Patrick A. Eyers

Validating Aurora B as an anti-cancer drug target

The Aurora kinases, a family of mitotic regulators, have received much attention as potential targets for novel anti-cancer therapeutics. Several Aurora kinase inhibitors have been described including ZM447439, which prevents chromosome alignment, spindle checkpoint function and cytokinesis. Subsequently, ZM447439-treated cells exit mitosis without dividing and lose viability. Because ZM447439 inhibits both Aurora A and B, we set out to determine which phenotypes are due to inhibition of which kinase. Using molecular genetic approaches, we show that inhibition of Aurora B kinase activity phenocopies ZM447439. Furthermore, a novel ZM compound, which is 100 times more selective for Aurora B over Aurora A in vitro, induces identical phenotypes. Importantly, inhibition of Aurora B kinase activity induces a penetrant anti-proliferative phenotype, indicating that Aurora B is an attractive anti-cancer drug target. Using molecular genetic and chemical-genetic approaches, we also probe the role of Aurora A kinase activity. We show that simultaneous repression of Aurora A plus induction of a catalytic mutant induces a monopolar phenotype. Consistently, another novel ZM-related inhibitor, which is 20 times as potent against Aurora A compared with ZM447439, induces a monopolar phenotype. Expression of a drug-resistant Aurora A mutant reverts this phenotype, demonstrating that Aurora A kinase activity is required for spindle bipolarity in human cells. Because small molecule-mediated inhibition of Aurora A and Aurora B yields distinct phenotypes, our observations indicate that the Auroras may present two avenues for anti-cancer drug discovery.

Identification of novel phosphorylation sites on Xenopus laevis aurora;A and analysis of phosphopeptide enrichment by immobilized;metal-affinity chromatography

Mass spectrometric analysis of proteolytically derived phosphopeptides has developed into a widespread technique for the identification of phosphorylated amino acids. Using liquid chromatography-electrospray ionization tandem mass spectrometry, 14 phosphorylation sites were identified on Xenopus laevis His6-Aurora A, a highly conserved regulator of centrosome maturation and cell division. These included seven novel phosphorylation sites, Ser-12, Thr-21, Thr-103, Ser-116, Thr-122, Tyr-155, and Thr-294, as well as the previously identified regulatory sites, Ser-53, Thr-295, and Ser-349. The identification of these novel phosphorylation sites will be important for future studies aimed at elucidating the mechanisms of Aurora A regulation by phosphorylation. Furthermore, we demonstrate that a “kinase-inactive” mutant of Aurora A, K169R, still retains 10% of activity of the wild-type enzyme in vitro along with occupancy of Thr-295 and Ser-12. However, mutation of Asp-281 to Ala completely abolishes activity of the enzyme and should therefore be used preferentially as a genuine kinase-dead construct. Because of the abundance of phosphorylated residues on His6-Aurora A, we found this protein to be an ideal tool for the characterization of immobilized metal-affinity chromatography (IMAC) as a method for phosphopeptide enrichment from complex mixtures. We present a detailed analysis of the binding and elution properties of both the phosphopeptides and unphosphorylated peptides of His6-Aurora A to Fe3+-IMAC before and after methyl esterification. Moreover, we demonstrate a significant difference in enrichment of phosphopeptides when different resins are used for Fe3+-IMAC and characterize the strengths and limitations of this methodology for the study of phosphoproteomics.

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains.

VX-680 Inhibits Aurora A and Aurora B Kinase Activity in Human Cells

VX-680, also known as MK-0457, is a member of a diverse group of small molecules that inhibit the Aurora kinases, and has shown significant potential as an anti-cancer agent. In keeping with many protein kinase inhibitors, this compound is not a monospecific agent, and its cellular specificity remains largely unknown. In cells, VX-680 blocks mitotic Histone H3 phosphorylation and induces polyploidy and apoptosis, consistent with inhibition of the mitotic protein kinase Aurora B. In this study, we have investigated the effects of VX-680 in proliferating human cancer cells, and demonstrate that it blocks the phosphorylation and activation of both Aurora A and B. Additionally, VX-680 suppresses the phosphorylation of specific substrates of each enzyme, including the Aurora A target TACC3 on Ser558. Exposure to VX-680 induces a monopolar spindle phenotype, delays mitotic progression and rapidly overrides the spindle assembly checkpoint in the presence of spindle poisons. VX-680 also exhibits potent cytotoxicity when compared to the well documented Aurora B inhibitor ZM447439. Taken together, these data identify Aurora A and Aurora B as dual intracellular targets of VX-680.

