Phase I study of MLN8237--investigational Aurora A kinase inhibitor--in relapsed/refractory multiple myeloma, non-Hodgkin lymphoma and chronic lymphocytic leukemia.

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.

Additive effects of vorinostat and MLN8237 in pediatric leukemia, medulloblastoma, and neuroblastoma cell lines.

PURPOSE: 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. METHODS: 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. RESULTS: The cells express the target of MLN8237, Aurora A. For each cell line, the single agent IC(50) 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. CONCLUSION: 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.

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.

Randomized Phase II Study of Paclitaxel plus Alisertib versus Paclitaxel plus Placebo as Second-Line Therapy for SCLC: Primary and Correlative Biomarker Analyses.

INTRODUCTION: We assessed the Aurora A kinase inhibitor, alisertib, plus paclitaxel (henceforth referred to as alisertib/paclitaxel) as second-line treatment for SCLC. METHODS: In this double-blind study, patients with relapsed or refractory SCLC were stratified by relapse type (sensitive versus resistant or refractory) and brain metastases and randomized 1:1 to alisertib/paclitaxel or placebo plus paclitaxel (henceforth referred to as placebo/paclitaxel) in 28-day cycles. The primary end point was progression-free survival (PFS). Associations of c-Myc expression in tumor tissue (prespecified) and genetic alterations in circulating tumor DNA (retrospective) with clinical outcome were evaluated. RESULTS: A total of 178 patients were enrolled (89 in each arm). The median PFS was 3.32 months with alisertib/paclitaxel versus 2.17 months with placebo/paclitaxel (hazard ratio [HR] = 0.77, 95% confidence limit [CI]: 0.557-1.067, p = 0.113 in the intent-to-treat population versus HR = 0.71, 95% CI: 0.509-0.985, p = 0.038 with corrected analysis applied). Among 140 patients with genetic alternations, patients with cell cycle regulator mutations (cyclin-dependent kinase 6 gene [CDK6], retinoblastoma-like 1 gene [RBL1], retinoblastoma-like 2 gene [RBL2], and retinoblastoma 1 gene [RB1]) had significantly improved PFS with alisertib/paclitaxel versus with placebo/paclitaxel (3.68 versus 1.80 months, respectively [HR = 0.395, 95% CI: 0.239-0.654, p = 0.0003]), and overall survival (7.20 versus 4.47 months, respectively [HR = 0.427, 95% CI: 0.259-0.704, p = 0.00085]). A subset of patients with c-Myc expression showed significantly improved PFS with alisertib/paclitaxel. The incidence of grade 3 or higher drug-related adverse events was 67% (58 patients) with alisertib/paclitaxel versus 22% (25 patients) with placebo/paclitaxel. Twelve patients (14%) versus 11 (12%) died on study, including four versus zero treatment-related deaths. CONCLUSIONS: Efficacy signals were seen with alisertib/paclitaxel in relapsed or refractory SCLC. c-Myc expression and mutations in cell cycle regulators may be potential predictive biomarkers of alisertib efficacy; further prospective validations are warranted.

Localization of human TACC3 to mitotic spindles is mediated by phosphorylation on Ser558 by Aurora A: a novel pharmacodynamic method for measuring Aurora A activity.

Aurora A is a serine/threonine protein kinase essential for normal mitotic progression. Aberrant increased expression of Aurora A, which occurs frequently in human cancers, results in abnormal mitoses leading to chromosome instability and possibly tumorigenesis. Consequently, Aurora A has received considerable attention as a potential target for anticancer therapeutic intervention. Aurora A coordinates several essential mitotic activities through phosphorylation of a variety of proteins, including TACC3, which modulates microtubule stabilization of the mitotic spindle. Recent studies identified a conserved serine in Xenopus (Ser(626)) and Drosophila (Ser(863)) TACC3 orthologues that is phosphorylated by Aurora A. We show that this conserved serine on human TACC3 (Ser(558)) is also phosphorylated by Aurora A. Moreover, phosphorylation of TACC3 by Aurora A in human cells is essential for its proper localization to centrosomes and proximal mitotic spindles. Inhibition of Aurora A with the selective small molecule inhibitor MLN8054 in cultured human tumor cells resulted in mislocalization of TACC3 away from mitotic spindles in a concentration-dependent manner. Furthermore, oral administration of MLN8054 to nude mice bearing HCT-116 human tumor xenografts caused a dose-dependent mislocalization of TACC3 away from spindle poles that correlated with tumor growth inhibition. As TACC3 localization to mitotic spindles depends on Aurora A-mediated phosphorylation, quantifying TACC3 mislocalization represents a novel pharmacodynamic approach for measuring Aurora A activity in cancer patients treated with inhibitors of Aurora A kinase.

