Marc Payton

Differential subcellular localization, expression and biological toxicity of BRCA1 and the splice variant BRCA1-Δ11b

The mechanism of BRCA1 tumor suppression in human breast and ovarian cells is the focus of intense investigation. In this report, full length BRCA1 (230 kDa) introduced into cells with CMV promoter constructs was nuclear when transgene expression was low whereas high expression resulted in cytoplasmic accumulation, aberrant nuclear and cell morphology. A nuclear localization signal (NLS) was mapped to BRCA1 amino acid positions 262 – 570. We describe a splice variant, BRCA1-Δ11b, missing the majority of exon 11 including the NLS. Exogenous BRCA1-Δ11b (110 kDa) was cytoplasmic and, unlike the full-length protein, overexpression of the protein encoded by the variant did not appear to be toxic. RNA probe titrations and RT – PCR demonstrated that BRCA1 and Δ11b transcripts are coexpressed in a wide variety of cells and tissues. Interestingly, BRCA1-Δ11b message was greatly reduced or absent in several breast and ovarian tumor lines relative to exon 11 transcripts and a Δ9,10 splice variant. Taken together our results suggest that full-length BRCA1 and BRCA1-Δ11b may have distinct roles in cell growth regulation and tumorigenesis.

Cyclin E2, a novel G1 cyclin that binds Cdk2 and is aberrantly expressed in human cancers.

ABSTRACT A novel cyclin gene was discovered by searching an expressed sequence tag database with a cyclin box profile. The human cyclin E2 gene encodes a 404-amino-acid protein that is most closely related to cyclin E. Cyclin E2 associates with Cdk2 in a functional kinase complex that is inhibited by both p27Kip1 and p21Cip1. The catalytic activity associated with cyclin E2 complexes is cell cycle regulated and peaks at the G1/S transition. Overexpression of cyclin E2 in mammalian cells accelerates G1, demonstrating that cyclin E2 may be rate limiting for G1 progression. Unlike cyclin E1, which is expressed in most proliferating normal and tumor cells, cyclin E2 levels were low to undetectable in nontransformed cells and increased significantly in tumor-derived cells. The discovery of a novel second cyclin E family member suggests that multiple unique cyclin E-CDK complexes regulate cell cycle progression.

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.

Cyclin E2, the cycle continues

The eukaryotic cell cycle is regulated by a family of serine/threonine protein kinases known as cyclin-dependent kinases (CDKs). The activation of a CDK is dependent on its association with a cyclin regulatory subunit. The formation of distinct cyclin-CDK complexes controls the progression through the first gap phase (G(1)) and initiation of DNA synthesis (S phase). These complexes are in turn regulated by protein phosphorylation and cyclin-dependent kinase inhibitors (CKIs). Cyclin E2 has emerged as the second member of the E-type cyclin family. Cyclin E2-associated kinase activity is regulated in a cell cycle dependent manner with peak activity at the G(1) to S transition. Ectopic expression of cyclin E2 in human cells accelerates G(1), suggesting that cyclin E2 is rate limiting for G(1) progression. Although the pattern and level of cyclin E2 expression in some primary tumor and normal tissue RNAs are distinct from cyclin E1, both E-type cyclins appear to have inherent functional redundancies. This functional redundancy has facilitated the rapid characterization of cyclin E2 and uncovered unique features associated with each E-type cyclin.

Deregulation of cyclin E2 expression and associated kinase activity in primary breast tumors.

The increased expression of G1 cyclins has been associated with the many types of human tumors. In primary solid tumors however, the expression and activity of cyclin E2, the newest member of the G1 cyclin family, is largely unknown. In this study we have analysed the expression of the E-type cyclins in primary solid tumors from breast, lung, uterus, ovary, colon, and rectal tissues. Relative gene expression was analysed by quantitative real-time reverse transcription polymerase chain reaction (Taqman). The levels of cyclin E1 and cyclin E2 were significantly elevated (23 vs 38%, respectively) in primary breast tumor samples relative to normal breast tissue controls. We also observed an inverse correlation between the expression of cyclin E1/E2 and estrogen receptor in breast tumors. Our results demonstrate that the expression and associated catalytic activity for both cyclin E1 and cyclin E2 is elevated in primary breast tumors when compared to normal breast tissue. The increased level of cyclin E2 in breast tumors suggests that, similar to cyclin E1, it may contribute to the pathogenesis of 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 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.

AMG 900, a potent inhibitor of aurora kinases causes pharmacodynamic changes in p-Histone H3 immunoreactivity in human tumor xenografts and proliferating mouse tissues

BackgroundThe Aurora family of serine-threonine kinases are essential regulators of cell division in mammalian cells. Aurora-A and -B expression and kinase activity is elevated in a variety of human cancers and is associated with high proliferation rates and poor prognosis. AMG 900 is a highly potent and selective pan-aurora kinase inhibitor that has entered clinical evaluation in adult patients with advanced cancers. In mice, oral administration of AMG 900 blocks the phosphorylation of histone H3 on serine-10 (p-Histone H3), a proximal substrate of aurora-B and inhibits the growth of multiple human tumor xenografts, including multidrug-resistant models.MethodsIn order to establish a preclinical pharmacokinetic-pharmacodynamic (PK-PD) relationship for AMG 900 that could be translated to the clinic, we used flow cytometry and laser scanning cytometry detection platforms to assess the effects on p-Histone H3 inhibition in terms of sensitivity, precision, and specificity, in human tumor xenografts in conjunction with mouse skin and bone marrow tissues. Mice with established COLO 205 tumors were administered AMG 900 at 3.75, 7.5, and 15 mg/kg and assessed after 3 hours.ResultsSignificant suppression of p-Histone H3 in mouse skin was only observed at 15 mg/kg (p <0.0001), whereas in mouse bone marrow and in tumor a dose-dependent inhibition was achieved at all three doses (p ≤0.00015). These studies demonstrate that AMG 900 inhibits p-Histone H3 in tumors and surrogate tissues (although tissues such as skin may be less sensitive for assessing PD effects). To further extend our work, we evaluated the feasibility of measuring p-Histone H3 using fine-needle aspirate (FNA) tumor xenograft biopsies. Treatment with AMG 900 significantly inhibited p-Histone H3 (>99% inhibition, p <0.0001) in COLO 205 tumors. Lastly, we illustrate this LSC-based approach can detect p-Histone H3 positive cells using mock FNAs from primary human breast tumor tissues.ConclusionPhosphorylation of histone H3 is a useful biomarker to determine the pharmacodynamics (PD) activity of AMG 900. FNA biopsies may be a viable approach for assessing AMG 900 PD effects in the clinic.