Introduction

Mutations that activate RAS/MEK/ERK or inactivate suppressors of this important pathway are commonly found in a variety of cancer types and are now being targeted for cancer therapy [1]. The activating NRASQ61R mutation is common in difficult to treat melanomas [2]. Recently, somatic NRAS mutations have begun to be identified in various vascular anomalies [3]. Notably, 10% of patients with pyogenic granuloma (PG), a vascular neoplasm, have non-inherited NRASQ61R mutation [4]. In other types of PG, KRAS and HRAS-activating mutations have been found [4, 5] leading to the possibility that hyperactive RAS/MAPK signaling is a common mechanism. The NRASQ61R mutation has also been found in patients with kaposiform lymphangiomatosis (KLA)[6] suggesting that RAS/MAPK/ERK may be an important pathway in this aggressive lymphatic malformation characterized by overgrowth of dysfunctional vessels and spindled endothelial cells. A recent case study also identified a somatic mutation in Casitas B-lineage lymphoma (CBL) proto-oncogene in one KLA patient who was effectively treated with a MEK inhibitor [7].

Hence, an emerging concept is that dysfunctional RAS signaling in endothelial cells may play a role in different types of vascular anomalies. However, while the pathways and effectors downstream of NRASQ61R in melanoma have been identified, the pathways that this mutation activates in endothelial cells are still largely unexplored. To address this question, we generated lentiviral constructs with doxycycline-regulated NRASQ61R or NRAS wild type (NRASWT). Each construct was introduced into human endothelial progenitor cells (EPC). EPCs isolated from human cord blood are excellent for modeling angiogenesis in vitro and in vivo[8]. NRASQ61R and NRASWT-expressing EPC were characterized in vitro and in vivo. Changes in EPC morphology, proliferation, migration, and downstream signaling pathways were assessed. Vascular morphogenesis was compared in a 3D in vitro angiogenesis model. A xenograft model was generated by taking NRASQ61R and NRASWT EPC enrobed in Matrigel® and injecting the cells under the skin of nude mice fed a doxycycline-containing diet. In a proof-of-concept study, inhibitors of the MAP Kinase and mTOR signaling pathways were tested to determine if they blocked changes in cell morphology.

Materials & methods

Generation of lentiviral constructs

DNA sequences-encoding NRASQ61R and NRASWT were subcloned into lentiviral vector pCW from Addgene (see Supplemental Fig. S1 for construct maps). The lentiviral constructs were packaged into viral particles and viral titer determined.

Cell culture, transduction and selection

Human endothelial progenitor cells (EPC) isolated from human cord blood were obtained from StemBiosys (CELLvo EPC). EPCs were plated onto fibronectin-coated plates and cultured in endothelial growth medium (EGM2) with 20% fetal bovine serum (FBS) (Lonza) and 1% penicillin–streptomycin–glutamine. Viral particles containing pCW-NRASQ61R or pCW-NRASWT were added to the EPC at a multiplicity of infection of 1. Hexadimethrine bromide (8 μg/ml) was also added to the media to increase the efficiency of infection. After 24 h, the media were replaced with puromycin-containing (0.5 μg /ml) media to select for cells that had taken up the constructs. The cells were cultured for 10 days in puromycin-containing medium and frozen back for future use. Doxycycline (Dox) was added to the media to induce NRASQ61R or NRASWT expression (1–2 μg/ml). For inhibitor studies, U0126 (10 μM) or rapamycin (15 nM), or vehicle (0.05% DMSO) was added and cell morphology (Circularity index) of the cells was measured at 48 h.

Cell morphology, proliferation & migration

Morphology of NRASQ61R and NRASWT EPC was photographed before (No Dox) and 24 and 48 h after addition of Dox. Circularity index of the cells, which is equal to 1 when cells are spheric and 0 when cell shape is spindled, was measured using ImageJ [9]. For proliferation analysis, cells were plated onto fibronectin-coated 96-well plates at 6000 cells per well with > 8 replicates. Cell growth was monitored for 3 days using an IncuCyte® ZOOM Live Cell Imaging System (Essen BioScience). Percentage of cell confluence was calculated to determine the cell growth rate. SRB assay was also used to assess cell proliferation. For the migration assay, cells were seeded at 1 × 105 cells/cm2 into an Ibidi Culture-Insert 2 Well system (Ibidi) and allowed to grow to confluency. The cells were pretreated with 2 mM hydroxyurea (Sigma) for 4 h to inhibit proliferation before creating the gap—The culture insert was gently removed using sterile tweezers, leaving a cell-free gap of approximatively 500 μm width, and medium was replaced with fresh medium containing 2 mM hydroxyurea. Images of the cell-free gap were captured at 0, 2, 4, 6, and 8 h using an EVOS-imaging system (Invitrogen). The cell-free area was measured over time using ImageJ software, and the value was expressed as percentage of the initial cell-free area (% gap closure area).

