Aurora Kinase Family Research Articles

Activity of Serono-AS703569, a Dual Inhibitor of Bcr-Abl and Aurora Kinases in Bcr-Abl Transformed Cells, Is Dependent On Aurora B Inhibition, and Is Not Affected by the Presence of the Highly Imatinib Resistant Bcr-Abl Mutation T315I.

Abstract 3247Poster Board III-1The tyrosine kinase inhibitor Imatinib is the gold standard in conventional treatment of CML. However, the emergence of resistance to IM remains a major problem. Alternative therapeutic strategies of IM-resistant BCR-ABL positive leukemias are urgently needed. One promising target for anticancer therapeutics is represented by the Aurora kinase family. These serine/threonine kinases are involved in regulating multiple steps of mitosis, including formation of bipolar spindle, chromosome alignment, spindle checkpoint function and cytokinesis.We report on studies accomplished with a small molecule inhibitor AS703569 (Merck Serono), which targets Bcr-Abl and Aurora kinases A-C. We could show that AS703569 exhibited strong anti-proliferative and pro-apoptotic activity against murine BaF3- cells ectopically expressing wild type (wt) or IM-resistant BCR-ABL mutants, including those harbouring the strongly resistant T315I mutation. This effect was observed already at rather low-AS703569 concentrations, at which Aurora- but not the Bcr-Abl kinase was inhibited. Furthermore, in cell cycle analysis we observed cells with a large 4N peak and DNA content more than 4N, indicating extensive polyploidisation, a consequence of continued cell cycle progression in the absence of cell division. Recent studies have revealed that this phenotype is based on suppression of Aurora B kinase activity, indicating that Aurora B inhibition is the major effect of AS703569 in Bcr-Abl positive cells. To confirm this assumption we designed MSCV based retroviruses encoding different point mutations in the Aurora B ATP binding site, which should lead to resistance against AS703569. By this strategy we were able to identify an AS703569 resistant mutant (Aurora B G216V). This mutant shows significant resistance in vitro and is able to augment the antiproliferative capacity of AS703569 in Bcr-Abl positive cells. Taken together, our data demonstrate that anti-proliferative effects of AS703569 in Bcr-Abl positive cells are primarily mediated by functional inhibition of Aurora kinases, especially of Aurora kinase B. Since Aurora kinases are clearly implicated in tumorgenesis, they will become a high potential therapeutic target for anticancer therapy. Disclosures:No relevant conflicts of interest to declare.

Inhibition of the aurora kinases suppresses in vitro NT2-D1 cell growth and tumorigenicity

The aurora kinase family members, Aurora-A, -B, and -C (listed as AURKA, AURKB and AURKC respectively in the HUGO Database), are serine/threonine kinases involved in the regulation of chromosome segregation and cytokinesis, and alterations in their expression are associated with malignant cell transformation and genomic instability. Deregulation of the expression of the aurora kinases has been shown to occur also in testicular germ cell tumors (TGCTs) identifying them as putative anticancer therapeutic targets. We here evaluated the in vitro effects of MK-0457, an aurora kinases inhibitor, on cell proliferation, cell cycle, ploidy, apoptosis, and tumorigenicity on the TGCT-derived cell line NT2-D1. Treatment with MK-0457 inhibited cell proliferation in a time- and dose-dependent manner, with IC(50)=17.2+/-3.3 nM. MK-0457 did not affect the expression of the three aurora kinases, but prevented their ability to phosphorylate substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK-0457-treated cells entered mitosis but were unable to complete it, presenting after short time the typical features of apoptotic cells. Cytofluorimetric analysis confirmed that the treatment with MK-0457 for 6 h induced NT2-D1 cells accumulation in the G(2)/M phase of the cell cycle and the subsequent appearance of sub-G(0) nuclei. The latter result was further supported by the detection of caspase-3 activation following 24-h treatment with the inhibitor. Finally, MK-0457 prevented the capability of the NT2-D1 cells to form colonies in soft agar. In conclusion, the above findings demonstrate that inhibition of aurora kinase activity is effective in reducing in vitro growth and tumorigenicity of NT2-D1 cells, and indicate its potential therapeutic value for TGCT treatment.

