mln-8237 and Neuroblastoma

Alisertib is an oral Aurora A kinase inhibitor with preclinical activity in neuroblastoma. Irinotecan and temozolomide have activity in patients with advanced neuroblastoma. The goal of this phase I study was to determine the maximum tolerated dose (MTD) of alisertib with irinotecan and temozolomide in this population.. Patients age 1 to 30 years with relapsed or refractory neuroblastoma were eligible. Patients received alisertib tablets at dose levels of 45, 60, and 80 mg/m(2) per day on days 1 to 7 along with irinotecan 50 mg/m(2) intravenously and temozolomide 100 mg/m(2) orally on days 1 to 5. Dose escalation of alisertib followed the rolling six design. Samples for pharmacokinetic and pharmacogenomic testing were obtained.. Twenty-three patients enrolled, and 22 were eligible and evaluable for dose escalation. A total of 244 courses were administered. The MTD for alisertib was 60 mg/m(2), with mandatory myeloid growth factor support and cephalosporin prophylaxis for diarrhea. Thrombocytopenia and neutropenia of any grade were seen in the majority of courses (84% and 69%, respectively). Diarrhea in 55% of courses and nausea in 54% of courses were the most common nonhematologic toxicities. The overall response rate was 31.8%, with a 50% response rate observed at the MTD. The median number of courses per patient was eight (range, two to 32). Progression-free survival rate at 2 years was 52.4%. Pharmacokinetic testing did not show evidence of drug-drug interaction between irinotecan and alisertib.. Alisertib 60 mg/m(2) per dose for 7 days is tolerable with a standard irinotecan and temozolomide backbone and has promising response and progression-free survival rates. A phase II trial of this regimen is ongoing.

Topics: Administration, Oral; Adolescent; Adult; Antineoplastic Combined Chemotherapy Protocols; Aurora Kinase A; Azepines; Camptothecin; Child; Child, Preschool; Dacarbazine; Disease Progression; Disease-Free Survival; Drug Administration Schedule; Drug Resistance, Neoplasm; Female; Genotype; Glucuronosyltransferase; Humans; Infant; Irinotecan; Kaplan-Meier Estimate; Male; Maximum Tolerated Dose; Neoplasm Recurrence, Local; Neuroblastoma; Phenotype; Protein Kinase Inhibitors; Pyrimidines; Tablets; Temozolomide; Time Factors; Treatment Outcome; United States; Young Adult

Neuroblastoma accounts for 15% of cancer-related deaths in children, highlighting an unmet need for novel therapies. Selinexor is a small molecule inhibitor of XPO1. XPO1 shuffles cargo proteins with a nuclear export sequence from the nucleus to the cytosol, many of which are essential for cancer growth and cell maintenance. We systematically tested the effect of selinexor against neuroblastoma cells in vitro and in vivo and used an advanced proteomic and phosphoproteomic screening approach to interrogate unknown mechanisms of action. We found that selinexor induced its cytotoxic effects in neuroblastoma through the predominantly nuclear accumulation of p53 and global activation of apoptosis pathways. Selinexor also induced p53 phosphorylation at site S315, which is one initiating step for p53 degradation. Since this phosphorylation step is undertaken mostly by aurora kinase A (AURKA), we used the clinically available AURKA inhibitor, alisertib, and found p53-mediated lethality could be further augmented in three orthotopic xenograft mouse models. These findings suggest a potential therapeutic benefit using selinexor and alisertib to synergistically increase p53-mediated cytotoxicity of high-risk neuroblastoma.

Topics: Animals; Apoptosis; Azepines; Cell Line, Tumor; Humans; Hydrazines; Karyopherins; Mice; Neuroblastoma; Proteomics; Pyrimidines; Receptors, Cytoplasmic and Nuclear; Triazoles; Tumor Suppressor Protein p53; Xenograft Model Antitumor Assays

Neuroblastoma is a severe childhood disease, accounting for ~10% of all infant cancers. The amplification of the MYCN gene, coding for the N-Myc transcription factor, is an essential marker correlated with tumor progression and poor prognosis. In neuroblastoma cells, the mitotic kinase Aurora-A (AURKA), also frequently overexpressed in cancer, prevents N-Myc degradation by directly binding to a highly conserved N-Myc region. As a result, elevated levels of N-Myc are observed. During recent years, it has been demonstrated that some ATP competitive inhibitors of AURKA also cause essential conformational changes in the structure of the activation loop of the kinase that prevents N-Myc binding, thus impairing the formation of the AURKA/N-Myc complex. In this study, starting from a screening of crystal structures of AURKA in complexes with known inhibitors, we identified additional compounds affecting the conformation of the kinase activation loop. We assessed the ability of such compounds to disrupt the interaction between AURKA and N-Myc in vitro, using Surface Plasmon Resonance competition assays, and in tumor cell lines overexpressing MYCN, by performing Proximity Ligation Assays. Finally, their effects on N-Myc cellular levels and cell viability were investigated. Our results identify PHA-680626 as an amphosteric inhibitor both in vitro and in MYCN overexpressing cell lines, thus expanding the repertoire of known conformational disrupting inhibitors of the AURKA/N-Myc complex and confirming that altering the conformation of the activation loop of AURKA with a small molecule is an effective strategy to destabilize the AURKA/N-Myc interaction in neuroblastoma cancer cells.