A framework for identification of actionable cancer genome dependencies in small cell lung cancer

Small cell lung cancer (SCLC) accounts for about 15% of all lung cancers. The prognosis of SCLC patients is devastating and no biologically targeted therapeutics are active in this tumor type. To develop a framework for development of specific SCLC-targeted drugs we conducted a combined genomic and pharmacological vulnerability screen in SCLC cell lines. We show that SCLC cell lines capture the genomic landscape of primary SCLC tumors and provide genetic predictors for activity of clinically relevant inhibitors by screening 267 compounds across 44 of these cell lines. We show Aurora kinase inhibitors are effective in SCLC cell lines bearing MYC amplification, which occur in 3–7% of SCLC patients. In MYC-amplified SCLC cells Aurora kinase inhibition associates with G2/M-arrest, inactivation of PI3-kinase (PI3K) signaling, and induction of apoptosis. Aurora dependency in SCLC primarily involved Aurora B, required its kinase activity, and was independent of depletion of cytoplasmic levels of MYC. Our study suggests that a fraction of SCLC patients may benefit from therapeutic inhibition of Aurora B. Thus, thorough chemical and genomic exploration of SCLC cell lines may provide starting points for further development of rational targeted therapeutic intervention in this deadly tumor type.

Drug-Resistant Aurora a Mutants for Cellular Target Validation of the Small Molecule Kinase Inhibitors Mln8054 and Mln8237.

The Aurora kinases regulate multiple aspects of mitotic progression, and their overexpression in diverse tumor types makes them appealing oncology targets. An intensive research effort over the past decade has led to the discovery of chemically distinct families of small molecule Aurora kinase inhibitors, many of which have demonstrated therapeutic potential in model systems. These agents are also important tools to help dissect signaling pathways that are orchestrated by Aurora kinases, and the antiproliferative target of pan-Aurora inhibitors such as VX-680 has been validated using chemical genetic techniques. In many cases the nonspecific nature of Aurora inhibitors toward unrelated kinases is well established, potentially broadening the spectrum of cancers to which these compounds might be applied. However, unambiguously demonstrating the molecular target(s) for clinical kinase inhibitors is an important challenge, one that is absolutely critical for deciphering the molecular basis of compound specificity, resistance, and efficacy. In this paper, we have investigated amino acid requirements for Aurora A sensitivity to the benzazepine-based Aurora inhibitor MLN8054 and the close analogue MLN8237, a second-generation compound that is in phase II clinical trials. A crystallographic analysis facilitated the design and biochemical investigation of a panel of resistant Aurora A mutants, a subset of which were then selected as candidate drug-resistance targets for further evaluation. Using inducible human cell lines, we show that cells expressing near-physiological levels of a functional but partially drug-resistant Aurora A T217D mutant survive in the presence of MLN8054 or MLN8237, authenticating Aurora A as a critical antiproliferative target of these compounds.

Discovery and exploitation of inhibitor-resistant Aurora and Polo kinase mutants for the analysis of mitotic networks

The Aurora and Polo-like kinases are central components of mitotic signaling pathways, and recent evidence suggests that substantial cross-talk exists between Aurora A and Plk1. In addition to their validation as novel anticancer agents, small molecule kinase inhibitors are increasingly important tools to help dissect clinically relevant protein phosphorylation networks. However, one major problem associated with kinase inhibitors is their promiscuity toward “off-target” members of the kinome, which makes interpretation of data obtained from complex cellular systems challenging. Additionally, the emergence of inhibitor resistance in patients makes it clear that an understanding of resistance mechanisms is essential to inform drug design. In this study, we exploited structural knowledge of the binding modes of VX-680, an Aurora kinase inhibitor, and BI 2536, a Polo-like kinase inhibitor, to design and evaluate drug-resistant kinase mutants. Using inducible stable human cell lines, we authenticated mitotic targets for both compounds and demonstrated that Aurora A mutants exhibit differential cellular sensitivity toward the inhibitors VX-680 and MLN8054. In addition, we validated Aurora B as an important anti-proliferative target for VX-680 in model human cancer cells. Finally, this chemical genetic approach allowed us to prove that Aurora A activation loop phosphorylation is controlled by a Plk1-mediated pathway in human cells.

