Endoglin Requirement for BMP9 Signaling in Endothelial Cells Reveals New Mechanism of Action for Selective Anti-Endoglin Antibodies

Abstract

Endoglin (ENG), a co-receptor for several TGFβ-family cytokines, is expressed in dividing endothelial cells alongside ALK1, the ACVRL1 gene product. ENG and ACVRL1 are both required for angiogenesis and mutations in either gene are associated with Hereditary Hemorrhagic Telangectasia, a rare genetic vascular disorder. ENG and ALK1 function in the same genetic pathway but the relative contribution of TGFβ and BMP9 to SMAD1/5/8 activation and the requirement of ENG as a co-mediator of SMAD phosphorylation in endothelial cells remain debated. Here, we show that BMP9 and TGFβ1 induce distinct SMAD phosphorylation responses in primary human endothelial cells and that, unlike BMP9, TGFβ only induces SMAD1/5/8 phosphorylation in a subset of immortalized mouse endothelial cell lines, but not in primary human endothelial cells. We also demonstrate, using siRNA depletion of ENG and novel anti-ENG antibodies, that ENG is required for BMP9/pSMAD1 signaling in all human and mouse endothelial cells tested. Finally, anti-ENG antibodies that interfere with BMP9/pSMAD1 signaling, but not with TGFβ1/pSMAD3 signaling, also decrease in vitro HUVEC endothelial tube formation and inhibit BMP9 binding to recombinant ENG in vitro. Our data demonstrate that BMP9 signaling inhibition is a key and previously unreported mechanism of action of TRC105, an anti-angiogenic anti-Endoglin antibody currently evaluated in clinical trials.

Safety, Pharmacokinetics, Pharmacodynamics, and Antitumor Activity of Dalantercept, an Activin Receptor–like Kinase-1 Ligand Trap, in Patients with Advanced Cancer

“…Also, in clinical development as an antitumor agent is a chimeric antibody (TRACON) capable of neutralizing endoglin (CD105; ref. 31), a proangiogenic protein in the TGFb superfamily that binds BMP9/BMP10 (22,45,46), with loss-of-function mutations resulting in another type of vascular dysplasia, HHT-1 (15,47). In patients with advanced, refractory tumors (31), anti-endoglin antibody was associated with infusion reactions and immunogenicity distinct from effects described for dalantercept in the present study, whereas other adverse effects such as anemia and telangiectasias were observed in both studies.…”

The role of endoglin in post-ischemic revascularization

“…Thus, endoglin may play a role in BMP signaling during angiogenesis. In support of this view, endoglin is necessary for BMP9 signaling in ECs involved in angiogenesis [151], and the blockade of BMP9 binding to ALK1 prevents endoglin binding to ALK1 and the subsequent angiogenic response [149].…”

“…BMP9 signals through the endoglin/ALK1 complex, and anti-ALK1 antibodies prevent the formation of the BMP9/ALK1/endoglin complexes and attenuate BMP9-dependent signaling and sprouting [149]. Additionally, endoglin appears to be necessary for BMP9 signaling through ALK1 in HUVECs, in which TGF-β is unable to activate Smad1/5/8 signaling on its own [151]. Similarly, contradictory findings regarding the effect of BMPs in angiogenesis have been reported.…”

Bone Morphogenetic Protein Signaling in Pulmonary Arterial Hypertension

“…The predominant type of mutation, the existence of mutation hot spots within the gene, and the effects of the mutations on expression and signaling of the receptor are currently unknown. However, evidence suggests that depletion of endoglin impaired BMP9-induced SMAD1/SMAD5/SMAD8 phosphorylation in human pulmonary artery endothelial cells [150]. Recently we reported that a soluble form of endoglin that may function as an anti-angiogenic ligand trap of BMP9 is present at elevated levels in the circulation of individuals with Group 1 PAH, suggesting an additional mechanism by which abnormalities in this protein may attenuate BMP9 signaling [123].…”

“…Signaling mediated by BMP9, BMP10, BMPR2, ALK1, and the type III co-receptor endoglin is specialized in the vascular endothelium and in embryonic endocardium owing to high abundance of these components [137]. Evidence suggests that BMPR2, ALK1, and endoglin are required for endothelial cell signaling and/or function in response to BMP9 or BMP10 [33,34,150,205]. …”