MLN8054, a small-molecule inhibitor of Aurora A, causes spindle pole and chromosome congression defects leading to aneuploidy

Aurora A kinase plays an essential role in the proper assembly and function of the mitotic spindle, as its perturbation causes defects in centrosome separation, spindle pole organization, and chromosome congression. Moreover, Aurora A disruption leads to cell death via a mechanism that involves aneuploidy generation. However, the link between the immediate functional consequences of Aurora A inhibition and the development of aneuploidy is not clearly defined. In this study, we delineate the sequence of events that lead to aneuploidy following Aurora A inhibition using MLN8054, a selective Aurora A small-molecule inhibitor. Human tumor cells treated with MLN8054 show a high incidence of abnormal mitotic spindles, often with unseparated centrosomes. Although these spindle defects result in mitotic delays, cells ultimately divide at a frequency near that of untreated cells. We show that many of the spindles in the dividing cells are bipolar, although they lack centrosomes at one or more spindle poles. MLN8054-treated cells frequently show alignment defects during metaphase, lagging chromosomes in anaphase, and chromatin bridges during telophase. Consistent with the chromosome segregation defects, cells treated with MLN8054 develop aneuploidy over time. Taken together, these results suggest that Aurora A inhibition kills tumor cells through the development of deleterious aneuploidy.     

The inhibition of Aurora A abrogates the mitotic delay induced by microtubule perturbing agents

The spindle assembly checkpoint functions during mitosis to ensure that chromosomes are properly aligned in mitotic cells prior to the onset of anaphase, thereby ensuring an equal segregation of genetic material to each daughter cell. Defects in the function of this checkpoint lead to aneuploidy, and eventually to cell death or senescence. The Aurora-related kinases, and in particular Aurora B, have been shown to play a role in regulating the spindle assembly checkpoint. In this study, we demonstrate that Aurora A activity is required for maintainance of the spindle assembly checkpoint mediated-mitotic delay induced by microtubule perturbing agents. Inhibition of Aurora A using MLN8054, a selective small-molecule inhibitor of Aurora A, in paclitaxel- or nocodazole-treated cells induces cells to become multinucleated. Using time-lapse microscopy, we demonstrate that the multinucleation phenotype arises via mitotic slippage, which is significantly accelerated upon Aurora A inhibition. Under these conditions, the spindle assembly checkpoint protein BubR1 remains localized to kinetochores prior to mitotic slippage. Moreover, we demonstrate that Aurora B remains active in these mitotic cells, indicating that the mitotic slippage induced by MLN8054 is most likely due to the inhibition of Aurora A. This finding was corroborated by demonstrating that Aurora A depletion using RNA interference in paclitaxel-treated cells also induces multinucleation. Taken together, these results suggest that Aurora A is necessary for the maintenance of the mitotic delay induced in response to microtubule-perturbing agents.     

A novel cell-based, high-content assay for phosphorylation of Lats2 by Aurora A

Aurora A kinase is a key regulator of mitosis, which is upregulated in several human cancers, making it a potential target for anticancer therapeutics. Consequently, robust medium- to high-throughput cell-based assays to measure Aurora A kinase activity are critical for the development of small-molecule inhibitors. Here the authors compare measurement of the phosphorylation of two Aurora A substrates previously used in high-content screening Aurora A assays, Aurora A itself and TACC3, with a novel substrate Lats2. Using antibodies directed against phosphorylated forms of Aurora A (pThr288), P-TACC3 (pSer558), and P-Lats2 (pSer83), the authors investigate their suitability in parallel for development of a cell-based assay using several reference Aurora inhibitors: MLN8054, VX680, and AZD1152-HQPA. They validate a combined assay of target-specific phosphorylation of Lats2 at the centrosome and an increase in mitotic index as a measure of Aurora A activity. The assay is both sensitive and robust and has acceptable assay performance for high-throughput screening or potency estimation from concentration-response assays. It has the advantage that it can be carried out using a commercially available monoclonal antibody against phospho-Lats2 and the widely available Cellomics ArrayScan HCS reader and thus represents a significant addition to the tools available for the identification of Aurora A specific inhibitors.     

