The high prevalence of KRAS mutations and importance of the RalGEF-Ral pathway downstream of activated K-Ras in pancreatic ductal adenocarcinoma (PDAC) emphasize the importance of identifying novel methods by which to therapeutically target these pathways. It was recently demonstrated that phosphorylation of RalA S194 by Aurora A kinase is critical for PDAC tumorigenesis. We sought to evaluate the Aurora A kinase-selective inhibitor MLN8237 as a potential indirect anti-RalA targeted therapy for PDAC. We utilized a site-specific phospho-S194 RalA antibody and determined that RalA S194 phosphorylation levels were elevated in a subset of PDAC cell lines and human tumors relative to unmatched normal controls. Effects of MLN8237 on anchorage-independent growth in PDAC cell lines and growth of patient-derived xenografts (PDX) were variable, with a subset of cell lines and PDX showing sensitivity. Surprisingly, RalA S194 phosphorylation levels in PDAC cell lines or PDX tumors did not correlate with MLN8237 responsiveness. However, we identified Ki67 as a possible early predictive biomarker for response to MLN8237 in PDAC. These results indicate that MLN8237 treatment may be effective for a subset of PDAC patients independent of RalA S194 phosphorylation. Ki67 may be an effective pharmacodynamic biomarker to identify response early in the course of treatment.
Aurora A kinas; RalA; pancreatic cancer; MLN8237; biomarker
Suppression of P53 transcriptional function mediates poor therapeutic response in cancer patients. AURKA and HDM2 are negative regulators of P53. Herein, we examined the role of AURKA in regulating HDM2 and its subsequent effects on P53 apoptotic function in gastric cancer (GC).
Primary tumors and in vitro GC cell models with overexpression or knockdown of AURKA were used. The role of AURKA in regulating HDM2 and cell survival coupled with P53 expression and activity were investigated.
Overexpression of AURKA enhanced HDM2 protein level; conversely, knockdown of endogenous AURKA decreased expression of HDM2 in AGS and SNU-1 cells. Dual co-immunoprecipitaion assay data indicated that AURKA was associated with HDM2 in a protein complex. The in vitro kinase assay using recombinant AURKA and HDM2 proteins followed by co-immunoprecipitaion revealed that AURKA directly interacts and phosphorylates HDM2 protein in vitro. The activation of HDM2 by AURKA led to induction of P53 ubiquitination and attenuation of cisplatin-induced activation of P53 in gastric cancer cells. Inhibition of AURKA using an investigational small molecule specific inhibitor, alisertib, decreased HDM2 protein level and induced P53 transcriptional activity. These effects markedly decreased cell survival in vitro and xenograft tumor growth in vivo. Notably, analysis of immunohistochemistry on tissue microarrays revealed significant overexpression of AURKA and HDM2 in human GC samples (P<0.05).
Collectively, our novel findings indicate that AURKA promotes tumor growth and cell survival through regulation of HDM2-induced ubiquitination and inhibition of P53.
AURKA; HDM2; Stomach; Cancer; P53; Ubiquitination; Esophagus
Background & Aims
Chronic inflammation contributes to the pathogenesis of gastric tumorigenesis. The Aurora kinase A gene (AURKA) is frequently amplified and overexpressed in gastrointestinal cancers. We investigated the roles of AURKA in inflammation and gastric tumorigenesis.
We used quantitative real-time reverse transcription PCR, immunofluorescence, immunohistochemistry, luciferase reporter, immunoblot, co-immunoprecipitation, and in vitro kinase assays to analyze AGS and MKN28 gastric cancer cells. We also analyzed Tff1−/− mice, growth of tumor xenografts, and human tissues.
We correlated increased expression of AURKA with increased levels of tumor necrosis factor-α and inflammation in the gastric mucosa of Tff1−/− mice (r = 0.62; P=.0001). MLN8237, an investigational small-molecule selective inhibitor of AURKA, reduced nuclear staining of NFκBp65 in human gastric cancer samples and mouse epithelial cells, suppressed NFκB reporter activity, and reduced the expression of NFκB target genes that regulate inflammation and cell survival. Inhibition of AURKA also reduced growth of xenograft tumors from human gastric cancer cells in mice and reversed the development of gastric tumors in Tff1−/− mice. AURKA was found to regulate NFκB activity by binding directly and phosphorylating IκBα in cells. Premalignant and malignant lesions from the gastric mucuosa of patients had increased levels of AURKA protein and nuclear NFκB, compared with healthy gastric tissue.
