Novel Drug Targets to Overcome De Novo Drug-Resistance In Multiple Myeloma

Human multiple myeloma (MM) still remains an incurable disease despite improved treatment regimens that include bortezomib, lenalidomide and thalidomide. New therapeutic targets are needed to further improve treatment outcomes. We have shown that targeting intracellular trafficking of proteins may sensitize cells to antitumor agents (Turner et al. 2009, Cancer Res, 69, 6899-905). We have previously demonstrated that topoisomerase II alpha (topo IIα) trafficking from the nucleus to the cytoplasm in myeloma cells occurs by a CRM1-dependent mechanism and resulting in drug resistance to topo II inhibitors (Engel et al. 2004, Exp Cell Res, 295, 421-31). We have also identified the nuclear export signals (NES) for topo IIα at amino acids 1017-28 (site 1) and 1054-66 (site 2) (Turner et al. 2004, J Cell Sci, 117, 3061-71). Blocking nuclear export of topo IIα with a CRM1 inhibitor or by siRNA has been shown to sensitize MM cells to topo II poisons (Turner et al. 2009, Cancer Res, 69, 6899-905). The NES amino acid sequence of topo IIα at 1017–1028 is a unique site. Even though this site 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. These factors allow the potential for the development of drugs that will exclusively block the NES of topo IIα and not affect the export of other nuclear proteins, as occurs with other known CRM1 inhibitors. Drug resistance to topo II inhibitors occurs when topo IIα is trafficked from the nucleus to the cytoplasm where it is no longer in contact with the DNA, and thus unable to induce cell death (Valkov et al 2000, Br J Haematol, 108, 331-45, Engel et al. 2004, Exp Cell Res, 295, 421-31). We therefore hypothesize that targeting a specific NES in topo IIα is an innovative treatment approach in MM and may allow a very focused and potent combination with topo II inhibitors, possibly overcoming de novo drug resistance in this malignancy. To date, we know of no agents that target the NES of a specific protein that are being developed to treat cancer. A computer generated hybrid molecule using the known three dimensional structure of yeast topo II and the human NES sequences of topo IIα was produced. Molecules were docked in silico using the NCI small molecule database (140,000 compounds). The molecules with the highest docking scores were obtained from NCI and assayed for IC50 values and synergy with the topo II inhibitor doxorubicin. All NES site 1 molecules tested showed activity, however, none of the NES site 2 molecules exhibited any anti-neoplastic activity with or without a topo II inhibitor. CT blue (Promega) robotic cell viability assays determined that several of the site 1 inhibitors had anti-proliferative activity. The IC50 values obtained from single drug cell viability assays in low density cells revealed two site 1 inhibitors compounds with IC50 values of 4.7 (NCI-36400) and 11.1 μM (NCI-35847). None of the site 1 inhibitors affected the viability of high-density cells (IC50>100 μM). Data from apoptosis assays indicate that three of the site 1 inhibitors (NCI-36400, NCI-35847, NCI-35024) that dock to NES site 1 do significantly (p<0.05) sensitize high density MM cells to doxorubicin. Immunofluorescence microscopy revealed an increase in topo IIα in the cell nucleus of cells treated for 20 hours with the three lead site 1 inhibitors. Nuclear-cytoplasmic fractionation revealed that the NES site 1 docking molecules prevent nuclear export of topo IIα. These compounds may lead to new chemotherapeutic treatments of myeloma.

No relevant conflicts of interest to declare.