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.Protein kinase inhibitors are prime examples of small molecules with the potential to revolutionize the treatment of chronic disease states such as inflammation and cancer (1, 2). For example, the discovery of inhibitors of the BCR-ABL kinase has transformed the survival rates of patients diagnosed with tyrosine kinase-driven leukemias (3). Moreover, inhibitors of many distinct protein kinases have emerged as indispensable biological tools, in part through their rapid and often reversible mode of action, but also because of their widespread availability and utility in a range of research settings. Remarkably, scientific conclusions drawn in many thousands of peer-reviewed research papers every year rely upon experiments conducted with kinase inhibitors, but in only a handful of studies is the important question of inhibitor specificity explicitly addressed (4–7). This is a vital issue because claims for specificity have rarely stood the test of time, yet a detailed knowledge of kinase inhibitor promiscuity would be beneficial in the clinic, where the simultaneous blockade of multiple signaling pathways can be exploited as an anticancer strategy (8).The vast majority of kinase inhibitors bind in the conserved ATP-binding site located between the N- and C-terminal lobes of the catalytic domain, where they prevent nucleotide binding or lock the kinase into a structurally inactive confirmation. Inhibitor structure-activity relationship trends, which are often gleaned from combined biochemical and structural analysis, can be mechanistically revealing, but often fail to adequately address the interconnected issues of specificity and chemical resistance. Indeed, the emergence of drug resistance in chronic myeloid leukemia patients is testament to the high mutagenic susceptibility of protein kinases either selected for, or induced by, inhibitor exposure in vivo, making the discovery of mechanistically distinct inhibitors as backup therapies vitally important (9, 10).In human cells, the key mitotic events of centrosome separation, bipolar spindle formation, and chromosome segregation are linked to the physical separation of the genomes during cytokinesis (11). These conserved signaling programs are controlled by dedicated mitotic protein kinases, which include two prominent human gene families, the Aurora kinases (comprising Aurora A, B, and C) and the Polo-like kinases (comprising Plk1–4), whose overexpression in a spectrum of cancers make them outstanding drug candidates (12). A more detailed knowledge of the substrates and physiological events regulated by Aurora and Polo signaling pathways has been facilitated by the development of potent inhibitors of both enzyme families (13, 14). These include clinical candidates such as the dual Aurora/tyrosine kinase inhibitors VX-680 (15, 16) and AT9283 (17) and the Aurora inhibitors MLN8054 (18) and AZD1152 (19). In addition, the clinically advanced Plk1–3 inhibitor BI 2536 has been well characterized in human cells (20) and cancer models (21).One of the frustrations associated with interpreting cellular data obtained with compounds such as VX-680 and BI 2536 is their unknown cellular selectivity. No kinome-wide data are available in public data bases for any kinase inhibitors, and it is likely that these compounds have multiple kinase and non-kinase targets in human cells. For example, VX-680 inhibits both Aurora A and B in human cells and tyrosine kinases such as ABL, Src, and Flt3 in vitro (15, 22), raising the question as to which, if any, of these targets are critical for phenotypes and anti-proliferative effects observed after drug exposure. In addition, Plk1 and Aurora A signaling functions are mutually dependent in proliferating human cells (23–26). This makes interpretation of experiments in which Aurora A or Plk1 inhibitors are employed potentially confusing because phenotypes assigned to one inhibitor target might actually be due to indirect inhibition of the other kinase. To begin to address these issues, we have investigated the cellular plasticity of kinase inhibition by both VX-680 and BI 2536. By evaluating drug-resistant Aurora A and B proteins in vitro and exploiting these mutants in stable human cell lines, we demonstrate that drug-resistant forms of these kinases can be used to prove that phenotypes arising from VX-680 exposure are actually due to inhibition of the predicted mitotic targets. We demonstrate that a VX-680-resistant Aurora A mutant remains sensitive to the distinct anti-proliferative agent MLN8054 in human cells and that Aurora B is the critical target of VX-680 that promotes cell death in a cancer cell model. Furthermore, by analyzing a Plk1 mutant with decreased sensitivity to BI 2536, we establish that a mitotic phenotype arising from exposure to this drug is indeed due to Plk1 inhibition and that, during mitosis, Plk1 controls Aurora A phosphorylation at the critical activating residue Thr²⁸⁸.     

