GENETIC CAUSES AND THERAPEUTIC INTERVENTIONS IN VASCULAR MALFORMATIONS

Vascular malformations are congenital anomalies classified by the vessel type affected: venous (VM), arteriovenous (AVM), lymphatic (LM), and capillary (CM) malformations. Depending on the vascular malformation and the anatomic location, patients experience life-threatening and costly morbidities (sepsis, hemorrhage, intracranial bleeding, coagulopathy, cardiac failure) throughout their lives. Military personnel are at risk of complication from undiagnosed and untreated malformations, and a loss of productivity due to a need to care for beneficiaries. Despite severe morbidities and costs, there is no Food and Drug Administration (FDA)-approved treatment, and current clinical interventions have shown only partial response and recurrence is common. Development of effective, biologically targeted therapies for vascular malformations is sorely needed, but hampered by limited understanding of the genetics and biology that underlie vascular malformation pathologies.Germline mutations have been identified in some vascular malformation subtypes, yet screening for germline variants has often failed to identify causative mutations. Recent studies have shown that post-zygotic mutations contribute to a significant portion of AVMs, VMs, and LMs. Thus, the somatic mutations are present only in affected tissues, specifically the malformation endothelial cells (ECs). We have isolated and characterized malformation ECs from LMs and VMs that are hyperproliferative compared to control ECs and recapitulate the malformation phenotypes in xenograft models. Initial whole exome sequencing of these ECs identified somatic mutations in both reported and unreported genes. We found that malformation ECs differentially activate downstream signaling pathways and respond to the chemotherapeutic agent, sirolimus, which may explain why there was incomplete and varied response to this drug commonly used in the clinical setting.We hypothesize that pathogenic genetic mutations can be identified in isolated vascular malformation ECs and that this genetic information can be used to understand phenotypes and therapeutic responses of vascular malformations.Aim 1: Identification of pathogenic mutations in endothelial cells isolated from vascular malformations.We hypothesize that malformation ECs can be used to screen for germline and somatic mutations. Whole exome sequencing and variant analysis will be performed on affected ECs from LMs, VMs, and AVMs. We demonstrate that this approach can identify known and novel pathogenic variants in malformation ECs. Putative pathological variants will be characterized and prioritized in expression studies of banked murine and human fetal tissues and a large cohort of vascular malformation tissues. The goal is to develop a precision medicine approach that is effective at identifying and characterizing the genetic causes of vascular malformations. Aim 2: Determine the effects of pathological variants on therapeutic responses in vascular malformations.Responses of LM and VM patients to current therapies are variable and temporary. We hypothesize that genetic variants influence therapeutic responses in vascular malformations. We will use malformation ECs with known and novel pathological variants to understand differential treatment responses. We will test classes of drugs that suppress RAS/MAPK and PI3K/AKT signaling, as these are the most commonly activated pathways in malformation ECs. We will also perform a high-throughput screen of greater than 1600 FDA-approved drugs. Therapeutic effects on malformation EC proliferation migration, differentiation, and xenograft modeling will be determined. The goal is to identify biologically-targeted therapies to be translated to the clinical setting.Aim 3: Determine the effects of pathogenic gene variants on vascular malformation pathogenesis.How genetic variants influence vascular malformation phenotype is unclear. We hypothesize that the gene mutated and variant location within the gene influences vascular malformation pathogenesis. We will perform structure/functions studies, endothelial bioassays, embryoid body assays and develop novel transgenic mouse models to understand how specific small nucleotide variants in genes of the RAS/MAPK and PI3K/AKT pathways, as well as variants identified in our WES screen influence vascular development. The goal is to better understand the relationship between genotype and phenotype in vascular malformations.For the proposed studies, the Partnering Principal Investigators (PIs) have brought together their expertise in developmental and pathogenic vascular biology (Dr. Shawber) and extensive clinical experience and translational research in vascular malformations (Dr. Wu). They have recruited Dr. Gresham Richter (Co-I), Director of Vascular Anomalies Center at Arkansas Children’s Hospital for his scientific and clinical expertise in vascular malformations, AVM specifically, and to increase the acquisition of vascular malformation tissues. The goal is to build on the strengths of the PIs and Co-I to establish a multi-institutional research program to understand the pathobiology of vascular malformations.