In analyses of gastric cancer cell lines, human tissue samples, and mouse models, we found AURKA to be upregulated during chronic inflammation to promote activation of NFκB and tumorigenesis. AURKA inhibitors might be developed as therapeutic agents for gastric cancer.
stomach cancer; mouse model; gene regulation; TNFα
Despite over 70,000 new cases of bladder cancer in the United States annually, patients with advanced disease have a poor prognosis due to limited treatment modalities. We evaluated Aurora kinase A, identified as an upregulated candidate molecule in bladder cancer, as a potential therapeutic target.
Gene expression in human bladder cancer samples was evaluated using RNA microarray and quantitative reverse-transcriptase PCR. Effects of the Aurora kinase A inhibitor MLN8237 (Millennium) on cell dynamics in malignant T24 and UM-UC-3 and papilloma-derived RT4 bladder cells were evaluated in vitro and in vivo in a mouse xenograft model.
A set of 13 genes involved in the mitotic spindle checkpoint, including Aurora kinases A and B, were upregulated in human urothelial carcinoma compared to normal urothelium. The Aurora kinase A inhibitor MLN8237 induced cell cycle arrest, aneuploidy, mitotic spindle failure, and apoptosis in the human bladder cancer cell lines T24 and UM-UC-3. MLN8237 also arrested tumor growth when administered orally over 4 weeks in a mouse bladder cancer xenograft model. Finally, in vitro sequential administration of MLN8237 with either paclitaxel or gemcitabine resulted in synergistic cytotoxic effects in T24 cells.
Mitotic spindle checkpoint dysfunction is a common characteristic of human urothelial carcinoma, and can be exploited with pharmacologic Aurora A inhibition. Given our demonstration of the ability of the Aurora A inhibitor MLN8237 to inhibit growth of bladder cancer in vitro and in vivo, we conclude that Aurora kinase inhibitors warrant further therapeutic investigation in bladder cancer.
Upper gastrointestinal adenocarcinomas (UGCs) respond poorly to current chemotherapeutic regimes. We and others have previously reported frequent AURKA gene amplification and mRNA and protein overexpression in UGCs. This study aimed to determine the therapeutic potential of Alisertib (MLN8237) alone and in combination with Docetaxel in UGCs.
Following treatment with Alisertib and/or Docetaxel, clonogenic cell survival, cell cycle analyses, Western blotting and tumor xenograft growth assays were carried out to measure cell survival, cell cycle progression, apoptotic protein expression and tumor xenograft volumes, respectively.
Using AGS, FLO-1 and OE33 UGC cell lines, with constitutive AURKA overexpression and variable-p53 status, we observed significantly enhanced inhibition of cancer cell survival with Alisertib and Docetaxel combination (p<0.001), as compared to single agent treatments. Cell cycle analyses after 48hr of treatment with Alisertib demonstrated a significant increase in the percentage of polyploidy in UGC cells (p<0.01) that was further enhanced by Docetaxel (p<0.001). Additionally, an increase in the percentage of cells in the sub-G1-phase that was observed with Alisertib (p<0.01) was significantly enhanced with the combination (p<0.001). Western blot analysis demonstrated higher induction of cleaved caspase 3 protein expression with the combined treatment, as compared to single agent treatments. In addition, FLO-1 and OE33 cell xenograft models demonstrated an enhanced anti-tumor activity for the Alisertib and Docetaxel combination, as compared to single agent treatments (p<0.001).
This study demonstrates that Alisertib, combined with Docetaxel, can mediate a better therapeutic outcome in UGCs cell lines.
AURKA; Alisertib; Docetaxel; stomach; esophagus; Cancer; Mitosis; Apoptosis
Aurora kinase A (AURKA) localizes to centrosomes and mitotic spindles where it mediates mitotic progression and chromosomal stability. Overexpression of AURKA is common in cancer, resulting in acquisition of alternate non-mitotic functions. In the current study, we identified a novel role for AURKA in regulating ovarian cancer cell dissemination and evaluated the efficacy of an AURKA-selective small molecule inhibitor, alisertib (MLN8237), as a single agent and combined with paclitaxel using an orthotopic xenograft model of epithelial ovarian cancer (EOC). Ovarian carcinoma cell lines were used to evaluate the effects of AURKA inhibition and overexpression on migration and adhesion. Pharmacologic or RNAi-mediated inhibition of AURKA significantly reduced ovarian carcinoma cell migration and adhesion and the activation-associated phosphorylation of the cytoskeletal regulatory protein SRC at tyrosine 416 (pSRCY416). Conversely, enforced expression of AURKA resulted in increased migration, adhesion and activation of SRC in cultured cells. In vivo tumor growth and dissemination were inhibited by alisertib treatment as a single agent. Moreover, combination of alisertib with paclitaxel, an agent commonly used in treatment of EOC, resulted in more potent inhibition of tumor growth and dissemination compared to either drug alone. Taken together, these findings support a role for AURKA in EOC dissemination by regulating migration and adhesion. They also point to the potential utility of combining AURKA inhibitors with taxanes as a therapeutic strategy for the treatment of EOC patients.
