No abstract

Aurora kinase A and B have essential and non-overlapping roles in mitosis, with elevated expression in a subset of human cancers, including acute myeloid leukemia (AML). In this study, pan-aurora kinase inhibitor (AKI) AMG 900 distinguishes itself as an anti-leukemic agent that is more uniformly potent against a panel of AML cell lines than are isoform-selective AKIs and classic AML drugs. AMG 900 inhibited AML cell growth by inducing polyploidization and/or apoptosis. AMG 900 and aurora-B selective inhibitor AZD1152-hQPA showed comparable cellular effects on AML lines that do not harbor a FLT3-ITD mutation. AMG 900 was active against P-glycoprotein-expressing AML cells resistant to AZD1152-hQPA and was effective at inducing expression of megakaryocyte-lineage markers (CD41, CD42) on human CHRF-288–11 cells and mouse Jak2V617F cells. In MOLM-13 cells, inhibition of p-histone H3 by AMG 900 was associated with polyploidy, extra centrosomes, accumulation of p53 protein, apoptosis, and cleavage of Bcl-2 protein. Co-administration of cytarabine (Ara-C) with AMG 900 potentiated cell killing in a subset of AML lines, with evidence of attenuated polyploidization. AMG 900 inhibited the proliferation of primary human bone marrow cells in culture, with a better proliferation recovery profile relative to classic anti-mitotic drug docetaxel. In vivo, AMG 900 significantly reduced tumor burden in a systemic MOLM-13 xenograft model where we demonstrate the utility of 3’-deoxy-3’−18F-fluorothymidine [18F]FLT positron emission tomographic (PET)–computed tomographic (CT) imaging to measure the anti-proliferative effects of AMG 900 in skeletal tissues in mice.

Sustained angiogenesis is essential for tumor growth as it provides the tumor with a network of blood vessels that supply both oxygen and essential nutrients. Limiting tumor-associated angiogenesis is a proven strategy for the treatment of human cancer. To date, the rapid detection and quantitation of tumor-associated endothelial cell (TAEC) proliferation has been challenging, largely due to the low frequency of endothelial cells (ECs) within the tumor microenvironment. In this report, we address this problem using a new multiparametric flow cytometry method capable of rapid and precise quantitation of proliferation by measuring bromodeoxyuridine (BrdUrd) uptake in mouse TAECs from established human tumor xenografts. We determined the basal proliferation labeling index of TAECs in two human tumor xenografts representing two distinct histologies, COLO 205 (colorectal cancer) and U-87 (glioblastoma). We then investigated the effects of two large-molecule antiangiogenic agents targeting different biochemical pathways. Blocking angiopoietinTie2 signaling with the peptide-Fc fusion protein, trebananib (AMG 386), inhibited proliferation of TAECs, whereas blocking Dll4-Notch signaling with an anti-Dll4-specific antibody induced hyperproliferation of TAECs. These pharmacodynamic studies highlight the sensitivity and utility of this flow cytometry-based method and demonstrate the value of this assay to rapidly assess the in vivo proliferative effects of angiogenesis-targeted agents on both the tumor and the associated vasculature.

