ARAF Recurrent Mutation Causes Central Conducting Lymphatic Anomaly Treatable with a MEK Inhibitor

Letters https://doi.org/10.1038/s41591-019-0479-2

ARAF recurrent mutation causes central conducting lymphatic anomaly treatable with a MEK inhibitor

Dong Li1, Michael E. March1, Alvaro Gutierrez-Uzquiza1,12, Charlly Kao1, Christoph Seiler2, Erin Pinto3, Leticia S. Matsuoka1, Mark R. Battig1, Elizabeth J. Bhoj1, Tara L. Wenger4, Lifeng Tian1, Nora Robinson1, Tiancheng Wang1, Yichuan Liu1, Brant M. Weinstein5, Matthew Swift6, Hyun Min Jung5, Courtney N. Kaminski1, Rosetta Chiavacci1, Jonathan A. Perkins7, Michael A. Levine8,9, Patrick M. A. Sleiman1,9, Patricia J. Hicks9, Janet T. Strausbaugh9, Jean B. Belasco9,10, Yoav Dori3,9 and Hakon Hakonarson 1,9,11*

The treatment of lymphatic anomaly, a rare devastating dis- rarity and overlapping of diagnostic criteria, has hampered the ease spectrum of mostly unknown etiologies, depends on development of innovative therapies6–9. GLA is defined as multifo- the patient manifestations1. Identifying the causal genes will cal lymphatic anomaly that has multiple areas of micro/macrocystic allow for developing affordable therapies in keeping with pre- lymphatic malformation and often involves bone destruction9–11. cision medicine implementation2. Here we identified a recur- CCLA, on the other hand, describes dysfunction of the thoracic rent gain-of-function ARAF mutation (c.640T>C:p.S214P) duct (TD) or cisterna chyli, leading to a retrograde flux of lymphatic in a 12-year-old boy with advanced anomalous lymphatic fluid or abnormal drainage of lymphatic fluid1,12,13. Both conditions disease unresponsive to conventional sirolimus therapy and can manifest with chylothorax, effusions, chylous ascites or lymph- in another, unrelated, adult patient. The mutation led to loss edema. The overlap of these apparently disparate disorders suggests of a conserved phosphorylation site. Cells transduced with that a common pathway rather than a common gene is responsible ARAF-S214P showed elevated ERK1/2 activity, enhanced for the various clinical syndromes, and implies that the distinction lymphangiogenic capacity, and disassembly of actin skeleton between entities may be artificial. Here we report the use of whole- and VE-cadherin junctions, which were rescued using the MEK exome sequencing (WES) to identify a recurrent missense muta- inhibitor trametinib. The functional relevance of the mutation tion in ARAF as the basis for a severely advanced lymphatic disease was also validated by recreating a lymphatic phenotype in characterized by a complex lymphatic anomaly in two unrelated a zebrafish model, with rescue of the anomalous phenotype patients. Our results provide a representative demonstration of how using a MEK inhibitor. Subsequent therapy of the lead pro- genetic classification presents a way to categorize complex medical band with a MEK inhibitor led to dramatic clinical improve- disorders, thereby guiding biologically based medical treatments, ment, with remodeling of the patient’s lymphatic system with which in our instance was life-saving. resolution of the lymphatic edema, marked improvement in The first tier of WES analyses of the known lymphatic anom- his pulmonary function tests, cessation of supplemental oxy- aly-associated genes was unrevealing, including mutation analysis gen requirements and near normalization of daily activities. of AKT1, PIK3CA, KRAS, HRAS, NRAS, BRAF, RAF1, PTPN11, Our results provide a representative demonstration of how SHOC2, CBL, RIT1 and SOS1. Subsequent gene prioritization knowledge of genetic classification and mechanistic under- revealed a novel X chromosomal ARAF mutation, c.640T>C:p. standing guides biologically based medical treatments, which S214P, in both patient P1, a male with CCLA (Fig. 1a,c–e; see in our instance was life-saving. Methods for a detailed clinical description), and patient P2, a female Although recent studies have demonstrated the benefit of siro- diagnosed with lymphangiomatosis in 2012 before the establish- limus in the treatment of generalized lymphatic anomaly (GLA) ment of the 2015 International Society for the Study of Vascular and central conducting lymphatic anomaly (CCLA)3–5, the absence Anomalies classification. The mutation affects a conserved phos- of clear clinical distinctions between these entities, due to their phorylation site, which putatively resulted in a gain-of-function

