Aurora Kinase Inhibitor MK-0457 Research Articles

Interaction of novel Aurora kinase inhibitor MK-0457 with human serum albumin: Insights into the dynamic behavior, binding mechanism, conformation and esterase activity of human serum albumin

MK-0457, a new pan-aurora kinase inhibitor, is in Phase II clinical development for the treatment of multiple tumor types and hematologic malignancies. The present work explored the dynamic behaviors and interaction mechanism of MK-0457 to human serum albumin (HSA) and the effect on the esterase-like activity and conformation of HSA by computer simulations and experiments. Docking and molecular dynamics trajectory analysis indicated that MK-0457 stably bound to Sudlow’s site 2 of HSA by multiple types of interaction forces. Competitive experiments further verified MK-0457 was bound at first to Sudlow’s site 2 and then the excess of drug was bound to Sudlow’s site 1. The steady-state fluorescence combined with ultraviolet–visible absorption and fluorescence lifetime measurements specified a static quenching mechanism with association constants of 104 M−1 reflecting moderate binding affinity of MK-0457 for HSA. The analysis of Rg values showed that the structure of HSA became loose due to MK-0457 binding, inducing slight conformational changes of HSA, which was consistent with the results obtained from circular dichroism, synchronous, and 3D fluorescence spectroscopy. The esterase-like activity of HSA showed that MK-0457 inhibits the catalytic activity of subdomain IIIA of HSA by binding to the vital residues TYR411. Atomic force microscopy images indicated that MK-0457 affects the molecular sizes of HSA by transforming the morphology of HSA from aggregation diploids to small monomers. This study is beneficial for understanding the biological action of MK-0457, providing additional information about the feasibility of its transport and accumulation in blood plasma.

Gadd45a transcriptional induction elicited by the Aurora kinase inhibitor MK-0457 in Bcr-Abl-expressing cells is driven by Oct-1 transcription factor

The advantage of Aurora kinase (AK) inhibitors in chronic myeloid leukemia (CML) therapy mostly arises from “off-target” effects on tyrosine kinase (TK) activity of wild type (wt) or mutated Bcr-Abl proteins which drive the disease resistance to imatinib (IM). We proved that the AK inhibitor MK-0457 induces the growth arrest DNA damage-inducible (Gadd) 45a through recruitment of octamer-binding (Oct)-1 transcription factor at a critical promoter region for gene transcription and covalent modifications of histone H3 (lysine 14 acetylation, lysine 9 de-methylation). Such epigenetic chromatin modifications may depict a general mechanism promoting the re-activation of tumor suppressor genes silenced by Bcr-Abl.

Aurora kinases are expressed in medullary thyroid carcinoma (MTC) and their inhibition suppresses in vitro growth and tumorigenicity of the MTC derived cell line TT

BackgroundThe Aurora kinase family members, Aurora-A, -B and -C, are involved in the regulation of mitosis, and alterations in their expression are associated with cell malignant transformation. To date no information on the expression of these proteins in medullary thyroid carcinoma (MTC) are available. We here investigated the expression of the Aurora kinases in human MTC tissues and their potential use as therapeutic targets.MethodsThe expression of the Aurora kinases in 26 MTC tissues at different TNM stages was analyzed at the mRNA level by quantitative RT-PCR. We then evaluated the effects of the Aurora kinase inhibitor MK-0457 on the MTC derived TT cell line proliferation, apoptosis, soft agar colony formation, cell cycle and ploidy.ResultsThe results showed the absence of correlation between tumor tissue levels of any Aurora kinase and tumor stage indicating the lack of prognostic value for these proteins. Treatment with MK-0457 inhibited TT cell proliferation in a time- and dose-dependent manner with IC50 = 49.8 ± 6.6 nM, as well as Aurora kinases phosphorylation of substrates relevant to the mitotic progression. Time-lapse experiments demonstrated that MK-0457-treated cells entered mitosis but were unable to complete it. Cytofluorimetric analysis confirmed that MK-0457 induced accumulation of cells with ≥ 4N DNA content without inducing apoptosis. Finally, MK-0457 prevented the capability of the TT cells to form colonies in soft agar.ConclusionsWe demonstrate that Aurora kinases inhibition hampered growth and tumorigenicity of TT cells, suggesting its potential therapeutic value for MTC treatment.