Aurora-C kinase supports mitotic progression in the absence of Aurora-B

Aurora family kinases regulate numerous mitotic processes, and their dysfunction or overexpression can cause aneuploidy, a contributing factor for tumorigenesis. In vertebrates, the Aurora-B kinase regulates kinetochore maturation, destabilization of improper kinetochore-microtubule attachments, the spindle assembly checkpoint, central spindle organization, and cytokinesis. A gene duplication event created the related Aurora-C kinase in mammals. While Aurora-C function is unclear, it has similar structural and localization properties as Aurora-B. Inhibition of either Aurora-B or Aurora-C function causes aneuploidy, while simultaneous inhibition of both causes a higher frequency of aneuploidy. To determine if Aurora-C and –B have overlapping or unique complementary functions during mitosis, we created a system where Aurora-B is replaced by wild-type or kinase-defective mutant Aurora-C in HeLa cells. In this model, Aurora-B protein levels and mitotic functions were suppressed including the regulation of kinetochore-microtubule attachments, the spindle assembly checkpoint, and cytokinesis. Wild-type, but not kinase-defective Aurora-C expression, was able to rescue these functions. Therefore, Aurora-C can perform these essential functions of Aurora-B in mitosis.

Clinical Experience with Aurora Kinase Inhibitors: A Review

The aurora kinase family of serine/threonine kinases comprises three members, designated auroras A, B, and C. Auroras A and B are essential components of the mitotic pathway, ensuring proper chromosome assembly, formation of the mitotic spindle, and cytokinesis. The role of aurora C is less clear. Overexpression of aurora A and B has been observed in several tumor types, and has been linked with a poor prognosis of cancer patients. Several small molecules targeting aurora kinases A and B or both have been evaluated preclinically and in early phase I trials. In this review we aim to summarize the most recent advances in the development of aurora kinase inhibitors, with a focus on the clinical data.

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.

The cell cycle as a therapeutic target against Trypanosoma brucei: Hesperadin inhibits Aurora kinase‐1 and blocks mitotic progression in bloodstream forms

Aurora kinase family members co-ordinate a range of events associated with mitosis and cytokinesis. Anti-cancer therapies are currently being developed against them. Here, we evaluate whether Aurora kinase-1 (TbAUK1) from pathogenic Trypanosoma brucei might be targeted in anti-parasitic therapies as well. Conditional knockdown of TbAUK1 within infected mice demonstrated its essential contribution to infection. An in vitro kinase assay was developed which used recombinant trypanosome histone H3 as a substrate. Tandem mass spectroscopy identified a novel phosphorylation site in the carboxyl-tail of recombinant trypanosome histone H3. Hesperadin, an inhibitor of human Aurora B, prevented the phosphorylation of substrate with IC(50) of 40 nM. Growth of cultured bloodstream forms was also sensitive to Hesperadin (IC(50) of 50 nM). Hesperadin blocked nuclear division and cytokinesis but not other aspects of the cell cycle. Consequently, growth arrested cells accumulated multiple kinetoplasts, flagella and nucleoli, similar to the effects of RNAi-dependent knockdown of TbAUK1 in cultured bloodstream forms cells. Molecular models predicted high-affinity binding of Hesperadin to both conserved and novel sites in TbAUK1. Collectively, these data demonstrate that cell cycle progression is essential for infections with T. brucei and that parasite Aurora kinases can be targeted with small-molecule inhibitors.

