Blocking a Topoisomerase IIα Nuclear Export Signal Sensitizes Human Multiple Myeloma Cells to Topoisomerase II Inhibitors.

We have previously demonstrated that topoisomerase IIa (topo IIa) is exported from the nucleus of high density human multiple myeloma (MM) cells by a CRM1-dependent mechanism (Engel et al, Exp. Cell Res. 295(2):421-31, 2004). We have also identified the nuclear export signals (NES) for topo IIa at amino acids 1017-28 (site 1) and 1054-66 (site 2) using mutated full-length FLAG-tagged topo IIa protein and immunofluorescence microscopy (Turner et al, J. Cell Sci. 117:3061-71, 2004). Drug resistance to topo II poisons occurs when topo II is trafficked to the cytoplasm where it is not in contact with the DNA and thus unable to induce cell death in response to topo II inhibitors. In addition, we have recently shown that blocking nuclear export of topo IIα with a CRM1 inhibitor or by siRNA sensitizes MM cells to topo II poisons (Turner et al, Cancer Res. In press).

The structure of S. cerevisiae topo II was used to create a model of human topo IIα using the program Protein Homology/analogy Recognition Engine (Phyre), available on the Web (http://www.imperial.ac.uk/phyre). The procedure for molecular docking involved selection of structural pockets of the NES in topo IIa that were suitable for interactions with drug-like small molecule inhibitors (SMI). Molecular docking simulations screened 140,000 small molecules (mw < 500) from the NCI database. The top scoring SMI for each of the two NES (20 total) were obtained from NCI and tested for induction of apoptosis (caspase 3) and anti-proliferative activity using CellTiter-Blue (Promega). Cell types used included human MM RPMI 8226 and NCI-H929 cells. SMI were tested both as single agents and in combination experiments with the topo II inhibitor doxorubicin. In addition, immunofluorescence microscopy for the intracellular location of topo IIα in SMI-treated MM cells was also performed.

Using computer-generated molecular modeling, we have identified four lead compounds from the NCI database that bind to the site 1 NES of topo IIα. These lead compounds are SMI that synergize with the topo II inhibitor doxorubicin. In vitro apoptosis assays indicate that these drugs may be effective as single agents or in combination with currently used cancer drugs that target topo II. These data may have potential clinical implications in the treatment of multiple myeloma.

Sullivan:Merck: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Merrion: Membership on an entity's Board of Directors or advisory committees.