The induction of polyploidy or apoptosis by the Aurora A kinase inhibitor MK8745 is p53-dependent

Aurora kinases are mitotic serine/threonine protein kinases and are attractive novel targets for anticancer therapy. Many small-molecule inhibitors of Aurora kinases are currently undergoing clinical trials. Aurora A kinase is essential for successful mitotic transition. MK8745 is a novel and selective small-molecule inhibitor of Aurora A kinase. MK8745 induced apoptotic cell death in a p53-dependent manner when tested in vitro in cell lines of multiple lineages. Cells expressing wild-type p53 showed a short delay in mitosis followed by cytokinesis, resulting in 2N cells along with apoptosis. However, cells lacking or with mutant p53 resulted in a prolonged arrest in mitosis followed by endoreduplication and polyploidy. Cytokinesis was completely inhibited in p53-deficient cells, as observed by the absence of 2N cell population. The induction of apoptosis in p53-proficient cells was associated with activation of caspase 3 and release of cytochrome c but was independent of p21. Exposure of p53 wild-type cells to MK8745 resulted in the induction of p53 phosphorylation (ser15) and an increase in p53 protein expression. p53-dependent apoptosis by MK8745 was further confirmed in HCT 116 p53^−/− cells transfected with wild-type p53. Transient knockdown of Aurora A by specific siRNA recapitulated these p53-dependent effects, with greater percent induction of apoptosis in p53 wild-type cells. In conclusion, our studies show p53 as a determining factor for induction of apoptosis vs. polyploidy upon inhibition of Aurora A.Key words: Aurora A kinase, polyploidy, apoptosis, p53, cell cycle     

Aurora A inhibition by MNL8054 promotes centriole elongation during Drosophila male meiosis

Aurora A kinase plays an important role in several aspects of cell division, including centrosome maturation and separation, a crucial step for the correct organization of the bipolar spindle. Although it has long been showed that this kinase accumulates at the centrosome throughout mitosis its precise contribution to centriole biogenesis and structure has until now not been reported. It is not surprising that so little is known, due to the small size of somatic centrioles, where only dramatic structural changes may be identified by careful electron microscopy analysis. Conversely, centrioles of Drosophila primary spermatocytes increase tenfold in length during the first prophase, thus making any change easily detectable. Therefore, we examined the consequence of the pharmacological inhibition of Aurora A by MLN8054 on centriole biogenesis during early Drosophila gametogenesis. Here, we show that depletion of this kinase results in longer centrioles, mainly during transition from prophase to prometaphase of the first meiosis. We also found abnormal ciliogenesis characterized by irregularly growing axonemal doublets. Our results represent the first documentation of a potential requirement of Aurora A in centriole integrity and elongation.     

Aurora Kinases as Targets in Drug-Resistant Neuroblastoma Cells

Aurora kinase inhibitors displayed activity in pre-clinical neuroblastoma models. Here, we studied the effects of the pan-aurora kinase inhibitor tozasertib (VX680, MK-0457) and the aurora kinase inhibitor alisertib (MLN8237) that shows some specificity for aurora kinase A over aurora kinase B in a panel of neuroblastoma cell lines with acquired drug resistance. Both compounds displayed anti-neuroblastoma activity in the nanomolar range. The anti-neuroblastoma mechanism included inhibition of aurora kinase signalling as indicated by decreased phosphorylation of the aurora kinase substrate histone H3, cell cycle inhibition in G2/M phase, and induction of apoptosis. The activity of alisertib but not of tozasertib was affected by ABCB1 expression. Aurora kinase inhibitors induced a p53 response and their activity was enhanced in combination with the MDM2 inhibitor and p53 activator nutlin-3 in p53 wild-type cells. In conclusion, aurora kinases are potential drug targets in therapy-refractory neuroblastoma, in particular for the vast majority of p53 wild-type cases.     

Polo and Aurora kinases: lessons derived from chemical biology

During the cell division cycle, mitotic entry, spindle assembly, chromosome segregation, and cytokinesis must all be carefully coordinated to ensure that the two daughter cells inherit all the genetic material required for further growth and development. Central to this coordination are several protein kinases including Aurora A, Aurora B, and the Polo-like kinase, Plk1. A number of small-molecule Aurora and Plk1 inhibitors have been developed because these kinases are seen as attractive anticancer drug targets. These inhibitors are now being widely used as chemical biology tools to understand how these kinases ensure faithful genome transmission.     

