Abstract
Purpose
Human multiple myeloma (MM) remains an incurable disease despite relatively effective treatments, including proteasome inhibitors, immunomodulator-based therapies, and high-dose chemotherapy with autologous stem cell rescue. New agents are needed to further improve treatment outcomes. In previous studies, we have shown that inhibitors of the nuclear export receptor XPO1, in combination with bortezomib, carfilzomib, doxorubicin, or melphalan, synergistically induced apoptosis in MM cells in vitro, in vivo and ex vivo without affecting non-myeloma cells. In early clinical trials, the oral, brain penetrating XPO1 inhibitor selinexor showed clear anti-myeloma activity however adverse events have been recorded, including nausea and anorexia. Our purpose was to investigate the use of oral KPT-8602, a novel small-molecule inhibitor of XPO1 with minimal brain penetration, which has been shown to have reduced toxicities in rodents and primates while maintaining potent anti-tumor effects.
Experimental Procedures
To test the efficacy of KPT-8602, we treated human MM cell lines (both parental and drug-resistant) with KPT-8602 ± currently used MM drugs, including bortezomib, carfilzomib, dexamethasone, doxorubicin, lenalidomide, melphalan, topotecan, and VP-16. Human MM cell lines assayed included RPMI-8226 (8226), NCI-H929 (H929), U266, and MM1.S, PI-resistant 8226-B25 and U266-PSR, doxorubicin-resistant 8226-Dox6 and 8226-Dox40, and melphalan-resistant 8226-LR5 and U266-LR6 cell lines. MM cells (2-4x106 cells/mL) were treated for 24 hours with KPT-8602 (300 nM), followed by treatment with one of the listed anti-MM agents for an additional 24 hours. MM cells were then assayed for cell viability (CellTiter-Blue, Promega). In addition, cells were treated with KPT-8602 ± anti-MM agents concurrently for 20 hours and assayed for apoptosis by flow cytometry. In vivo testing was done in NOD/SCID-g mice by intradermal injection of U266 MM cells. Treatment started 2 weeks after tumor challenge with KPT-8602 (10 mg/kg) ± melphalan (1 or 3 mg/kg) 2X/week (Tuesday, Friday) or with KPT-8602 alone 5X weekly (10 mg/kg) (Monday-Friday). A parallel experiment was run using the clinical XPO1 inhibitor KPT-330 (selinexor). Ex vivo testing was performed on MM cells from newly diagnosed/relapsed patient bone marrow aspirates with KPT-8602 ± bortezomib, carfilzomib, dexamethasone, doxorubicin, lenalidomide, melphalan, topotecan, or VP16. CD138+/light-chain+ cells were assayed for apoptosis by flow cytometry.
Results
Viability assay showed that KPT-8602 had low IC50values (~140 nM) as a single agent and functioned synergistically with bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16. (CI values < 1.0). This synergistic effect was less pronounced in myeloma cells when KPT-8602 was used in combination with dexamethasone or lenalidomide. KPT-8602 ± bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 combination therapy also induced apoptosis in all MM cell lines tested, including drug-resistant cell lines, as shown by caspase 3 cleavage and flow cytometric analyses. NOD/SCID-gamma mouse tumor growth was reduced and survival increased in KPT-8602/melphalan-treated mice when compared to single-agent controls. In addition, mice treated with KPT-8602 5X weekly had significantly reduced tumor growth and increased survival when compared to 2X weekly drug administration. No toxicity was observed in KPT-8602-treated mice as determined by weight loss in both the 2X and 5X groups. In patient bone marrow biopsies, the combination of KPT-8602 ± bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 was more effective than single agents at inducing apoptosis in CD138+/LC+ MM cells in both newly diagnosed and relapsed/refractory patient samples.
Conclusions
We found that the novel XPO1 inhibitor KPT-8602 sensitizes MM cells to bortezomib, carfilzomib, doxorubicin, melphalan, topotecan, and VP16 as shown by apoptosis in parental and drug-resistant cell lines and by cell viability assays. Sensitization was found to be synergistic. In addition, KPT-8602 was effective in treatment of human MM tumors in mice as a single agent or in combination with melphalan and was effective when combined with several MM drugs in MM cell lines and MM patient bone marrow aspirates. KPT-8602 may be a potential candidate for future clinical trials.
Disclosures
Shacham: Karyopharm: Employment, Equity Ownership. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties.
Cross-Resistance of CD95- and Drug-Induced Apoptosis as a Consequence of Deficient Activation of Caspases (ICE/Ced-3 Proteases)