Western blot analysis of signaling pathways

Cell lysates were prepared after 2, 7, and 14 days of Dox treatment or from no Dox control plates. 30 μg of protein was loaded onto a 4–12% Bis/Tris polyacrylamide gel. After separation of the proteins by electrophoresis, the proteins were transferred to a PVDF membrane by western transfer. The membranes were blocked in 5% nonfat dry milk overnight at 4 °C. Primary antibodies against NRASQ61R (227658, 1:400; Abcam), NRAS (77392, 1:1000; Abcam), phospho-ERK (4370, 1:2000; Cell Signaling), ERK (9102, 1:1000; Cell Signaling), phospho-AKT (Ser473)(4060, 1:2000; Cell Signaling), AKT (9272, 1:1000; Cell Signaling), phospho-S6 Ser 235/236 (4858, Cell Signaling), S6 (2217, Cell Signaling), and actin (C4, 1:10,000; Seven Hills Bioreagents) were added for 1 h. After washing 5 times in TBST for 5 min, secondary antibodies (anti-rabbit, anti-goat, anti-mouse; Calbiochem) were added for 1 h at room temperature. Blots were washed, and Millipore Immobilon was used for the chemiluminescent detection. Images of blots were obtained using the LAS 4000 imaging system (Fujifilm).

3D lumen formation assay

3D lumen formation assay was performed as previously described [10]. Briefly, Cytodex® 3 microcarrier beads (Sigma) were incubated with NRASQ61R or NRASWT EPC at a concentration of 400 cells/ bead for 4 h at 37 °C. The following day, coated beads were resuspended in 2 mg/mL of fibrinogen (Sigma) solution containing 0.15 U/mL of aprotinin (Sigma) at a concentration of 500 beads/ml. Then 0.625 U/mL of thrombin (Sigma) and 0.5 ml beads/fibrinogen suspension were added per well of a 24-well plate and incubated at 37 °C for 20 min to allow fibrin clotting. The gels were overlaid with human dermal fibroblasts at 2 × 104 cells/well and cultured in EBM2 supplemented with 20% FBS and with or without 2ug/mL doxycycline. Medium was replaced every other day. Images were acquired at indicated time points using the EVOS cell-imaging system (Invitrogen), and the vascular area was analyzed with Fiji (Image J) software [11]. Immunofluorescent staining on fibrin gels was performed upon overnight fixation in 4% paraformaldehyde at 4 °C. Fibrin gels were incubated overnight at 4 °C with biotinylated UEA-1 (20 µg/ml, Vector Laboratories) followed by incubation with Texas Red® Streptavidin (Vector Laboratories; 5 µg/ml) for 4 h at room temperature. Nuclei were visualized with DAPI (4′,6-diamidino-2-phenylindole). Z-stack images were acquired on a Nikon A1R laser-scanning confocal microscope, and surface reconstruction of vascular structures was performed using Imaris Imaging Analysis software (Bitplane).

Generation of mouse xenograft model & evaluation of vascular growth

All animal experiments were approved by the Institutional Animal Care and Use Committee at Cincinnati Children’s Hospital in an Association for Assessment and Accreditation of Laboratory Animal Care-approved facility. Following expansion in tissue culture, 2.5 × 106 human EPCs expressing NRASQ61R or NRASWT were exposed to doxycycline for 48 h (no doxycycline as control) and suspended in 200 μL Matrigel® (Corning) and subcutaneously injected into both flanks of 6 to 7-week-old male athymic nu/nu male mice (Envigo). Lesions were dissected and weighed 8 days later, then fixed in 4% paraformaldehyde, and paraffin embedded. Paraffin Sects. (5 μm) were stained with hematoxylin and eosin (H&E). Five random images per section were captured using an EVOS microscope (Life Technologies), and vascular area (% of field area) was quantified using ImageJ software as previously reported [12].