Aurora kinase A; SRC; ovarian cancer; migration; alisertib; adhesion
Patients with Neurofibromatosis Type 1 (NF1) develop malignant peripheral nerve sheath tumors (MPNST) which are often inoperable and do not respond well to current chemotherapies or radiation. The goal of this study was to utilize comprehensive gene expression analysis to identify novel therapeutic targets.
Nerve Schwann cells and/or their precursors are the tumorigenic cell types in MPNST due to the loss of the NF1 gene, which encodes the RasGAP protein neurofibromin. Therefore, we created a transgenic mouse model, CNP-HRas12V, expressing constitutively-active HRas in Schwann cells and defined a Ras-induced gene expression signature to drive a Bayesian factor regression model analysis of differentially expressed genes in mouse and human neurofibromas and MPNSTs. We tested functional significance of Aurora kinase over-expression in MPNST in vitro and in vivo using Aurora kinase shRNAs and compounds that inhibit Aurora kinase.
We identified 2000 genes with probability of linkage to nerve Ras signaling of which 339 were significantly differentially expressed in mouse and human NF1-related tumor samples relative to normal nerves, including Aurora kinase A (AURKA). AURKA was dramatically over-expressed and genomically amplified in MPNSTs but not neurofibromas. Aurora kinase shRNAs and Aurora kinase inhibitors blocked MPNST cell growth in vitro. Furthermore, an AURKA selective inhibitor, MLN8237, stabilized tumor volume and significantly increased survival of mice with MPNST xenografts.
Integrative cross-species transcriptome analyses combined with preclinical testing has provided an effective method for identifying candidates for molecular-targeted therapeutics. Blocking Aurora kinases may be a viable treatment platform for MPNST.
Neurofibroma; NF1; MPNST; Ras; Aurora Kinase
Purpose Amplification or over-expression of the mitotic Aurora A kinase (AAK) has been reported in several heme-lymphatic malignancies. MLN8237 (alisertib) is a novel inhibitor of AAK that is being developed for the treatment of advanced malignancies. The objectives of this phase I study were to establish the safety, tolerability, and pharmacokinetic profiles of escalating doses of MLN8237 in patients with relapsed or refractory heme-lymphatic malignancies. Methods Sequential cohorts of patients received MLN8237 orally as either a powder-in-capsule (PIC) or enteric-coated tablet (ECT) formulation. Patients received MLN8237 PIC 25–90 mg for 14 or 21 consecutive days plus 14 or 7 days’ rest, respectively, or MLN8237 ECT, at a starting dose of 40 mg/day once-daily (QD) for 14 days plus 14 days’ rest, all in 28-day cycles. Subsequent cohorts received MLN8237 ECT 30–50 mg twice-daily (BID) for 7 days plus 14 days’ rest in 21-day cycles. Results Fifty-eight patients were enrolled (PIC n = 28, ECT n = 30). The most frequent grade ≥3 drug-related toxicities were neutropenia (45 %), thrombocytopenia (28 %), anemia (19 %), and leukopenia (19 %). The maximum tolerated dose on the ECT 7-day schedule was 50 mg BID. The terminal half-life of MLN8237 was approximately 19 h. Six (13 %) patients achieved partial responses and 13 (28 %) stable disease. Conclusion The recommended phase II dose of MLN8237 ECT is 50 mg BID for 7 days in 21-day cycles, which is currently being evaluated as a single agent in phase II/III trials in patients with peripheral T-cell lymphoma.
Electronic supplementary material
The online version of this article (doi:10.1007/s10637-013-0050-9) contains supplementary material, which is available to authorized users.