Spindle checkpoint proteins Mad1 and Mad2 are required for cytostatic factor–mediated metaphase arrest

In cells containing disrupted spindles, the spindle assembly checkpoint arrests the cell cycle in metaphase. The budding uninhibited by benzimidazole (Bub) 1, mitotic arrest-deficient (Mad) 1, and Mad2 proteins promote this checkpoint through sustained inhibition of the anaphase-promoting complex/cyclosome. Vertebrate oocytes undergoing meiotic maturation arrest in metaphase of meiosis II due to a cytoplasmic activity termed cytostatic factor (CSF), which appears not to be regulated by spindle dynamics. Here, we show that microinjection of Mad1 or Mad2 protein into early Xenopus laevis embryos causes metaphase arrest like that caused by Mos. Microinjection of antibodies to either Mad1 or Mad2 into maturing oocytes blocks the establishment of CSF arrest in meiosis II, and immunodepletion of either protein blocked the establishment of CSF arrest by Mos in egg extracts. A Mad2 mutant unable to oligomerize (Mad2 R133A) did not cause cell cycle arrest in blastomeres or in egg extracts. Once CSF arrest has been established, maintenance of metaphase arrest requires Mad1, but not Mad2 or Bub1. These results suggest a model in which CSF arrest by Mos is mediated by the Mad1 and Mad2 proteins in a manner distinct from the spindle checkpoint.

Spindle Pole Regulation by a Discrete Eg5-Interacting Domain in TPX2

Targeting protein for Xklp2 (TPX2) activates the Ser/Thr kinase Aurora A in mitosis and targets it to the mitotic spindle [1, 2]. These effects on Aurora A are mediated by the N-terminal domain of TPX2, whereas a C-terminal fragment has been reported to affect microtubule nucleation [3]. Using the Xenopus system, we identified a novel role of TPX2 during mitosis. Injection of TPX2 or its C terminus (TPX2-CT) into blastomeres of two-cell embryos led to potent cleavage arrest. Despite cleavage arrest, TPX2-injected embryos biochemically undergo multiple rounds of DNA synthesis and mitosis, and arrested blastomeres have abnormal spindles, clustered centrosomes, and an apparent failure of cytokinesis. In Xenopus S3 cells, transfection of TPX2-FL causes spindle collapse, whereas TPX2-CT blocks pole segregation, resulting in apposing spindle poles with no evident displacement of Aurora A. Analysis of TPX2-CT deletion peptides revealed that only constructs able to interact with the class 5 kinesin-like motor protein Eg5 induce the spindle phenotypes. Importantly, injection of Eg5 into TPX2-CT-arrested blastomeres causes resumption of cleavage. These results define a discrete domain within the C terminus of TPX2 that exerts a novel Eg5-dependent function in spindle pole segregation.

Dawn of the dead: protein pseudokinases signal new adventures in cell biology

Recent studies of proteins containing kinase-like domains that lack catalytic residue(s) classically required for phosphotransfer, termed pseudokinases, have uncovered important roles in cell signalling across the kingdoms of life. Additionally, mutations within pseudokinase domains are known to underlie human diseases, suggesting that these proteins may represent new and unexplored therapeutic targets. To date, few pseudokinases have been studied in intricate detail, but as described in the present article and in the subsequent papers in this issue of Biochemical Society Transactions, several new studies have provided an advanced template and an improved framework for interrogating the roles of pseudokinases in signal transduction. In the present article, we review landmarks in the establishment of this field of study, highlight some experimental challenges and propose a simple scheme for definition of these domains based on their primary sequences, rather than experimentally defined nucleotide-binding or catalytic activities.