Synthesis and identification of 2,4-bisanilinopyrimidines bearing 2,2,6,6-tetramethylpiperidine-N-oxyl as potential Aurora A inhibitors

The Aurora kinases are a family of serine/threonine kinases that interact with components of the mitotic apparatus and serve as potential therapeutic targets in oncology. Herein, we reported a series of 2,4-bisanilinopyrimidines bearing 2,2,6,6-tetramethylpiperidine-N-oxyl with selective inhibition of Aurora A in either enzymatic assays or cellular phenotypic assays, and displaying more potent anti-proliferation compared with that of VX-680. The most potent compound 10a forms better interaction with Aurora A than Aurora B in molecular docking. Mechanistic studies revealed that 10a disrupt the spindle formation, block the cell cycle progression in the G2/M phase and induce apoptosis in HeLa cell. These results suggested that the produced series of compounds are potential anticancer agents for further development as selective Aurora A inhibitors.     

Aurora kinases: structure, functions and their association with cancer

Background: Aurora kinases are a recently discovered family of kinases (A, B & C) consisting of highly conserved serine\threonine protein kinases found to be involved in multiple mitotic events: regulation of spindle assembly checkpoint pathway, function of centrosomes and cytoskeleton, and cytokinesis. Aberrant expression of Aurora kinases may lead to cancer. For this reason the Aurora kinases are potential targets in the treatment of cancer. In this review we discuss the biology of these kinases: structure, function, regulation and association with cancer. Methods and Results: A literature search. Conclusion: Many of the multiple functions of mitosis are mediated by the Aurora kinases. Their aberrant expression can lead to the deregulation of cell division and cancer. For this reason, the Aurora kinases are currently one of the most interesting targets for cancer therapy. Some Aurora kinase inhibitors in the clinic have proven effectively on a wide range of tumor types. The clinical data are very encouraging and promising for development of novel class of structurally different Aurora kinase inhibitors. Hopefully the Aurora kinases will be potentially useful in drug targeted cancer treatment.     

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.     

Characterization of a highly selective inhibitor of the Aurora kinases

Aurora kinases play an essential role in mitosis and cell cycle regulation. In recent years Aurora kinases have proved popular cancer targets and many inhibitors have been developed. The majority of these clinical candidates are multi-targeted, rendering them inappropriate as tools for studying Aurora kinase mediated signaling. Here we report discovery of a highly selective inhibitor of Aurora kinases A, B and C, with potent cellular activity and minimal off-target activity (PLK4). The X-ray co-crystal structure of Aurora A in complex with compound 2 is reported, and provides insights into the structural determinants of ligand binding and selectivity.     

Synthesis and biological evaluation of nitroxide labeled pyrimidines as Aurora kinase inhibitors

To find novel effective Aurora kinases inhibitors, a series of structurally interesting nitroxide labeled pyrimidines were synthesized and evaluated their anti-proliferative and Aurora kinases inhibitory activities. Among them, butyl 2-(3-((5-fluoro-2-((4-((1-oxyl-2,2,6,6-tetramethylpiperidin-4-yl)carbamoyl) phenyl) amino)pyrimidin-4-yl)amino)-1H-pyrazol-5-yl)acetate (22) possessed the most potent anti-proliferative effects against four carcinoma cell lines with IC₅₀ values in range of 0.89–11.41?μM, and kinases inhibition against Aurora A and B with the IC₅₀ values were 9.3 and 2.8?nM, respectively. Furthermore, compound 22 blocked the phosphorylation of Aurora A (T288), Aurora B (Thr232) and HisH3, decreased the expression of proteins TPX2, Eg5 and Bora, as well as disrupted the mitotic spindle formation in HeLa cells. Molecular docking studies indicated that compound 22 well interact with both Aurora A and B. The results showed that compound 22 is a potential anticancer agent as promising pan-Aurora kinase inhibitor.     

