Small-molecule Inhibitor Of Aurora Research Articles

Noninvasive MRI Native T1 Mapping Detects Response to MYCN-targeted Therapies in the Th-MYCN Model of Neuroblastoma.

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 MYCN protein, such as small-molecule inhibitors of Aurora A and mTOR, are currently being evaluated in early phase clinical trials in children with high-risk MYCN-driven disease, with limited ability to evaluate conventional pharmacodynamic biomarkers of response. T1 mapping is an MRI scan that measures the proton spin-lattice relaxation time T1. Using a multiparametric MRI-pathologic cross-correlative approach and computational pathology methodologies including a machine learning-based algorithm for the automatic detection and classification of neuroblasts, we show here that T1 mapping is sensitive to the rich histopathologic heterogeneity of neuroblastoma in the Th-MYCN transgenic model. Regions with high native T1 corresponded to regions dense in proliferative undifferentiated neuroblasts, whereas regions characterized by low T1 were rich in apoptotic or differentiating neuroblasts. Reductions in tumor-native T1 represented a sensitive biomarker of response to treatment-induced apoptosis with two MYCN-targeted small-molecule inhibitors, Aurora A kinase inhibitor alisertib (MLN8237) and mTOR inhibitor vistusertib (AZD2014). Overall, we demonstrate the potential of T1 mapping, a scan readily available on most clinical MRI scanners, to assess response to therapy and guide clinical trials for children with neuroblastoma. The study reinforces the potential role of MRI-based functional imaging in delivering precision medicine to children with neuroblastoma. SIGNIFICANCE: This study shows that MRI-based functional imaging can detect apoptotic responses to MYCN-targeted small-molecule inhibitors in a genetically engineered murine model of MYCN-driven neuroblastoma.

Phase II study of alisertib as a single agent in recurrent or progressive atypical teratoid rhabdoid tumors.

10542 Background: We conducted a Phase II study of alisertib, small-molecule inhibitor of Aurora A kinase, as single-agent treatment in patients < 22 y with recurrent or progressive atypical teratoid rhabdoid tumors (ATRT) (NCT02114229). Methods: Patients received alisertib once daily [80 mg/m2 (enteric-coated tablets) or 60 mg/m2 (liquid)] on Days 1–7 of a 21-day cycle for 2 y or until progressive disease (PD). Therapy was considered promising if ≥10 patients were without PD by MR imaging at 12 wk. Molecular groups were determined using Infinium Methylation EPIC BeadChips and the Heidelberg classifier. Alisertib plasma concentrations were measured in cycle 1, on Days 1 (single dose) and 7 (steady state) and analyzed using population-based modeling. Results: Data from 30 patients representing all 3 molecular groups [SHH (10/26), MYC (10/26), TYR (6/26), unknown (4/26)] was analyzed. One patient remains on therapy. The study did not meet the efficacy end point as only 8/29 patients were without PD after 12 wk, including 1 with partial response. Progression-free survival (PFS) was 31%±8.2% at 6 months and 15.8%±6.5% at 1 y. One- year overall survival (OS) was 42.1%±9.2%. One patient remained on treatment for > 12 months, and another for > 18 months. The median treatment duration was 44 days (range, 2–653 days). There was no difference in OS (p = 0.096) or PFS (p = 0.98) by molecular groups. Neutropenia was the most common adverse effect (77%). After single-dose alisertib, we observed higher mean maximum concentration (Cmax) 10.1±3.0 µM and faster time to Cmax (Tmax = 1.2±0.7 h) in the 22 patients who received liquid formulation than those who received tablets (Cmax = 5.7±2.4 µM, Tmax = 3.4±1.4 h). Drug exposure did not differ between the formulations (AUC0-∞ = 58.6±25 h·µM). Average apparent oral clearance was 2.32 L/h per m2, which was about half that reported in adults. Serial CSF samples were collected in 2 patients; the CSF/plasma AUC ratios were 1.2%-2.7%. Conclusions: Although the study did not meet the efficacy end point, alisertib was well tolerated as a single agent in children with recurrent ATRT. A third of the patients demonstrated disease stabilization for > 6 months. Treatment response beyond 1 y was seen in 2 patients Clinical trial information: NCT02114229.

A phase II clinical trial of the Aurora and angiogenic kinase inhibitor ENMD-2076 for previously treated, advanced, or metastatic triple-negative breast cancer

BackgroundTriple-negative breast cancer (TNBC) remains an aggressive breast cancer subtype with limited treatment options. ENMD-2076 is a small-molecule inhibitor of Aurora and angiogenic kinases with proapoptotic and antiproliferative activity in preclinical models of TNBC.MethodsThis dual-institution, single-arm, two-stage, phase II clinical trial enrolled patients with locally advanced or metastatic TNBC previously treated with one to three prior lines of chemotherapy in the advanced setting. Patients were treated with ENMD-2076 250 mg orally once daily with continuous dosing in 4-week cycles until disease progression or unacceptable toxicity occurred. The primary endpoint was 6-month clinical benefit rate (CBR), and secondary endpoints included progression-free survival, pharmacokinetic profile, safety, and biologic correlates in archival and fresh serial tumor biopsies in a subset of patients.ResultsForty-one patients were enrolled. The 6-month CBR was 16.7% (95% CI, 6–32.8%) and included two partial responses. The 4-month CBR was 27.8% (95% CI, 14–45.2%), and the average duration of benefit was 6.5 cycles. Common adverse events included hypertension, fatigue, diarrhea, and nausea. Treatment with ENMD-2076 resulted in a decrease in cellular proliferation and microvessel density and an increase in p53 and p73 expression, consistent with preclinical observations.ConclusionsSingle-agent ENMD-2076 treatment resulted in partial response or clinical benefit lasting more than 6 months in 16.7% of patients with pretreated, advanced, or metastatic TNBC. These results support the development of predictive biomarkers using archival and fresh tumor tissue, as well as consideration of mechanism-based combination strategies.Trial registrationClinicalTrials.gov, NCT01639248. Registered on July 12, 2012.

