Functional assessment of two variants of unknown significance in TEK by endothelium-specific expression in zebrafish embryos

Abstract

Vascular malformations are most often caused by somatic mutations of the PI3K/mTOR and the RAS signaling pathways, which can be identified in the affected tissue. Venous malformations commonly harbor PIK3CA and TEK mutations, whereas arteriovenous malformations are usually caused by BRAF, RAS, or MAP2K1 mutations. Correct identification of the underlying mutation is of increasing importance, since targeted treatments are becoming more and more relevant, especially in patients with extensive vascular malformations. However, variants of unknown significance are often identified and their pathogenicity and response to targeted therapy cannot be precisely predicted. Here we show, that zebrafish embryos can be used to rapidly assess the pathogenicity of novel variants of unknown significance in TEK, encoding for the receptor TIE2, present on endothelial cells of venous malformations. Endothelium-specific overexpression of TEK mutations leads to robust induction of venous malformations whereas MAP2K1 mutations cause arteriovenous malformations in our zebrafish model. TEK mutations are often found as double mutations in cis; using our model, we show that double mutations have an additive effect in inducing venous malformations compared to the respective single variants. The clinically established mTOR-inhibitor sirolimus (rapamycin) efficiently abrogates the development of venous malformations in this zebrafish model. In summary, endothelium-specific overexpression of patient-derived TEK variants in the zebrafish model allows assessment of their pathogenic significance as well as testing of candidate drugs in a personalized and mutation-specific approach.

Somatic RIT1 indels identified in arteriovenous malformations hyperactivate RAS-MAPK signaling and are amenable to MEK inhibition

“…To further assess the impact of the novel RIT1 variants on vascular development, we used an approach that mimics the endothelial mosaicism that occurs in patients. In our previous work, we assessed various TEK mutations by endothelial-specific mosaic expression in zebrafish embryos and observed the development of venous malformations 18 . In the current work, we observed that RIT1 mutations induced AVM-like lesions in zebrafish embryos, further confirming that overactivation of RAS-MAPK signaling caused by RIT1 indels has a deleterious effect on vascular development.…”

“…From the 10-somite stage or from 48 hpf on, embryos of the treatment group were transferred in E3 Medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , 0.33 mM Mg 2 SO 4 ) containing 0.2 mM 1-phenyl 2-thiourea (PTU; Sigma, Taufkirchen, Germany, P7629) and MEK1/2 inhibitor Trametinib (MedChemExpress, GSK1120212; 10 mg) using 100× stock solutions dissolved in dimethyl sulfoxide (DMSO; Sigma, D2650). The treatment dose of trametinib was chosen at 100 nM, according to our previous publication 18 , and by repeating of the toxicity assay in zebrafish embryos. Embryos of the control group were raised in E3 medium with 0.2 mM PTU and DMSO (equal amount to the treatment group).…”

“…Having shown previously that the RIT1 indels identified in AVM patients induced strong activation of the RAS-MAPK pathway, we next assessed whether their expression can lead to aberrant vascular development in the tail vasculature of zebrafish embryos. The zebrafish is an established model for the study of vascular development 16 that has also been applied to translational research 8,17,18 . To this end, we used plasmids, which contain the transcriptional upstream activating sequence (UAS) that controls the expression of wildtype or variant RIT1 linked to GFP via the self-cleaving peptide P2A.…”

“…These plasmids were injected in the one-cell stage of Tg(fli1a:Gal4; UAS:RFP) embryos. While the plasmid integrated randomly into the DNA of cells of the zebrafish embryo, expression of RIT1-P2A-GFP was limited to endothelial cells that expressed Gal4 under the control of the endothelial fli1a promoter (Fig 3A) 18 . Using this approach, we observed AVM-like lesions in the zebrafish embryo tail.…”