Blockade of Nuclear Export Protein CRM1 (chromosomal region maintenance 1, XPO1) by a Novel, Potent and Selective CRM1 Inhibitor KPT-185 Induces Significant Antitumor Activity Against Human Multiple Myeloma

Abstract 2913

CRM1 (chromosomal region maintenance 1, XPO1) is the major export protein regulating degradation of key tumor suppressors by transporting them from nucleus to cytoplasm, thus abrogating their function. Highly expressed CRM1 is associated with poor prognosis in several solid tumors, and inhibition of CRM1 restores function of tumor suppressors such as p53, p21 and FOXO and IκB (cellular antagonist of NF-κB). Furthermore, CRM1 knockdown enhances sensitivity of human multiple myeloma (MM) cell lines to topoisomerase II inhibitor (Cancer Res 2009 :69, 17). In this study, we investigated a novel selective CRM1 inhibitor, KPT-185, in human MM cells. Gene expression profiling analysis using GEO database showed that CRM1 expression is significantly increased in CD138+ cells from MM patients versus monoclonal gammopathy of undetermined significance (MGUS) patients or normal donors (p<0.05). Importantly, KPT-185 inhibits cell proliferation against drug sensitive and resistant MM cell lines with either wild type or mutated p53 status (n=7) in a dose-dependent manner (ED₅₀: 5–100nM), evidenced by potently decreased [³H]-thymidine incorporation. MTT assay also demonstrated a significant reduction in cell viability (ED₅₀: 20–120nM), associated with induction of apoptosis by annexin V/PI analysis. The percentage of apoptotic cells increased in a dose-dependent fashion in MM1.S, H929, U266, and RPMI-8226 cells, accompanied by increased activities of caspase 3/7 and 8. Cell cycle analysis confirmed that KPT185 inhibited cell proliferation, associated with 1.4-fold increased G₀/G₁ phase and a 4.5-fold decreased S phase at 24hr in MM.1S. Immunoblotting analysis next demonstrated that KPT-185-altered cell cycle was associated with increased p53 and p21, as well as decreased cyclin D1, cyclin E, CDK2, CDK4, and CDK6. KPT185 significantly induced cytotoxicity against CD138+ cells purified from MM patients (n=12) including newly diagnosed, advanced stage and relapsed patients. The median ED₅₀ was 20 nM and 291 nM against patient MM cells, as determined by cell proliferation and viability assay, respectively. To evaluate the effects of the bone marrow (BM) microenvironment on KPT-185 treatment, BM stromal cells (BMSCs) derived from MM patients and HS-5 stromal cell line were co-cultured with MM cells, in the presence of serial dilutions of KPT-185. KPT-185 blocked MM cell growth induced by adhesion to BMSCs (p<0.05), indicating that KPT-185 could overcome BMSC-induced MM cell proliferation. KPT-185 also inhibited VEGF and MIP1 β secretion induced by coculture of BMSCs and IL-6-dependent ANBL6 (p<0.05). When MM1.S and INA6 cells were co-cultured with osteoclasts, KPT185 also demonstrated potent cytotoxicity against MM cells. Finally, treatments with KPT-185 and dexamethasone or lenalidomide synergistically induced cytotoxicity against MM1.S, MM1.R, U266, and H929 cells (CI <1.0). KPT-185 combined with melphalan or bortezomib induced additive or synergistic effects against MM cells. Together, results from these preclinical studies establish CRM1 as a promising novel target in MM and strongly support clinical evaluation of KPT-185, alone or in combination with conventional or novel anti-MM agents, to improve patient outcome in MM.