Abstract 2066: Combination therapy of human multiple myeloma using proteosome and CRM1 inhibitors.
Authors: Turner Joel G. , Dawson Jana L. , Cubitt Christopher L. , Kauffman Michael , Shacham Sharon , Sullivan Daniel M. 
Published: August 2014

Purpose. Despite improved treatments using proteosome inhibitor- and immunomodulator-based (thalidomide) therapies human multiple myeloma (MM) still remains an incurable disease. New therapeutic targets or unique drug-combinations are needed to further improve treatment outcomes. Our purpose was to investigate the use of small-molecule inhibitors of the nuclear export (SINE) in combination with proteosome inhibitors (PI), bortezomib (BTZ) and carfilzomib (CFZ) as a potential treatment for MM both in vitro and ex vivo. Methods. Previously, we reported that blocking nuclear export of topo IIα with SINE sensitized MM cells to topo II poisons. In this study, we used SINE inhibitors developed by Karyopharm Therapeutics, KPT-185, -249 and a new clinical compound KPT-330. In addition we investigated the use of KOS-2464 (BMS), an effective and relatively non-toxic analog of the known CRM1 inhibitor LMB. To test the efficacy of these inhibitors, we treated human MM cell lines, RPMI-8226, U266B1 and NCI-H929 with SINE +/- PI. MM cell lines or MM patient bone marrow aspirates were placed at high-density conditions and treated for 20 hours with KPT-drugs (300 nM) or KOS-2464 (10 nM), either concurrently or sequentially with 10-40 nM BTZ or CFZ. Cells were then assayed for cell viability by an automated CellTiter-Blue (Promega) analysis. Additional experiments were performed to investigate time-course of drug action and dose-dependence. In addition, treated cell lines or CD138+/light chain+ patient MM cells were assayed for induction of apoptosis by cleaved caspase 3 staining and flow cytometry. To determine if the SINE 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. In addition, CRM1 expression levels were measured in all treated cell lines by both Western blot and quantitative PCR. Results. SINE used in combination with PI were highly effective against both MM cell lines and patient MM cells. We found that SINE 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 all SINE tested had low IC50 values (nanomolar) as single agents and functioned synergistically with BTZ and CFZ (CI &lt;0.5). SINE/PI combination therapy also induced apoptosis in MM cell cultures and patient MM cells (ex vivo), as shown by caspase 3 cleavage and flow cytometric analyses. Conclusions. We found that specific, small-molecule CRM1 inhibitors, sensitized MM cells to BTZ and CFZ 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 combination therapy may be an effective treatment for multiple myeloma.

Citation Format: Joel G. Turner, Jana L. Dawson, Christopher L. Cubitt, Michael Kauffman, Sharon Shacham, Daniel M. Sullivan. Combination therapy of human multiple myeloma using proteosome and CRM1 inhibitors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2066. doi:10.1158/1538-7445.AM2013-2066