A high-throughput liposome substrate assay with automated lipid extraction process for PI 3-kinase.

The signaling pathways involving lipid kinase class I phosphatidylinositol 3-kinases (PI 3-kinases) regulate cell growth, proliferation, and survival. Class I PI 3-kinases catalyze the conversion of PI (4,5)P(2) to PI (3,4,5)P(3), which acts as a lipid second messenger to activate mitogenic signaling cascades. Recently, p110alpha, a class IA PI 3-kinase, was found to be mutated frequently in many human cancers. Therefore, it is increasingly studied as an anticancer drug target. Traditionally, PI 3-kinase activities have been studied using liposome substrates. This method, however, is hampered significantly by the labor-intensive manual lipid extraction followed by a low-throughput thin-layer chromatography analysis. The authors describe a high-throughput liposome substrate-based assay based on an automated lipid extraction method that allows them to study PI 3-kinase enzyme mechanism and quantitatively measure inhibitor activity using liposome substrates in a high-throughput mode. This improved assay format can easily be extended to study other classes of phosphoinositide lipid kinases.

Homeodomain-interacting protein kinase 1 modulates Daxx localization, phosphorylation, and transcriptional activity.

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.

Aurora A kinase inhibition enhances oncolytic herpes virotherapy through cytotoxic synergy and innate cellular immune modulation.

Malignant peripheral nerve sheath tumor (MPNST) and neuroblastoma models respond to the investigational small molecule Aurora A kinase inhibitor, alisertib. We previously reported that MPNST and neuroblastomas are also susceptible to oncolytic herpes virus (oHSV) therapy. Herein, we show that combination of alisertib and HSV1716, a virus derived from HSV-1 and attenuated by deletion of RL1, exhibits significantly increased antitumor efficacy compared to either monotherapy. Alisertib and HSV1716 reduced tumor growth and increased survival in two xenograft models of MPNST and neuroblastoma. We found the enhanced antitumor effect was due to multiple mechanisms that likely each contribute to the combination effect. First, oncolytic herpes virus increased the sensitivity of uninfected cells to alisertib cytotoxicity, a process we term virus-induced therapeutic adjuvant (VITA). Second, alisertib increased peak virus production and slowed virus clearance from tumors, both likely a consequence of it preventing virus-mediated increase of intratumoral NK cells. We also found that alisertib inhibited virus-induced accumulation of intratumoral myeloid derived suppressor cells, which normally are protumorigenic. Our data suggest that clinical trials of the combination of oHSV and alisertib are warranted in patients with neuroblastoma or MPNST.

Connecting the Dots: Therapy-Induced Senescence and a Tumor-Suppressive Immune Microenvironment.

BACKGROUND: Tumor cell senescence is a common outcome of anticancer therapy. Here we investigated how therapy-induced senescence (TIS) affects tumor-infiltrating leukocytes (TILs) and the efficacy of immunotherapy in melanoma. METHODS: Tumor senescence was induced by AURKA or CDK4/6 inhibitors (AURKAi, CDK4/6i). Transcriptomes of six mouse tumors with differential response to AURKAi were analyzed by RNA sequencing, and TILs were characterized by flow cytometry. Chemokine RNA and protein expression were determined by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Therapeutic response was queried in immunodeficient mice, in mice with CCL5-deficient tumors, and in mice cotreated with CD137 agonist to activate TILs. CCL5 expression in reference to TIS and markers of TILs was studied in human melanoma tumors using patient-derived xenografts (n = 3 patients, n = 3 mice each), in AURKAi clinical trial samples (n = 3 patients, before/after therapy), and in The Cancer Genome Atlas (n = 278). All statistical tests were two-sided. RESULTS: AURKAi response was associated with induction of the immune transcriptome (P = 3.5 x 10-29) while resistance inversely correlated with TIL numbers (Spearman r = -0.87, P < .001). AURKAi and CDK4/6i promoted the recruitment of TILs by inducing CCL5 secretion in melanoma cells (P ≤ .005) in an NF-κB-dependent manner. Therapeutic response to AURKAi was impaired in immunodeficient compared with immunocompetent mice (0% vs 67% tumors regressed, P = .01) and in mice bearing CCL5-deficient vs control tumors (P = .61 vs P = .02); however, AURKAi response was greatly enhanced in mice also receiving T-cell-activating immunotherapy (P < .001). In human tumors, CCL5 expression was also induced by AURKAi (P ≤ .02) and CDK4/6i (P = .01) and was associated with increased immune marker expression (P = 1.40 x 10-93). CONCLUSIONS: Senescent melanoma cells secret CCL5, which promotes recruitment of TILs. Combining TIS with immunotherapy that enhances tumor cell killing by TILs is a promising novel approach to improve melanoma outcomes.