A chemical-genetic approach for functional analysis of plant protein kinases

Plant genomes encode hundreds of protein kinases, yet only for a small fraction of them precise functions and phosphorylation targets have been identified. Recently, we applied a chemical-genetic approach to sensitize the tomato serine/threonine kinase Pto to analogs of PP1, an ATP-competitive and cell-permeable small-molecule inhibitor. The Pto kinase confers resistance to Pst bacteria by activating immune responses upon specific recognition of bacterial effectors. By using PP1 analogs in combination with the analog-sensitive Pto, we shed new light on the role of Pto kinase activity in effector recognition and signal transduction. Here we broaden the use of this chemical-genetic approach to another defense-related plant protein kinase, the MAP kinase LeMPK3. In addition, we show that analog-sensitive but not wild-type kinases are able to use unnatural N⁶-modified ATP analogs as phosphodonors that can be exploited for tagging direct phosphorylation targets of the kinase of interest. Thus, sensitization of kinases to analogs of the small-molecule inhibitor PP1 and ATP can be an effective tool for the discovery of cellular functions and phosphorylation substrates of plant protein kinases.Key words: chemical genetics, gatekeeper, LeMPK3, protein kinase, Pto, small-molecule inhibitor     

The induction of polyploidy or apoptosis by the Aurora A kinase inhibitor MK8745 is p53-dependent

Aurora kinases are mitotic serine/threonine protein kinases and are attractive novel targets for anticancer therapy. Many small-molecule inhibitors of Aurora kinases are currently undergoing clinical trials. Aurora A kinase is essential for successful mitotic transition. MK8745 is a novel and selective small-molecule inhibitor of Aurora A kinase. MK8745 induced apoptotic cell death in a p53-dependent manner when tested in vitro in cell lines of multiple lineages. Cells expressing wild-type p53 showed a short delay in mitosis followed by cytokinesis, resulting in 2N cells along with apoptosis. However, cells lacking or with mutant p53 resulted in a prolonged arrest in mitosis followed by endoreduplication and polyploidy. Cytokinesis was completely inhibited in p53-deficient cells, as observed by the absence of 2N cell population. The induction of apoptosis in p53-proficient cells was associated with activation of caspase 3 and release of cytochrome c but was independent of p21. Exposure of p53 wild-type cells to MK8745 resulted in the induction of p53 phosphorylation (ser15) and an increase in p53 protein expression. p53-dependent apoptosis by MK8745 was further confirmed in HCT 116 p53-/- cells transfected with wild-type p53. Transient knockdown of Aurora A by specific siRNA recapitulated these p53- dependent effects, with greater percent induction of apoptosis in p53 wild-type cells. In conclusion, our studies show p53 as a determining factor for induction of apoptosis vs. polyploidy upon inhibition of Aurora A.     

Discovery and optimization of novel benzothiophene-3-carboxamides as highly potent inhibitors of Aurora kinases A and B

Aurora kinases as regulators of cell division have become promising therapeutic targets recently. Here we report novel, low molecular weight benzothiophene-3-carboxamide derivatives designed and optimized for inhibiting Aurora kinases. The most effective compound 36 inhibits Aurora kinases in vitro in the nanomolar range and diminishes HCT 116 cell viability blocking cytokinesis and inducing apoptosis. According to western blot analysis, the lead molecule inhibits Aurora kinases equipotently to VX-680 (Tozasertib) and similarly synergizes with other targeted drugs.     

3-Hydroxyflavone inhibits endogenous Aurora B and induces growth inhibition of cancer cell line

The Aurora kinases play a critical role in mitosis and have been suggested as promising targets for cancer therapy due to their frequent overexpression in a variety of tumors. Compared with established inhibitors of cell division such as the anti-tubulins, novel agents target mitotic enzymes and show similar efficacy but with fewer side effects. Several small-molecule inhibitors of Aurora kinases have been developed as anticancer agents, some of which have progressed to early clinical evaluation. Here we identified 3-hydroxyflavone as a novel Aurora B inhibitor through high throughput screening. 3-Hydroxyflavone showed potent inhibition to Aurora B with the IC₅₀ on a nanomolar basis in the enzyme-based kinase activity assay. In the cell-based western blotting analysis, 3-hydroxyflavone dramatically decreased the phosphorylation level of Histone H3 on the site of serine 10, demonstrating the potent endogenous Aurora B activity inhibition in cell level. The followed cell image analysis provided the consist result. To make it clear whether 3-hydroxyflavone inhibited Aurora B by direct binding or not, SPR analysis was carried out to measure the affinity of interaction between Aurora B protein and 3-hydroxyflavone and the result proved the binding with high affinity. Usually Aurora activity suppression induced cancer cell proliferation inhibition. Colony formation and cell viability with/without treatment of 3-hydroxyflavone were measured using CCK-8. The growth suppression under 3-hydroxyflavone present and the growth recovery after being released gave strong evidence that presence of 3-hydroxyflavone efficiently inhibited the fast growth of cancer cells.     

