Therapeutic Potential of ALK1 Activating Drugs in HHT Models

PROJECT SUMMARY/ABSTRACT (DESCRIPTION) Hereditary hemorrhagic telangiectasia (HHT) is a highly debilitating and potentially life-threatening systemic vascular dysplasia characterized by arteriovenous malformation (AVM) development in several tissues. HHT is caused by loss-of-function mutations in ALK1, ENG, and Smad4, three signal transducers of the TGF-β family ligands, BMP9 and BMP10. ALK1, an endothelial BMP type I receptor, interacts with a BMP type II receptor and the co-receptor ENG to activate Smad1/5/8, mobilize Smad4, and control specific gene expression programs involved in vascular maintenance. In collaboration with Diagonal Therapeutics (DGL), we obtained a clustering bispecific antibody (BsAb) that binds the extracellular domains of ALK1 and the BMP type II receptor, BMPRII. We found that DGL's BsAb (DGL-BsAb) increased ALK1 and BMPRII levels at the cell surface and produced a sustained Smad1/5/8 activating response in endothelial cells (ECs) in vitro. Strikingly, DGL-BsAb prevented and reversed retinal AVMs in mice treated with BMP9/10 blocking antibodies (BMP9/10ib mice), a model of HHT. In addition, DGL-BsAb activated Smad1/5/8 signaling in blood outgrowth ECs isolated from HHT patients carrying ALK1 mutations. The overall goal of this application is to determine whether ALK1-BMPRII receptor activation rescues Smad1/5/8 signaling and treats the different HHT pathologies in pertinent mouse models, as well as to study the mechanisms of ALK1-BMPRII receptor activation upon binding to DGL-BsAb. In Aim 1, we will assess the anti-AVM properties of ALK1-BMPRII receptor activation in the retinal vasculature of BMP9/10ib and ENG inducible EC-specific knockout (iECKO) mice. The endothelial and mural cell defects, hypervascularization, vessel dilation, and arteriovenous specification disruption accompanying AVM pathology in these two HHT mouse models will also be investigated. Aim 2 will determine the efficacy of ALK1-BMPRII receptor activation in treating systemic vascular defects in newborn and adult HHT mouse models. We will assess liver, lung, and brain vascular defects, bleeding, and anemia following DGL-BsAb treatment in newborn BMP9/10ib and ENG iECKO mice and adult HHT mutant ALK1 knock-in mice. Transcriptomic analyses will be performed in liver ECs to identify the anti-AVM gene expression signatures of DGL-BsAb treatment in HHT mice. Aim 3 will identify the mechanisms and downstream signaling pathways of ALK1-BMPRII receptor activation by DGL-BsAb. We will first confirm the proper target engagement by DGL-BsAb by determining whether Alk1 and Bmpr2 knockdown precludes DGL-BsAb-mediated Smad1/5/8 signaling activation in ECs and AVM blockade in HHT mice. We will then analyze whether DGL-BsAb controls ALK1-BMPRII interaction, stabilization, and endocytosis. Lastly, we will use RNA-seq in HUVECs to compare the gene expression signatures of DGL-BsAb and BMP9 treatments to identify the specific signaling pathways associated with ALK1-BMPRII activation.