Abstract PD3-16: Clinical safety and efficacy of the aurora and angiogenic kinase inhibitor ENMD-2076 in previously treated, locally advanced or metastatic triple-negative breast cancer

Abstract Background: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype defined by the lack of expression of the estrogen and progesterone receptors and lack of HER2 over-expression. ENMD-2076 is an orally bioavailable small molecule inhibitor of Aurora and angiogenic kinases with pro-apoptotic and antiproliferative activity in preclinical models of TNBC. Methods: This two institution, single-arm, two-stage, phase II clinical trial enrolled patients with locally advanced or metastatic TNBC refractory to 1-3 prior lines of chemotherapy in the advanced setting. Patients had ECOG PS ≤ 1, measureable disease by RECIST 1.1 and no evidence of brain metastasis. Patients were treated with ENMD-2076 250 mg PO daily with continuous dosing in 4-week cycles until disease progression or unacceptable toxicity occurred. The primary end point was 6-month clinical benefit rate (6-CBR) and secondary endpoints included time to progression (TTP), PK profile, safety and biologic correlatives in archival and fresh serial tumor biopsies in a subset of patients. Results: Between July 2012 and October 2016, 41 patients were enrolled (median age 54; range 30-73; female 40; male 1). Patients received a mean 1.7 prior lines of chemotherapy for locally advanced unresectable or metastatic disease and 80.5% received prior neoadjuvant or adjuvant chemotherapy (N=33). Thirty-six patients were evaluable per protocol for the primary efficacy analysis. Five patients (12.2%) were not included in the efficacy analysis due to: adverse events (AE) leading to discontinuation prior to objective efficacy assessment (N=3), not meeting eligibility criteria on day 1 (N=1) and withdraw of consent in cycle 1 (N=1). The study proceeded to the second stage of enrollment based on observing three 6-CBR events in Stage 1 (N=18 patients). The 6-CBR in the overall trial was 16.7% (95% exact CI: 6%-32.8%; 2 patients with PR and 4 patients with SD > 6 mos). The median duration of response or clinical benefit in these patients was 32 weeks (8 cycles). 4-CBR was 27.8% (95% exact CI: 14%-45.2%). Dose reduction occurred in 8 patients (20%) for fatigue, hypertension and proteinuria. The most common grade 3 treatment-related adverse events were hypertension (37.5%) and fatigue (10%). One patient experienced grade 4 hypertension. Analysis of serial tumor biopsies prior to and following 2 weeks of ENMD-2076 (N=8 patients), demonstrated a treatment-induced decrease in cellular proliferation (Ki-67) and microvessel density (CD34) as assessed by IHC. Immunofluorescence performed on a subset of samples demonstrated an increase in p53-family member expression following treatment, consistent with changes observed in preclinical TNBC patient-derived tumor xenograft models. Conclusions: ENMD-2076 has durable clinical activity in a subset of patients with pretreated, advanced or metastatic triple-negative breast cancer. Predictive biomarker development using archival and fresh tumor tissue is underway. Exploration of lower doses of ENMD-2076 in future clinical trials may improve tolerability. Citation Format: Diamond JR, Eckhardt SG, Pitts TM, van Bokhoven A, Aisner D, Gustafson DL, Capasso A, Elias AD, Storniolo AM, Schneider BP, Gao D, Tentler JJ, Borges VF, Miller KD. Clinical safety and efficacy of the aurora and angiogenic kinase inhibitor ENMD-2076 in previously treated, locally advanced or metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-16.

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.

Alisertib added to rituximab and vincristine is synthetic lethal and potentially curative in mice with aggressive DLBCL co-overexpressing MYC and BCL2.

