The radiosensitizing effect of the aurora kinase inhibitors, ENMD-2076, on canine mast cell tumours in vitro.
ABSTRACT: ENMD-2076 is an aurora kinase inhibitor that also has multi-target tyrosine kinase inhibitor properties. In this study, the mRNA and the protein expression of aurora-A and aurora-B were evaluated in three canine mast cell tumour cell lines. Dose-dependent cytotoxicity was seen in the cells treated, and it affected the cell cycle with cells in the G2/M phase being selectively killed. The cells were also evaluated for radiosensitivity with/without ENMD-2076, and radiosensitization was seen after 3 Gy and 6 Gy exposures with ENMD-2076 for 48 h. Protein expression of caspase-3 was gradually increased, and the expression intensity was highest at 24 h post irradiation in cells without ENMD-2076 treatment, which indicates that radiation exposure with ENMD-2076-induced cell death faster than radiation treatment alone. Our study results suggest the potential usefulness of treating canine mast cell tumours with aurora kinase inhibitors alone or in conjunction with radiation therapy.
Project description:ENMD-2076, an aurora-A kinase inhibitor with anti-angiogenic properties, has shown activity in solid and hematologic malignancies. We investigated oral ENMD-2076 in an open-label, single-arm phase II study using 275?mg daily on a 28-day cycle in patients with advanced soft-tissue sarcomas (STS) receiving ?1 line of prior therapy. Primary endpoint was 6-month progression-free survival (PFS) with ?15% indicating no interest, and ?40% indicating further interest in ENMD-2076. Secondary/exploratory endpoints included clinical benefit (CBR ?6-months) and objective response (ORR) rates, PFS, OS, safety, and whole-exome sequencing (WES) for potentially associated biomarkers. Overall, 23/25 (92%) patients receiving ENMD-2076 were efficacy evaluable with median follow-up of 14 months (range 2.2-39.5). Common subtypes were leiomyosarcoma (n?=?10), undifferentiated pleomorphic sarcoma (n?=?3), angiosarcoma (n?=?3), and alveolar soft-part sarcoma (n?=?3). The 6-month PFS was 20.8% (95% CI:3.2-38.4) with a CBR of 17% (95% CI:1.55-33.23) and ORR of 9% (95% CI:3.08-20.46). Median PFS was 2.5 months (95% CI:2.20-4.47) and OS was 14.1 months (95% CI:6.07-20.07). The most common high-grade treatment-related adverse event was hypertension (60%). WES identified PTPRB mutations in 3/4 patients (p?=?0.018) benefiting from ENMD-2076. Although this study failed to meet its primary endpoint, occasional responses and prolonged stable disease was noted. ENMD-2076 evaluation in PTPRB mutated tumors and/or angiosarcoma is warranted.
Project description:The Aurora kinases are a family of conserved serine-threonine kinases with key roles in mitotic cell division. As with other promising anticancer targets, patient selection strategies to identify a responsive subtype will likely be required for successful clinical development of Aurora kinase inhibitors. The purpose of this study was to evaluate the antitumor activity of the Aurora and angiogenic kinase inhibitor ENMD-2076 against preclinical models of breast cancer with identification of candidate predictive biomarkers.Twenty-nine breast cancer cell lines were exposed to ENMD-2076 and the effects on proliferation, apoptosis, and cell-cycle distribution were evaluated. In vitro activity was confirmed in MDA-MB-468 and MDA-MB-231 triple-negative breast cancer xenografts. Systematic gene expression analysis was used to identify up- and downregulated pathways in the sensitive and resistant cell lines, including within the triple-negative breast cancer subset.ENMD-2076 showed antiproliferative activity against breast cancer cell lines, with more robust activity against cell lines lacking estrogen receptor expression and those without increased HER2 expression. Within the triple-negative breast cancer subset, cell lines with a p53 mutation and increased p53 expression were more sensitive to the cytotoxic and proapoptotic effects of ENMD-2076 exposure than cell lines with decreased p53 expression.ENMD-2076 exhibited robust anticancer activity against models of triple-negative breast cancer and the candidate predictive biomarkers identified in this study may be useful in selecting patients for Aurora kinase inhibitors in the future.
