Tammy L. Bush

Preclinical Evaluation of AMG 900, a Novel Potent and Highly Selective Pan-Aurora Kinase Inhibitor with Activity in Taxane-Resistant Tumor Cell Lines

In mammalian cells, the aurora kinases (aurora-A, -B, and -C) play essential roles in regulating cell division. The expression of aurora-A and -B is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis, making them attractive targets for anticancer therapy. AMG 900 is an orally bioavailable, potent, and highly selective pan-aurora kinase inhibitor that is active in taxane-resistant tumor cell lines. In tumor cells, AMG 900 inhibited autophosphorylation of aurora-A and -B as well as phosphorylation of histone H3 on Ser(10), a proximal substrate of aurora-B. The predominant cellular response of tumor cells to AMG 900 treatment was aborted cell division without a prolonged mitotic arrest, which ultimately resulted in cell death. AMG 900 inhibited the proliferation of 26 tumor cell lines, including cell lines resistant to the antimitotic drug paclitaxel and to other aurora kinase inhibitors (AZD1152, MK-0457, and PHA-739358), at low nanomolar concentrations. Furthermore, AMG 900 was active in an AZD1152-resistant HCT116 variant cell line that harbors an aurora-B mutation (W221L). Oral administration of AMG 900 blocked the phosphorylation of histone H3 in a dose-dependent manner and significantly inhibited the growth of HCT116 tumor xenografts. Importantly, AMG 900 was broadly active in multiple xenograft models, including 3 multidrug-resistant xenograft models, representing 5 tumor types. AMG 900 has entered clinical evaluation in adult patients with advanced cancers and has the potential to treat tumors refractory to anticancer drugs such as the taxanes.

Discovery and Evaluation of Dual CDK1 and CDK2 Inhibitors

In eukaryotic cells, cyclin-dependent kinase (CDK) complexes regulate the temporal progression of cells through the cell cycle. Deregulation in the cell cycle is an essential component in the evolution of cancer. Here, we validate CDK1 and CDK2 as potential therapeutic targets using novel selective small-molecule inhibitors of cyclin B1/CDK1 and cyclin E2/CDK2 enzyme complexes (CDKi). Flow cytometry-based methods were developed to assess intracellular retinoblastoma (Rb) phosphorylation to show inhibition of the CDK pathway. Tumor cells treated with CDK inhibitors showed an overall decrease in cell proliferation, accumulation of cells in G1 and G2, and apoptosis in a cell line-specific manner. Although CDK inhibitors activate p53, the inhibitors were equipotent in arresting the cell cycle in isogenic breast and colon tumor cells lacking p53, suggesting the response is independent of p53. In vivo, the CDK inhibitors prevented the growth of colon and prostate tumors, blocked proliferation of tumor cells, and inhibited Rb phosphorylation. The discovery and evaluation of novel potent and selective CDK1 and CDK2 inhibitors will help delineate the role that CDK complexes play in regulating tumorigenesis.

Broad Antitumor Activity in Breast Cancer Xenografts by Motesanib, a Highly Selective, Oral Inhibitor of Vascular Endothelial Growth Factor, Platelet-Derived Growth Factor, and Kit Receptors

Purpose: Angiogenesis plays a critical role in breast cancer development and progression. Vascular endothelial growth factor (VEGF) is a potent angiogenic factor that regulates endothelial cell proliferation and survival. We investigated the effects of motesanib, a novel, oral inhibitor of VEGF receptors 1, 2, and 3; platelet-derived growth factor receptor; and Kit receptor, on the growth of xenografts representing various human breast cancer subtypes. Experimental Design: Athymic nude mice were implanted with MCF-7 (luminal) or MDA-MB-231 (mesenchymal) tumor fragments or Cal-51 (mixed/progenitor) tumor cells. Once tumors were established, animals were randomized to receive increasing doses of motesanib alone or motesanib plus cytotoxic chemotherapy (docetaxel, doxorubicin, or tamoxifen). Results: Across all three xenograft models, motesanib treatment resulted in significant dose-dependent reductions in tumor growth, compared with vehicle-treated controls, and in marked reductions in viable tumor fraction and blood vessel density. No significant effect on body weight was observed with compound treatment compared with control-treated animals. Motesanib did not affect the proliferation of tumor cells in vitro. There was a significantly greater reduction in xenograft tumor growth when motesanib was combined with docetaxel (MDA-MB-231 tumors) or with the estrogen receptor modulator tamoxifen (MCF-7 tumors), compared with either treatment alone, but not when combined with doxorubicin (Cal-51 tumors). Conclusions: Treatment with motesanib alone or in combination with chemotherapy inhibits tumor growth in vivo in various models of human breast cancer. These data suggest that motesanib may have broad utility in the treatment of human breast cancer.