Pearson correlation coefficient for expression analysis of the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) demonstrated Aurora A and B are highly correlated with MYC in DLBCL and mantle cell lymphoma (MCL), while both Auroras correlate with BCL2 only in DLBCL. Auroras are up-regulated by MYC dysregulation with associated aneuploidy and resistance to microtubule targeted agents such as vincristine. Myc and Bcl2 are differentially expressed in U-2932, TMD-8, OCI-Ly10 and Granta-519, but only U-2932 cells over-express mutated p53. Alisertib [MLN8237 or M], a highly selective small molecule inhibitor of Aurora A kinase, was synergistic with vincristine [VCR] and rituximab [R] for inhibition of cell proliferation, abrogation of cell cycle checkpoints and enhanced apoptosis versus single agent or doublet therapy. A DLBCL (U-2932) mouse model showed tumor growth inhibition (TGI) of ∼10–20% (p = 0.001) for M, VCR and M-VCR respectively, while R alone showed ∼50% TGI (p = 0.001). M-R and VCR-R led to tumor regression [TR], but relapsed 10 days after discontinuing therapy. In contrast, M-VCR-R demonstrated TR with no relapse >40 days after stopping therapy with a Kaplan-Meier survival of 100%. Genes that are modulated by M-VCR-R (CENP-C, Auroras) play a role in centromere-kinetochore function in an attempt to maintain mitosis in the presence of synthetic lethality. Together, our data suggest that the interaction between alisertib plus VCR plus rituximab is synergistic and synthetic lethal in Myc and Bcl-2 co-expressing DLBCL. Alisertib plus vincristine plus rituximab [M-VCR-R] may represent a new strategy for DLBCL therapy.

Aurora A Kinase Is Required For Hematopoiesis and Couples Polyploidization With Terminal Differentiation In Megakaryocytes Through Phosphorylation Of NF-E2

We have recently shown that small molecule inhibitors of Aurora A kinase (AURKA) including dimethyfasudil and MLN827 induce polyploidization and differentiation of normal and malignant megakaryocytes, as evidenced by increased DNA content and upregulation of the cell surface markers CD41 and CD42. Furthermore, our pre-clinical studies demonstrate that MLN8237 shows potent anti-leukemia activity and anti-myelofibrotic activity in MPNs by promoting polyploidization and terminal differentiation of abnormal megakaryocytes. To determine the mechanism by which AURKA inhibitors ameliorate the leukemic and myelofibrotic phenotypes, we have examined the functional requirement for Aurka in adult hematopoiesis. To circumvent the embryonic lethality of the germline knockout and study the requirement for AURKA in adult hematopoiesis, we induced deletion in Aurka floxed mice with Mx1-Cre. Complete loss of AURKA caused a rapid and profound defect in hematopoiesis, with the mice developing pancytopenia and marked hypocellularity of the bone marrow. Notably, we observe an increase in the proportion of CD41 and CD42-positive megakaryocytes in contrast to the deficiency of myeloid and lymphoid cells in the bone marrow. To determine whether the observed defects are cell autonomous, we transplanted Aurkafl/fl, Mx1-Cre bone marrow cells to lethally irradiated recipients, confirmed engraftment, and induced deletion with pIpC. Upon AURKA deletion, the transplanted mice develop an identical phenotype to the one observed in the conditional knockout mice, demonstrating a cell autonomous requirement for AURKA during hematopoiesis. Moreover, in competitive transplantation experiments, Aurka-/- cells are selectively lost and fail to contribute to hematopoiesis. Mechanistically, loss of AURKA led to a significant degree of apoptosis in hematopoietic cells, likely due to mitotic catastrophe resulting from impaired chromosome segregation. To bypass the requirement for AURKA in progenitor cells and examine AURKA function in the megakaryocyte lineage, we deleted AURKA in megakaryocytes ex vivo by infecting Aurka floxed bone marrow with MSCV-Cre. We found that deletion of AURKA resulted in increased CD41 and CD42 expression as well as increased ploidy of the megakaryocyte fraction. Together these results are consistent with a selective differentiation effect of AURKA deficiency on megakaryocytes.To investigate whether AURKA modulates differentiation of megakaryocytes through interactions with lineage specific transcription factors, we performed co-immunoprecipitation experiments between AURKA and megakaryocyte transcription factors containing consensus AURKA phosphorylation sites in primary megakaryocytes. Results confirmed a robust interaction between AURKA and p45 NF-E2. Additionally, we found by an in vitro kinase assay that AURKA phosphorylates p45 NF-E2 on the S170 residue, as the S170A mutant of NF-E2 cannot be phosphorylated by AURKA. To explore the functional importance of NF-E2 phosphorylation at S170, we created a phosphomimetic mutant, S170D, and assayed its ability to induce megakaryocyte differentiation in primary cultures. While overexpression of wild-type NF-E2 significantly increased the expression of the megakaryocyte differentiation markers CD41 and CD42, overexpression of the S170D mutant was markedly less effective in promoting megakaryocyte differentiation, indicating that the loss of phosphorylation promotes NF-E2 activity and that NF-E2 activity may be suppressed by AURKA phosphorylation. Finally, to determine if AURKA inhibitors promote megakaryocyte differentiation by allowing activation of NF-E2, we knocked-down down NF-E2 in megakaryocytic cell lines and subsequently treated with diMF or MLN8237. Strikingly, cells with NF-E2 knocked-down displayed significantly less CD41 and CD42 expression in response to AURKA inhibitor treatment compared to control knockdowns. This experiment demonstrates that NF-E2 is required for the differentiation effect caused by AURKA inhibition. Taken together, our data show that Aurora A kinase is required for adult hematopoiesis and that Aurora A regulates NF-E2 function during megakaryocyte differentiation. Our results also reveal that inhibition of AURKA kinase activity couples polyploidization and terminal differentiation of megakaryocytes. Disclosures:Crispino: Sanofi: Research Funding.