A Common but Overlooked Mechanism of BCR-ABL1 Kinase Inhibitor Resistance in Chronic Myeloid Leukemia.

Abstract 2179Poster Board II-156Point mutations in the BCR-ABL1 protein are the most commonly reported mechanism of resistance to imatinib mesylate (Gleevec or STI-571) treatment in patients with chronic myelogenous leukemia (CML). We have reported that some patients with imatinib-resistant CML express an alternatively spliced BCR-ABL1 mRNA with a 35-bp insertion (BCR-ABL135INS), but the prevalence and effect of this mutation have not been well delineated. We sought to improve understanding of this mutation in order to provide insight into mechanisms of resistance to tyrosine kinase inhibitors. Using a sensitive PCR method, we determined the prevalence of the alternatively spliced BCR-ABL135INS mRNA in a group of 168 patients with chronic-phase CML resistant to imatinib. Expression of truncated protein was confirmed by Western blot in patient samples. The effects of various kinase inhibitors on human K562 CML cells transfected with BCR-ABL135INS cDNA were tested by measuring cell survival, caspase-3 activation, and the BIM activation pathway. In our analysis of BCR-ABL1 genotypes in 168 imatinib-resistant chronic-phase CML patients, (56%) of patients expressed various levels of BCR-ABL135INS whereas only (25%) had detectable point mutations in BCR-ABL1. Expression of this truncated BCR-ABL1 fusion protein was also confirmed by immunoprecipitation at levels proportional to those predicted by mRNA quantification. In vitro, BCR-ABL135INS expression conferred resistance to the tyrosine kinase inhibitors imatinib, dasatinib, and nilotinib in a concentration-dependent fashion, but had no effect on the protein-synthesis inhibitor homoharringtonine (HHT or omacetaxine) or the aurora kinase inhibitor MK-0457 (VX-680). The combination of imatinib with nilotinib or HHT was synergistic in overcoming BCR-ABL135INS-induced resistance. Moreover, the resistance to imatinib induced by BCR-ABL135INS was associated with loss of BIM expression. This loss of BIM was overcome by pretreatment with HHT. In conclusion, these findings emphasize the importance of the overlooked alternatively spliced BCR-ABL135INS found in imatinib-resistant CML patients. Optimal patient management in the future will likely require periodic testing for the expression of BCR-ABL-135INS mRNA during therapy. This may help in predicting various levels of resistance or the potential for eliminating the disease by adjusting the treatment strategy. Disclosures:No relevant conflicts of interest to declare.

Targets and effectors of the cellular response to aurora kinase inhibitor MK-0457 (VX-680) in imatinib sensitive and resistant chronic myelogenous leukemia

MK-0457 inhibits aurora, BCR-ABL and other kinases and may be clinically active in imatinib resistant leukemia. To define mediators of MK-0457 responsiveness, kinase inhibitory profiles were examined in multiple cell models of imatinib sensitive and resistant disease. Aurora and BCR-ABL kinase inhibition were consistently measured at 20–100 nM and 2–10 μM MK-0457, respectively, but expression of T315I-BCR-ABL and overexpression of Lyn kinase reduced MK-0457 sensitivity. Aurora kinase inhibition was associated with cell cycle restriction and p53 induction and p53-null cells were far less responsive to MK-0457, requiring BCR-ABL inhibitory concentrations for apoptotic activity. In wild-type p53 expressing CML cells MK-0457 sensitivity was modulation by alterations in p53 levels through HDM-2 inhibition and gene silencing. MK-0457 suppressed aurora kinase activity and induced apoptosis in imatinib resistant clinical specimens expressing T315I and other BCR-ABL mutations without effecting BCR-ABL kinase activity. Together, these results suggest that MK-0457 apoptotic activity in CML cells is primarily associated with aurora kinase inhibition but can be altered by multiple molecular changes associated with disease progression or acquisition of imatinib resistance.