Aurora A Regulates Prometaphase Progression by Inhibiting the Ability of RASSF1A to Suppress APC-Cdc20 Activity

The Aurora (Ipl) kinase family plays important roles in the regulation of mitosis and tumorigenesis. The tumor suppressor RASSF1A controls mitotic progression by regulating anaphase-promoting complex (APC)-Cdc20 activity and microtubule stability, but the mechanism by which this action is regulated has not been previously established. Here, we show that Aurora A and B associate with and phosphorylate RASSF1A on serine 203 in vivo at different times and in different subcellular compartments during mitosis. Notably, both depletion of Aurora A by RNA interference and expression of a nonphosphorylatable RASSF1A (S203A) mutant gene led to a marked delay in prometaphase progression. This is likely because of the failure of RASSF1A to dissociate from Cdc20, constitutive inhibition of APC-Cdc20, and accumulation of mitotic cyclins. In contrast, the delay in prometaphase progression caused by Aurora A depletion was largely normalized by phosphomimetic RASSF1A (S203D). Finally, RASSF1A phosphorylation on serine 203 was up-regulated in Aurora A-overexpressing human tumors. These findings indicate that Aurora A plays a critical role in RASSF1A-APC-Cdc20 regulatory mechanisms that control normal prometaphase progression and that are involved in tumorigenesis. [Cancer Res 2009;69(6):2314-23.

Mechanism of MK-0457 efficacy against BCR–ABL positive leukemia cells

Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance. MK-0457 is a small molecule inhibitor of the Aurora kinase family, but the mechanism of MK-0457 has not been evaluated. In this study, the gene expression profiles and intracellular signaling of chronic myeloid leukemia (CML) cell line K562 exposed to imatinib or MK-0457. MK-0457 induced cell growth inhibition in K562 cells. In gene expression profiles, there was an increase of 938 genes in imatinib and 895 genes in MK-0457 and 638 genes overlapped. In contrast, there was a decrease of 597 genes in imatinib and 582 genes in MK-0457 and 406 genes overlapped. These down-regulated genes include heat shock proteins (HSPs). These results indicate that MK-0457 is effective in CML cells by the down-regulation of HSPs which may relate to BCR–ABL stability, and offer new information regarding the molecular basis of strategy against to CML.

Aurora kinase inhibitors synergize with paclitaxel to induce apoptosis in ovarian cancer cells

BackgroundA large percentage of patients with recurrent ovarian cancer develop resistance to the taxane class of chemotherapeutics. While mechanisms of resistance are being discovered, novel treatment options and a better understanding of disease resistance are sorely needed. The mitotic kinase Aurora-A directly regulates cellular processes targeted by the taxanes and is overexpressed in several malignancies, including ovarian cancer. Recent data has shown that overexpression of Aurora-A can confer resistance to the taxane paclitaxel.MethodsWe used expression profiling of ovarian tumor samples to determine the most significantly overexpressed genes. In this study we sought to determine if chemical inhibition of the Aurora kinase family using VE-465 could synergize with paclitaxel to induce apoptosis in paclitaxel-resistant and sensitive ovarian cancer cells.ResultsAurora-A kinase and TPX2, an activator of Aurora-A, are two of the most significantly overexpressed genes in ovarian carcinomas. We show that inhibition of the Aurora kinases prevents phosphorylation of a mitotic marker and demonstrate a dose-dependent increase of apoptosis in treated ovarian cancer cells. We demonstrate at low doses that are specific to Aurora-A, VE-465 synergizes with paclitaxel to induce 4.5-fold greater apoptosis than paclitaxel alone in 1A9 cells. Higher doses are needed to induce apoptosis in paclitaxel-resistant PTX10 cells.ConclusionOur results show that VE-465 is a potent killer of taxane resistant ovarian cancer cells and can synergize with paclitaxel at low doses. These data suggest patients whose tumors exhibit high Aurora-A expression may benefit from a combination therapy of taxanes and Aurora-A inhibition.