Abstract A282: Antitumor activity of alisertib (MLN8237), an investigational small molecule Aurora A kinase inhibitor, as a single agent and in combination with paclitaxel, in experimental models of small cell lung cancer.

Abstract Alisertib (MLN8237) is a highly selective, potent small molecule inhibitor of Aurora A kinase that is being developed for the treatment of patients with advanced malignancies, including small cell lung cancer (SCLC). SCLC accounts for approximately 13-15% of all lung cancers diagnosed and represents a major unmet medical need. SCLC is an aggressive disease with a median survival of 2-4 months if left untreated. Despite SCLC often being chemosensitive initially, with response rates between 60 - 80%, relapse is almost universal. Here, we assessed the antitumor effects of alisertib in vitro in various SCLC cell lines and in vivo in primary human SCLC xenograft models. The growth inhibitory effect of alisertib was determined in a number of SCLC cell lines. The effect on growth of these lines was measured by two independent methods, after treating these cells for 96 hours with varying concentrations of alisertib. The viability assays used were CyQuant direct, which measures cell proliferation by quantifying DNA content, and PrestoBlue, which determines cell viability by measuring the ability of cells to reduce resazurin. Cell growth was inhibited by alisertib in all lines tested with concentrations producing a 50% inhibition (IC50) ranging from 9 to 34 nM and 8 to 26 nM in the CyQuant and PrestoBlue assays, respectively. The antitumor activity of alisertib as a single agent was also evaluated in a number of primary human SCLC xenografts. In all tumor models tested, alisertib resulted in significant tumor growth inhibition at 20 mg/kg, administered both QD and BID. Alisertib dosed at 20 mg/kg BID in the CTG-0198 model led to regressions in tumor size during the period of dosing. The antitumor activity of alisertib was also tested in combination with a number of standard of care agents including paclitaxel. The combination of alisertib and paclitaxel provided enhanced antitumor activity in comparison to either agent alone in a number of models. These results demonstrate that alisertib effectively inhibits growth of experimental human SCLC models in cell culture, and displays effective antitumor activity in multiple primary human SCLC xenografts, both alone and in combination with paclitaxel. This work provides preclinical support for the further clinical evaluation of alisertib in SCLC. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A282. Citation Format: Derek Blair, Mengkun Zhang, Michael D. Smith, Carmin Szynal, Jeffrey A. Ecsedy, Huifeng Niu. Antitumor activity of alisertib (MLN8237), an investigational small molecule Aurora A kinase inhibitor, as a single agent and in combination with paclitaxel, in experimental models of small cell lung cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A282.

Abstract C265: Significant in vivo activity of ENMD-2076, a novel multi-targeted kinase inhibitor, towards xenograft models of human hepatocellular carcinomas .

Abstract Background: Hepatocellular carcinoma (HCC) is a primary malignancy of the liver. It represents the third leading cause of cancer deaths worldwide, with over 500,000 people affected each year. The incidence of HCC is particularly high in China, accounting for more than 50% of the global incidences. The development of targeted anticancer agents effective against HCC for patients who do not tolerate, or have failed or relapsed from sorafenib treatment would meet a major unmet medical need. ENMD-2076 is an orally bioavailable small molecule inhibitor of Aurora A and angiogenic kinases including VEGFR 2 and FGFR 1 with pro-apoptotic, antiproliferative and angiogenic activities against a variety of human cancers. ENMD-2076 has been tested in multiple Phase I and II clinical trials, with partial responses (PR) and prolonged progression free survival (PFS) observed in multiple cancers including liver cancer. Objective: This in vivo study was designed to compare the efficacy of ENMD-2076 with that of standard of care agents including sorafenib, doxorubicin, and 5-FU in 3 different cell-line derived human HCC xenograft models. The tolerability and efficacy of combinations of ENMD-2076 with doxorubicin or 5-FU were also determined. Experimental Design: Three different human HCC cell lines, SMMC-7721, QGY-7703 and HepG 2 were used for establishing subcutaneous tumor xenograft models in nude mice. After tumors grew to more than 100 mm3, mice were randomly assigned into one of the groups receiving vehicle, sorafenib, ENMD-2076 alone or in combination with chemotherapy agents, including doxorubicin or 5-FU, respectively, for 20 days. Results: In all HCC models tested, ENMD-2076 induced tumor growth inhibition significantly greater than that of sorafenib. When treated with ENMD-2076 at 100 mg/kg, the tumor growth inhibition (TGI) rates were 73.67%, 76.59% or 80.12% in the SMMC-7721, QGY-7703 or HepG 2 models, respectively, which were significantly higher than those observed in sorafenib treated groups (48.63%, 51.43%, or 57.60%, respectively;P<0.01 in all three models). Tumor growth inhibition rates were similarly higher in the ENMD-2076 treated groups than in groups treated with doxorubicin at 1 mg/kg and 5-FU at 30 mg/kg. The combined treatment of ENMD-2076 with either doxorubicin or 5-FU was well tolerated. Higher TGI rates were observed in combination groups compared to single agents of chemotherapies but were not statistically different. Conclusion: ENMD-2076 showed robust antitumor activity against three cell line-derived xenograft models of HCC superior to that of sorafenib, doxorubicin, and 5FU, supporting clinical investigation of this agent in HCC patients who do not tolerate, or have failed or relapsed from other systemic treatment such as sorafenib, doxorubicin or 5 FU. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C265. Citation Format: Peipei Yin, Hongqi Lu, Mark R. Bray, Bingsheng Li, Ken Ren. Significant in vivo activity of ENMD-2076, a novel multi-targeted kinase inhibitor, towards xenograft models of human hepatocellular carcinomas . [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C265.