Combination Therapy with Histone Deacetylase Inhibitor Vorinostat Plus Aurora Kinase Inhibitor MK-0457 Leads to Enhanced Lymphoma Cell Killing with Stabilization of p53 and Repression of C-Myc, hTERT, and Myc-Responsive miRNAs

Background: Patients with relapsed or refractory Hodgkin (HL) and Non Hodgkin Lymphoma (NHL) have few therapeutic options after salvage therapy and transplant, and new agents are thus needed. MK-0457 is a novel aurora kinase inhibitor (AKI) which leads to polyploidy and G2 arrest by disregulating mitosis. We demonstrate that the addition of vorinostat, a broad spectrum HDACi, leads to the reactivation of proapoptotic and DNA damage response elements leading to enhanced lymphoma cell death. A panel of HL and NHL cell lines was studied with either drug or the combination using MTS, Annexin V, and flow cytometry studies.Results: Vorinostat alone at a dose range of 0.5 – 3 mM reduces cell growth by at least 50% in all lines tested. For example, 1.5 mM vorinostat results in averages of 50% growth reduction and 30% apoptotic cells at 48 hours in the L540 HL cell line. MK-0457 alone at 100 nM – 500 nM results in up to a 50% reduction in cell growth and 20% apoptosis in the tested cell lines. However, the combination of 1.5 mM vorinostat and 100 nM MK-0457 results in 50 – 70% apoptosis of the L540 and KM-H2 Hodgkin cell lines at 48 hours. Cell cycle analyses by FACS show an increased percentage of cells in G2/M with few cells in sub-G1, whereas in combination with vorinostat there is a significant increase in the sub G1 population. Real-time PCR analysis and immunoblotting of L540 cells treated with either single agent or in combination suggest an epigenetic as well as a protein acetylation mechanism for this activity. Prosurvival genes such as bcl-XL and hTERT are downregulated five-fold by combination drug treatment, while the proapoptotic BAK gene is upregulated 1.5 – 2-fold. The p53 tumor suppressor is stabilized both by increased acetylation in response to vorinostat as well as by reduced inhibitory phosphorylation at Ser315 by MK-0457-inhibited aurora kinase, leading to a corresponding increase in the p53 target genes p21 and Noxa. Protein and mRNA levels of c-myc are reduced. Repression of microRNAs (miRNAs) in the myc-regulated polycistronic cluster of miRNAs of chromosome 13, such as miR-17.5p, -17.3p, and 18, is demonstrated with vorinostat and TSA.Conclusions: The HDACi vorinostat leads to both transcriptional and post-transcriptional changes which create a pro-apoptotic milieu, sensitizing the cell to centrosome-acting agents such as the AKI, MK-0457.[We acknowledge Merck Inc for providing Vorinostat, MK-0457, and research support.]<

Vorinostat synergistically potentiates MK-0457 lethality in chronic myelogenous leukemia cells sensitive and resistant to imatinib mesylate

AbstractInteractions between the dual Bcr/Abl and aurora kinase inhibitor MK-0457 and the histone deacetylase inhibitor vorinostat were examined in Bcr/Abl+ leukemia cells, including those resistant to imatinib mesylate (IM), particularly those with the T315I mutation. Coadministration of vorinostat dramatically increased MK-0457 lethality in K562 and LAMA84 cells. Notably, the MK-0457/vorinostat regimen was highly active against primary CD34+ chronic myelogenous leukemia (CML) cells and Ba/F3 cells bearing various Bcr/Abl mutations (ie, T315I, E255K, and M351T), as well as IM-resistant K562 cells exhibiting Bcr/Abl-independent, Lyn-dependent resistance. These events were associated with inactivation and down-regulation of wild-type (wt) and mutated Bcr/Abl (particularly T315I). Moreover, treatment with MK-0457 resulted in accumulation of cells with 4N or more DNA content, whereas coadministration of vorinostat markedly enhanced aurora kinase inhibition by MK-0457, and preferentially killed polyploid cells. Furthermore, vorinostat also interacted with a selective inhibitor of aurora kinase A and B to potentiate apoptosis without modifying Bcr/Abl activity. Finally, vorinostat markedly induced Bim expression, while blockade of Bim induction by siRNA dramatically diminished the capacity of this agent to potentiate MK-0457 lethality. Together, these findings indicate that vorinostat strikingly increases MK-0457 activity against IM-sensitive and -resistant CML cells through inactivation of Bcr/Abl and aurora kinases, as well as by induction of Bim.

