Abstract B176: Novel inhibitors of nuclear export as a treatment for multiple myeloma.
Authors: Turner Joel G. , Dawson Jana L. , Shachem Sharon , Kaufman Michael , Sullivan Daniel M. 
Published: June 2012

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 small-molecule inhibitors of the nuclear export receptor CRM1 and whether these inhibitors 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 CRM1 inhibitors may sensitize MM cells to topo II poisons. In this study, we investigated novel, small molecule (&lt;500 MW), potent, drug-like, highly selective inhibitors of nuclear export (SINE) that specifically block CRM1 (provided by Karyopharm Therapeutics). To test the efficacy of these inhibitors, we treated human MM cell lines, RPMI-8226 and NCI-H929, with the CRM1 inhibitors +/− the topo IIα drug doxorubicin. MM cells were placed at high-density drug resistant conditions and were treated for 20 hours with each CRM1 inhibitor (at concentrations of 100 nM, 300 nM and 1 μM), followed by treatment with 2 μM doxorubicin for four hours. MM 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 inhibitors 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: CRM1 inhibitors were used in combination with the topo IIα drug doxorubicin to overcome intrinsic drug resistance in MM cells. We found that the CRM1 inhibitors 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 the lead CRM1 inhibitor tested had low IC50 values (∼140 nM) as single agent and functioned synergistically with doxorubicin (combination index 0.071). This synergistic effect was less pronounced in myeloma cells when CRM1 inhibitors were used in combination with melphalan, bortezomib, dexamethasone, or lenalidomide (combination index &gt; 0.65). CRM1-doxorubicin combination therapy also induced apoptosis in MM cell cultures, as shown by caspase 3 cleavage and flow cytometric analyses.

Conclusions: We found that specific, small-molecule CRM1 inhibitors sensitize MM cells to doxorubicin 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. These compounds may be potential candidates for future clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B176.