Tripolin A, a Novel Small-Molecule Inhibitor of Aurora A Kinase, Reveals New Regulation of HURP's Distribution on Microtubules

Mitotic regulators exhibiting gain of function in tumor cells are considered useful cancer therapeutic targets for the development of small-molecule inhibitors. The human Aurora kinases are a family of such targets. In this study, from a panel of 105 potential small-molecule inhibitors, two compounds Tripolin A and Tripolin B, inhibited Aurora A kinase activity in vitro. In human cells however, only Tripolin A acted as an Aurora A inhibitor. We combined in vitro, in vivo single cell and in silico studies to demonstrate the biological action of Tripolin A, a non-ATP competitive inhibitor. Tripolin A reduced the localization of pAurora A on spindle microtubules (MTs), affected centrosome integrity, spindle formation and length, as well as MT dynamics in interphase, consistent with Aurora A inhibition by RNAi or other specific inhibitors, such as MLN8054 or MLN8237. Interestingly, Tripolin A affected the gradient distribution towards the chromosomes, but not the MT binding of HURP (Hepatoma Up-Regulated Protein), a MT-associated protein (MAP) and substrate of the Aurora A kinase. Therefore Tripolin A reveals a new way of regulating mitotic MT stabilizers through Aurora A phosphorylation. Tripolin A is predicted to bind Aurora A similarly but not identical to MLN8054, therefore it could be used to dissect pathways orchestrated by Aurora kinases as well as a scaffold for further inhibitor development.

256 Mechanism-based Preclinical PK/PD/E Relationships for Alisertib, an Investigational Small-molecule Inhibitor of Aurora A Kinase, in a Range of Human Solid Tumor Xenografts

256 Mechanism-based Preclinical PK/PD/E Relationships for Alisertib, an Investigational Small-molecule Inhibitor of Aurora A Kinase, in a Range of Human Solid Tumor Xenografts

Abstract 3684: Targeting Aurora kinase in peripheral T-cell non-Hodgkin's lymphomas

Abstract Aurora kinases (A and B) are oncogenic serine/threonine (S/T) kinases that play central roles in the mitotic phase of the eukaryotic cell cycle. Over-expression of Auroras during dysregulated cell cycling and their aberrant activation over-rides mitotic and spindle check points leading to anueploidy in many human cancers make Auroras attractive therapeutic targets. Gene expression profiling in mantle cell lymphoma [MCL] have shown the Auroras are over-expressed and is a poor prognostic marker within the ‘proliferative’ signature. PTCL (NOS) are a heterogeneous group of rare non-Hodgkin's lymphomas (NHL) derived from activated CD4+ or CD8+ T cells. PTCL and anaplastic large cell lymphoma (ALCL) both of T-cell phenotype and ALK negative have an aggressive clinical course with an inferior event free and overall survival relative to B-cell NHL counterparts. The biological explanation for the difference is unknown. Patients with PTCL have a poor prognosis due to a very aggressive disease course coupled to a lack of effective therapies. Therefore, the development of novel and effective treatments based on biologically validated targets is urgently needed for this disease. We hypothesized and investigated that (1). Auroras are over-expressed in human PTCL-NOS, (2). Aurora ATP-site small molecule inhibitor (SMI) is effective in inhibiting cell proliferation and promoting apoptosis in cell culture and (3). Aurora SMI will be safe and effective in treating patients with relapsed aggressive T-cell NHL in early phase clinical trials. In this study, we demonstrate that both aurora A and B are highly over-expressed in PTCL patients and cell lines. MLN8237 (alisertib), an ATP-site small molecule inhibitor of Aurora A had potent activity in inhibiting phosphorylation of Aurora A and histone H3 in PTCL cell lines. Cells treated with MLN8237 exhibited endoreduplication confirming the mechanism of action of an Aurora inhibitor. Also, treatment of PTCL cell lines with MLN8237 induced apoptosis in a dose and time dependent manner (flow cytometry and PARP-cleavage) and inhibited cell proliferation with an IC50 < 0.1µM. Preliminary data from a phase II clinical trial using single-agent MLN8237 in patients with relapsed refractory aggressive NHL has demonstrated notable activity with 4 confirmed responses in 6 evaluable PTCL patients. Cytokine profiling on day 1 (pre-treatment) and day 8 (post-treatment) showed a profile consistent with aurora A activity. Together the data suggest inhibition of Auroras offers a promising treatment strategy for patients with aggressive T-cell NHL [Funded by the Lymphoma SPORE, P50 CAB0805501A1]. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3684. doi:1538-7445.AM2012-3684

Characterization of Alisertib (MLN8237), an Investigational Small-Molecule Inhibitor of Aurora A Kinase Using Novel In Vivo Pharmacodynamic Assays