Immunohistochemistry

Paraffin sections of the xenografts were stained with UEA I (Ulex europaeus Agglutinin I;1:100, Vector Laboratories). This was followed by peroxidase-conjugated secondary antibody and staining with diaminobenzidine peroxidase (horseradish peroxidase) substrate (Vector Laboratories). Slides were counterstained using hematoxylin (Vector Laboratories). Images were captured using a Nikon microscope.

Immunofluorescence staining for VE-cadherin

Immunofluorescence staining was performed with an antibody to VE-cadherin (555661, 1:50; BD Pharmingen) was performed on fixed monolayers incubated overnight at 4C. Secondary antibody was Alexa Fluor 488 anti-mouse labeled IgG (A21121, 1:200, Invitrogen). Slides were covered with a coverslip with Prolong Gold with DAPI (Invitrogen), and images were acquired on Nikon A1R laser-scanning confocal microscope.

Statistical analysis

Data are expressed as mean ± standard deviation (SD) and analyzed by Welch’s t test or parametric one-way ANOVA after normal distribution, and equal variance was assessed. One-way ANOVA with post hoc (Tukey or Fisher) test was used for multiple comparisons. For data with correlated observations, a repeated measures two-way ANOVA was used, followed by post hoc (Bonferroni) test. All calculations were performed using GraphPad Prism. Differences were considered significant for P value < 0.05.

Results

Expression of NRAS Q61R in human endothelial cells induces altered morphology and hyperactive MAPK pathway

Lentiviral constructs were generated with doxycycline-inducible expression of mutant NRASQ61R or NRASWT as a control (Supplemental Fig. S1). Each construct was transduced into EPC. EPCs were grown in endothelial growth medium, and morphology was analyzed 24 h and 48 h after doxycycline was added to the medium. Cell shape in EPC-expressing NRASQ61R overtly changed from the typical endothelial cobblestone-like morphology to an elongated/spindled shape (Fig. 1A and Supplemental Fig. S2). The morphological change was analyzed by measuring the circularity index which equals 1 when cells are spheric and 0 when cell shape is spindled. The circularity index was significantly lower (p < 0.0001 at both 24 and 48 h) in EPC-expressing NRASQ61R compared to NRASWT (Fig. 1B). Furthermore, in EPC-expressing NRASQ61R, the circularity index decreased at 24 h and 48 h, when compared to no Dox (t = 0 h) (Fig. 1C).

Fig. 1
figure 1

Morphology of human endothelial progenitor cells (EPC)-expressing NRASWT and NRASQ61R. Panel A: Representative images of EPC monolayer with no Doxycycline (No Dox) and 24 and 48 h after addition of Doxycycline to the cell culture media. n = 4 independent experiments. Scale bar = 200 μm Panel B and C: Circularity index measurements for EPC-expressing NRASWT and NRASQ61R in No Dox and Dox-containing media at 24 and 48 h. In B, measurements for each cell is shown. One-way Anova followed by Tukey’s test for multiple comparisons. In C, data points are mean ± SD; n = 4 independent experiments. Two-way Anova followed by Bonferroni test for multiple comparisons, p values are at 24 and 48 h

Full size image

Next, to confirm expression of NRASQ61R and NRASWT, we performed immunoblotting with an antibody specific for the mutant NRASQ61R and an antibody that detects both NRASWT and NRASQ61R, hereafter referred to as total NRAS. Upon doxycycline administration for 2, 7, and 14 days, NRASQ61R expression was elevated, and ERK and AKT (Ser473) were phosphorylated (Fig. 2A). The proliferative and migratory capacity of EPC expressing the mutant NRASQ61R or NRASWT were measured. Using an Incucyte® system, NRASQ61R EPC (with doxycycline) showed a higher proliferation rate than NRASWT EPC (on doxycycline) and no dox NRASQ61R EPC (Fig. 2B). Proliferation was also measured using SRB (Sulforhodamine B) assay, and similar results were seen (Supplemental Fig. S3). Cell migration was measured with a gap closure assay upon removal of an insert that generates a gap in the cell monolayer. EPC-expressing NRASQ61R (treated with doxycycline) migrated faster and closed the gap faster than NRASWT (with and without doxycycline) (Fig. 2C, D). These data show that EPC-expressing NRASQ61R developed an abnormal morphology, increased p-ERK, enhanced proliferation, and migration compared to NRASWT cells.

