Abstract
Introduction
In multiple myeloma (MM), de novo drug resistance to topoisomerase (topo) II poisons occurs at high cell densities due to trafficking of topo IIα from the nucleus to the cytoplasm where it is no longer in contact with the DNA and thus unable to induce cell death (Turner et al. 2009, Cancer Res, 69, 6899-905, Engel et al. 2004, Exp Cell Res, 295, 421-31). We have previously demonstrated that topo IIα is exported from the nucleus of human myeloma cells by a CRM1-dependent mechanism ( Valkov et al 2000, Br J Haematol, 108, 331-45, Engel et al. 2004, Exp Cell Res, 295, 421-31) and we have also identified the nuclear export signals (NES) for topo IIα at amino acids 1017-28 (site A) and 1054-66 (site B) ( Turner et al. 2004, J Cell Sci, 117, 3061-71). Blocking nuclear export with a CRM1 inhibitor or by siRNA has been shown to sensitize drug-resistant MM cells to topo II poisons ((Turner et al. 2009, Cancer Res, 69, 6899-905, Turner et al 2013, J. of Cancer, In press). Even though the active NES site (site A) of topo IIα conforms to the hydrophobic amino acid motif for an NES, the amino acid sequence does not occur in any other human protein. In addition, this NES is in a pocket formed by the three-dimensional structure of the topo IIα protein. This specificity of the NES could lead to the development of drugs that may exclusively block the NES of topo IIα and not affect the CRM 1-dependent export of other nuclear proteins.
Methods
In this study, our aims were to; 1) generate an atomic homology model of human topo IIα to identify lead compounds targeting the NES of topo IIα (nuclear export signal inhibitors, NESi), 2) determine if the compounds bind specifically to topo IIα and inhibit nuclear export by immunofluorescence microscopy, nuclear-cytoplasmic fractionation, immunoprecipitation, proximity ligation assay, and Biacore kinetic/affinity assay, and 3) test the anticancer activity of these compounds in multiple myeloma and normal cells with and without a topo IIα inhibitor.
Results and Conclusions
We generated an atomic homology model of human topo IIα to provide the basis for in silico molecular docking screenings of 139,735 small molecules (MW < 500) to identify lead compounds or NESi targeting the NES of topo IIα. The highest docking score NESi compounds (46 total) were assayed for their ability to induce apoptosis in drug-resistant human H929 myeloma cells when used in combination with the topo II inhibitor doxorubicin. We isolated four lead compounds that induced apoptosis (caspase 3 cleavage) when used with doxorubicin and that inhibited nuclear export of topo IIα, as shown by immuno-fluorescence microscopy and nuclear-cytoplasmic fractionation. Immunoprecipitation, proximity ligation assay and Biacore kinetic/affinity assay showed that the most potent lead compound, NCI-9138, inhibited binding of topo IIα to the export receptor CRM1. Inhibition was specific to topo IIα because p53 trafficking was unaffected and the inhibitor did not affect topo IIα protein expression or topo IIα function (decatenation). The NESi were found to sensitize drug-resistant human myeloma cells to doxorubicin but did not affect normal fibroblast cell lines (Flow2000 or WI-38) or human PBMCs. These topo IIα-specific nuclear export inhibitors may potentially lead to a new approach in circumventing drug resistance in multiple myeloma.
Disclosures:
No relevant conflicts of interest to declare.
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