Small-molecule inhibitors of Aurora A (AAK) and B (ABK) kinases, which play important roles in mitosis, are currently being pursued in oncology clinical trials. We developed three novel assays to quantitatively measure biomarkers of AAK inhibition in vivo. Here, we describe preclinical characterization of alisertib (MLN8237), a selective AAK inhibitor, incorporating these novel pharmacodynamic assays. We investigated the selectivity of alisertib for AAK and ABK and studied the antitumor and antiproliferative activity of alisertib in vitro and in vivo. Novel assays were used to assess chromosome alignment and mitotic spindle bipolarity in human tumor xenografts using immunofluorescent detection of DNA and alpha-tubulin, respectively. In addition, 18F-3'-fluoro-3'-deoxy-l-thymidine positron emission tomography (FLT-PET) was used to noninvasively measure effects of alisertib on in vivo tumor cell proliferation. Alisertib inhibited AAK over ABK with a selectivity of more than 200-fold in cells and produced a dose-dependent decrease in bipolar and aligned chromosomes in the HCT-116 xenograft model, a phenotype consistent with AAK inhibition. Alisertib inhibited proliferation of human tumor cell lines in vitro and produced tumor growth inhibition in solid tumor xenograft models and regressions in in vivo lymphoma models. In addition, a dose of alisertib that caused tumor stasis, as measured by volume, resulted in a decrease in FLT uptake, suggesting that noninvasive imaging could provide value over traditional measurements of response. Alisertib is a selective and potent inhibitor of AAK. The novel methods of measuring Aurora A pathway inhibition and application of tumor imaging described here may be valuable for clinical evaluation of small-molecule inhibitors.

Phase I Assessment of New Mechanism-Based Pharmacodynamic Biomarkers for MLN8054, a Small-Molecule Inhibitor of Aurora A Kinase

The mitotic kinase Aurora A is an important therapeutic target for cancer therapy. This study evaluated new mechanism-based pharmacodynamic biomarkers in cancer patients in two phase I studies of MLN8054, a small-molecule inhibitor of Aurora A kinase. Patients with advanced solid tumors received MLN8054 orally for 7 consecutive days in escalating dose cohorts, with skin and tumor biopsies obtained before and after dosing. Skin biopsies were evaluated for increased mitotic cells within the basal epithelium. Tumor biopsies were assessed for accumulation of mitotic cells within proliferative tumor regions. Several patients in the highest dose cohorts showed marked increases in the skin mitotic index after dosing. Although some tumors exhibited increases in mitotic cells after dosing, others displayed decreases, a variable outcome consistent with dual mechanisms of mitotic arrest and mitotic slippage induced by antimitotics in tumors. To provide a clearer picture, mitotic cell chromosome alignment and spindle bipolarity, new biomarkers of Aurora A inhibition that act independently of mitotic arrest or slippage, were assessed in the tumor biopsies. Several patients, primarily in the highest dose cohorts, had marked decreases in the percentage of mitotic cells with aligned chromosomes and bipolar spindles after dosing. Evidence existed for an exposure-effect relationship for mitotic cells with defects in chromosome alignment and spindle bipolarity that indicated a biologically active dose range. Outcomes of pharmacodynamic assays from skin and tumor biopsies were concordant in several patients. Together, these new pharmacodynamic assays provide evidence for Aurora A inhibition by MLN8054 in patient skin and tumor tissues.

Phase I Study of the Selective Aurora A Kinase Inhibitor MLN8054 in Patients with Advanced Solid Tumors: Safety, Pharmacokinetics, and Pharmacodynamics

This phase I trial examined the safety, pharmacokinetics, and pharmacodynamics of MLN8054, an oral, selective, small-molecule inhibitor of Aurora A kinase. Patients with advanced solid tumors received increasing doses of MLN8054 in 28-day cycles until dose-limiting toxicity (DLT) was seen in ≥2 of 3-6 patients in a cohort. For the 10-mg and 20-mg cohorts, treatment was administered once daily on days 1 to 5 and 8 to 12. Patients in later cohorts (25, 35, 45, 55, 60, 70, and 80 mg/day) were treated four times daily on days 1 to 14, with the largest dose at bedtime (QID-14D) to mitigate benzodiazepine-like effects possibly associated with peak plasma concentrations. Patients (n = 43) received a median of 1 cycle (range, 1-10). DLT of somnolence was first noted in the 20-mg cohort. Two DLTs of somnolence (n = 1) and transaminitis (n = 1) were seen at QID-14D 80 mg. Grade 2 oral mucositis (n = 1), predicted to be a mechanistic effect, was observed only at QID-14D 80 mg. MLN8054 exposure levels were roughly linear with dose; terminal half-life was 30 to 40 hours. Pharmacodynamic analyses of skin and tumor mitotic indices, mitotic cell chromosome alignment, and spindle bipolarity provided evidence of Aurora A inhibition. MLN8054 dosing for 10 to 14 days in 28-day cycles was feasible. Somnolence and transaminitis were DLTs. Pharmacodynamic analyses in mitotic cells of both skin and tumor provided proof of mechanism for Aurora A kinase inhibition. A more potent, selective, second-generation Aurora A kinase inhibitor, MLN8237, is in clinical development.