The Analysis of HDAC Inhibitor, Vorinostat Efficacy against Wild Type and BCR-ABL Mutant Positive Leukemia Cells in Monotherapy and in Combination with a Pan-Aurora Kinase Inhibitor, MK-0457

The use of imatinib, an ABL tyrosine kinase inhibitor, has led to a dramatic change in the management of BCR-ABL positive leukemia patients. However, the resistance to imatinib mediated by mutations in the BCR-ABL domain has become a major problem in the treatment. Histone deacetylase (HDAC) inhibitors have been shown to mediate the regulation of gene expression, induce cell growth, cell differentiation and apoptosis of tumor cells. Vorinostat (suberoylamide hydroxamic acid:SAHA) is a hydroxamic acid based polar HDAC inhibitor. Vorinostat have shown efficacy in a wide range of cancers such as cutaneous T-cell lymphoma (CTCL). However, efficacy of vorinostat against the BCR-ABL mutants has fully not known. Here we report on the studies performed against murine Ba/F3 cell line which was transfected wild type (Wt) p210 and p185 BCR-ABL or imatinib resistant BCR-ABL mutants such as G250E, Q252H, Y253F, E255K, M294V, T315I, T315A, F317L, F317V, M351T, H396P and T315I(p185). 48 hours treatment of vorinostat exhibits cell growth inhibition and proapoptotic activity murine Ba/F3 cells ectopically expressing Wt and imatinib resistant BCR-ABL mutants including T315I mutation in a dose dependent manner. IC50 of these cell lines are Wt(720nM), G250E(625nM), Q252H(220nM), Y253F(525nM), E255K(685nM), M294V(785nM), T315I(500nM), T315A(715nM), F317L(560nM), F317V(565nM), M351T(375nM) and H396P(485nM). Aurora kinases play a pivotal role in the regulator of mitotic processes during cell division. MK-0457 is a small molecule inhibitor of the Aurora kinase family and was found to be active against the cells from BCR-ABL positive patients with T315I mutation in clinical trial. Because vorinostat also depleted BCR-ABL, as well as induced apoptosis and sensitized BCR-ABL-expressing leukemia cells, we examined whether vorinostat and MK-0457 enhances the apoptosis in imatinib resistant BCR-ABL-expressing cells. 48 hours treatment of MK-0457 exhibits cell growth inhibition of Ba/F3 cells ectopically expressing Wt and imatinib resistant BCR-ABL mutants including T315I mutation. IC50 of MK-0457 is Wt(215nM), G250E(205nM), Q252H(185nM), Y253F(245nM), E255K(185nM), M294V(238nM), T315I(205nM), T315A(165nM), F317L(200nM), F317V(200nM), M351T(225nM) and H396P(195nM). We examined the intracellular signaling by using these cell lines. We found that caspase 3, and poly (ADPribose) polymerase (PARP) were activated after MK-0457 treatment in a dose dependent manner. Phosphorylation of BCR-ABL and Crk-L which is downstream target of BCR-ABL was reduced after MK-0457 treatment. We found that combination of vorinostat and MK-0457 synergistically cell growth inhibition of Wt and BCR-ABL mutants Ba/F3 cells in 48 hours treatment. Phosphorylation of Crk-L was reduced after vorinostat and MK-0457 treatment. Caspase 3 and PARP activation were also synergistically increased after vorinostat and MK-0457 treatment. We evaluated the activity of MK-0457 and vorinostat in primary BCR-ABL positive acute lymphoblastic leukemia (ALL) cells with the T315I mutation. We found that MK-0457 potently induced cell growth inhibition of primary T315I cells in 48 hours treatment. Moreover, combination of vorinostat and MK-0457 synergistically increased the cell growth inhibition in primary T315I cells. This study demonstrate monotherapy of vorinostat and the combination of vorinostat and MK-0457 are more potent efficacy not only wild type BCR-ABL but also imatinib resistant BCR-ABL mutants cells and represents a promising new strategy for treatment of imatinib resistant BCR-ABL positive leukemias, including those harboring the T315I mutation.