Phase I-III Leukemia and lymphomas; Novel antitumor agent; Cell cycle mechanisms of anticancer drug action; Aurora A kinase inhibitor; MLN8237
Novel therapies are urgently needed to improve clinical outcomes for patients with acute myeloid leukemia (AML). The investigational drug alisertib (MLN8237) is a novel Aurora A kinase inhibitor being studied in multiple Phase I and II studies. We investigated the preclinical efficacy and pharmacodynamics of alisertib in AML cell lines, primary AML cells, and mouse models of AML. Here we report that alisertib disrupted cell viability, diminished clonogenic survival, induced expression of the FOXO3a targets p27 and BIM, and triggered apoptosis. A link between Aurora A expression and sensitivity to ara-C was established, suggesting that Aurora A inhibition may be a promising strategy to increase the efficacy of ara-C. Accordingly, alisertib significantly potentiated the anti-leukemic activity of ara-C in both AML cell lines and primary blasts. Targeted FOXO3a knockdown significantly blunted the pro-apoptotic effects of the alisertib/ara-C combination, indicating that it is an important regulator of sensitivity to these agents. In vivo studies demonstrated that alisertib significantly augmented the efficacy of ara-C without affecting its pharmacokinetic profile and led to the induction of p27 and BIM. Our collective data indicate that targeting Aurora A with alisertib represents a novel approach to increase the efficacy of ara-C that warrants further investigation.
Aurora kinase A; acute myeloid leukemia; apoptosis; cytarabine; FOXO
Histone deacetylase (HDAC) inhibitors, such as vorinostat, decrease Aurora kinase activity by a variety of mechanisms. Vorinostat and MLN8237, a selective Aurora A kinase inhibitor, disrupt the spindle assembly and the mitotic checkpoint at different points, suggesting that the combination could have increased antitumor activity. The purpose of this study was to determine the cytotoxicity of vorinostat and MLN8237 in pediatric tumor cell lines.
Cell survival was measured after 72 h of drug treatment using a modified methyl tetrazolium assay. For drug combination experiments, cells were exposed to medium alone (controls), single drug alone, or to different concentrations of the combination of the two drugs, for a total of 36 concentration pairs per plate. The interaction of the drug combination was analyzed using the universal response surface approach.
The cells express the target of MLN8237, Aurora A. For each cell line, the single agent IC50 for MLN8237 and for vorinostat was in the clinically relevant range. Both drugs inhibited cell survival in a concentration-dependent fashion. At concentrations of MLN8237 exceeding approximately 1 μM, there was a paradoxical increase in viability signal in all three lines that may be explained by inhibition of Aurora B kinase. The combination of MLN8237 and vorinostat showed additive cytotoxicity in all three cell lines and nearly abrogated the paradoxical increase in survival noted at high single-agent MLN8237 concentrations.
MLN8237 and vorinostat are active in vitro against cancer cell lines. These results provide important preclinical support for the development of future clinical studies of MLN8237and vorinostat.
Vorinostat; Histone deacetylase inhibitor; MLN8237; Aurora kinase inhibitor; Cytotoxicity
Esophageal adenocarcinomas (EACs) are poorly responsive to chemotherapeutics. This study aimed to determine the levels of Aurora kinas A (AURKA) and the therapeutic potential of MLN8237, an investigational AURKA inhibitor, alone and in combination with Cisplatin. Using quantitative real time polymerase chain reaction we detected frequent AURKA gene amplification (15/34, 44%) and mRNA overexpression (37/44, 84%) in EACs (p<0.01). Immunohistochemistry analysis demonstrated overexpression of AURKA in more than two-thirds of EACs tissue samples (92/132, 70%) (p<0.001). Using FLO-1, OE19 and OE33 EAC cell lines, with constitutive AURKA overexpression and mutant-p53, we observed inhibition of colony formation with a single treatment of 0.5μM MLN8237 (p<0.05). This effect was further enhanced in combination with 2.5μM Cisplatin (p<0.001). 24hrs after treatment with the MLN8237 or MLN8237 and Cisplatin, cell cycle analyses demonstrated a sharp increase in the percentage of polyploid cells (p<0.001). This was followed by an increase in the percentage of cells in the sub-G1-phase at 72hrs, concordant with the occurrence of cell death (p<0.001). Western blot analysis demonstrated higher induction of TAp73β, PUMA, NOXA, cleaved caspase 3 and cleaved PARP with the combined treatment, as compared to a single agent treatment. Using xenograft models, we demonstrated an enhanced anti-tumor role for the MLN8237 and Cisplatin combination, as compared to single agent treatments (p<0.001). In conclusion, this study demonstrates frequent overexpression of AURKA and suggests that MLN8237 could be an effective anti-tumor agent, which can be combined with CDDP for a better therapeutic outcome in EACs.
