CRM1 Is Highly Expressed in Myeloma Plasma Cells and Its Inhibition by KPT-SINE Induces Cytotoxicity by Increasing p53 in the Nucleus of Multiple Myeloma (MM) Cells

CRM1 (XPO1, exportin) is a nuclear export protein which controls the nuclear-cytoplasmic localization of multiple tumor suppressor proteins and cell proliferation pathways including p53, p21, PI3K/AKT/FOXO, Wnt/ß-catenin/APC, topoisomerase II, and NF-κB/I-κB. Transport of nuclear proteins to the cytoplasm can render them ineffective as tumor suppressors or as targets for chemotherapy. Small molecule, selective inhibitors of nuclear export (SINE) that block CRM1-dependent nuclear export can force the nuclear retention of tumor suppressor proteins, thus rendering cancer cells more susceptible to apoptosis and responsive to other chemotherapy. In this study we evaluated CRM1 as a potential target in MM and the effect of SINE on the activity of established anti-myeloma agents currently in use in treatment of MM. KPT-276 is the lead CRM1 inhibitor being investigated which will be submitted for IND in 2012.

To evaluate expression of CRM1, bone marrow aspirates from MM patients and tonsil tissue from normal patients were enriched for plasma cells (PC) and proteins from cell lysates were separated by SDS-PAGE followed by immunoblotting with CRM1 antibodies. In functional experiments, isolated fresh MM PCs from patients, and NCI-H929, MM1.S, MM1.R and RPMI-8226 cell lines were cultured in RPMI-1640 with 10–15% serum. Cells were treated for 24–72 hrs with CRM1 inhibitors KPT-SINE compounds with or without bortezomib and dexamethasone and were analyzed for cytotoxicity by MTT assay. Drug concentrations for combination experiments were chosen to be at or below IC50 for each individual drug. Apoptosis induction in primary MM cells and cell lines was studied by Annexin V labeling and flow cytometry. Cell lysates from primary MM PCs and cell lines were prepared after treatment with KPT-SINE and were used to determine the expression of p53 and CRM1.

Primary MM plasma cells derived from naïve, previously untreated patients show 4–20 fold higher CRM1 protein expression, compared to normal peripheral blood mononuclear cells (PBMCs) and normal tonsilar PCs. Dose response analysis of KPT-SINE compounds in myeloma cell lines showed potent activity with IC50s in the range of 10–100nM. The lead compound KPT-276 had an IC50 of <100 nM in NCI-H929, MM1.S, MM1.R and RPMI-8226 cells. Functional studies in MM patient plasma cells showed that in vitro inhibition of CRM1 with related SINEs KPT-185, −225 or −276 increase apoptosis induction as measured by Annexin V assay. In addition, the inhibition of CRM1 with KPT-SINE results in a dose-dependent increase in levels of nuclear as well as total p53 in MM patient plasma cells within 48 hrs. When combined with proteasome inhibitors like bortezomib and/or dexamethasone, KPT-SINE compounds potently increase the cellular cytotoxicity of these drugs in MM cell lines. Mechanism of activity of drug combinations is under investigation in MM cell lines and MM patient plasma cells.

MM plasma cells express CRM1 that is functionally active and therefore is a valid target in the treatment of myeloma. Moreover, higher expression of CRM1 in malignant plasma cells compared to normal PBMCs and normal PCs suggests possibility of therapeutic index. Early mechanistic studies indicate that CRM1 inhibition can lead to an increased expression of p53 (and other tumor suppressors) and its nuclear localization in myeloma cells and therefore might serve as a mechanism for the activity of CRM1 inhibitors in MM. Potentiation of cytotoxicity of bortezomib and dexamethasone by KPT-SINE suggests that these drugs might be useful in treating MM refractory to currently used agents and provide rationale for combining inhibitors of nuclear transport with other drugs.

Off Label Use: KPT-SINE family of drugs are not approved for the treatment of multiple myeloma. These drugs have a novel mechanism and are in pre-clinical development for the treatment of several malignancies. McCauley:Karyopharm Therapeutic Inc.: Employment. Shacham:Karyopharm Therapeutics Inc.: Employment. Kauffman:Karyopharm Therapeutics Inc.: Employment. Jakubowiak:Exelixis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Consultancy, Honoraria, Speakers Bureau; Ortho Biotech: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.