BACKGROUND: Aurora kinases (AK) A and B play essential roles in multiple stages of mitosis and are frequently overexpressed in a subset of human cancers, including ovarian cancer (OC). AMG 900, a potent and highly selective small molecule inhibitor of AKs, showed promising single-agent activity in heavily pretreated patients with advanced OC in a Phase 1b clinical trial. In this study, we report the preclinical effects of AMG 900 in a panel of well-characterized human cancer cell lines representing clinically-relevant OC subtypes. METHODS: The anti-proliferative effects of AMG 900 were evaluated using a 5-day cell count assay. Cell lines were classified as sensitive to AMG 900 when lethality was > 15% at 10 nM. Molecular markers were profiled including TP53 mutation status, AURKA, CCNE1, MYC copy number, and p53, p21 and cyclin E1 protein by reverse phase protein array. Flow cytometry and imaging methods were used to evaluate the mechanism of action of AMG 900 alone and in combination with chemotherapy. The combination of AMG 900 plus docetaxel was evaluated in an IGROV-1 ovarian endometrioid carcinoma xenograft model. RESULTS: One third of the cell lines (11 of 35) were classified as sensitive to AMG 900 and showed enrichment for TP53 mutations and serous OC subtype. However, 10 of 24 resistant cell lines harbored TP53 mutations, indicating that TP53 mutational status alone was not sufficient for predicting AMG 900 sensitivity. Inhibition of AK activity by AMG 900 in OC cells resulted in aborted cell division leading to polyploidy and cell death (suggestive of aurora-B dominant phenotype). Re-plating of remnant cells after AMG 900 treatment showed an attenuation of cell regrowth, where TP53mut IGROV-1 cells showed minimal regrowth compared to TP53wt OVCAR-5 cells. AMG 900 inhibited proliferation at low nanomolar concentrations in the majority of OC cell lines and enhanced the effects of paclitaxel, carboplatin, and doxorubicin in IGROV-1 cells. In tumor-bearing mice, administration of AMG 900 at 7.5 mg/kg (PO) for two days per week or docetaxel at 10 mg/kg (IP) weekly for four cycles significantly inhibited the growth of IGROV-1 tumor xenografts (P < .0001 vs. vehicle alone). Notably, co-administration of AMG 900 with docetaxel enhanced efficacy and induced a delay in tumor regrowth compared to docetaxel alone. Single-agent-treated mice showed minimal body weight loss (BWL), whereas combination-treated mice showed moderate BWL (average < 10%) that was largely reversible (2 of 12 animals removed due to toxicity). CONCLUSIONS: AMG 900 alone or in combination with chemotherapy such as paclitaxel may be a promising clinical strategy to treat patients with ovarian cancer. Citation Format: Ondrej Kalous, Dylan Conklin, Kanthinh Manivong, William Wayne, Kelly Hanestad, Jude Canon, Robert Loberg, Gregory Friberg, Erick Gamelin, Florian D. Vogl, Gloria Juan, Angela Coxon, Dennis Slamon, Richard Finn, Marc Payton. Preclinical characterization of AMG 900, a pan-aurora kinase inhibitor, alone and in combination with taxanes in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3008.

Inhibition of angiogenesis is a proven cancer treatment strategy as an adequate blood supply is critical for tumor growth. Two of the key pathways regulating tumor angiogenesis are angiopoietin/Tie2 and Dll4/Notch. Inhibition of the angiopoietin/Tie2 pathway has been shown to reduce blood vessel density and inhibit tumor growth in mouse xenograft models. Inhibition of Dll4 also prevents tumor xenograft growth but does so by promoting a non-productive vascular network via excessive endothelial branching and sprouting. In this study, we examined the effects of combined inhibition of these pathways in models of angiogenesis and tumor growth. To inhibit the angiopoietin pathway we used AMG 386, a selective angiopoietin 1/2-neutralizing peptibody that prevents the interaction between these two angiopoietins and the Tie2 receptor. To inhibit Dll4 we used a neutralizing monoclonal antibody. The combination of AMG 386 and anti-Dll4 led to enhanced antitumor activity compared with either agent alone in four xenograft models (U-87 glioma, Calu-6 lung, Colo205 colorectal and MiaPaca pancreatic tumors). All treatments were well tolerated and no weight loss was observed. Histological analysis of U-87 tumors following one week of treatment revealed an increase in vessel area with anti-Dll4 alone and the combined treatment with AMG 386 showed a similar increase. At these same doses, AMG 386 reduced anti-Dll4-induced tumor associated endothelial cell proliferation following 24 hours of treatment. We also examined AMG 386/anti-Dll4 treatment in models of angiogenesis. In the rat cornea, individual anti-Dll4 and AMG 386 treatment had distinct morphological effects on VEGF-induced vessel formation. Vessel number and area were decreased following AMG 386 treatment, whereas vessel cross-branching and terminal tufting were observed following anti-Dll4 treatment. After combined treatment, vessels were truncated but the branching/tufting phenotype was preserved. Similar effects were seen in the mouse neonatal retina, where AMG 386 and anti-Dll4 treatment alone resulted in the expected distinct vascular phenotypes; AMG 386 treatment reduced vessel density and inhibited radial expansion, while anti-Dll4 treatment resulted in marked branching and thickening, and showed full radial expansion. In this model, combined treatment led to clear evidence of both mechanisms of action: increased branching and reduced radial expansion were observed in all retinas examined. Taken together, these data suggest that combined inhibition of the angiopoietin/Tie2 and Dll4/Notch pathways does not interfere with their individual mechanisms of action and furthermore, leads to enhanced efficacy in preclinical models. Therefore, the combination of these agents may have the potential to provide an expanded therapeutic opportunity to inhibit tumor angiogenesis beyond either individual agent alone. 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 LB-311. doi:1538-7445.AM2012-LB-311