The transcription factor SMAD4 is is the only shared partner of two signalling pathways (TGFß and BMP), and as such, it will play a key role in the balance of these two pathways. SMAD4 mutations have been involved in three cardiovascular diseases that we will call SMAD4pathies: 1) Hereditary haemorrhagic telangiectasia (HHT), a vascular disorder with epitaxies and arteriovenous malformations, 2) the Myhre syndrome (MS), a developmental disorder with cardiomyopathy, septal defects, and aortic coarctation and 3) Thoracic aortic aneurysm and dissection (TAAD) which is a life-threatening disease characterized by an enlargement of the aortic wall. These SMAD4 mutations can either be found all over the gene in HHT or in a specific localization in MS. Our work suggests that MS SMAD4 mutations lead to a gain of function (GOF) through monoubiquitination. Conversely, the SMAD4 mutation described in TAAD has been proposed to lead to a loss of function (LOF) due to polyubiquitination. In HHT, our working hypothesis would be that of a LOF in accordance to the ACVRL1 and ENG mutations found in HHT. Together, these data support that SMAD4 mutations are directly linked to cardiovascular diseases, however the role of SMAD4 in the development of these diseases is not known. The aim of this ambitious project is to decipher the role of SMAD4 in these three diseases. Throughout the project, we will use state-of-the-art approaches to answer the following questions: 1) Can we identify new SMAD4 mutations using genomic approaches in HHT and TAAD? (task 1-2), 2) How does SMAD4 mediate distinct TGFß/BMP signalling pathways? For this we will isolate/generate primary SMAD4 mutated cells from these 3 diseases (task3), identify their functional significance (task 4), define whether SMAD4 mutations are LOF or GOF in each disease (task 5), identify the specific cellular transcriptomic profile related to each disease (tasks 6-7), 3) How do SMAD4 mutations in vivo affect the cardiovascular system? for this, we will analyse the cardiac phenotype of the MS KI mouse model (CMVCre;Smad4I500/+) and challenge it with an aortic dissection model (tasks 8 and 9). This project is based on strong preliminary results: 1) We have identified in TAAD 3 new very rare variants of SMAD4. 2) We have identified 31 HHT cases with SMAD4 mutations among which, 9 unpublished SMAD4 mutations. 3) We have generated a CMVCre;Smad4+/ile500 KI mouse model for the MS. As expected this mutation leads to an increase in SMAD4 expression and our preliminary results support that this mouse model mimics the MS phenotype at the skeletal, ocular and heart levels. The deliverables of this project are: new SMAD4 mutations and new genes in a TAAD and HHT and analysis of the link between HHT and TAAD, generation of cells carrying SMAD4 mutations (vascular smooth muscle cells (VSMCs), endothelial cells (ECs) and CRISPR-Cas9 modified fibroblasts), functional characterization of SMAD4 mutations (GOF versus LOF), roles of the BMP versus TGFß signalling in SMAD4pathies and in particular in MS, new direct and indirect target genes of SMAD4 in cells carrying SMAD4 mutations (RNA- and ChIP-Seq analyses) and characterization of the KI mouse model for MS and its susceptibility to develop TAAD. This collaborative research project involves two teams with highly complementary and synergistic skills pooled to reach the project’s objectives. The team of C. Le Goff in Paris and of S. Bailly in Grenoble are internationally recognized for their genetic findings in TAAD and MS and in TGFß/BMP signalling in vascular remodelling in HHT, respectively. In conclusion, our ambition is to generate three distinct SMAD4 related gene signatures and to understand the impaired biological functions in a disease specific context. A better knowledge of these SMAD4pathies will offer novel perspectives in the management and treatment of these patients.

Heparan sulfate (HS) are complex polysaccharides abundantly found in extracellular matrices and cell surfaces. These polysaccharides participate to major cellular processes through their ability to bind and modulate a wide array of signalling proteins. HS/ligands interactions occur through saccharide domains (termed S-domains) of specific sulfation pattern, present within the polysaccharide. Assembly of such functional domains is orchestrated by a complex biosynthesis machinery and their structure is further regulated at the cell surface by post-synthetic modifying enzymes, including extracellular sulfatases of the Sulf family. Sulfs specifically target HS S-domains and catalyze the selective removal of 6-O-sulfate groups, which are required for the recognition of many proteins. Although structurally subtle, these modifications have great functional consequences, and Sulfs have emerged as critical regulators of HS activity, in physiological processes such as embryogenesis and tissue regeneration, and in diseases such as cancer. There are two identified isoforms of Sulfs, Sulf-1 and Sulf-2, which share a very similar molecular organization. They are composed of two regions that are essential for enzyme activity: the catalytic domain (CAT-D), which includes the enzyme active site and is well conserved amongst sulfatases, and a highly basic, hydrophilic domain (Hyd-D), which is responsible for recognition and binding to HS substrates and is a unique feature of the Sulfs. However, despite increasing interest, Sulfs still remain poorly understood. During our recent studies of these enzymes, we have shed light on an original processive desulfation mechanism and on remarkable structural features. Based on these data, the SULF@AS project proposes to deliver an integrated study of the human isoforms HSulf-1 and HSulf-2, combining biochemical and biophysical approaches to characterize their structure and post-translational modifications ; in vitro, in cellulo and in vivo functional analysis to determine their substrate specificities and respective role during tumour progression ; and the development of specific inhibitors based on HS mimetics. This project should provide major insights into the regulatory role played by these enzymes in many biological processes and deliver the structural basis for the development of therapeutic strategies targeting HSulfs.