Topics: Adenosine Triphosphate; Antineoplastic Agents; Aurora Kinase A; Azepines; Benzazepines; Binding Sites; Binding, Competitive; Cell Line; Drug Evaluation, Preclinical; Humans; N-Myc Proto-Oncogene Protein; Neuroblastoma; Protein Conformation; Protein Kinase Inhibitors; Pyrazoles; Pyrimidines; Pyrroles; Surface Plasmon Resonance

Noninvasive early indicators of treatment response are crucial to the successful delivery of precision medicine in children with cancer. Neuroblastoma is a common solid tumor of young children that arises from anomalies in neural crest development. Therapeutic approaches aiming to destabilize

Topics: Algorithms; Animals; Azepines; Benzamides; Child; Female; Humans; Machine Learning; Male; Mice; Mice, Transgenic; Morpholines; Multiparametric Magnetic Resonance Imaging; N-Myc Proto-Oncogene Protein; Neuroblastoma; Precision Medicine; Protein Kinase Inhibitors; Pyrimidines; Time Factors; TOR Serine-Threonine Kinases; Treatment Outcome

A majority of cases of high-risk neuroblastoma, an embryonal childhood cancer, are driven by MYC or MYCN-driven oncogenic signaling. While considered to be directly "undruggable" therapeutically, MYC and MYCN can be repressed transcriptionally by inhibition of Bromodomain-containing protein 4 (BRD4) or destabilized posttranslationally by inhibition of Aurora Kinase A (AURKA). Preclinical and early-phase clinical studies of BRD4 and AURKA inhibitors, however, show limited efficacy against neuroblastoma when used alone. We report our studies on the concomitant use of the BRD4 inhibitor I-BET151 and AURKA inhibitor alisertib. We show that, in vitro, the drugs act synergistically to inhibit viability in four models of high-risk neuroblastoma. We demonstrate that this synergy is driven, in part, by the ability of I-BET151 to mitigate reflexive upregulation of AURKA, MYC, and MYCN in response to AURKA inhibition. We then demonstrate that I-BET151 and alisertib are effective in prolonging survival in four xenograft neuroblastoma models in vivo, and this efficacy is augmented by the addition of the antitubule chemotherapeutic vincristine. These data suggest that epigenetic and posttranslational inhibition of MYC/MYCN-driven pathways may have significant clinical efficacy against neuroblastoma.

Topics: Animals; Antineoplastic Combined Chemotherapy Protocols; Aurora Kinase A; Azepines; Cell Cycle Proteins; Cell Line, Tumor; Drug Synergism; Gene Expression Regulation, Neoplastic; Heterocyclic Compounds, 4 or More Rings; Humans; Mice, SCID; N-Myc Proto-Oncogene Protein; Neuroblastoma; Nuclear Proteins; Pyrimidines; Survival Rate; Transcription Factors; Vincristine; Xenograft Model Antitumor Assays

Neuroblastoma is a biologically and clinically heterogeneous pediatric malignancy that includes a high-risk subset for which new therapeutic agents are urgently required. As well as MYCN amplification, activating point mutations of ALK and NRAS are associated with high-risk and relapsing neuroblastoma. As both ALK and RAS signal through the MEK/ERK pathway, we sought to evaluate two previously reported inhibitors of ETS-related transcription factors, which are transcriptional mediators of the Ras-MEK/ERK pathway in other cancers. Here we show that YK-4-279 suppressed growth and triggered apoptosis in nine neuroblastoma cell lines, while BRD32048, another ETV1 inhibitor, was ineffective. These results suggest that YK-4-279 acts independently of ETS-related transcription factors. Further analysis reveals that YK-4-279 induces mitotic arrest in prometaphase, resulting in subsequent cell death. Mechanistically, we show that YK-4-279 inhibits the formation of kinetochore microtubules, with treated cells showing a broad range of abnormalities including multipolar, fragmented and unseparated spindles, together leading to disrupted progression through mitosis. Notably, YK-4-279 does not affect microtubule acetylation, unlike the conventional mitotic poisons paclitaxel and vincristine. Consistent with this, we demonstrate that YK-4-279 overcomes vincristine-induced resistance in two neuroblastoma cell-line models. Furthermore, combinations of YK-4-279 with vincristine, paclitaxel or the Aurora kinase A inhibitor MLN8237/Alisertib show strong synergy, particularly at low doses. Thus, YK-4-279 could potentially be used as a single-agent or in combination therapies for the treatment of high-risk and relapsing neuroblastoma, as well as other cancers.

