Casein Kinase II Modulates Topoisomerase II Alpha Nuclear Export and Drug Sensitivity in Multiple Myeloma

Abstract 4032

Nuclear export of topoisomerase II alpha (topo IIα) to the cytoplasm results in de novo resistance to topo IIα inhibitors in hematological malignancies. We have previously demonstrated that topo IIα is exported from the nucleus of human multiple myeloma cells by a CRM1-dependent mechanism at densities similar to those in myeloma patient bone marrow (Engel et al, Exp. Cell Res. 295:421-31, 2004). We have also identified the nuclear export signals for topo IIα at amino acids 1017–1028 and 1054–1066 using mutated full-length FLAG-topo IIα protein and immunofluorescence microscopy, (Turner et al, J. Cell Sci. 117:3061-71, 2004). In addition, blocking nuclear export with a CRM1 inhibitor or by using CRM1-siRNA sensitizes myeloma cells to topo II poisons (Turner et al. Cancer Res. 69:6899-905, 2009). Therefore, preventing nuclear export may sensitize myeloma cells to topo II inhibitors.

To determine what signals CRM1-mediated nuclear export of topo IIα we investigated the phosphorylation status of topo IIα, both in the nucleus and in the cytoplasm, using proteomic technologies. Topo IIα was isolated from the nuclei and cytoplasm of human myeloma cells by immunoprecipitation. Purified topo IIα fractions were analyzed by Orbitrap mass spectroscopy. We found that serine 1524 was highly phosphorylated in the cytoplasmic fraction. Using site-directed mutagenesis we converted serine 1524 to an alanine to determine if mutated FLAG-tagged topo IIα export was reduced as compared to wild-type FLAG-tagged topo IIα. Serine 1524 is a CK2 phosphorylation site, therefore, we tested a recently published and highly specific inhibitor of CK2; CX-4945 (Ferguson et al, FEBS Letters 585:104–110, 2011). This drug was used in combination with the topo IIα inhibitor doxorubicin and assayed for apoptosis by caspase 3 cleavage and flow cytometry both in vitro using myeloma cell lines and ex vivo using patient bone marrow mononuclear cells.

Using mass spectroscopy we found that cytoplasmic but not nuclear topo IIα was phosphorylated at serine 1524, a published CK2 motif. Using site-directed mutagenesis we converted serine 1524 to an alanine and found that mutated FLAG-topo IIα export was reduced, as compared to wild-type FLAG- topo IIα. The CK2 inhibitor CX-4945 induced apoptosis in both myeloma cell lines and in myeloma patient bone marrow aspirates. In addition, we found that CX-4945 prevented nuclear export of topo IIα in high-density cells. These data were duplicated using a CK2 specific siRNA to knockdown CK2 expression (90% knockdown). Blocking nuclear export of topo IIα with the CK2 inhibitor CX-4945 or CK2-specific siRNA sensitized drug-resistant myeloma cells to the topo II poison doxorubicin. These data may have potential clinical implications in the treatment of multiple myeloma.