Project description:BACKGROUND:Glioblastoma is the most common type of malignant brain tumor. Bioinformatics technology and structure biology were effectively and systematically used to identify specific targets in malignant tumors and screen potential drugs. RESULTS:GBM patients have higher AURKA and KDR mRNA expression compared with normal samples. Then, we identified a small molecular compound, ENMD-2076, could effectively inhibit Aurora kinase A and VEGFR-2 (encoded by KDR) activities. ENMD-2076 is predicted without toxic properties and also has absorption and gratifying brain/blood barrier penetration ability. Further results demonstrated that ENMD-2076 could significantly inhibit GBM cell lines proliferation and vitality, it also suppressed GBM cells migration and invasion. ENMD-2076 induced glioblastoma cell cycle arrest in G2-M phase and apoptosis by inhibiting PI3K/AKT/mTOR signaling pathways. Additionally, ENMD-2076 prolonged the median survival time of tumor-bearing rats and restrained growth rate of tumor volume in vivo. CONCLUSIONS:Our findings reveal that ENMD-2076 is a promising drug in dealing with glioblastoma and have a perspective application. METHODS:We show that AURKA and KDR genes are hub driver genes in glioblastoma with bioinformatics technology including WGCNA analysis, PPI network, GO, KEGG analysis and GSEA analysis. After identifying a compound via virtual screening analysis, further experiments were carried out to examine the anti-glioblastoma activities of the compound in vivo and in vitro.
Project description:ENMD-2076 is a unique orally bioavailable Aurora kinase and VEGFR inhibitor. The purpose of this phase 1 study of ENMD-2076 was to determine the MTD, pharmacokinetic, and pharmacodynamic profiles and preliminary antitumor activity.Patients with refractory advanced solid malignancies were treated with ENMD-2076 orally with continuous once daily dosing. Doses from 60 to 200 mg/m(2) were evaluated using a standard 3 (to 4) + 3 design. Pharmacokinetic parameters were studied on days 1, 28, and 30 to 35 of cycle 1. Expanded MTD cohorts included patients with ovarian cancer, colorectal cancer, and refractory solid tumors.A total of 67 patients (46 F, 21M; ages 30-76) entered the study. Dose levels of 60, 80, 120, 200, and 160 mg/m(2) were evaluated. Two patients experienced grade 3 hypertension at 200 mg/m(2), and additional grade 3 neutropenia events limited tolerability at this dose. An intermediate dose of 160 mg/m(2) was determined to be the MTD. The most common drug-related adverse events included hypertension, nausea/vomiting, and fatigue. The pharmacokinetics of ENMD-2076 were characterized by a rapid absorption phase (T(max) 3-7.8 hours), a t(1/2) of 27.3 to 38.3 hours after a single dose, and dose proportional exposure. Decreased plasma sVEGFR2 was observed posttreatment. Two patients with platinum refractory/resistant ovarian cancer had RECIST partial responses.ENMD-2076 was well tolerated, had a linear pharmacokinetic profile, and showed promising antitumor activity, particularly in ovarian cancer. The recommended phase 2 dose of ENMD-2076 is 160 mg/m(2) administered orally once daily with continuous dosing.
Project description:BACKGROUND:Triple-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. METHODS:This 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. RESULTS:Forty-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. CONCLUSIONS:Single-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 REGISTRATION:ClinicalTrials.gov, NCT01639248 . Registered on July 12, 2012.