A pediatric phase I trial and pharmacokinetic study of MLN8237, an oral selective small molecule inhibitor of aurora a kinase: A Children's Oncology Group Phase I Consortium study.

9529 Background: MLN8237, a selective small molecule inhibitor of Aurora A Kinase, has activity in a broad range of in vitro and in vivo preclinical models of pediatric cancer. We performed a phase I trial to determine the dose limiting toxicities (DLT), maximum tolerated dose (MTD), and pharmacokinetics (PK) of MLN8237 in children with refractory solid tumors. Methods: MLN8237 was administered orally either once daily (part A1) or divided twice daily (part A2) for 7 days, every 21 days. Using the rolling-six design, four dose levels (45, 60, 80, 100 mg/m2/d) were evaluated on the once daily regimen, and subsequently two dose levels (60 and 80 mg/m2/d) on the twice-daily regimen. Pharmacokinetic studies were performed with the initial dose, with trough drug concentrations also being obtained at steady-state. Results: 37 patients were enrolled, 32 were fully evaluable for toxicity [median (range) age 12.5 yrs (4.8 - 21.6)]: 21 on part A1 and 11 on part A2. In part A1, 1/6 pts developed DLT (grade 3 mucositis) at 45 mg/m2 and 1/6 developed DLT (grade 4 mood alteration) at 80 mg/m2. At 100 mg/m2, 3/4 patients developed DLT (neutropenia –thrombocytopenia), thus exceeding the MTD. In part A2, 2/6 patients developed dose limiting myelosuppression at 80 mg/m2; one of whom also experienced grade 3 mucositis. 1/5 patient experienced DLT (grade 3 alkaline phosphatase) at 60 mg/m2. Of note, 5/11 patients experienced hand-foot-skin syndrome (grade 1 - 3) on the bid schedule versus 1/21 (grade 2) on qd schedule. A high degree of inter- patient variability in systemic exposure was observed. Average trough concentrations exceeded 1 μ M, the efficacious concentration in preclinical studies. The mean (±SD) steady state MLN8237 trough concentrations at the 45, 60, 80 and 100 mg/m2/d dose levels were 5.1 ± 5.7, 4.9 ± 6.3, 3.7 ± 3.8, and 5.5 ± 4.9 μ M, respectively. Conclusions: Although the divided dose regimen appears to be well tolerated in adults, children experienced more myelosuppression and hand-foot-skin syndrome with divided versus once daily dosing. Therefore the recommended pediatric phase 2 dose and schedule of MLN8237 is 80 mg/m2/d administered once daily for 7 days. No significant financial relationships to disclose.

Abstract 5032: MK8745, an Aurora A kinase-specific inhibitor induces apoptosis in a p53-dependent manner

Abstract Aurora kinases are mitotic serine/threonine protein kinases and are attractive novel targets for anticancer therapy. Many small molecule inhibitors are being developed and few are currently undergoing clinical trials. Aurora A kinase is essential for the successful mitotic transition. MK8745 is a novel and selective small molecule inhibitor of Aurora A kinase. Treatment of tumor cells with MK8745 (1 to 5 µM) resulted in accumulation of cells in mitosis with an increase in Aurora A expression. Despite this increase in Aurora A in the cells, dose dependent inhibition of Aurora A kinase activity was confirmed by an in vitro kinase assay using a PLK1, a specific substrate of Aurora A kinase. Also, treatment of HCT116 (p53−/−) cells ectopically overexpressing wild type p53 with MK8745 showed a decrease in phospho p53 (ser315), another known substrate of Aurora A. We then explored the mechanism by which MK8745 induces proliferation inhibition in cell lines of multiple lineages such as colon, melanoma, sarcoma and pancreatic according to p53 status. In general, the cell lines expressing wild type p53 showed a short delay in mitosis followed by cytokinesis with significant apoptosis (15%) upon exposure to MK8745. However, cell lines with null or mutant p53 underwent endoreduplication resulting in 60% polyploidy after a prolonged delay in mitosis with no apoptosis. This was further confirmed in the isogenic HCT-116 cell lines such that HCT p53−/− cells underwent polyploidy, whereas HCT p53 +/+ and the HCT p21 −/− cells underwent apoptosis. Under all of these conditions phospho histone H3 (Ser10) was not inhibited, indicating these phenotypic effects are a result of Aurora A inhibition and not Aurora B. P53 dependence for apoptosis was also confirmed in p53 null cells overexpressing wild type p53. Transient knock down of Aurora A kinase by specific SiRNA resulted in high levels of apoptosis and again showed p53 dependence. These results suggest that determining the p53 status may be important when using Aurora A kinase targeted therapies in the clinic. Further studies are underway to determine the role of p53 in the induction of apoptosis that results from inhibition of Aurora A kinase. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5032.