Fig. 2
figure 2

Signaling pathways and angiogenic properties of human endothelial progenitor cells (EPC) expressing NRASWT and NRASQ61R. Panel A: Cell lysates were analyzed 2, 7 and 14 days after doxycycline (Dox) was added to the cell culture media. Antibodies specific to the mutated form of NRAS (NRASQ61R), wild-type NRAS (total), phospho-ERK, phospho-AKT Ser473, total ERK, total AKT, and actin were used. Representative blot of n = 3 independent repeats. Panel B: Proliferation of endothelial cells was measured using an Incucyte® system in endothelial basal medium (EBM2) containing 20% fetal bovine serum (FBS). Data points are mean ± SD; n = 8 wells. Two-way Anova followed by Bonferroni test for multiple comparisons. P value < 0.0001 for times ≥ 36 h. Panel C: Representative images of migration of endothelial cells at t = 0 and t = 10 h after the wound was made. Scale bar: 200 μm. Panel D: Migration area of endothelial cells was measured every 2 h from 0 to 10 h after creating the gap. Percentage gap closure was determined. Data points are mean ± SD; shown is one representative of n = 2 independent experiments with 3–4 wells analyzed. Two-way Anova followed by Bonferroni test for multiple comparisons. P value < 0.05 at 6, 8, 10 h

Full size image

Expression of NRASQ61R in human endothelial cells induces formation of dilated vascular channels in a 3D fibrin gel lumen formation assay

To investigate the vasculogenic potential of EPC-expressing NRASQ61R, we employed an in vitro 3D (three-dimensional) fibrin gel system [10] that we previously employed to mimic venous malformation morphogenesis [13]. When control NRASWT human EPC-coated beads were embedded in fibrin gels topped with fibroblasts, they formed longitudinal lumenized vessels (Fig. 3A). Conversely, EPC-expressing NRASQ61R formed dilated hollow vascular structures (Fig. 3A and Supplemental Video S1-2).

Fig. 3
figure 3

Human endothelial progenitor cells (EPC)-expressing NRASQ61R form enlarged vascular structures in a 3D lumen formation assay. Panel A: Imaris-generated 3D structural models of vascular channels formed by human EPC-expressing NRASWT and NRASQ61R stained for Ulex europaeus agglutinin-I (UEA) on day 7. Scale Bars = 200  μμ. Panel B: Experimental protocol schematic (top), Doxycycline was added 48 h prior starting the 3D lumen formation assay. Representative 2D images of vascular structures (bottom, left) and quantification of vascular area (normalized by bead number) at day 7 (bottom, right). Data points are mean ± SD; n = 3 independent experiments. Welch’s t test. Panel C: Experimental protocol schematic, Doxycycline was not added until day 10 (top). Representative 2D images of vascular structures (bottom, left) and quantification of vascular area at days 10, 13, and 21 (bottom, right). Data points are mean ± SD; n = 3 independent experiments. Two-way Anova followed by Bonferroni test for multiple comparisons. P value < 0.0001 at day 21. Scale Bars in Panel B and C = 100 μμ

Full size image

To further determine the consequences of the NRASQ61R expression on the lumen formation, we pretreated cell monolayer with doxycycline for 48 h prior embedding the cells in the 3D fibrin gel, and doxycycline was added to the medium on top of the gel. At day 7, analysis revealed that NRASQ61R EPC formed dilated vascular channels and showed significantly increased vascular area compared to NRASWT (p < 0.0001) (Fig. 3B).

To model changes in lumen morphogenesis in a normal vessel – upon expression of NRASQ61R, we performed the 3D lumen formation assay with NRASQ61R and control NRASWT EPC without exposing cells to doxycycline. By day 10 without doxycycline administration, lumens formed as in normal EPC (Fig. 3C). After day 10, doxycycline was added to the gel and correlated analysis at days 13 and 21 revealed vascular area expansion due to NRASQ61R expression, while area did not change with expression of NRASWT. These data show that EPC-expressing NRASQ61R in the 3D fibrin gel can phenocopy dilated vascular lesions as detected in vascular anomaly patients with hyperactive NRAS mutations.