The novel Aurora A kinase inhibitor MLN8237 is active in resistant chronic myeloid leukaemia and significantly increases the efficacy of nilotinib

Novel therapies are urgently needed to prevent and treat tyrosine kinase inhibitor resistance in chronic myeloid leukaemia (CML). MLN8237 is a novel Aurora A kinase inhibitor under investigation in multiple phase I and II studies. Here we report that MLN8237 possessed equipotent activity against Ba/F3 cells and primary CML cells expressing unmutated and mutated forms of breakpoint cluster region-Abelson kinase (BCR-ABL). Notably, this agent retained high activity against the T315I and E255K BCR-ABL mutations, which confer the greatest degree of resistance to standard therapy. MLN8237 treatment disrupted cell cycle kinetics, induced apoptosis, caused a dose-dependent reduction in the expression of the large inhibitor of apoptosis protein Apollon, and produced a morphological phenotype consistent with Aurora A kinase inhibition. In contrast to other Aurora kinase inhibitors, MLN8237 did not significantly affect BCR-ABL activity. Moreover, inhibition of Aurora A with MLN8237 significantly increased the in vitro and in vivo efficacy of nilotinib. Targeted knockdown of Apollon sensitized CML cells to nilotinib-induced apoptosis, indicating that this is an important factor underlying MLN8237's ability to increase the efficacy of nilotinib. Our collective data demonstrate that this combination strategy represents a novel therapeutic approach for refractory CML that has the potential to suppress the emergence of T315I mutated CML clones.     

Cooperative effects of Janus and Aurora kinase inhibition by CEP701 in cells expressing Jak2V617F

The Janus kinase 2 mutant V617F occurs with high frequency in myeloproliferative neoplasms. Further mutations affecting the Janus kinase family have been discovered mostly in leukaemias and in myeloproliferative neoplasms. Owing to their involvement in neoplasia, inflammatory diseases and in the immune response, Janus kinases are promising targets for kinase inhibitor therapy in these disease settings. Various quantitative assays including two newly developed screening assays were used to characterize the function of different small‐molecule compounds in cells expressing Jak2V617F. A detailed comparative analysis of different Janus kinase inhibitors in our quantitative assays and the subsequent characterization of additional activities demonstrated for the first time that the most potent Jak2 inhibitor in our study, CEP701, also targets Aurora kinases. CEP701 shows a unique combination of both activities which is not found in other compounds also targeting Jak2. Furthermore, colony forming cell assays showed that Janus kinase 2 inhibitors preferentially suppressed the growth of erythroid colonies, whereas inhibitors of Aurora kinases preferentially blocked myeloid colony growth. CEP701 demonstrated a combined suppression of both colony types. Moreover, we show that combined application of a Janus and an Aurora kinase inhibitor recapitulated the effect observed for CEP701 but might allow for more flexibility in combining both activities in clinical settings, e.g. in the treatment of myeloproliferative neoplasms. The newly developed screening assays are high throughput compatible and allow an easy detection of new compounds with Janus kinase 2 inhibitory activity.     

BimEL is phosphorylated at mitosis by Aurora A and targeted for degradation by βTrCP1

Bcl-2-interacting mediator of cell death (Bim) is a pro-apoptotic B-cell lymphoma 2 family member implicated in numerous apoptotic stimuli. In particular, Bim is required for cell death mediated by antimitotic agents, however, mitotic regulation of Bim remains poorly understood. Here, we show that the major splice variant of Bim, BimEL, is regulated during mitosis by the Aurora A kinase and protein phosphatase 2A (PP2A). We observed that BimEL is phosphorylated by Aurora A early in mitosis and reversed by PP2A after mitotic exit. Aurora A phosphorylation stimulated binding of BimEL to the F-box protein beta-transducin repeat containing E3 ubiquitin protein ligase and promoted ubiquitination and degradation of BimEL. These findings describe a novel mechanism by which the oncogenic kinase Aurora A promotes cell survival during mitosis by downregulating proapoptotic signals. Notably, we observed that knockdown of Bim significantly increased resistance of cells to the Aurora A inhibitor MLN8054. Inhibitors of Aurora A are currently under investigation as cancer chemotherapeutics and our findings suggest that efficacy of this class of drugs may function in part by enhancing apoptotic activity of BimEL.