to Identification of Potential Sites of Drug Resistance Mutations: Aurora Kinases, MEK1 Camptothecin and the PI3Ks The Aurora kinases are a family of serine/threonine kinases that are key regulators of eukaryotic cell mitosis. There are three Aurora kinases in humans that have been characterized to date: Aurora A, Aurora B and Aurora C. Aurora A is localized to centrosomes and spindle poles during various phases of mitosis and is closely associated with centrosome maturation. Krishnamurty and Maly Page 8 ACS Chem Biol. Author manuscript, available in PMC 2011 January 15. NIH PA Author Manuscript NIH PA Author Manuscript NIH PA Author Manuscript Aurora B localizes to microtubules and is responsible for histone H3 phosphorylation as well as spindle assembly checkpoint and cytokinesis.
Aurora C is thought to be a chromosome passenger, but little more is known about this third class of mitotic serine/ threonine kinases. Overexpression of these enzymes is apparent in several human cancers, thus, these kinases have become popular targets for anti cancer therapies. Baicalein A number of ATP competitive inhibitors of the Aurora kinases have been discovered that block such cellular actions as chromosome alignment, SAC and cell division. Some of these inhibitors include the small molecules ZM447439, VX 680 and Hesperadin. ZM447439 is a quinazoline based inhibitor, which is 20 fold more potent against Aurora B than Aurora A. Mammalian cells that are treated with ZM447439 enter mitosis but have a perturbed spindle assembly and chromosome alignment, inhibiting cytokinesis.
VX 680, a pyrimidinyl based compound is a potent inhibitor of both Aurora A and B in cells. VX 680 is highly effective in blocking cell cycle progression and inducing apoptosis in a variety of developing tumors. In addition, VX 680 has been shown to have anti tumor activity in rodent xenograft models. Hesperadin acts much like ZM447439, inhibiting chromosome alignment and segregation in the cell. While no Aurora kinase inhibitors have yet been approved for clinical use, the lessons learned from the emergence of drug resistance to BCR ABL and EGFR inhibitors stress the importance of anticipating which specific mutations, and their consequent effects, may arise. To this end, Girdler and co workers developed a novel genetic screen to identify cell lines that are resistant to the Aurora kinase inhibitor ZM447439.
A key component of this screen is the use of HCT 116 cells, which are a hypermutagenic cell line due to a defect in DNA mismatch repair. In addition, these cells express low levels of drug transporters, which reduces the likelihood of resistance occurring through this mechanism. HCT 116 cells were treated with a 1 M cytotoxic concentration of ZM447439 over a three week span. Seven cell lines were generated from those cells that maintained strong colony growth in the presence of ZM447439, with several of these cell lines maintaining high cell numbers in the presence of increasing concentrations of the drug. In comparison to parental control cells, two of the resistant cell lines maintained both cell division and histone H3 phosphorylation, indicating that Aurora B kinase was indeed active, and a mutation in this kinase might be the source of drug resistance. Sequencing of Aurora cDNAs from the seven drug resistant clones showed that all