Generation of a murine xenograft model for vascular anomalies with human endothelial mutant NRASQ61R cells

Human EPC-expressing NRASWT or NRASQ61R were expanded in doxycycline-containing cell culture medium, suspended in 200μl of Matrigel® and subcutaneously injected into immune-deficient mice fed with doxycycline-containing chow (Fig. 4A). We evaluated if they could recapitulate the histological features of vascular anomalies with hyperactive NRAS mutations. Murine lesion xenograft explants (plugs) containing doxycycline-treated NRASQ61R EPC were visibly vascularized on day 8 and their weight was significantly (p < 0.05) higher compared to the other experimental control groups (Fig. 4B). Hematoxylin and eosin staining of xenograft sections revealed numerous dilated vascular channels in mice injected with EPC-expressing NRASQ61R on Dox, while EPC-expressing NRASWT (treated with doxycycline), NRASQ61R, and NRASWT not on Dox generated only minimal vascularization (Fig. 4C). The vascular area in doxycycline-treated NRASQ61R EPC-xenograft lesions was significantly higher than control EPC xenografts (p < 0.05) (Fig. 4C), and in general, vessels were bigger in size compared to the control groups (Supplemental Fig. S4). Lesional ectatic channels in the xenografts showed binding to the endothelial human-specific lectin Ulex europaeus agglutinin-I (UEAI) (Fig. 4D). This confirms that vascular channels in the xenografts were mostly generated by the human EPC rather than invading mouse vessels. All together, these results show that NRASQ61R expression induces formation of enlarged vessels using this xenograft model.

Fig. 4
figure 4

Human endothelial progenitor cells (EPC)-expressing NRASQ61R form enlarged and perfused vascular structures in a murine xenograft model. Panel A: Experimental protocol showing time of cell injection and Doxycycline treatment period, and time of harvest of xenograft plugs. Panel B: Pictures and graph of weight measurements of the xenograft plugs after removal from mice at day 8. Data points are mean ± SD; n = 2–6 mice with 2 plugs. One-way Anova followed by Fisher test for multiple comparisons. Panel C: Histology of xenograft plugs after removal from mice at day 8. Tissue sections were stained with hematoxylin and eosin. Graph shows vascular area measured from histology. One-way Anova followed by Fisher test for multiple comparisons. Panel D: Immunostaining of xenograft plug sections for Ulex europaeus agglutinin-I (UEA) that is expressed by human endothelial cells and not mouse endothelial cells. Scale bars = 1 cm in B, 100 μm in C and D

Full size image

Treatment of mutant NRASQ61R endothelial cells with MAPK/ERK and mTOR inhibitors

A proof-of-concept study was performed to verify that NRASQ61R EPC could be used to determine the role of signaling pathways and to test drugs. First, we verified that the MAPK/ERK inhibitor U0126 at 10 μM prevented the phosphorylation of ERK and that mTOR inhibitor rapamycin at 15 nM reduced phosphorylation of S6. These two drugs were effective on both NRASQ61R and NRASWT EPC (Fig. 5A). Next, measurement of the cell shape was performed with doxycycline and U0126 or rapamycin for 48 h. Analysis of cell circularity index showed that U0126 prevented the shift to a spindle-shaped morphology in the NRASQ61R EPC, whereas mTOR inhibitor rapamycin did not (Fig. 5B, C). These results show that NRASQ61R expression induces cell shape change towards a spindle morphology and that MAPK/ERK inhibition is effective at preventing this change. This would suggest MAPK/ERK pathway is involved in the cell shape change in the NRAS mutant cells.

Fig. 5
figure 5

Treatment of endothelial progenitor cells (EPC) expressing NRASQ61R with MAPK/ERK and mTOR inhibitors. Panel A: Western blot analysis of phospho-ERK, ERK, phospho-S6, S6, and actin 48 h hours after addition of doxycycline and either vehicle (DMSO control), MAPK/ERK inhibitor U0126 (U; 10 μM), or rapamycin (R; 15 nM). Panel B: Representative images of EPC monolayers with no Doxycycline (No Dox), plus Doxycycline, and either vehicle (DMSO control), MAPK/ERK inhibitor U0126 (10  μM), or rapamycin (15 nM). Scale bar = 200 μm. Panel C: Circularity index measurements for EPC-expressing NRASWT and NRASQ61R in Dox-containing media at 48 h and DMSO control (-), MAPK/ERK inhibitor U0126 (U; 10 μM), or rapamycin (R; 15 nM). Data are expressed in % change relative to No Dox control. One-way Anova followed by Tukey’s test for multiple comparisons. ns: p > 0.05

Full size image

Discussion

Somatic-activating mutations in RAS or loss-of-function mutations in genes that suppress RAS signaling are common in tumors [1] and have now been identified in patients with vascular anomalies [3,4,5]. While the NRASQ61R mutation has been found in tissue from PG and KLA patients [4, 6], its role in the pathogenesis of these diseases is unclear. Whether the mutation by itself is sufficient to drive the disease or additional mutations or environmental factors are needed is unknown. Our results show that NRASQ61R expression in human endothelial cells induces abnormal cell morphology and increases the rate of proliferation and migration in vitro. Our in vitro angiogenesis model lends further support as NRASQ61R-expressing endothelial cells formed enlarged abnormal structures compared to NRASWT cells. Furthermore, in a xenograft model, NRASQ61R endothelial cells formed enlarged channels with increased vascular area and size.

