Roselyne Castonguay

Soluble Endoglin Specifically Binds Bone Morphogenetic Proteins 9 and 10 via Its Orphan Domain, Inhibits Blood Vessel Formation, and Suppresses Tumor Growth

Endoglin (CD105), a transmembrane protein of the transforming growth factor β superfamily, plays a crucial role in angiogenesis. Mutations in endoglin result in the vascular defect known as hereditary hemorrhagic telangiectasia (HHT1). The soluble form of endoglin was suggested to contribute to the pathogenesis of preeclampsia. To obtain further insight into its function, we cloned, expressed, purified, and characterized the extracellular domain (ECD) of mouse and human endoglin fused to an immunoglobulin Fc domain. We found that mouse and human endoglin ECD-Fc bound directly, specifically, and with high affinity to bone morphogenetic proteins 9 and 10 (BMP9 and BMP10) in surface plasmon resonance (Biacore) and cell-based assays. We performed a function mapping analysis of the different domains of endoglin by examining their contributions to the selectivity and biological activity of the protein. The BMP9/BMP10 binding site was localized to the orphan domain of human endoglin composed of the amino acid sequence 26–359. We established that endoglin and type II receptors bind to overlapping sites on BMP9. In the in vivo chick chorioallantoic membrane assay, the mouse and the truncated human endoglin ECD-Fc both significantly reduced VEGF-induced vessel formation. Finally, murine endoglin ECD-Fc acted as an anti-angiogenic factor that decreased blood vessel sprouting in VEGF/FGF-induced angiogenesis in in vivo angioreactors and reduced the tumor burden in the colon-26 mouse tumor model. Together our findings indicate an important role of soluble endoglin ECD in the regulation of angiogenesis and highlight efficacy of endoglin-Fc as a potential anti-angiogenesis therapeutic agent.

ALK1-Fc Inhibits Multiple Mediators of Angiogenesis and Suppresses Tumor Growth

Activin receptor–like kinase-1 (ALK1) is a type I, endothelial cell–specific member of the transforming growth factor-β superfamily of receptors known to play an essential role in modulating angiogenesis and vessel maintenance. In the present study, we sought to examine the angiogenic and tumorigenic effects mediated upon the inhibition of ALK1 signaling using a soluble chimeric protein (ALK1-Fc). Of 29 transforming growth factor-β–related ligands screened by surface plasmon resonance, only bone morphogenetic protein (BMP9) and BMP10 displayed high-affinity binding to ALK1-Fc. In cell-based assays, ALK1-Fc inhibited BMP9-mediated Id-1 expression in human umbilical vein endothelial cells and inhibited cord formation by these cells on a Matrigel substrate. In a chick chorioallantoic membrane assay, ALK1-Fc reduced vascular endothelial growth factor–, fibroblast growth factor–, and BMP10-mediated vessel formation. The growth of B16 melanoma explants was also inhibited significantly by ALK1-Fc in this assay. Finally, ALK1-Fc treatment reduced tumor burden in mice receiving orthotopic grafts of MCF7 mammary adenocarcinoma cells. These data show the efficacy of chimeric ALK1-Fc proteins in mitigating vessel formation and support the view that ALK1-Fc is a powerful antiangiogenic agent capable of blocking vascularization. Mol Cancer Ther; 9(2); 379–

Characterization of the ligand binding functionality of the extracellular domain of activin receptor type IIB

The single transmembrane domain serine/threonine kinase activin receptor type IIB (ActRIIB) has been proposed to bind key regulators of skeletal muscle mass development, including the ligands GDF-8 (myostatin) and GDF-11 (BMP-11). Here we provide a detailed kinetic characterization of ActRIIB binding to several low and high affinity ligands using a soluble activin receptor type IIB-Fc chimera (ActRIIB.Fc). We show that both GDF-8 and GDF-11 bind the extracellular domain of ActRIIB with affinities comparable with those of activin A, a known high affinity ActRIIB ligand, whereas BMP-2 and BMP-7 affinities for ActRIIB are at least 100-fold lower. Using site-directed mutagenesis, we demonstrate that ActRIIB binds GDF-11 and activin A in different ways such as, for example, substitutions in ActRIIB Leu79 effectively abolish ActRIIB binding to activin A yet not to GDF-11. Native ActRIIB has four isoforms that differ in the length of the C-terminal portion of their extracellular domains. We demonstrate that the C terminus of the ActRIIB extracellular domain is crucial for maintaining biological activity of the ActRIIB.Fc receptor chimera. In addition, we show that glycosylation of ActRIIB is not required for binding to activin A or GDF-11. Together, our findings reveal binding specificity and activity determinants of the ActRIIB receptor that combine to effect specificity in the activation of distinct signaling pathways.

Abstract B5: ACE‐041, a soluble activin receptor‐like kinase‐1 fusion protein, is a novel antiangiogenic agent

The therapeutic targeting of angiogenesis has been associated with clinical benefit for a number of tumors including colorectal carcinoma, non‐small cell lung cancer, breast cancer, renal cell carcinoma and glioblastoma. Activin receptor‐like kinase‐1, an endothelium specific member of the TGFβ‐type I receptor family, is believed to play a role in modulating angiogenesis. Homozygous inactivation of Alk1 in mice leads to embryonic lethality resulting from severe vascular abnormalities, while heterozygous mice exhibit chronic hemorrhaging and arteriovenous malformation similar to hereditary hemorrhagic telangiectasia‐2 (HHT‐2) patients harboring mutations in ALK1. To examine the potential of ALK1 inhibition as a novel anti‐angiogenic therapy for cancer, we developed a soluble form of the ALK1 receptor (ACE‐041) by fusing the extracellular region of human ALK1 to the Fc of human IgG1. ACE‐041 was shown to bind only BMP‐9 and BMP‐10 out of 29 ligands tested in TGFβ family. ALK1 inhibition blocks SMAD 1/5/8 signaling and blocks in vitro cord formation of endothelial cells. ACE‐041 inhibited in vivo blood vessel formation using Matrigel plug assay and chick chorioallantoic assay (CAM). Moreover, ACE‐041 decreased tumor volume in a modified tumor CAM assay and in an orthotopic breast cancer model induced by MDA‐MD‐231 and MCF‐7 mammary adenocarcinoma cells. These data suggest that ALK1 inhibition may be a promising therapeutic strategy for patients with breast and other solid tumors and support advancement of ACE‐041 into clinical trials. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B5.