Co-treatment with aurora kinase inhibitor MK-0457 and pan-histone deacetylase inhibitor vorinostat: A novel targeted treatment for AML and CML

11054 Background: MK-0457 (M) is a pan-Aurora kinase inhibitor that also inhibits Bcr-Abl, including mutant Bcr-AblT315I. Aurora kinases are chaperoned by hsp90. Vorinostat (V) is a pan-histone deacetylase inhibitor that induces growth arrest, differentiation and apoptosis of acute leukemia cells. By inducing acetylation and inhibition of hsp90, V depletes the levels of hsp90 chaperoned proteins. Methods: Mouse BaF3 cells with ectopic expression of wild-type Bcr-Abl or mutant Bcr-AblE255K or Bcr-AblT315I, cultured human AML HL-60, OCI-AML3, and CML K562, as well as primary AML and imatinib-refractory CML cells were treated with M (50 to 250 nM) and/or V (0.5 to 2.0 μM) for 24 to 48 hours. Cell cycle status, DNA endoreduplication (PI staining) and apoptosis (Annexin/PI staining) were determined by flow cytometry. Intracellular levels of Aurora A and B, survivin, unmutated and mutant Bcr-Abl, p-STAT5, p-CrkL and Bim were determined by immunoblot analyses. Results: Treatment with M decreased phosphorylated (P)-serine 10 on histone H3 and p-survivin, as well as induced DNA endo-reduplication and apoptosis of the cultured AML cells. M induced more apoptosis of BaF3 cells with either the wild-type or mutant Bcr-AblE255K or Bcr-AblT315I, versus the control BaF3 cells. Co-treatment with M and V versus either agent alone resulted in more attenuation of the levels and activity of Aurora kinases and synergistically induced apoptosis of the cultured AML cells. Co-treatment with V and M markedly attenuated the levels of p-Bcr-Abl, p-STAT5, p-CrkL and Aurora kinases, as well as increased Bim and synergistically induced apoptosis of K562 cells. Co-treatment with M and V also induced synergistic apoptosis of BaF3/Bcr-Abl, BaF3/Bcr-AblE255K and BaF3/Bcr- AblT315I cells. M and V versus either agent alone, caused more loss of cell viability of primary AML and imatinib-refractory CML cells. The combination was also less toxic to normal CD34+ progenitor cells. Finally, co-treatment with M and V also inhibited leukemia growth and prolonged survival of NOD-SCID mice engrafted with OCI-AML3 cells. Conclusions: Combined treatment with M and V is highly active and should be tested in patients with AML or resistant CML, especially with Bcr-AblT315I. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Merck & Co. Novartis Institute for Biomedical Research Inc. Merck & Co., Novartis, Novartis Institute for Biomedical Research Inc.

The Bcr-Abl tyrosine kinase inhibitor imatinib and promising new agents against Philadelphia chromosome-positive leukemias

Chronic myeloid leukemia (CML) was the first human malignant disease to be linked to a single, acquired genetic abnormality. Identification of the Bcr-Abl kinase fusion protein and its pivotal role in the pathogenesis of CML provided new opportunities to develop molecular-targeted therapies. Imatinib mesylate (IM, Gleevec, Novartis Pharmaceuticals, Basel, Switzerland), which specifically inhibits the autophosphorylation of the Abl TK, has improved the treatment of CML. However, resistance is often reported in patients with advanced-stage disease. Several novel TK inhibitors have been developed that override IM resistance mechanisms caused by point mutations within the Abl kinase domain. Inhibitors of Abl TK are divided into two main groups, namely, ATP-competitive and ATP noncompetitive inhibitors. The ATP-competitive inhibitors fall into two subclasses, the Src/Abl inhibitors, and the 2-phenylaminopyrimidine-based compounds. Dasatinib (formerly BMS-354825), AP23464, SKI-606, and PD166326 are classified as Src/Abl inhibitors, while nilotinib (AMN107) and INNO-406 (NS-187) belong to the latter subclass of inhibitors. Of these agents, dasatinib and nilotinib underwent clinical trials earlier than the others and favorable results are now accumulating. Clinical studies of the other compounds, including SKI-606 and INNO-406, have been performed in rapid succession. Because of their strong affinities for the ATP-binding site compared to IM, most ATP-competitive inhibitors may be effective in IM-resistant patients. However, an ATP-competitive inhibitor that can inhibit the phosphorylation of T315I Bcr-Abl has not yet been developed. Instead, ATP noncompetitive inhibitors, such as ON012380, Aurora kinase inhibitor MK0457 (VX-680), and p38 MAP kinase inhibitor BIRB-796, have been developed to address this problem. This review provides an update on the underlying pathophysiologies of disease progression and IM resistance, and discusses the development of new targeted TK inhibitors for managing CML and the importance of future strategies targeting CML stem cells.