Topics: Antimitotic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Aurora Kinase A; Azepines; Cell Cycle; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Synergism; Humans; Indoles; Inhibitory Concentration 50; Kinetochores; Mitosis; Neuroblastoma; Paclitaxel; Prometaphase; Protein Kinase Inhibitors; Pyrimidines; RNA Interference; Signal Transduction; Spindle Apparatus; Time Factors; Transfection; Tumor Suppressor Protein p53; Vincristine

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.

Topics: Animals; Antineoplastic Agents; Aurora Kinase A; Azepines; Blotting, Western; Cell Line, Tumor; Combined Modality Therapy; Cytotoxicity, Immunologic; Female; Flow Cytometry; Herpesvirus 1, Human; Humans; Immunity, Innate; Immunohistochemistry; Mice; Mice, Nude; Neurilemmoma; Neuroblastoma; Oncolytic Virotherapy; Pyrimidines; Xenograft Model Antitumor Assays

Aurora kinase A (AURKA) overexpression is associated with poor prognosis in neuroblastoma and has been described to upregulate VEGF in gastric cancer cells. However, the exact role of AURKA in the regulation of neuroblastoma tumorigenesis remains unknown. We hypothesize that AURKA-mediated stabilization of N-Myc may affect VEGF expression and angiogenesis in neuroblastoma. Therefore, we sought to determine whether inhibition of AURKA modulates neuroblastoma angiogenesis.. Cell viability and anchorage-independent growth were determined after silencing AURKA or after treatment with MLN8237, AURKA inhibitor. Immunofluorescence was used to determine N-Myc localization. Human umbilical vein endothelial cells (HUVECs) were used to assess angiogenesis in vitro. Real time-PCR and ELISA were performed to determine VEGF transcription and secretion, respectively.. Knockdown of AURKA significantly reduced cell proliferation and inhibited anchorage-independent growth. It also decreased N-Myc protein levels and nuclear localization. AURKA inhibition also decreased HUVECs tubule formation along with VEGF transcription and secretion. Similarly, MLN8237 treatment decreased neuroblastoma tumorigenicity in vitro.. Our findings demonstrate that AURKA plays a critical role in neuroblastoma angiogenesis. AURKA regulates nuclear translocation of N-Myc in neuroblastoma cells, thus potentially affecting cell proliferation, anchorage-independent cell growth, and angiogenesis. Targeting AURKA might provide a novel therapeutic strategy in treating aggressive neuroblastomas.

Topics: Antineoplastic Agents; Aurora Kinase A; Azepines; Cell Adhesion; Cell Division; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Human Umbilical Vein Endothelial Cells; Humans; In Vitro Techniques; Molecular Targeted Therapy; Neoplasm Proteins; Neovascularization, Pathologic; Neuroblastoma; Protein Kinase Inhibitors; Protein Transport; Proto-Oncogene Proteins c-myc; Pyrimidines; RNA, Neoplasm; RNA, Small Interfering; Tumor Stem Cell Assay; Vascular Endothelial Growth Factor A

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

Topics: Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; Aurora Kinase A; Aurora Kinase B; Azepines; Brain Neoplasms; Cell Cycle Checkpoints; Cell Proliferation; Drug Resistance, Neoplasm; Drug Synergism; Gene Expression Regulation, Neoplastic; Histones; Humans; Imidazoles; Neuroblastoma; Phosphorylation; Piperazines; Primary Cell Culture; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-mdm2; Pyrimidines; Signal Transduction; Tumor Suppressor Protein p53

Amplification of MYCN is a driver mutation in a subset of human neuroendocrine tumors, including neuroblastoma. No small molecules that target N-Myc, the protein encoded by MYCN, are clinically available. N-Myc forms a complex with the Aurora-A kinase, which protects N-Myc from proteasomal degradation. Although stabilization of N-Myc does not require the catalytic activity of Aurora-A, we show here that two Aurora-A inhibitors, MLN8054 and MLN8237, disrupt the Aurora-A/N-Myc complex and promote degradation of N-Myc mediated by the Fbxw7 ubiquitin ligase. Disruption of the Aurora-A/N-Myc complex inhibits N-Myc-dependent transcription, correlating with tumor regression and prolonged survival in a mouse model of MYCN-driven neuroblastoma. We conclude that Aurora-A is an accessible target that makes destabilization of N-Myc a viable therapeutic strategy.

Topics: Animals; Antineoplastic Agents; Aurora Kinase A; Aurora Kinases; Azepines; Benzazepines; Cell Cycle Proteins; Cell Line, Tumor; Cyclohexanecarboxylic Acids; F-Box Proteins; F-Box-WD Repeat-Containing Protein 7; Humans; Mice; Neuroblastoma; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-myc; Pyrimidines; Thiazoles; Ubiquitin-Protein Ligases

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 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.. 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.

Topics: Antineoplastic Agents; Aurora Kinase B; Aurora Kinases; Azepines; Cell Line, Tumor; Cell Survival; Drug Interactions; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia; Medulloblastoma; Neuroblastoma; Protein Kinase Inhibitors; Protein Serine-Threonine Kinases; Pyrimidines; Vorinostat