Analysis of Protein Biology to Increase Efficacy of Aurora Kinase Inhibitors for Multiple Myeloma Therapy.

Despite improvement in therapeutic regimes which have extended survival of patients with multiple myeloma, the search for novel targets to further advance treatment options continues. It is critical to the success of targeted cancer therapy that the rational selection of drugs be derived from the molecular and cellular biology of protein targets. Furthermore, studies have indicated that focused combination drug therapy could produce remarkable improvement in patient outcomes. Aurora kinases A/B/C (AURK), components of the centrosome and important for cell cycle function, are promising new targets in the treatment of numerous types of cancer. We have previously validated AURKs as a therapeutic target in pre-clinical models of multiple myeloma. In this study our aim is to better define the biology of AURKs in both normal and malignant cells, with the goal of identifying rational strategies for improving the therapeutic potential of Aurora kinase inhibitors (AKIs) as anti-cancer agents. We use a systematic two-pronged approach to map post-translational modifications and novel protein-protein interactions that define the interactome of AURKs. We also examine whether Aurora kinase interactions and posttranslational modifications are significantly different in cancer cells compared to normal cells. Increased understanding of Aurora kinase biology will help develop mechanisms to intensify the anti-cancer effects of AKIs. SUMOylation is a recently discovered posttranslational modification of numerous proteins that has been linked to many cancer types. A small ubiquitin-like modifier (SUMO) group is conjugated at the canonical ubiquitin site of a protein to alter its function, stability or sub-cellular localization. We believe that Aurora kinases may be differentially SUMOylated in cancer cells compared to normal cells, and that SUMOylation of Aurora kinases in cancer cells may alter their function and their susceptibility to AKI therapy. Here we have determined putative SUMOylation sites of all three AURKs in silico and also have evidence of AURK SUMOylation using an in vitro assay and also in human HEK293 cells. Furthermore, the gene encoding the central SUMO conjugating enzyme - UBC9 - is expressed at higher levels in CD138+ malignant plasma cells compared to other CD138−bone marrow cells in myeloma patients. As part of our strategy, we have also used Tandem Affinity Purification (TAP) technology to identify novel protein-protein interactions of Aurora kinases. In the initial stage, we have established stable expressing clones in a human cancer cell line and a normal line that express TAP-tagged AURKA/B or C. In pilot experiments we have successfully isolated pure recombinant AURKA along with interacting proteins using the TAP method. Using mass spectrometry (MS/MS) protein ID, we have identified putative protein interaction partners with AURKA which have implications in general cancer biology and centrosomal structure. In the future, these interactions will be validated using conventional biochemical methods before being tested in myeloma cell lines.