by reference, each in its entirety, to the same extent as if each were incorporated by reference individually.

While there has been described and illustrated specific embodiments of a topoisomerase II nuclear export inhibitor, it will be apparent to those skilled in the art that variations and modifications are possible without deviating from the broad spirit and principle of the present invention. It is also to be understood that the following claims are intended to cover all of the generic and specific features of the invention herein described, and all statements of the scope of the invention which, as a matter of language, might be said to fall therebetween.

Claims

What is claimed is:

1. A method of treating an oncogenic disease, comprising administering a therapeutically effective amount of a topoisomerase II nuclear export signal inhibitor to a patient; wherein the topoisomerase II nuclear export signal inhibitor is NCI-9138, NCI-155877, or an having the backbone structure

wherein the oncogenic disease is multiple myeloma, breast cancer, ovarian cancer, testicular cancer, lung cancer, bronchogenic lung cancer, gastric cancer, soft tissue and osteogenic sarcomas, Ewing's sarcoma, malignant lymphoma (Hodgkin's and non-Hodgkin's), acute myeloblastic leukemia, acute lymphoblastic leukemia, or Kaposi's sarcoma.

2. The method of claim 1 , wherein the topoisomerase II nuclear export signal inhibitor is NCI-36400, NCI-35847, NCI-80640, NCI-9138, NCI-155877, NCI-203305, NCI- 281703, or NCI-35024.

3. The method of claim 2, wherein the topoisomerase II nuclear export signal inhibitor is NCI-35847, NCI-80640, NCI-9138, or NCI-15587.

4. The method of claim 1 , wherein the topoisomerase II nuclear export signal inhibitor is administered at 2 μΜ to 25 μ .

5. The method of claim 1 , further comprising co-administering the topoisomerase II nuclear export signal inhibitor with a topoisomerase II inhibitor.

6. The method of claim 5, wherein the topoisomerase II inhibitor is administered concurrently, at between 1.6 to 24 hours after administration of the topoisomerase II nuclear export signal inhibitor, or at about 4 hours after administration of the topoisomerase II nuclear export signal inhibitor.

7. The method of claim 5, wherein the topoisomerase II inhibitor is amsacrine, etoposide, etoposide phosphate, teniposide, doxorubicin, epirubicin, daunomycin, mitoxantrone, and idarubicin.

. The method of claim 5, wherein the topoisomerase II inhibitor is doxorubicin.

. The method of claim 5, wherein the topoisomerase II inhibitor is doxorubicin administered at 2 μΜ, bortezomib administered at 10 n , dexamethasone administered at 10 μΜ, lenalidomide administered at 10 μΜ, melphalan administered at 10 μΜ, or topotecan administered at 10 μΜ.

0. A method of treating an oncogenic disease, comprising administering a therapeutically effective amount of a topoisomerase II inhibitor and a therapeutically effective amount of a topoisomerase II nuclear export signal inhibitor to a patient; wherein the oncogenic disease is multiple myeloma; and wherein the topoisomerase II nuclear export signal inhibitor is

having a structure of formula I or II

^R

wherein R, is Br, CI,

47

wherein R₂ is

11. The method of claim 10, wherein the topoisomerase II nuclear export signal inhibitor is administered at 2 μΜ to 25 μΜ.

12. The method of claim 10, wherein the topoisomerase II inhibitor is administered concurrently, at between 1.6 to 24 hours after administration of the topoisomerase II nuclear export signal inhibitor, or at about 4 hours after administration of the topoisomerase II nuclear export signal inhibitor.

13. The method of claim 10, wherein the topoisomerase II inhibitor is amsacrine, etoposide, etoposide phosphate, teniposide, doxorubicin, epirubicin, daunomycin, mitoxantrone, and idarubicin.

14. The method of claim 13, wherein the topoisomerase II inhibitor is doxorubicin.

15. The method of claim 13, wherein the topoisomerase II inhibitor is doxorubicin administered at 2 μΜ, bortezomib administered at 10 nM, dexamethasone administered at 10 μΜ, lenalidomide administered at 10 μΜ, melphalan administered at 10 μΜ, or topotecan administered at 10 μΜ.

16. A method of inhibiting topoisomerase II export from a cell nucleus, comprising contacting a cell with a therapeutically effective amount of a topoisomerase II nuclear export signal inhibitor; wherein the topoisomerase II nuclear export signal inhibitor is NCI-9138, NCI-155877, or an having the backbone structure

17. The method of claim 16, wherein the topoisomerase II nuclear export signal inhibitor is NCI-36400, NCI-35847, NCI-80640, NCI-9138, NCI-155877, NCI-203305, NCI-

18. The method of claim 17, wherein the topoisomerase II nuclear export signal inhibitor is NCI-35847, NCI-80640, NCI-9138, or NCI-15587.

19. The method of claim 16, wherein the topoisomerase II nuclear export signal inhibitor is administered at between 2 μΜ and 25 μ .