Identification of Aurora-A as a Direct Target of E2F3 during G2/M Cell Cycle Progression

Aurora-A is a centrosome kinase and plays a pivotal role in G(2)/M cell cycle progression. Expression of Aurora-A is cell cycle-dependent. Levels of Aurora-A mRNA and protein are low in G(1)/S, accumulate during G(2)/M, and decrease rapidly after mitosis. Previous studies have shown regulation of the Aurora-A protein level during the cell cycle through the ubiquitin-proteasome pathway. However, the mechanism of transcriptional regulation of Aurora-A remains largely unknown. Here, we demonstrated that E2F3 modulates Aurora-A mRNA expression during the cell cycle. Ectopic expression of E2F3 induces Aurora-A expression. Stable knockdown of E2F3 decreases mRNA and protein levels of Aurora-A and delays G(2)/M entry. Further, E2F3 directly binds to Aurora-A promoter and stimulates the promoter activity. Deletion and mutation analyses of the Aurora-A promoter revealed that a region located 96-bp upstream of the transcription initiation site is critical for the activation of the promoter by E2F3. In addition, expression of E2F3 positively correlates with the protein level of Aurora-A in human ovarian cancer examined. These results indicate for the first time that Aurora-A is transcriptionally regulated by E2F3 during the cell cycle and that E2F3 is a causal factor for up-regulation of Aurora-A in a subset of human ovarian cancer. Thus, the E2F3-Aurora-A axis could be an important target for cancer intervention.

Targeting Aurora Kinase with MK-0457 Inhibits Ovarian Cancer Growth

The Aurora kinase family plays pivotal roles in mitotic integrity and cell cycle. We sought to determine the effects of inhibiting Aurora kinase on ovarian cancer growth in an orthotopic mouse model using a small molecule pan-Aurora kinase inhibitor, MK-0457. We examined cell cycle regulatory effects and ascertained the therapeutic efficacy of Aurora kinase inhibition both alone and combined with docetaxel using both in vitro and in vivo ovarian cancer models. In vitro cytotoxicity assays with HeyA8 and SKOV3ip1 cells revealed >10-fold greater docetaxel cytotoxicity in combination with MK-0457. After in vivo dose kinetics were determined using phospho-histone H3 status, therapy experiments with the chemosensitive HeyA8 and SKOV3ip1 as well as the chemoresistant HeyA8-MDR and A2780-CP20 models showed that Aurora kinase inhibition alone significantly reduced tumor burden compared with controls (P values<0.01). Combination treatment with docetaxel resulted in significantly improved reduction in tumor growth beyond that afforded by docetaxel alone (P <or= 0.03). Proliferating cell nuclear antigen immunohistochemistry revealed that MK-0457 alone and in combination with docetaxel significantly reduced cellular proliferation (P values<0.001). Compared with controls, treatment with MK-0457 alone and in combination with docetaxel also significantly increased tumor cell apoptosis by approximately 3-fold (P<0.01). Remarkably, compared with docetaxel monotherapy, MK-0457 combined with docetaxel resulted in significantly increased tumor cell apoptosis. Aurora kinase inhibition significantly reduces tumor burden and cell proliferation and increases tumor cell apoptosis in this preclinical orthotopic model of ovarian cancer. The role of Aurora kinase inhibition in ovarian cancer merits further investigation in clinical trials.

Generation and Characterization of Novel Monoclonal Antibodies Against Human Aurora-A

The mitotic kinase Aurora-A is essential for mitotic progression, including centrosome maturation, mitotic spindle formation, and faithful segregation of chromosomes to daughter cells. Several lines of evidences also suggest that the mammalian aurora kinase family proteins play a role in tumorigenesis. We have previously shown that human Aurora-A was ubiquitinated and negatively regulated by an early mitotic checkpoint protein, Chfr (checkpoint protein with FHA and RING domain). Here, we established several mouse anti-Aurora-A monoclonal antibodies (MAb). GST-tagged human Aurora-A was expressed in BL21 and used as an antigen to immunize mice. Three different hybridomas were obtained and antibodies produced by these hybridomas were analyzed. The results reveal that these antibodies specifically recognize endogenous Aurora-A in both immunoblotting and immunofluroscence experiments. They are useful tools for further analysis of human Aurora-A.