Scientific Rationale Supporting the Clinical Development Strategy for the Investigational Aurora A Kinase Inhibitor Alisertib in Cancer.

Alisertib (MLN8237) is a selective small molecule inhibitor of Aurora A kinase that is being developed in multiple cancer indications as a single agent and in combination with other therapies. A significant amount of research has elucidated a role for Aurora A in orchestrating numerous activities of cells transiting through mitosis and has begun to shed light on potential non-mitotic roles for Aurora A as well. These biological insights laid the foundation for multiple clinical trials evaluating the antitumor activity of alisertib in both solid cancers and heme-lymphatic malignancies. Several key facets of Aurora A biology as well as empirical data collected in experimental systems and early clinical trials have directed the development of alisertib toward certain cancer types, including neuroblastoma, small cell lung cancer, neuroendocrine prostate cancer, atypical teratoid/rhabdoid tumors, and breast cancer among others. In addition, these scientific insights provided the rationale for combining alisertib with other therapies, including microtubule perturbing agents, such as taxanes, EGFR inhibitors, hormonal therapies, platinums, and HDAC inhibitors among others. Here, we link the key aspects of the current clinical development of alisertib to the originating scientific rationale and provide an overview of the alisertib clinical experience to date.

Combining an Aurora Kinase Inhibitor and a Death Receptor Ligand/Agonist Antibody Triggers Apoptosis in Melanoma Cells and Prevents Tumor Growth in Preclinical Mouse Models.

PURPOSE: Preclinical studies show that inhibition of aurora kinases in melanoma tumors induces senescence and reduces tumor growth, but does not cause tumor regression. Additional preclinical models are needed to identify agents that will synergize with aurora kinase inhibitors to induce tumor regression. EXPERIMENTAL DESIGN: We combined treatment with an aurora kinase A inhibitor, MLN8237, with agents that activate death receptors (Apo2L/TRAIL or death receptor 5 agonists) and monitored the ability of this treatment to induce tumor apoptosis and melanoma tumor regression using human cell lines and patient-derived xenograft (PDX) mouse models. RESULTS: We found that this combined treatment led to apoptosis and markedly reduced cell viability. Mechanistic analysis showed that the induction of tumor cell senescence in response to the AURKA inhibitor resulted in a decreased display of Apo2L/TRAIL decoy receptors and increased display of one Apo2L/TRAIL receptor (death receptor 5), resulting in enhanced response to death receptor ligand/agonists. When death receptors were activated in senescent tumor cells, both intrinsic and extrinsic apoptotic pathways were induced independent of BRAF, NRAS, or p53 mutation status. Senescent tumor cells exhibited BID-mediated mitochondrial depolarization in response to Apo2L/TRAIL treatment. In addition, senescent tumor cells had a lower apoptotic threshold due to decreased XIAP and survivin expression. Melanoma tumor xenografts of one human cell line and one PDX displayed total blockage of tumor growth when treated with MLN8237 combined with DR5 agonist antibody. CONCLUSIONS: These findings provide a strong rationale for combining senescence-inducing therapeutics with death receptor agonists for improved cancer treatment.

Combined inhibition of MEK and Aurora A kinase in KRAS/PIK3CA double-mutant colorectal cancer models.

Aurora A kinase and MEK inhibitors induce different, and potentially complementary, effects on the cell cycle of malignant cells, suggesting a rational basis for utilizing these agents in combination. In this work, the combination of an Aurora A kinase and MEK inhibitor was evaluated in pre-clinical colorectal cancer models, with a focus on identifying a subpopulation in which it might be most effective. Increased synergistic activity of the drug combination was identified in colorectal cancer cell lines with concomitant KRAS and PIK3CA mutations. Anti-proliferative effects were observed upon treatment of these double-mutant cell lines with the drug combination, and tumor growth inhibition was observed in double-mutant human tumor xenografts, though effects were variable within this subset. Additional evaluation suggests that degree of G2/M delay and p53 mutation status affect apoptotic activity induced by combination therapy with an Aurora A kinase and MEK inhibitor in KRAS and PIK3CA mutant colorectal cancer. Overall, in vitro and in vivo testing was unable to identify a subset of colorectal cancer that was consistently responsive to the combination of a MEK and Aurora A kinase inhibitor.