Our studies showed that expression of NRASQ61R induced a spindle-shaped endothelial cell morphology that is seen in the histopathology of KLA patients [14]. Changes in cell morphology to acquire elongations or spindled shape have also been reported in endothelial cells-expressing PIK3CA or TEK constitutive active mutations [15] modeling venous malformation and in a SMAD4 loss-of-function murine model of arteriovenous malformations [16]. It is possible that these morphological changes are a pre-requisite for the formation of vascular malformations with dilated vascular channels. NRASQ61R endothelial cells had enhanced proliferation and migration, as was MAPK/ERK and PI3 Kinase/AKT pathway activation. These are not unexpected given the role of these key signaling pathways in cell morphology, division, and migration. In a 3D in vitro angiogenesis model, NRASQ61R endothelial cells formed abnormally large channels compared to NRASWT cells which formed narrow-hollowed longitudinal channels. Of note, a recent study showed abnormal morphogenesis and failure to form elongated tubes in endothelial cells-expressing mutant HRASV12 [17]. A similar in vitro 3D model for venous malformation was successfully used to establish the efficacy of PI3K/mTOR inhibitors in preventing lumen formation and enlargement [18]. These 3D systems could be employed in the future as a tool to study the mechanisms leading to malformed vascular lumens and to screen for drugs that could normalize the NRASQ61R phenotype.

When injected under the skin of nude mice the NRASQ61R endothelial cells formed enlarged bloody channels not unlike the pathology of lesions in PG and KLA patients [14]. While animal models for vascular anomalies with the NRASQ61R mutation have not been reported yet, other groups have recently modeled in mouse vascular anomalies associated with KRAS activating mutations [19, 20]. Somatic-activating mutations in KRAS have been found in patients with brain arteriovenous malformations and targeted endothelial expression of KRASG12D in mice generated a mouse model with pathology similar to the patients [19]. The KRASG12V mutation has been found in Gorham-Stout Disease (GSD), where ectopic overgrowth of lymphatic vessels into the bones is associated with bone loss–this was modeled using Prox1-CreERT2 mice to target KRASG12V and mimicked many key features of human GSD [20].

Although signaling of the RAS family members is similar because of the highly conserved effector domains, the specificity of the different isoforms is just starting to be explored in the vascular endothelium. Clearly, RAS signaling is implicated in the manifestation of vascular anomalies, and as such it will be a critical area for future discovery.

The mTOR inhibitory drug rapamycin has become a standard therapy for many patients with complex vascular anomalies; however, it often only produces a partial response and patients can rebound if therapy is withdrawn [21]. Hence, there is a great need for new therapeutic agents to be developed. Pre-clinical models for vascular anomalies are vital to identify new targets and test treatments. Therefore, in a proof-of-concept study, we evaluated whether a well-known MAPK/ERK inhibitor (U0126) or rapamycin prevented the shift to a spindle-shaped morphology in the NRASQ61R mutant cells. The study clearly showed that U0126 prevented the spindle morphology shift in the mutant cells whereas rapamycin did not. A MEK inhibitor was effective in a recent KLA patient with a somatic mutation in Casitas B-lineage lymphoma (CBL) proto-oncogene [7]. In the future, these NRASQ61R EPC models should prove useful to identify new therapeutic targets. Testing drug combinations will also be important as this may allow for lower doses to be used with improved efficacy and reduced side effects compared to one agent alone. In one KLA patient, multimodal therapy was much more effective than single treatment [22].

In summary, NRASQ61R expression in human endothelial cells recapitulated many of the key characteristics seen in the lesions of vascular anomaly patients with RAS mutations. This provides support for NRASQ61R as a key driver in the etiology of vascular anomalies. The in vitro and in vivo models reported can be used to gain a better understanding of the pathways driving the pathogenesis of mutant NRAS-induced diseases. These models should also facilitate pre-clinical testing of new treatments and combinational therapies.