Abstract 1771: KOS-2464, a novel CRM1 inhibitor and a potential drug for multiple myeloma and leukemia
Authors: Turner Joel G. , Dawson Jana L. , Sullivan Daniel M. 
Published: August 2012

Abstract
Purpose: Human multiple myeloma (MM) remains an incurable disease despite improved treatments that include bortezomib- and lenalidomide-based therapies and high-dose chemotherapy with autologous stem cell rescue. New therapeutic targets are needed to further improve treatment outcomes. Our purpose was to investigate the use of a small-molecule inhibitor (KOS-2464) of the nuclear export receptor CRM1 and whether this inhibitor sensitized drug-resistant MM to topoisomerase IIα (topo IIα) inhibitors. Experimental Procedures: In previous studies, we have shown that MM cells possess intrinsic drug resistance to topo II inhibitors due to a CRM1-mediated export of topo IIα in high cell density microenvironments, such as found in the diseased bone marrow. Blocking nuclear export of topo IIα with a CRM1 inhibitor may sensitize MM cells to topo II poisons. In this study, we investigated a novel, small molecule (585 MW), analog of the known CRM1 inhibitor LMB. To test the efficacy of this inhibitor, we treated human MM cell lines, RPMI-8226 and NCI-H929 and AML cell lines Kg1a and HL-60, with KOS-2464 +/− the topo IIα inhibitors doxorubicin (dox) or daunomycin. MM cells were placed at high-density drug resistant conditions and were treated for 20 hours with KOS-2464 (at concentrations of 1 nM, 10 nM and 100 nM), followed by treatment with 2 µM dox or daunomycin for four hours. Cells were then assayed for cell viability by an automated CellTiter-Blue (Promega) analysis. In addition, treated cells were assayed for induction of apoptosis by cleaved caspase 3 staining and flow cytometry. To determine if the CRM1 inhibitor blocked CRM1-mediated export, treated cells were separated into nuclear and cytoplasmic fractions and assayed for export of p53 and topo IIα. Immunofluorescent microscopy was used to confirm fractionation results. Results: KOS-2464 was used in combination with the topo IIα drugs dox and daunomycin to overcome intrinsic drug resistance in MM and AML cells. We found that KOS-2464 blocked nuclear export of both p53 and topo IIα as shown by nuclear/cytoplasmic fractionation and also by immunocytochemical staining. Using the CellTiter-Blue assay, we found that KOS-2464 had low IC50 values (2 nM) as single agent and functioned synergistically with dox (combination index 0.268). KOS-2464/dox combination therapy also induced apoptosis in MM cell cultures, as shown by caspase 3 cleavage and flow cytometric analyses. Conclusions: We found that a specific, small-molecule CRM1 inhibitor KOS-2464, sensitized MM and AML cells to dox and daunomycin as shown by apoptosis and cell viability assays. Sensitization was found to be synergistic. In addition, CRM1 inhibiting activity was verified by nuclear/cytoplasmic fractionation and immunocytochemical staining in MM cells. This compound may be a potential candidate for future clinical trials.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1771. doi:1538-7445.AM2012-1771