Project description:PURPOSE:Triple-negative breast cancer (TNBC) is a subtype associated with poor prognosis and for which there are limited therapeutic options. The purpose of this study was to evaluate the efficacy of ENMD-2076 in p53-mutated TNBC patient-derived xenograft (PDX) models and describe patterns of terminal cell fate in models demonstrating sensitivity, intrinsic resistance, and acquired resistance to ENMD-2076. EXPERIMENTAL DESIGN:p53-mutated, TNBC PDX models were treated with ENMD-2076 and evaluated for mechanisms of sensitivity or resistance to treatment. Correlative tissue testing was performed on tumor tissue to assess for markers of proliferation, apoptosis, senescence, and pathways of resistance after treatment and at the time of acquired resistance. RESULTS:Sensitivity to ENMD-2076 200?mg/kg daily was associated with induction of apoptosis while models exhibiting intrinsic or acquired resistance to treatment presented with a senescent phenotype. Response to ENMD-2076 was accompanied by an increase in p53 and p73 levels, even within the background of mutant p53. Treatment with ENMD-2076 resulted in a decrease in pAurA and an increase in pHH3. We observed a TNBC subtype switch from the luminal androgen receptor to the basal-like subtype at acquired resistance. CONCLUSION:ENMD-2076 has antitumor activity in preclinical models of p53-mutated TNBC. Increased levels of p53 and p73 correlated with sensitivity whereas senescence was associated with resistance to ENMD-2076. The novel finding of a TNBC subtype switch at time of acquired resistance may provide mechanistic insights into the biologic effects of selective pressure of anticancer treatments on TNBC. ENMD-2076 is currently being evaluated in a Phase 2 clinical trial in patients with metastatic, previously treated TNBC where these biologic correlates can be further explored.
Project description:Cancer is a disease caused by several factors characterized by uncontrolled cell division, growth, and survival. ENMD-2076, is a novel orally active small molecule multikinase inhibitor targeting angiogenesis, proliferation, and the cell cycle. It is selectively active against the mitotic kinases aurora A and B, and kinases responsible for angiogenesis including VEGFR2/KDR and FGFR1 and 2. ENMD-2076 has been shown to inhibit tumor growth and prevent angiogenesis in vitro and in vivo in preclinical cancer models. Moreover, in a phase I trial, ENMD-2076 was well tolerated, exhibited a linear pharmacokinetic profile, and showed a promising antitumor activity in a number of solid tumors. In this study, we show that ENMD-2076 has antiproliferative effects, causes cell cycle arrest, and has activity in preclinical models of colorectal cancer (CRC), including patient-derived xenograft (PDX) models. Forty-seven human CRC cell lines were exposed in vitro to ENMD-2076 and analyzed for effects on cell cycle, apoptosis, and downstream effector proteins. The drug was then tested against 20 human CRC PDX models to further evaluate in-vivo antitumor activity. We show that ENMD-2076 exhibits a broad range of activity against a large panel of CRC cell lines with varying molecular characteristics. Mechanistically, ENMD-2076 exposure resulted in a G2/M cell cycle arrest, an increase in aneuploidy, and cell death in responsive cell lines. In addition, ENMD-2076 treatment resulted in a promising antitumor activity in CRC PDX models. These results support the continued development of ENMD-2076 in CRC including further exploration of rational combinations.