Mdm2 and aurora kinase a inhibitors synergize to block melanoma growth by driving apoptosis and immune clearance of tumor cells.

Therapeutics that induce cancer cell senescence can block cell proliferation and promote immune rejection. However, the risk of tumor relapse due to senescence escape may remain high due to the long lifespan of senescent cells that are not cleared. Here, we show how combining a senescence-inducing inhibitor of the mitotic kinase Aurora A (AURKA) with an MDM2 antagonist activates p53 in senescent tumors harboring wild-type 53. In the model studied, this effect is accompanied by proliferation arrest, mitochondrial depolarization, apoptosis, and immune clearance of cancer cells by antitumor leukocytes in a manner reliant upon Ccl5, Ccl1, and Cxcl9. The AURKA/MDM2 combination therapy shows adequate bioavailability and low toxicity to the host. Moreover, the prominent response of patient-derived melanoma tumors to coadministered MDM2 and AURKA inhibitors offers a sound rationale for clinical evaluation. Taken together, our work provides a preclinical proof of concept for a combination treatment that leverages both senescence and immune surveillance to therapeutic ends.

MLN8054 and Alisertib (MLN8237): Discovery of Selective Oral Aurora A Inhibitors.

The Aurora kinases are essential for cell mitosis, and the dysregulation of Aurora A and B have been linked to the etiology of human cancers. Investigational agents MLN8054 (8) and alisertib (MLN8237, 10) have been identified as high affinity, selective, orally bioavailable inhibitors of Aurora A that have advanced into human clinical trials. Alisertib (10) is currently being evaluated in multiple Phase II and III clinical trials in hematological malignancies and solid tumors.

Response to MLN8237 in pancreatic cancer is not dependent on RalA phosphorylation.

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 (AAK) is critical for PDAC tumorigenesis. We sought to evaluate the AAK-selective inhibitor MLN8237 as a potential indirect anti-RalA-targeted therapy for PDAC. We used 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 patients with PDAC independent of RalA S194 phosphorylation. Ki67 may be an effective pharmacodynamic biomarker to identify response early in the course of treatment.

The selective Aurora-A kinase inhibitor MLN8237 (alisertib) potently inhibits proliferation of glioblastoma neurosphere tumor stem-like cells and potentiates the effects of temozolomide and ionizing radiation.

The selective Aurora-A kinase inhibitor MLN8237 is in clinical trials for hematologic malignancies, ovarian cancer and other solid tumors. We previously showed that MLN8237 is potently antiproliferative toward standard monolayer-cultured glioblastoma cells. We have now investigated the effect of MLN8237 with and without temozolomide or ionizing radiation on the proliferation of glioblastoma tumor stem-like cells (neurospheres) using soft agar colony formation assays and normal human astrocytes by MTT assay. Western blotting was utilized to compare MLN8237 IC50s to cellular Aurora-A and phosphoThr(288)Aurora-A levels. MLN8237 was more potently antiproliferative to neurosphere cells than to standard monolayer glioma cells, and was non-toxic to normal human astrocytes. Western blot analysis revealed that MLN8237 treatment inhibits phosphoThr(288)Aurora-A levels providing proof of drug target-hit in glioblastoma cells. Furthermore, phosphoThr(288)Aurora-A levels partially predicted the antiproliferative efficacy of MLN8237. We also found that Aurora-A inhibition by MLN8237 was synergistic with temozolomide and potentiated the effects of ionizing radiation on colony formation in neurosphere glioblastoma tumor stem-like cells. These results further support the potential of Aurora-A inhibitors as primary chemotherapy agents or biologic response modifiers in glioblastoma patients.

Translational exposure-efficacy modeling to optimize the dose and schedule of taxanes combined with the investigational Aurora A kinase inhibitor MLN8237 (alisertib).