Emerging cancer therapeutic opportunities by inhibiting mitotic kinases

Among cellular kinases, several cell cycle protein kinases play critical roles in mitotic entry and chromosome segregation. Inhibition of these proteins frequently results in dramatic mitotic arrest and subsequent apoptosis. Most drug discovery efforts have been directed against members of the cyclin-dependent kinase (CDK), Aurora and Polo-like kinase families. Inhibition of these proteins with small molecules has emerged as a powerful research tool and their clinical use is currently being tested in phase I and phase II trials for cancer therapy. New unexplored kinases or new protein domains distinct to the kinase pocket are now being evaluated for the next generation of mitotic drugs. The therapeutic value of inhibiting these kinases will improve with the availability of new specific and potent inhibitors, but it will also rely on a better knowledge of the physiological requirement for these proteins in normal and tumor cell cycles.

Aurora Kinase Family: A New Target for Anticancer Drug

Aurora kinases (AK) are the name given to a family of Serine/threonine (Ser/Thr) protein kinases. These proteins represent a novel family of kinases crucial for cell cycle control. The cell division process is one of the hallmarks of every living organism. Within the complete cell-cycle process, mitosis constitutes one of the most critical steps. The main purpose of mitosis is to segregate sister chromatics into two daughters cells. It is a complex biologic process, and errors in this mechanism can lead to genomic instability, a condition associated with tumorigenesis. This process is tightly regulated by several proteins, some of them acting as check-points that ultimately ensure the correct temporal and spatial coordination of this critical biologic process. Among this network of mitotic regulators, AK play a critical role in cellular division by controlling chromatid segregation. Three AK family members have been identified in mammalian cells: A, B, and C. These proteins are implicated in several vital events in mitosis. In experimental models, overexpression of AK can induce spindle defects, chromosome mis-segregation, and malignant transformation. Conversely, downregulation of AK expression cause mitotic arrest and apoptosis in tumor cell lines. The expression levels of human AK are increased in certain types of cancer including breast, colon, pancreatic, ovarian, and gastric tumors. This observation has lent an interest to this family of kinases as potential drug targets for development of new anticancer therapies. This review focuses in recent progress in the role of AK in tumorogenesis and the development of new anticancer drug against AK proteins. This manuscript also includes some relevant patents as well.

Discovery of Selective Aminothiazole Aurora Kinase Inhibitors

Aurora family kinases regulate important events during mitosis including centrosome maturation and separation, mitotic spindle assembly, and chromosome segregation. Misregulation of Aurora kinases due to genetic amplification and protein overexpression results in aneuploidy and may contribute to tumorigenesis. Here we report the discovery of new small molecule aminothiazole inhibitors of Aurora kinases with exceptional kinase selectivity and report a 1.7 A cocrystal structure with the Aurora B:INCENP complex from Xenopus laevis. The compounds recapitulate the hallmarks of Aurora kinase inhibition, including decreased histone H3 serine 10 phosphorylation, failure to complete cytokinesis, and endoreduplication.

Aurora kinases

Aurora kinases

Aurora kinase inhibitors: identification and preclinical validation of their biomarkers

Aurora kinases are key regulators of mitosis and inhibitors being developed by a wide range of pharmaceutical and biotechnology companies for the treatment of cancer. Tumor cells respond differentially on inhibition of different Aurora kinase family members and these differences have to be considered in the clinical development of small-molecule inhibitors with respect to the chosen indications, the schedules or the selection of appropriate end points and they should also guide the development of biomarkers. Preclinical validation of potential biomarkers for Aurora kinase inhibitors led to a first application in clinical trials, as exemplified for the phosphorylation of histone H3 to follow Aurora-B inhibition. This review discusses the criteria for translation into the clinic and the value of pharmacodynamic biomarkers and their potential, but also their limitations to be used as surrogate markers for clinical end points.

PHA-739358, a potent inhibitor of Aurora kinases with a selective target inhibition profile relevant to cancer

PHA-739358 is a small-molecule 3-aminopyrazole derivative with strong activity against Aurora kinases and cross-reactivities with some receptor tyrosine kinases relevant for cancer. PHA-739358 inhibits all Aurora kinase family members and shows a dominant Aurora B kinase inhibition-related cellular phenotype and mechanism of action in cells in vitro and in vivo. p53 status-dependent endoreduplication is observed upon treatment of cells with PHA-739358, and phosphorylation of histone H3 in Ser(10) is inhibited. The compound has significant antitumor activity in different xenografts and spontaneous and transgenic animal tumor models and shows a favorable pharmacokinetic and safety profile. In vivo target modulation is observed as assessed by the inhibition of the phosphorylation of histone H3, which has been validated preclinically as a candidate biomarker for the clinical phase. Pharmacokinetics/pharmacodynamics modeling was used to define drug potency and to support the prediction of active clinical doses and schedules. We conclude that PHA-739358, which is currently tested in clinical trials, has great therapeutic potential in anticancer therapy in a wide range of cancers.