AURKA; Cisplatin; Barrett’s; Esophageal; Cancer; Mitosis; Apoptosis; p53; p73
Aurora A is critical for mitosis and is overexpressed in several neoplasms. Its overexpression transforms cultured cells, and both its overexpression and knockdown cause genomic instability. In transgenic mice, Aurora A haploinsufficiency, not overexpression, leads to increased malignant tumor formation. Aurora A thus appears to have both tumor-promoting and tumor-suppressor functions. Here, we report that Aurora A protein, measured by quantitative western blotting, is differentially expressed in major glioma types in lineage-specific patterns. Aurora A protein levels in WHO grade II oligodendrogliomas (n = 16) and grade III anaplastic oligodendrogliomas (n = 16) are generally low, similar to control epilepsy cerebral tissue (n = 11). In contrast, pilocytic astrocytomas (n = 6) and ependymomas (n = 12) express high Aurora A levels. Among grade II to grade III astrocytomas (n = 7, n = 14, respectively) and grade IV glioblastomas (n = 31), Aurora A protein increases with increasing tumor grade. We also found that Aurora A expression is induced by hypoxia in cultured glioblastoma cells and is overexpressed in hypoxic regions of glioblastoma tumors. Retrospective Kaplan-Meier analysis revealed that both lower Aurora A protein measured by quantitative western blot (n = 31) and Aurora A mRNA levels measured by real-time quantitative RT-PCR (n = 58) are significantly associated with poorer patient survival in glioblastoma. Furthermore, we report that the selective Aurora A inhibitor MLN8237 is potently cytotoxic to glioblastoma cells, and that MLN8237 cytotoxicty is potentiated by ionizing radiation. MLN8237 also appeared to induce senescence and differentiation of glioblastoma cells. Thus, in addition to being significantly associated with survival in glioblastoma, Aurora A is a potential new drug target for the treatment of glioblastoma and possibly other glial neoplasms.
Aurora A; glioblastoma; MLN8237; differential expression; patient survival; radiation; diffuse astrocytoma; oligodendroglioma; ependymoma; pilocytic astrocytoma
Aurora A kinase is critical in assembly and function of the mitotic spindle. It is overexpressed in various tumor types and implicated in oncogenesis and tumor progression. This trial evaluated the dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) of MLN8054, a selective small-molecule inhibitor of Aurora A kinase.
In this first-in-human, dose-escalation study, MLN8054 was given orally for 7, 14, or 21 days followed by a 14-day treatment-free period. Escalating cohorts of 3–6 patients with advanced solid tumors were treated until DLT was seen in ≥2 patients in a cohort. Serial blood samples were collected for pharmacokinetics and skin biopsies were collected for pharmacodynamics.
Sixty-one patients received 5, 10, 20, 30 or 40 mg once daily for 7 days; 25, 35, 45 or 55 mg/day in four divided doses (QID) for 7 days; or 55, 60, 70 or 80 mg/day plus methylphenidate or modafinil with daytime doses (QID/M) for 7–21 days. DLTs of reversible grade 3 benzodiazepine-like effects defined the estimated MTD of 60 mg QID/M for 14 days. MLN8054 was absorbed rapidly, exposure was dose-proportional, and terminal half-life was 30-40 hours. Three patients had stable disease for >6 cycles.
MLN8054 dosing for up to 14 days of a 28-day cycle was feasible. Reversible somnolence was dose limiting and prevented achievement of plasma concentrations predicted necessary for target modulation. A recommended dose for investigation in phase 2 trials was not established. A second-generation Aurora A kinase inhibitor is in development.
MLN8054; Aurora A kinase; dose-limiting toxicity; pharmacokinetics; pharmacodynamics
To gain a greater understanding of the potential of the Aurora kinase A inhibitor MLN8237 in the treatment of pediatric malignancies.
The activity of MLN8237 was evaluated against 28 neuroblastoma and Ewing sarcoma cell lines, and its in vivo efficacy was studied over a range of doses against 12 pediatric tumor xenograft models. Pharmacokinetic, pharmacodynamic, and genomic studies were undertaken.