Aurora A kinase orchestrates multiple key activities, allowing cells to transit successfully into and through mitosis. MLN8237 (alisertib) is a selective Aurora A inhibitor that is being evaluated as an anticancer agent in multiple solid tumors and heme-lymphatic malignancies. The antitumor activity of MLN8237 when combined with docetaxel or paclitaxel was evaluated in in vivo models of triple-negative breast cancer grown in immunocompromised mice. Additive and synergistic antitumor activity occurred at multiple doses of MLN8237 and taxanes. Moreover, significant tumor growth delay relative to the single agents was achieved after discontinuing treatment; notably, durable complete responses were observed in some mice. The tumor growth inhibition data generated with multiple dose levels of MLN8237 and paclitaxel were used to generate an exposure-efficacy model. Exposures of MLN8237 and paclitaxel achieved in patients were mapped onto the model after correcting for mouse-to-human variation in plasma protein binding and maximum tolerated exposures. This allowed rank ordering of various combination doses of MLN8237 and paclitaxel to predict which pair would lead to the greatest antitumor activity in clinical studies. The model predicted that 60 and 80 mg/m(2) of paclitaxel (every week) in patients lead to similar levels of efficacy, consistent with clinical observations in some cancer indications. The model also supported using the highest dose of MLN8237 that can be achieved, regardless of whether it is combined with 60 or 80 mg/m(2) of paciltaxel. The modeling approaches applied in these studies can be used to guide dose-schedule optimization for combination therapies using other therapeutic agents.

The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell-cycle progression in malignant bladder cancer cells in vitro and in vivo.

PURPOSE: Despite more than 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. EXPERIMENTAL DESIGN: 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. RESULTS: A set of 13 genes involved in the mitotic spindle checkpoint, including Aurora kinases A and B, were upregulated in human urothelial carcinoma compared with 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. CONCLUSIONS: 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.

Preclinical pharmacokinetic/pharmacodynamic/efficacy relationships for alisertib, an investigational small-molecule inhibitor of Aurora A kinase.

PURPOSE: Alisertib (MLN8237) is an investigational inhibitor of Aurora A kinase (AAK). Aurora A plays an essential role in the regulation of spindle assembly and chromosome alignment during mitosis. Inhibition of Aurora A by alisertib in tissue culture has previously been demonstrated to lead to improper chromosomal alignment and disruption of spindle organization, resulting in a transient mitotic delay. The spindle organization defects induced by alisertib have been used to develop a pharmacodynamic (PD) assay for Aurora A inhibition based on the percentage of mitotic cells with proper chromosomal alignment at the metaphase plate (% aligned spindles, abbreviated as AS). The transient mitotic delay that occurs with AAK inhibition permits the use of the mitotic index (the fraction of cells in the population currently undergoing mitosis, abbreviated as MI) as an additional PD assay. When the two PD assays were used in Phase I clinical trials, the reduction in AS was strongly correlated with dose levels and exposures in patients from single time point PD measurements; however, MI failed to show any correlation. To further understand this clinical finding, we constructed PK/PD/efficacy models for AS and MI that can precisely capture the temporal dynamics of the PD markers from in vivo xenograft studies. METHODS: A PK/PD study was conducted using a single oral dose of alisertib at 3, 10, and 20 mg/kg in HCT-116 xenografts implanted subcutaneously in mice. An extravascular, two-compartmental pharmacokinetic (PK) model was used to describe the drug kinetics. Consistent with the mechanistic hypothesis for AAK inhibition, the PD biomarkers such as AS and MI were fitted to PK using a direct response inhibitory sigmoid model and an indirect response turnover model, respectively. The antitumor activity of alisertib dosed orally for 21 days with different dose levels and schedules was evaluated. RESULTS: The PK/PD models showed a fast, sustained response for AS after alisertib administration, whereas MI exhibited a slow, transient response. The PK/efficacy relationship for alisertib in HCT-116 xenografts closely corresponds to the PK/PD relationship for the PD markers, with all three IC50s in close agreement (303, 270, and 280 nM, respectively). CONCLUSION: The PK/PD and PK/efficacy models show that both AS and MI are equally relevant as mechanism-based PD markers to capture drug activity. However, of the two PD markers, the fast, sustained response of AS makes it the only clinically viable PD marker for defining a dose-response relationship, as its maximal effect can be captured from a wider time window with a single PD sampling; while the window to capture dose-related MI response is narrower.

Ras-driven transcriptome analysis identifies aurora kinase A as a potential malignant peripheral nerve sheath tumor therapeutic target.

PURPOSE: 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 use comprehensive gene expression analysis to identify novel therapeutic targets. EXPERIMENTAL DESIGN: Nerve Schwann cells and/or their precursors are the tumorigenic cell types in MPNST because of 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 overexpression in MPNST in vitro and in vivo using Aurora kinase short hairpin RNAs (shRNA) and compounds that inhibit Aurora kinase. RESULTS: We identified 2,000 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 overexpressed 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. CONCLUSION: 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.