MLN8054, an Inhibitor of Aurora A Kinase, Induces Senescence in Human Tumor Cells Both In vitro and In vivo

Aurora A kinase is a serine/threonine protein kinase responsible for regulating several mitotic processes including centrosome separation, spindle assembly, and chromosome segregation. Small molecule inhibitors of Aurora A kinase are being pursued as novel anticancer agents, some of which have entered clinical trials. Despite the progress in developing these agents, terminal outcomes associated with Aurora A inhibition are not fully understood. Although evidence exists that Aurora A inhibition leads to apoptosis, other therapeutically relevant cell fates have not been reported. Here, we used the small molecule inhibitor MLN8054 to show that inhibition of Aurora A induces tumor cell senescence both in vitro and in vivo. Treatment of human tumor cells grown in culture with MLN8054 showed a number of morphologic and biochemical changes associated with senescence. These include increased staining of senescence-associated beta-galactosidase, increased nuclear and cell body size, vacuolated cellular morphology, upregulation/stabilization of p53, p21, and hypophosphorylated pRb. To determine if Aurora A inhibition induces senescence in vivo, HCT-116 xenograft-bearing animals were dosed orally with MLN8054 for 3 weeks. In the MLN8054-treated animals, increased senescence-associated beta-galactosidase activity was detected in tissue sections starting on day 15. In addition, DNA and tubulin staining of tumor tissue showed a significant increase in nuclear and cell body area, consistent with a senescent phenotype. Taken together, this data shows that senescence is a terminal outcome of Aurora A inhibition and supports the evaluation of senescence biomarkers in clinic samples.

Aurora A is a negative prognostic factor and a new therapeutic target in human neuroblastoma

We studied expression of the Aurora A gene and its clinical significance in a cohort of neuroblastoma patients. In addition, we investigated the antitumor activity of MLN8054, a novel small-molecule inhibitor of Aurora A kinase, on cultured NB cell lines in vitro. Aurora A mRNA expression was assessed by quantitative real-time PCR in tumor tissue specimens from 67 patients at diagnosis and in 9 human neuroblastoma cell lines. Western blot assays for Aurora A protein were done on tumor tissue of 53 patients. The results were correlated with various prognostic factors of neuroblastoma. Aurora A mRNA and protein expression were identified in 9 of 9 neuroblastoma cell lines. Overexpression of Aurora A mRNA in neuroblastoma tumor tissue is associated with high risk (P = 0.019), high-stage (International Neuroblastoma Staging System III and IV) tumors (P = 0.007), unfavorable histology (P = 0.007), MYCN amplification (P = 0.017), disease relapse (P = 0.019), and decreased progression-free survival (P < 0.0001) but not correlated with the age at diagnosis (P = 0.877). Similarly, Aurora A protein expression also significantly correlated with high risk (P = 0.011), high stage (P = 0.0028), unfavorable histology (P = 0.0006), MYCN amplification (P = 0.0029), and disease relapse (P = 0.044). Small interfering RNA-mediated knockdown of the endogenous Aurora A gene causes a proliferation defect and enhances chemosensitivity in human neuroblastoma cell lines. In support of these observations, the Aurora A kinase inhibitor, MLN8054, markedly inhibited growth of cultured neuroblastoma cell lines through an apoptosis-dependent pathway. Overexpression of Aurora A is associated with disease progression in neuroblastoma. Inhibition of this kinase is a promising modality for neuroblastoma treatment.

Effect of Aurora A kinase inhibitor MLN8237 combined with rituximab on antitumor activity in preclinical B-cell non-Hodgkin's lymphoma models

8553 Background: Aurora A kinase is a serine/threonine protein kinase that is essential for the successful transit of cells through mitosis. MLN8237 is a selective small molecule inhibitor of Aurora A kinase that has demonstrated anti-tumor activity in animal models of solid human tumors. In this study we explored the anti-tumor effect of MLN8237 in vivo in pre-clinical models of human Diffuse Large B-cell Lymphoma (DLBCL) both as a single agent and in combination with the anti-CD20 monoclonal antibody Rituximab. Methods: Three human DLBCL models were examined in SCID mice. In two of the models (Ly19 &amp; WSU) the tumor cells expressed a constitutively active luciferase, enabling tumor burden analysis in either a subcutaneous or disseminated setting. The third model was a primary DLBCL recently obtained from a patient. Tumor bearing animals were treated for 21 days with MLN8237 (QD PO dosing at 3–20mg/kg), Rituximab (Q7D IV dosing at 10 mg/kg), or the two agents combined. Tumor burden was measured in the disseminated models as a function of luciferase-induced photon flux, and in the subcutaneous models using vernier calipers. Results: MLN8237 induced anti-tumor activity that was dose-dependent in all three models. In LY19 disseminated model, 3 mg/kg of MLN8237 combined with Rituximab induced synergistic anti-tumor activity (n=2); while 10 mg/kg MLN8237 (dosed QD) combined with Rituximab was additive. Importantly, combining MLN8237 with Rituximab led to complete cures in 100% of the animals. In the WSU model, combining MLN8237(3–10mg/kg) with Rituximab resulted in additive tumor growth inhibition. The mean survival endpoint was significantly longer (p=0.003 and &lt;0.001 respectively) in the combination group when compared to the individual group. In the primary lymphoma model, MLN8237(10–20mg/kg) caused a significant anti-tumor effect during treatment period (TGI = 83–95%). Combining MLN8237 and Rituximab in primary model resulted in additive anti-tumor effect. Conclusions: MLN8237 combined with Rituximab was found to reduce tumor burden in an additive and/or synergistic mechanism in multiple DLBCL tumor models. MLN8237 is currently being tested as a single agent in a phase I clinical trail in patients with DLBCL. [Table: see text]