Specificity and structure of a high affinity activin receptor-like kinase 1 (ALK1) signaling complex.

Activin receptor-like kinase 1 (ALK1), an endothelial cell-specific type I receptor of the TGF-ß superfamily, is an important regulator of normal blood vessel development as well as pathological tumor angiogenesis. As such, ALK1 is an important therapeutic target. Thus, several ALK1-directed agents are currently in clinical trials as anti-angiogenic cancer therapeutics. Given the biological and clinical importance of the ALK1 signaling pathway, we sought to elucidate the biophysical and structural basis underlying ALK1 signaling. The TGF-ß family ligands BMP9 and BMP10 as well as the three type II TGF-ß family receptors ActRIIA, ActRIIB, and BMPRII have been implicated in ALK1 signaling. Here, we provide a kinetic and thermodynamic analysis of BMP9 and BMP10 interactions with ALK1 and type II receptors. Our data show that BMP9 displays a significant discrimination in type II receptor binding, whereas BMP10 does not. We also report the crystal structure of a fully assembled ternary complex of BMP9 with the extracellular domains of ALK1 and ActRIIB. The structure reveals that the high specificity of ALK1 for BMP9/10 is determined by a novel orientation of ALK1 with respect to BMP9, which leads to a unique set of receptor-ligand interactions. In addition, the structure explains how BMP9 discriminates between low and high affinity type II receptors. Taken together, our findings provide structural and mechanistic insights into ALK1 signaling that could serve as a basis for novel anti-angiogenic therapies.

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 Leu(79) 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.

A novel therapeutic approach to treating obesity through modulation of TGFß signaling.

Obesity results from disproportionately high energy intake relative to energy expenditure. Many therapeutic strategies have focused on the intake side of the equation, including pharmaceutical targeting of appetite and digestion. An alternative approach is to increase energy expenditure through physical activity or adaptive thermogenesis. A pharmacological way to increase muscle mass and hence exercise capacity is through inhibition of the activin receptor type IIB (ActRIIB). Muscle mass and strength is regulated, at least in part, by growth factors that signal via ActRIIB. Administration of a soluble ActRIIB protein comprised of a form of the extracellular domain of ActRIIB fused to a human Fc (ActRIIB-Fc) results in a substantial muscle mass increase in normal mice. However, ActRIIB is also present on and mediates the action of growth factors in adipose tissue, although the function of this system is poorly understood. In the current study, we report the effect of ActRIIB-Fc to suppress diet-induced obesity and linked metabolic dysfunctions in mice fed a high-fat diet. ActRIIB-Fc induced a brown fat-like thermogenic gene program in epididymal white fat, as shown by robustly increased expression of the thermogenic genes uncoupling protein 1 and peroxisomal proliferator-activated receptor-γ coactivator 1α. Finally, we identified multiple ligands capable of reducing thermogenesis that represent likely target ligands for the ActRIIB-Fc effects on the white fat depots. These data demonstrate that novel therapeutic ActRIIB-Fc improves obesity and obesity-linked metabolic disease by both increasing skeletal muscle mass and by inducing a gene program of thermogenesis in the white adipose tissues.

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-beta 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-beta-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.

Treatment with a soluble receptor for activin improves bone mass and structure in the axial and appendicular skeleton of female cynomolgus macaques (Macaca fascicularis).

A recent study suggests that activin inhibits bone matrix mineralization, whereas treatment of mice with a soluble form of the activin type IIA receptor markedly increases bone mass and strength. To further extend these observations, we determined the skeletal effects of inhibiting activin signaling through the ActRIIA receptor in a large animal model with a hormonal profile and bone metabolism similar to humans. Ten female cynomolgus monkeys (Macaca fascicularis) were divided into two weight-matched groups and treated biweekly, for 3 months, with either a subcutaneous injection 10 mg/kg of a soluble form of the ActRIIA receptor fused with the Fc portion of human IgG(1) (ACE-011) or vehicle (VEH). Bone mineral density (BMD), micro-architecture, compressive mechanical properties, and ash fraction were assessed at the end of the treatment period. BMD was significantly higher in ACE-011 treated individuals compared to VEH: +13% (p=0.003) in the 5th lumbar vertebral body and +15% (p=0.05) in the distal femur. In addition, trabecular volumetric bone density at the distal femur was 72% (p=0.0004) higher than the VEH-treated group. Monkeys treated with ACE-011 also had a significantly higher L5 vertebral body trabecular bone volume (p=0.002) and compressive mechanical properties. Ash fraction of L4 trabecular bone cores did not differ between groups. These results demonstrate that treatment with a soluble form of ActRIIA (ACE-011) enhances bone mass and bone strength in cynomolgus monkeys, and provide strong rationale for exploring the use of ACE-011 to prevent and/or treat skeletal fragility.

A soluble activin receptor type IIb prevents the effects of androgen deprivation on body composition and bone health.

Androgen deprivation, a consequence of hypogonadism, certain cancer treatments, or normal aging in men, leads to loss of muscle mass, increased adiposity, and osteoporosis. In the present study, using a soluble chimeric form of activin receptor type IIB (ActRIIB) we sought to offset the adverse effects of androgen deprivation on muscle, adipose tissue, and bone. Castrated (ORX) or sham-operated (SHAM) mice received either TBS [vehicle-treated (VEH)] or systemic administration of ActRIIB-mFc, a soluble fusion protein comprised of a form of the extracellular domain of ActRIIB fused to a murine IgG2aFc subunit. In vivo body composition imaging demonstrated that ActRIIB-mFc treatment results in increased lean tissue mass of 23% in SHAM mice [19.02 +/- 0.42 g (VEH) versus 23.43 +/- 0.35 g (ActRIIB-mFc), P < 0.00001] and 26% in ORX mice [15.59 +/- 0.26 g (VEH) versus 19.78 +/- 0.26 g (ActRIIB-mFc), P < 0.00001]. Treatment also caused a decrease in adiposity of 30% in SHAM mice [5.03 +/- 0.48 g (VEH) versus 3.53 +/- 0.19 g (ActRIIB-mFc), NS] and 36% in ORX mice [7.12 +/- 0.53 g (VEH) versus 4.57 +/- 0.28 g (ActRIIB-mFc), P < 0.001]. These changes were also accompanied by altered serum levels of leptin, adiponectin, and insulin, as well as by prevention of steatosis (fatty liver) in ActRIIB-mFc-treated ORX mice. Finally, ActRIIB-mFc prevented loss of bone mass in ORX mice as assessed by whole body dual x-ray absorptiometry and micro-computed tomography of proximal tibias. The data demonstrate that treatment with ActRIIB-mFc restored muscle mass, adiposity, and bone quality to normal levels in a mouse model of androgen deprivation, thereby alleviating multiple adverse consequences of such therapy.