Somatic MAP2K1 Mutations in Arteriovenous Malformation Constitutively Activate the RAS/MAPK Pathway
Jeremy A. Goss, MD, Patrick J. Smits, PhD, Mohammed H. Alomari, MD, Arin K. Greene, MD, MMSc.
Boston Children's Hospital, Boston, MA, USA.
PurposeArteriovenous malformation (AVM) is a locally destructive congenital vascular anomaly that enlarges over time. We previously reported that AVMs contain somatic mutations in the MAP2K1 gene, and that the mutation is isolated to the endothelial cell (EC). The purpose of this study was to determine the effects of MAP2K1 mutations in ECs on its signaling pathway.
Methods Human AVM-ECs were collected during a clinically-indicated procedure. MAP2K1 mutations (K57N) were confirmed in the ECs using droplet digital PCR (ddPCR). We also engineered wild-type endothelial colony forming cells (ECFCs) with the MAP2K1 (K57N) mutation. Patient-derived MAP2K1-AVM ECs and engineered MAP2K1-ECFCs were compared to control ECs: human white adipose (HWAT-ECs), ECFCs + empty vector, and ECFCs + wild-type MAP2K1 vector. Western blot analysis was used to assess cell signaling along the RAS/MAPK pathway (e.g., baseline and phosphorylated MEK1 and ERK1/2). Densitometry quantification was performed with Image Studio™ Lite (Version 5.2).
Human MAP2K1-AVM ECs had consistent baseline MEK1 and ERK1/2 expression with controls; however, MAP2K1-AVM ECs produced 176% more active/phosphorylated ERK1/2 than non-mutant ECs. Similarly, ECFCs engineered to overexpress mutant MAP2K1 demonstrated 433% more phosphorylated ERK1/2 expression than control ECFCs.
MAP2K1 mutations in both human-derived AVM ECs as well as in engineered ECs activate the RAS/MAPK signaling pathway by increasing phosphorylation of ERK1/2, which is the downstream target of MAP2K1. Pharmacotherapy that inhibits the activating function of the mutation may inhibit the growth of AVMs.
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