Discovery of a potent, selective, and orally bioavailable pyridinyl-pyrimidine phthalazine aurora kinase inhibitor.

The discovery of aurora kinases as essential regulators of cell division has led to intense interest in identifying small molecule aurora kinase inhibitors for the potential treatment of cancer. A high-throughput screening effort identified pyridinyl-pyrimidine 6a as a moderately potent dual inhibitor of aurora kinases -A and -B. Optimization of this hit resulted in an anthranilamide lead (6j) that possessed improved enzyme and cellular activity and exhibited a high level of kinase selectivity. However, this anthranilamide and subsequent analogues suffered from a lack of oral bioavailability. Converting the internally hydrogen-bonded six-membered pseudo-ring of the anthranilamide to a phthalazine (8a-b) led to a dramatic improvement in oral bioavailability (38-61%F) while maintaining the potency and selectivity characteristics of the anthranilamide series. In a COLO 205 tumor pharmacodynamic assay measuring phosphorylation of the aurora-B substrate histone H3 at serine 10 (p-histone H3), oral administration of 8b at 50 mg/kg demonstrated significant reduction in tumor p-histone H3 for at least 6 h.

AMG 900, a small molecule inhibitor of aurora kinases, potentiates the activity of microtubule-targeting agents in human metastatic breast cancer models

Breast cancer is the most prevalent malignancy affecting women and ranks second in cancer-related deaths, in which death occurs primarily from metastatic disease. Triple-negative breast cancer (TNBC) is a more aggressive and metastatic subtype of breast cancer that is initially responsive to treatment of microtubule-targeting agents (MTA) such as taxanes. Recently, we reported the characterization of AMG 900, an orally bioavailable, potent, and highly selective pan-Aurora kinase inhibitor that is active in multidrug-resistant cell lines. In this report, we investigate the activity of AMG 900 alone and in combination with two distinct classes of MTAs (taxanes and epothilones) in multidrug-resistant TNBC cell lines and xenografts. In TNBC cells, AMG 900 inhibited phosphorylation of histone H3 on Ser10, a proximal substrate of Aurora-B, and induced polyploidy and apoptosis. Furthermore, AMG 900 potentiated the antiproliferative effects of paclitaxel and ixabepilone at low nanomolar concentrations. In mice, AMG 900 significantly inhibited the growth of MDA-MB-231 (F11; parental), MDA-MB-231 (F11) PTX-r (paclitaxel-resistant variant), and DU4475 xenografts. The combination of AMG 900 with docetaxel enhanced tumor inhibition in MDA-MB-231 (F11) xenografts compared with either monotherapy. Notably, combining AMG 900 with ixabepilone resulted in regressions of MDA-MB-231 (F11) PTX-r xenografts, in which more than 50% of the tumors failed to regrow 75 days after the cessation of drug treatment. These findings suggest that AMG 900, alone and in combination with MTAs, may be an effective intervention strategy for the treatment of metastatic breast cancer and provide potential therapeutic options for patients with multidrug-resistant tumors. Mol Cancer Ther; 12(11); 2356–66. ©2013 AACR.

Discovery of triazine-benzimidazoles as selective inhibitors of mTOR.

mTOR is part of the PI3K/AKT pathway and is a central regulator of cell growth and survival. Since many cancers display mutations linked to the mTOR signaling pathway, mTOR has emerged as an important target for oncology therapy. Herein, we report the discovery of triazine benzimidazole inhibitors that inhibit mTOR kinase activity with up to 200-fold selectivity over the structurally homologous kinase PI3Kα. When tested in a panel of cancer cell lines displaying various mutations, a selective inhibitor from this series inhibited cellular proliferation with a mean IC(50) of 0.41 μM. Lead compound 42 demonstrated up to 83% inhibition of mTOR substrate phosphorylation in a murine pharmacodynamic model.

Discovery and optimization of potent and selective imidazopyridine and imidazopyridazine mTOR inhibitors

mTOR is a critical regulator of cellular signaling downstream of multiple growth factors. The mTOR/PI3K/AKT pathway is frequently mutated in human cancers and is thus an important oncology target. Herein we report the evolution of our program to discover ATP-competitive mTOR inhibitors that demonstrate improved pharmacokinetic properties and selectivity compared to our previous leads. Through targeted SAR and structure-guided design, new imidazopyridine and imidazopyridazine scaffolds were identified that demonstrated superior inhibition of mTOR in cellular assays, selectivity over the closely related PIKK family and improved in vivo clearance over our previously reported benzimidazole series.