Activity of the aurora kinase inhibitor MK-0457 (VX-680) in combination with taxotere

14069 Background: MK-0457 (VX-680) is a reversible kinase inhibitor that targets Aurora A, B and C (Ki’s of 0.7, 18 and 4.6 nM, respectively). Clinical trials are on-going in patients with solid tumors and hematologic malignancies. This study evaluated the in vitro combination of MK-0457 and Taxotere (TXT) to determine if inhibition of Aurora kinases, which are critical for cytokinesis and spindle assembly may synergize with a microtubule targeted agent. Methods: A panel of cancer cell lines (that were either wild-type, mutant, or null for p53) and a matched pair of A549 cell lines either expressing p53 shRNA or vector control (A549p53- and A549p53+) were evaluated for sensitivity to MK-0457 and/or TXT. Viability was measured by Cell Titer Glo (Promega). Cell cycle profiles were determined by FACS analysis, and colony formation (CFU) assays were performed in soft agar and on plastic. Combination effects were evaluated by the Bliss Independence method. Results: Single agent IC50’s ranged from 50 nM to &gt; 5 μM for MK-0457 and 0.4 to 10 nM for TXT. FACS analysis of DNA content showed MK-0457 induced G2/M arrest and polyploidy, characteristic of Aurora kinase inhibition. The effects of MK-0457 and TXT combination ranged from antagonism to synergy and were concentration, cell context, and regimen dependent. In 3–4 day viability assays simultaneous combination of MK-0457 + TXT did not show synergy, however sequential exposure showed synergy at low concentrations of both agents. In contrast to the short-term viability assays, simultaneous exposure to MK-0457 + TXT in long term survival assays (i.e. CFU on plastic or soft agar) resulted in enhanced cell death compared to the single agents. Conclusions: The synergy observed for MK-0457 + TXT, especially in long term survival assays, supported the evaluation of this combination in xenograft models (described in B. Furey et al., ASCO 2006). No significant financial relationships to disclose.

Synergistic Activity of the Aurora Kinase Inhibitor MK-0457 (VX-680) with Idarubicin, Ara-C, and Inhibitors of BCR-ABL.

MK-0457 (VX-680) is a reversible small molecule kinase inhibitor that targets Aurora A, B, and C with Ki values of 0.7, 18, and 4.6 nM, respectively. MK-0457 also inhibits Flt3 (Ki = 30 nM), and both the wild type and the T315I mutant of BCR-ABL (Ki = 30 and 40 nM, respectively). Clinical trials are ongoing in patients with solid tumors and hematological malignancies. Recent data show that MK-0457 is active in patients against subtypes of AML, BCR-ABL T315I mutant CML, and Philadelphia positive (Ph+) ALL. To support multi-agent clinical trials, the activity of MK-0457 in combination with idarubicin, Ara-C, and BCR-ABL inhibitors was investigated. The viability of a panel of AML, ALL, and CML cell lines was assessed following single agent and either simultaneous or sequential combinations of agents. Combination effects were evaluated using the Bliss Independence Model. MK-0457 as a single agent markedly inhibited leukemia cell viability (at 72 hrs) with an IC50 range of 20–300 nM for MV4-11, Molt-4, Molm-13, K562, LAMA-84, MEG-01, and KU812F cells. Additionally, MK-0457 inhibited the viability of BaF3 cells transformed by wild type, T315I, or Y253F mutants of BCR-ABL with similar IC50s (approximately 300 nM). The sequential combination of MK-0457 followed by either idarubicin or Ara-C showed greater synergy than simultaneous combinations in a cell line dependent manner. MK-0457 displayed strong synergy in simultaneous combination with Gleevec (imatinib mesylate) in a panel of human CML-derived cell lines and BaF3 cells expressing wild type BCR-ABL. MK-0457 enhanced the Gleevec-mediated cell death of K562 leukemia cells as evidenced by increased caspase activity, PARP cleavage, and induction of the sub-G1 population. At concentrations where synergy was observed by cell viability analysis, the MK-0457/Gleevec combination resulted predominantly in aneuploidy and G2/M arrest, consistent with inhibition of Aurora kinases by MK-0457. These results support the clinical evaluation of MK-0457 combined with idarubicin and Ara-C in AML and with BCR-ABL inhibitors in CML and Ph+ ALL.