a-patient-derived-xenograft-model-for-venous-malformation

A Patient-Derived Xenograft Model for Venous Malformation

Venous malformation (VM) is a vascular anomaly that arises from impaired development of the venous network resulting in dilated and often dysfunctional ...

Cincinnati Children's Hospital Medical Center

Vascular malformations are defects caused by the abnormal growth of the vasculature. Among them, venous malformation (VM) is an anomaly characterized by slow-flow vascular lesions with abnormally shaped veins, typically in sponge-like configuration. VMs can expand over years causing disfigurement, obstruction of vital structures, thrombosis, bleeding, and pain. Treatments have been very limited and primarily based on supportive care, compression garments, sclerotherapy, and/or surgical resection. Sirolimus treatment has recently shown efficacy in some patients with complicated vascular anomalies, including VMs. Activating somatic TIE2 gene mutations have been identified in up to 60% of VMs and PIK3CA mutations have been found in another 25%. Here, we report a xenograft model of VM that reflects the patients' mutation heterogeneity. First, we established a protocol to isolate and expand in culture endothelial cells (VM-EC) from VM tissue or VM blood of nine patients. In these cells, we identified somatic mutations of TIE2, PIK3CA, or a combination of both. Both TIE2 and PIK3CA mutations induced constitutive AKT activation, while TIE2 mutations also showed high MAPK-ERK signaling. Finally, VM-EC implanted into immune-deficient mice generated lesions with ectatic blood-filled channels with scarce smooth muscle cell coverage, similar to patients' VM. This VM xenograft model could be instrumental to test the therapeutic efficacy of Sirolimus in the presence of the different TIE2 or PIK3CA mutations or to test for efficacy of additional compounds in targeting the specific mutated protein(s), thus enabling development of personalized treatment options for VM patients.

A xenograft model for venous malformation.

Objective- Venous malformations (VMs) arise from developmental defects of the vasculature and are characterized by massively enlarged and tortuous venous channels. VMs grow commensurately leading to deformity, obstruction of vital structures, bleeding, and pain. Most VMs are associated with the activating mutation L914F in the endothelial cell (EC) tyrosine kinase receptor TIE2. Therapeutic options for VM are limited and ineffective while therapy with the mammalian target of rapamycin inhibitor rapamycin shows moderate efficacy. Here, we investigated novel therapeutic targets promoting VM regression. Approach and Results- We performed an unbiased screen of Food and Drug Administration-approved drugs in human umbilical vein ECs expressing the TIE2-L914F mutation (HUVEC-TIE2-L914F). Three ABL (Abelson) kinase inhibitors prevented cell proliferation of HUVEC-TIE2-L914F. Moreover, c-ABL, common target of these inhibitors, was highly phosphorylated in HUVEC-TIE2-L914F and VM patient-derived ECs with activating TIE2 mutations. Knockdown of c-ABL/ARG in HUVEC-TIE2-L914F reduced cell proliferation and vascularity of murine VM. Combination treatment with the ABL kinase inhibitor ponatinib and rapamycin caused VM regression in a xenograft model based on injection of HUVEC-TIE2-L914F. A reduced dose of this drug combination was effective in this VM murine model with minimal side effects. The drug combination was antiproliferative, enhanced cell apoptosis and vascular channel regression both in vivo and in a 3-dimensional fibrin gel assay. Conclusions- This is the first report of a combination therapy with ponatinib and rapamycin promoting regression of VM. Mechanistically, the drug combination enhanced AKT inhibition compared with single drug treatment and reduced PLCγ (phospholipase C) and ERK (extracellular signal-regulated kinase) activity.

Ponatinib Combined With Rapamycin Causes Regression of Murine Venous Malformation.

Abnormalities in controlling key aspects of angiogenesis including vascular cell migration, lumen formation and vessel maturation are hallmarks of vascular anomalies including venous malformation (VM). Gain-of-function mutations in the tyrosine kinase receptor TIE2 can cause VM and induce a ligand-independent hyperactivation of TIE2. Despite these important findings, the TIE2-dependent mechanisms triggering enlarged vascular lesions are not well understood. Herein we studied TIE2 p.L914F, the most frequent mutation identified in VM patients. We report that endothelial cells harboring a TIE2-L914F mutation display abnormal cell migration due to a loss of front-rear polarity as demonstrated by a non-polarized Golgi apparatus. Utilizing a three-dimensional fibrin-matrix based model we show that TIE2-L914F mutant cells form enlarged lumens mimicking vascular lesions present in VM patients, independently of exogenous growth factors. Moreover, these abnormal vascular channels demonstrate a dysregulated expression pattern of apico-basal polarity markers Podocalyxin and Collagen IV. Furthermore, in this system we recapitulated another pathological feature of VM, the paucity of pericytes around ectatic veins. The presented data emphasize the value of this in vitro model as a powerful tool for the discovery of cellular and molecular signals contributing to abnormal vascular development and subsequent identification of novel therapeutic approaches.

Constitutive Active Mutant TIE2 Induces Enlarged Vascular Lumen Formation with Loss of Apico-basal Polarity and Pericyte Recruitment.

Capillary lymphatic venous malformations (CLVM) are complex vascular anomalies characterized by aberrant and enlarged lymphatic and blood vessels. CLVM appear during fetal development and enlarge after birth, causing life-long complications such as coagulopathy, pulmonary embolism, chronic pain, and disfigurement. Treatment includes surgical debulking, amputation, and recurrent sclerotherapy. Somatic, mosaic mutations in the 110-kD catalytic α-subunit of phosphoinositide-3-kinase (PIK3CA) gene have been previously identified in affected tissues from CLVM patients; however, the cell population harboring the mutation is still unknown. In this study, we hypothesized that endothelial cells (EC) carry the PIK3CA mutations and play a major role in the cellular origin of CLVM. We isolated EC from the lesions of seven patients with CLVM and identified PIK3CA hotspot mutations. The CLVM EC exhibited constitutive phosphorylation of the PI3K effector AKT as well as hyperproliferation and increased resistance to cell death compared to normal EC. Inhibitors of PIK3CA (BYL719) and AKT (ARQ092) attenuated the proliferation of CLVM EC in a dose-dependent manner. A xenograft model of CLVM was developed by injecting patient-derived EC into the flanks of immunocompromised mice. CLVM EC formed lesions with enlarged lymphatic and vascular channels, recapitulating the patient histology. EC subpopulations were further obtained by both immunomagnetic separation into lymphatic EC (LEC) and vascular EC (VEC) and generation of clonal populations. By sequencing these subpopulations, we determined that both LEC and VEC from the same patient express the PIK3CA mutation, exhibit increased AKT activation and can form lymphatic or vascular lesions in mouse.

Article	Year
A xenograft model for venous malformation. Angiogenesis\| PubMed ID: 29786783	2018
Ponatinib Combined With Rapamycin Causes Regression of Murine Venous Malformation. Arteriosclerosis, thrombosis, and vascular biology\| PubMed ID: 30626204	2019
Constitutive Active Mutant TIE2 Induces Enlarged Vascular Lumen Formation with Loss of Apico-basal Polarity and Pericyte Recruitment. Scientific reports\| PubMed ID: 31451744	2019
Constitutively active PIK3CA mutations are expressed by lymphatic and vascular endothelial cells in capillary lymphatic venous malformation. Angiogenesis\| PubMed ID: 32350708	2020

Jillian Goines

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