A PI3K-RAC1 signaling axis downstream of mutant TIE2 promotes endothelial dysfunction in venous malformation

Abstract

Background: Activating mutations in the endothelial receptor tyrosine kinase TIE2 are a cause of venous malformations (VMs), primarily through dysregulated PI3K/AKT signaling. However, the downstream mechanisms linking constitutively active mutant TIE2 to enlarged vascular lumens, endothelial dysfunction and VM remain incompletely understood. Here, we investigated the role of RAC1 signaling in mutant TIE2 p.L914F driven VM. Methods: Human umbilical vein endothelial cells (HUVECs) expressing wild-type TIE2 or constitutively active mutant TIE2-L914F were analyzed using RAC1 GTP pull-down assays, immunoblotting, and endothelial cell (EC) 2D and 3D functional assays. The role of RAC1 was evaluated using the RAC1 inhibitor NSC23766 in vitro and in vivo xenograft model of VM. Results & Conclusions: ECs expressing mutant TIE2-L914F showed increased active GTP-bound RAC1. TIE2-L914F ECs exhibited cytoskeletal changes with enhanced lamellipodia formation and maturation, accompanied by increased migration. Pharmacological inhibition of RAC1 significantly attenuated the aberrant vascular lumen area and migration in the mutant TIE2 ECs. Mechanistically, we showed that RAC1 hyperactivation is PI3K-dependent but not MEK/ERK-dependent. Consistent with these findings, RAC1 inhibition significantly reduced vascular lesion formation in a xenograft model of VM with TIE2-L914F ECs. Our study identifies RAC1 as a downstream mediator of TIE2-L914F driven endothelial dysfunction. These results establish a previously unrecognized PI3K-RAC1 signaling axis in the pathogenesis of TIE2-mutated VMs.