Abstract 725: Protein kinase-CK2 inhibition as a potential treatment for multiple myeloma

Abstract

Abstract Hypothesis Nuclear export of topo IIα to the cytoplasm results in de novo resistance to topo IIα inhibitors in hematological malignancies. Background 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 fluorescence 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. Methods 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 three recently published and highly specific inhibitors of CK2; quinilizarin, resorufin and hematein. These drugs were used in combination with the topo IIα inhibitor doxorubicin and assayed for apoptosis by caspase 3 cleavage and flow cytometry. Results 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. Of the tested CK2 inhibitors, hematein, quinalizarin and resorufin, we found that the CK2 inhibitor quinalizarin (1,2,5,8-tetrahydroxyanthracene-9,10-dione) produced significantly more apoptosis in the nanomolar range than both resorufin and hematein. In addition, we found that quinalizarin 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). Conclusion We found that blocking nuclear export of topo IIα with the CK2 inhibitor quinalizarin or CK2-specific siRNA sensitized drug-resistant myeloma cells to the topo II poisons doxorubicin and VP-16. These data may have potential clinical implications in the treatment of multiple myeloma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 725.