In vitro neuroblastoma cell lines were generally more sensitive to MLN8237 than Ewing sarcoma lines. MLN8237 demonstrated significant activity in vivo against solid tumor models at the maximum tolerated dose (MTD); however, only 2 of 6 neuroblastoma models had objective responses at 0.25MTD. In contrast, MLN8237 induced objective responses at its MTD and at 0.5MTD in three ALL models and in two out of three at 0.25MTD. Pharmacokinetic studies at 0.5MTD demonstrated a Tmax of 0.5 h, Cmax of 24.8 μM, AUC(0–24) of 60.3 μM h, and 12 h trough level of 1.2 μM. Mitotic indices increased 6–12 h after MLN8237 administration. AURKA copy number variation was frequent in xenografts, and expression was highly correlated with copy number.
Objective responses were more frequent in tumors with decreased AURKA copy number (5/8) compared to those with increased gene copy number (2/14). This report confirms the significant activity against both solid tumor and ALL xenografts at the MTD, with a steep dose response. These data support clinical development of MLN8237 in childhood cancer. Because of the steep dose–response relationship, such studies should target achieving trough levels of 1 μM or higher for sustained periods of treatment.
Electronic supplementary material
The online version of this article (doi:10.1007/s00280-011-1618-8) contains supplementary material, which is available to authorized users.
Preclinical testing; Developmental therapeutics; MLN8237; Pediatric cancer
We investigated the role of Aurora kinase A (AURKA) in regulating p73-dependent apoptosis utilizing p53-deficient cancer cell lines; H1299, TE7, and HCT116p53−/−. Overexpression of AURKA led to down-regulation of the TAp73-induced activation of the p53/p73-dependent luciferase reporter plasmid (pG13-luc). The reduction in the TAp73 transcription activity was confirmed by measuring the activity of luciferase reporters for p21/WAF1, and Puma. The siRNA knockdown of endogenous AURKA reversed these effects and Western blot analysis demonstrated a significant increase in the protein level of TAp73 and its downstream transcription targets; Puma, Noxa, and p21/WAF1. The co-expression of AURKA together with TAp73 inhibited the activation of the pG13-luc, Puma-luc, and p21/WAF1-luc reporter plasmids with reduction in the protein levels of TAp73 transcription targets. Treatment with AURKA-selective small molecule inhibitor, MLN8054 (Millennium Pharmaceuticals, Cambridge, MA) led to a significant increase in the activities of pG13-luc, Puma-luc, and p21/WAF1-luc reporter plasmids. This effect was accompanied by a significant increase in the mRNA and protein levels of several TAp73 transcription targets; p21/WAF1, PUMA, and NOXA. Flow cytometry cell cycle analysis, following MLN8054 treatment showed more than a two-fold increase in cell death. The apoptotic outcome was corroborated by showing an increase in cleaved caspase-3 protein levels by Western blot. Using TUNEL assay, we demonstrated that the expression of dominant-negative mutant TAp73 expression plasmid (p73DD) counteracted the MLN8054-induced cell death. Taken together, our results indicate that AURKA regulates TAp73-dependent apoptosis and highlight the potential of the AURKA inhibitor MLN8054 in treating cancers that are defective in p53 signaling.
AURKA; MLN8054; p73; apoptosis; cancer
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.
We describe an interaction between homeodomain-interacting protein kinase 1 (HIPK1) and Daxx, two transcriptional regulators important in transducing growth-regulatory signals. We demonstrate that HIPK1 is ubiquitously expressed in mice and humans and localizes predominantly to the nucleus. Daxx normally resides within the nucleus in promyelocytic leukemia protein (PML) oncogenic domains (PODs), where it physically interacts with PML. Under certain circumstances, Daxx is relocalized from PODs to chromatin, where it then acts as a transcriptional repressor through an association with histone deacetylase (HDAC1). We propose two novel mechanisms for regulating the activity of Daxx, both mediated by HIPK1. First, HIPK1 physically interacts with Daxx in cells and consequently relocalizes Daxx from PODs. Daxx relocalization disrupts its interaction with PML and augments its interaction with HDAC1, likely influencing Daxx activity. Although the relocalization of Daxx from PODs is phosphorylation independent, an active HIPK1 kinase domain is required, suggesting that HIPK1 autophosphorylation is important in this interaction. Second, HIPK1 phosphorylates Daxx on Ser 669, and phosphorylation of this site is important in modulating the ability of Daxx to function as a transcriptional repressor. Mutation of Daxx Ser 669 to Ala results in increased repression in three of four transcriptional reporters, suggesting that phosphorylation by HIPK1 diminishes Daxx transcriptional repression of specific promoters. Taken together, our results indicate that HIPK1 and Daxx collaborate in regulating transcription.