Activity of panitumumab alone or with chemotherapy in non-small cell lung carcinoma cell lines expressing mutant epidermal growth factor receptor

Epidermal growth factor receptor (EGFR) kinase domain mutations cause hyperresponsiveness to ligand and hypersensitivity to small-molecule tyrosine kinase inhibitors. However, little is known about how these mutations respond to antibodies against EGFR. We investigated the activity of panitumumab, a fully human anti-EGFR monoclonal antibody, in vitro in mutant EGFR-expressing non-small cell lung carcinoma (NSCLC) cells and in vivo with chemotherapy in xenograft models. Mutant EGFR-expressing NSCLC cells (NCI-H1975 [L858R+T790M] and NCI-H1650 [Δ746-750]) and CHO cells were treated with panitumumab before EGF stimulation to assess the inhibition of EGFR autophosphorylation. Established tumors were treated with panitumumab (25, 100, or 500 μg/mouse twice a week) alone or with docetaxel (10 or 20 mg/kg once a week) or cisplatin (7.5 mg/kg once a week). Antitumor activity and levels of proliferation markers were analyzed. Treatment of mutant EGFR-expressing CHO and NSCLC cells with panitumumab inhibited ligand-dependent autophosphorylation. In NCI-H1975 and NCI-H1650 xenografts, treatment with panitumumab alone or with cisplatin inhibited tumor growth compared with control (P < 0.0003). With panitumumab plus docetaxel, enhanced antitumor activity was seen in both xenografts versus panitumumab alone. Panitumumab treatment alone decreased Ki-67 and phospho- mitogen-activated protein kinase (pMAPK) staining in both xenografts compared with control. Docetaxel enhanced panitumumab activity in NCI-H1650 xenografts (decreased Ki-67 and pMAPK staining by >60%) when compared with either agent alone. Panitumumab inhibits ligand-induced EGFR phosphorylation, tumor growth, and markers of proliferation alone or with docetaxel in NSCLC cell lines that express clinically observed EGFR kinase domain mutations, including the small-molecule tyrosine kinase inhibitor-resistant T790M mutation. [Mol Cancer Ther 2009;8(6):1536–46]

Discovery of N-(4-(3-(2-Aminopyrimidin-4-yl)pyridin-2-yloxy)phenyl)-4-(4-methylthiophen-2-yl)phthalazin-1-amine (AMG 900), A Highly Selective, Orally Bioavailable Inhibitor of Aurora Kinases with Activity against Multidrug-Resistant Cancer Cell Lines

Efforts to improve upon the physical properties and metabolic stability of Aurora kinase inhibitor 14a revealed that potency against multidrug-resistant cell lines was compromised by increased polarity. Despite its high in vitro metabolic intrinsic clearance, 23r (AMG 900) showed acceptable pharmacokinetic properties and robust pharmacodynamic activity. Projecting from in vitro data to in vivo target coverage was not practical due to disjunctions between enzyme and cell data, complex and apparently contradictory indicators of binding kinetics, and unmeasurable free fraction in plasma. In contrast, it was straightforward to relate pharmacokinetics to pharmacodynamics and efficacy by following the time above a threshold concentration. On the basis of its oral route of administration, a selectivity profile that favors Aurora-driven pharmacology and its activity against multidrug-resistant cell lines, 23r was identified as a potential best-in-class Aurora kinase inhibitor. In phase 1 dose expansion studies with G-CSF support, 23r has shown promising single agent activity.

Motesanib inhibits Kit mutations associated with gastrointestinal stromal tumors

BackgroundActivating mutations in Kit receptor tyrosine kinase or the related platelet-derived growth factor receptor (PDGFR) play an important role in the pathogenesis of gastrointestinal stromal tumors (GIST).MethodsThis study investigated the activity of motesanib, an inhibitor of vascular endothelial growth factor receptors (VEGFR) 1, 2, and 3; PDGFR; and Kit, against primary activating Kit mutants and mutants associated with secondary resistance to imatinib. Single- and double-mutant isoforms of Kit were evaluated for their sensitivity to motesanib or imatinib in autophosphorylation assays and in Ba/F3 cell proliferation assays.ResultsMotesanib inhibited Kit autophosphorylation in CHO cell lines expressing primary activating mutations in exon 9 (AYins503-504, IC50 = 18 nM) and exon 11 (V560 D, IC50 = 5 nM; Δ552-559, IC50 = 1 nM). Motesanib also demonstrated activity against kinase domain mutations conferring imatinib resistance (V560D/V654A, IC50 = 77 nM; V560D/T670I, IC50 = 277 nM; Y823 D, IC50 = 64 nM) but failed to inhibit the imatinib-resistant D816V mutant (IC50 > 3000 nM). Motesanib suppressed the proliferation of Ba/F3 cells expressing Kit mutants with IC50 values in good agreement with those observed in the autophosphorylation assays.ConclusionsIn conclusion, our data suggest that motesanib possesses inhibitory activity against primary Kit mutations and some imatinib-resistant secondary mutations.