Project description:BACKGROUND: Hepatocellular carcinoma (HCC) represents a highly vascularized tumor entity and the process of angiogenesis is essential for the growth of HCC. Importantly, the pro-angiogenic transcription factors HIF-1alpha and STAT3 have been implicated in HCC progression, thus representing interesting targets for molecular targeted therapy. We hypothesized that therapeutic inhibition of HIF-1alpha could be achieved by using a novel tubulin-binding agent (ENMD-1198). ENMD-1198 is an analog of 2-methoxyestradiol (2ME2) with antiproliferative and antiangiogenic activity. METHODS: The human HCC cell lines HUH-7 and HepG2 were used for experiments. Effects of ENMD-1198 on constitutive and inducible (hypoxia, growth factors) activation of signaling cascades, including HIF-1alpha and STAT3, were investigated by Western blotting. Changes in VEGF expression were determined by real-time PCR. Effects of ENMD-1198 on cancer cell migration and invasion were evaluated in in vitro-assays. The growth-inhibitory effects of ENMD-1198 (200 mg/kg/day) were determined in a subcutaneous tumor model (HUH-7). RESULTS: ENMD-1198 inhibited the phosphorylation of MAPK/Erk, PI-3K/Akt and FAK. Moreover, activation of HIF-1alpha and STAT3 was dramatically reduced by ENMD-1198, which resulted in lower VEGF mRNA expression (P < 0.05). In addition, tumor cell migratory and invasive properties were significantly inhibited (P < 0.05, for both). In vivo, treatment with ENMD-1198 led to a significant reduction in tumor growth, tumor vascularization, and numbers of proliferating tumor cells (P < 0.05 for all). CONCLUSION: The novel microtubule destabilizing agent ENMD-1198 is suitable for inhibiting HIF-1alpha and STAT3 in human HCC cells and leads to reduced tumor growth and vascularization in vivo. Hence, inhibition of HIF-1alpha and STAT3 could prove valuable for therapy of hepatocellular carcinoma.
Project description:BACKGROUND:The WW domain-containing oxidoreductase (WWOX) tumor suppressor gene is frequently lost in a variety of solid and hematopoietic malignancies in humans. Dysregulation of WWOX has been implicated as playing a key role in tumor cell survival, DNA damage repair, and genomic stability. The purpose of this study was to characterize WWOX expression in spontaneous canine mast cell tumors (MCTs) and malignant cell lines and investigate the potential contribution of WWOX loss on malignant mast cell behavior. METHODS/RESULTS:WWOX expression is decreased in primary canine MCTs and malignant mast cell lines compared to normal canine bone marrow-cultured mast cells. In transformed canine mastocytoma cell lines, overexpression of WWOX or WWOX knockdown had no effect on mast cell viability. Inhibition of WWOX enhanced clonogenic survival following treatment with ionizing radiation in the C2 mast cell line. Lastly, immunohistochemistry for WWOX was performed using a canine MCT tissue microarray, demonstrating that WWOX staining intensity and percent of cells staining for WWOX is decreased in high-grade MCTs compared to low-grade MCTs. CONCLUSIONS:These data suggest that WWOX expression is attenuated or lost in primary canine MCTs and malignant mast cell lines. Given the observed increase in clonogenic survival in WWOX-deficient C2 mast cells treated with ionizing radiation, further investigation of WWOX and its role in mediating the DNA damage response in malignant mast cells is warranted.
Project description:Clinical studies using definitive-intent stereotactic radiation therapy (SRT) for the local treatment of canine osteosarcoma (OSA) have shown canine patients achieving similar median survival times as the current standard of care (amputation and adjuvant chemotherapy). Despite this, there remains an unacceptable high risk of pathologic fracture following radiation treatment. Zoledronic acid (ZA) and parathyroid hormone (PTH) are therapeutic candidates for decreasing this fracture risk post-irradiation. Due to differing mechanisms, we hypothesized that the combined treatment with ZA and PTH would significantly improve bone healing more than ZA or PTH treatment alone. Using an orthotopic model of canine osteosarcoma in athymic rats, we evaluated bone healing following clinically-relevant doses of radiation therapy (12 Gy x 3 fractions, 36 Gy total). Groups included 36 Gy SRT only, 36 Gy SRT plus ZA, 36 Gy SRT plus ZA and PTH, 36 Gy SRT plus PTH, and 36 Gy SRT plus localized PTH treatment. Our study showed significant increases in bone volume and increased polar moments of inertia (in the distal femoral metaphysis) 8 weeks after radiation in the combined (ZA/PTH) treatment group as compared to radiation treatment alone. Histomorphometric analysis revealed evidence of active mineralization at the study endpoint as well as successful tumor-cell kill across all treatment groups. This work provides further evidence for the expanding potential indications for ZA and PTH therapy, including post-irradiated bone disease due to osteosarcoma.