Abstract
Multiple myeloma (MM) is an incurable bone marrow derived plasma cell malignancy. Despite significant improvements in treating patients suffering from this disease, MM remains uniformly fatal owing to intrinsic or acquired drug resistance. Thus, additional modalities for treating MM are required. In this study, we examined the anti-tumor activity of MLN8237, a small molecule Aurora-A kinase inhibitor, in experimental models of MM. Aurora-A is a mitotic kinase that localizes to centrosomes and the proximal mitotic spindle and functions in mitotic spindle formation and in regulating chromatid congression and segregation. Aurora-A gene amplification and protein overexpression is a common event in many cancers, and has been experimentally linked to genetic instability and tumorigenesis. In MM, increased Aurora-A gene expression has previously been correlated with centrosome amplification and a worsened disease prognosis. Thus, inhibition of Aurora A in MM may prove to be therapeutically beneficial. Here we show that Aurora-A protein is highly expressed in eight distinct MM cell lines. The affect of Aurora-A inhibition in these cell lines was examined in cytotoxicity (MTT viability) and proliferation (3[H]thymidine incorporation) assays by treating with MLN8237 (0.25 mM −32 mM) for 24, 48 and 72h. Although there was no significant inhibition of cell viability and proliferation at 24h, a marked effect occurred 48 and 72h after compound addition at concentrations as low as 0.25 mM. Interestingly, the melphalan resistant line (LR5) and Doxorubucin resistant line (Dox40) were among the least sensitive to MLN8237 induced cell cytotoxicity. The affect of MLN8237 on peripheral blood mononuclear cells (PBMCs) from healthy donors was also examined at the same concentrations and exposure time used for the MM cell lines. In healthy PBMCs, MLN8237 did not induce cytotoxicity as measured by the MTT assay, but there was a significant inhibition of proliferation at 48 and 72h as measured by the 3[H]thymidine incorporation assay at concentrations above 4uM. To delineate the mechanisms of cytotoxicity and growth inhibitory activity of MLN8237, apoptotic markers and cell cycle profiles were examined in the MM cell lines. Fluorescence conjugated-Annexin V and propidium iodide (PI) co-staining of MM cell lines after culturing in the presence or absence of MLN8237 at 1 mM (IC50) for 24, 48 and 72h demonstrated that MLN8237 induces apoptosis in these lines. This finding was corroborated by demonstrating increased capase-9 expression by Western blot analysis. Cell cycle analysis by flow cytometry demonstrated that MLN8237 results in an accumulation of tetraploid cells, presumably by abrogating G2/M progression. These results suggest that MLN8237 represents a possible novel agent for treating MM patients. Additional studies are ongoing to assess the anti-tumor effects of MLN8237 alone and in combination with other therapeutic agents in xenograft models of MM.
Mio depletion links mTOR regulation to Aurora A and Plk1 activation at mitotic centrosomes