Targeting Neuropilin‐2 in Lymphatic Malformation

Abstract

Lymphatic malformations (LM) are sporadic, non‐familial congenital lesions consisting of dilated, abnormal lymphatic vessels that can cause severe lymphedema, disfigurement, organ dysfunction, and in some cases morbidity. These vascular anomalies grow slowly but progress over time and recur after surgery; no effective drugs or preventions currently exist for LM. Genome sequencing identified that approximately 80% of LM patients have somatic activating mutations in the PIK3CA gene. Lymphatic endothelial cells (LEC) expressing mutant PIK3CAH1047Rinduce both macrocystic and microcystic LM in mouse models. Our goal is to understand the cellular and molecular processes that regulate LM growth in order to develop novel therapeutic strategies for patients with LM. Neuropilin‐2 (NRP2) is an essential co‐receptor for VEGFC/VEGFR3 signaling through the PI3K/AKT pathway which is crucial for LEC viability and motility, yet NRP2 also mediates inhibitory signals from its Semaphorin‐3F (SEMA3F) ligand to attenuate activation of AKT and induce cytostasis. Our results using immunohistochemistry to detect NRP2 protein in paraffin‐embedded human samples indicate that NRP2 is highly upregulated in LM lesions compared to normal lymphatic vessels. LM‐LEC primary cell lines, derived separately from several patients, show increased NRP2 levels and hyper‐activated AKT compared to primary LEC derived from healthy human dermis. Additionally, LEC isolated from the skin of transgenic mice (prox1‐creERT2;Nrp2 f/f) specifically lacking Nrp2 in lymphatic vessels following tamoxifen induction (LEC‐Nrp2‐KO) show suppressed proliferation and migration following VEGF‐C stimulation compared to normal LEC isolated from wildtype mice. Importantly, SEMA3F exogenous protein, in a dose‐dependent manner, inhibits the proliferation of normal LEC and LM‐LEC and attenuates the phosphorylation of AKT. Taken together, our study suggests that NRP2 may be an important target for therapy in LM patients. Future studies using SEMA3F protein or specific antibodies targeting the VEGF‐binding domain of NRP2 will be tested in preclinical trials.