Mechanism of Action of MK-0457 Efficacy in Monotherapy and in Combination with Vorinostat in Wild Type and T315I Mutant BCR/ABL Positive Leukemia Cells.

Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance in patients with BCR/ABL-positive leukemia. MK-0457 is a small molecule inhibitor of the Aurora kinase family with potent cross-reactivity towards wild type and T315I BCR/ABL and FLT-3, and the compound has shown activity in preclinical models of solid tumors and FLT-3 driven leukemia. MK-0457 is presently being evaluated in a clinical trial in chronic myelogenous leukemia (CML) patients with imatinib resistance such as T315I mutation, but the mechanism of MK-0457 is not fully evaluated. In this study, the gene expression profiles of CML cell line, K562, exposed to imatinib or MK-0457 were analyzed and compared. When their gene expression profiles were compared, 937 genes in imatinib and 895 genes in MK-0457 were increased and 625 genes were overlapped. In contrast, 597 genes in imatinib and 537 genes in MK-0457 were decreased and 396 genes were overlapped. These down regulated genes included heat shock proteins (HSPs) suggesting these results may relate to the BCR/ABL stability. Next, we examined the intracellular signaling of MK-0457 in BCR/ABL positive cells with T315I mutation by using BAF-3 p185 BCR/ABL (p185) and BAF-3 BCR/ABL T315I (T315I) cell lines. MK-0457 potently induced apoptosis of p185 cells and T315I cells in 72 hours treatment. IC50 of MK-0457 was 100 nM (p185) and 250 nM (T315I). We found that caspase 3, and poly (ADP-ribose) polymerase (PARP) were activated and BCR/ABL phosphorylation was reduced after MK-0457 treatment in a dose dependent manner. It has been reported that histone deacetylase inhibitor (e.g., vorinostat) also depleted BCR/ABL, as well as induced apoptosis and sensitized BCR/ABL-expressing leukemia cells to apoptosis induced by imatinib. These data suggest that MK-0457 may be synergistic with histone deacetylase inhibitors in light of recent publications that have shown that vorinostat induces acetylation of the heat shock protein (HSP), depletes the HPS90 client proteins, including BCR/ABL, and enhances the imatinib-induced apoptosis in BCR/ABL-expressing cells. We found that combination of MK-0457 and vorinostat synergistically increased the apoptosis of p185 cells and T315I cells in 72 hours treatment. Caspase 3 and PARP activation were also synergistically increased after vorinostat and MK-0457 treatment. We examined the intracellular signaling by using these cell lines. Phosphorylation of BCR/ABL, signal-transducing activators of transcription 5 (STAT5), one of the src kinase, Lyn, Crk-L were reduced after treatment with MK-0457 + vorinostat. We also found that cyclin D1 was decreased after MK-0457 or vorinostat and MK-0457 treatment. Histone H4 is also acetylated after vorinostat treatment, but phosphorylation of BCR/ABL was not reduced directly. We evaluated the activity of MK-0457 and vorinostat in primary Ph positive acute lymphoblastic leukemia cells with the T315I mutation. We found that MK-0457 potently induced cell growth inhibition of primary T315I cells in 72 hours treatment. Moreover, combination of vorinostat and MK-0457 synergistically increased the extent of apoptosis in primary T315I cells. This study provides further insight into the mechanism of action of MK-0457 in wild type and T315I mutant BCR-ABL cells and provides mechanistic rational for the synergistic induction of apoptosis observed for the combination of MK-0457 + vorinostat.