Aurora kinases play an important role in cell division by controlling chromatid segregation. Aberrant functions of aurora kinases result in genetic instability, a condition often seen in cancers. While Aurora A is important in the alignment of the sister chromatids, Aurora B, a spindle fiber associated kinase, heavily influences the equal division of sister chromatids and along with the Aurora C kinase also assists in a regulated cell division. Inhibition of either of the Aurora family kinases affects chromosomal alignment and segregation during the course of cell division resulting in polyploidy, cell growth arrest and ultimately cell death. VEGF has been implicated in the increased angiogenesis in MM patients. Increase in VEGF levels leads to upregulation in the Ras/Mek/Erk pathway and increased angiogenesis, cell proliferation and decreased apoptosis. Inhibiting this pathway has shown to induce apoptosis in MM cells. We therefore investigated the role of a small molecule inhibitor A-1014907 that inhibits both aurora kinases and VEGF stimulated Ras/Mek/Erk pathway.
A-1014907 was synthesized and provided by Abbott Laboratories Ltd. Stock solutions were made in DMSO, and subsequently diluted in RPMI-1640 medium for use. MM cell lines were cultured in RPMI 1640 containing 10% fetal bovine serum, penicillin, and streptomycin. Cytotoxicity was measured using the MTT viability assay and proliferation using thymidine uptake. Apoptosis was measured using flow cytometry upon cell staining with Annexin V-FITC and propidium iodide (PI) for cell lines and patient cells. Immunoblotting was done on cell extracts at various time points following incubation with the drug in order to study the cell signaling pathways.
A-1014907 was able to induce cytotoxicity and inhibit proliferation in all MM cell lines tested with IC50 values between 50–100nM. Similar extents of inhibition of proliferation was also observed when MM cells were co-cultured with bone marrow stromal cells or HUVEC cells or tumor promoting cytokines IL6, IGF and VEGF. The increase in cytotoxicity was due to apoptotic cell death observed in both MM cell lines and patient cells. Cell cycle assays demonstrated that A-1014907 was able to induce cell cycle arrest at the G2/M stage of cell cycle followed by polyploidy indicative of Aurora B inhibition. We also observed an increase over time the proportion of cells in sub G0/G1 stage indicative of cell death. Western blots were performed to understand the mechanism of action of A-1014907. We observed that A-1014907 was able to significantly down regulate Aurora B activity as measured by pHistone H3 (Ser 10) down regulation. This was accompanied by up regulation of p21 and down regulation of CDK4 and cyclin E both indicative of G2/M arrest. We also observed the ability of A-1014907 to inhibit the Ras/Mek/Erk pathway by measuring levels of pErk post treatment with the drug. A-1014907 potently inhibited pErk levels and this down regulation was also observed when MM cells were co-cultured with either VEGF or HUVEC cells. Furthermore, A-1014907 at sub IC50 doses induced synergistic cell death of MM cell lines when combined with sub IC50 doses of dexamethasone
A-1014907 clearly inhibits the Ras/Mek/Erk pathway and aurora B activity and induces apoptosis in MM cell lines and patient cells. We are currently using siRNA to aurora A in combination with A-1014907. We are also examining the mechanism of action of dexamethasone in combination with A-1014907. All this will help to design clinical trials with A-1014907 either alone or in combination with other anti-MM agents in MM patients.
Kumar:Celgene: Consultancy, Research Funding; Merck: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Research Funding; Novartis: Research Funding; Genzyme: Research Funding; Cephalon: Research Funding.
Asterisk with author names denotes non-ASH members.