1Center for Applied Genomics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 2Zebrafish Core Facility, The Children’s Hospital of Philadelphia Research Institute, Philadelphia, PA, USA. 3Center for Lymphatic Imaging and Interventions, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 4Division of Craniofacial Medicine, Seattle Children’s Hospital, Seattle, WA, USA. 5Division of Developmental Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA. 6Department of Oncology, Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington DC, USA. 7Division of Otolaryngology–Head and Neck Surgery, Seattle Children’s Hospital, Seattle, WA, USA. 8Division of Endocrinology and Diabetes, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 9Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. 10Division of Oncology, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 11Divisions of Human Genetics and Pulmonary Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA. 12Present address: Department of Biochemistry and Molecular Biology II, School of Pharmacy, Complutense University, Madrid, Spain. *e-mail: [email protected]

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a bc f Patient P1 Patient P2

Mother of patient P2

Daughter of patient P2 de ARAF c.640T>C ARAF c.640T>C (p.S214P) (p.S214P) in patient P2

g ARAF c.640T>C (p.S214P) CR1 CR2 CR3

Human Rhesus Mouse Dog Elephant Xenopus tropicalis Zebrafish B-RAF C-RAF

Fig. 1 | Clinical images in the lead proband with lymphatic anomaly and molecular analysis. a, The coronal slice of a T2-weighted non-contrast lymphangiogram, demonstrating a large pericardial effusion (arrow). b, The maximal intensity projection of a dynamic contrast-enhanced magnetic resonance lymphangiogram in a healthy control person, showing a normal TD coursing towards the left innominate vein. c, The maximal intensity projection of a dynamic contrast-enhanced magnetic resonance lymphangiogram in P1, showing dilated lumber lymphatic networks with retrograde liver hilar flow (arrowhead) and a dilated and tortuous TD (arrow) coursing towards the innominate vein on the left and also supplying retrograde perfusion to the mediastinum and pericardium (box). d, The contrast lymphangiogram of the boxed region in c, demonstrating dilated and tortuous distal TD with retrograde flow towards the mediastinum, pericardium and lungs through dilated lymphatic networks originating at the distal TD (arrows). e, The coronal maximal intensity projection of the pelvis and genitalia, demonstrating multiple dilated ducts (arrowhead) originating in bilateral groin lymph nodes and supplying retrograde flow into the penis and scrotum (arrow). f, The pedigrees and genotypes of a recurrent mutation, c.640T>C (p.S214P), in ARAF identified in unrelated kindreds. g, The schematic topology of the ARAF protein, where the asterisk indicates the position of the p.S214P mutation in CR2. The Ser 214 residue is highly conserved across vertebrate species and all RAF isoforms.

(GoF) effect as the residue Ser 214 is a paralogous regulatory site significantly greater activation of ERK1/2, as measured by increased in its homologous protein C-RAF (also known as RAF1) for inhibi- phosphorylation, compared with HEK293T cells expressing wild- tion by 14-3-3 proteins. This missense mutation was absent from type (WT) ARAF (Fig. 2a,b). Phosphorylation of AKT, p70S6K, 1000 Genomes Project, ESP6500SI, ExAC v0.3, gnomAD v2.1 or mTOR and p38 (another family of MAP kinases) was not altered by additional exome-sequencing data from more than 5,000 samples ARAF-S214P (Fig. 2b). Similar results were obtained in HeLa cells that we had in our in-house database. Sanger sequencing of blood- (Extended Data Fig. 1) and in primary human dermal lymphatic derived DNA from P1 and both parents confirmed that this X-linked endothelial cells (HDLECs) (Fig. 2c). This marked overactivation ARAF mutation occurred as a somatic heterozygous event as shown was also present even in the absence of cytokines or growth factors in the male patient (Fig. 1f). Sanger sequencing of the ARAF muta- (Extended Data Fig. 1). tion in P2, her unaffected daughter and mother confirmed the HDLECs expressing ARAF-S214P manifest enhanced lymphan- mutation was present only in P2 (Fig. 1f). The father was unavail- giogenic capacity compared with HDLECs expressing ARAF-WT, able for sequencing; however, as her father had no reported respira- as measured by the number of sprouts and the sprout length in the tory symptoms it remains likely that the ARAF mutation arose as a three-dimensional lymphatic spheroid sprouting assay conducted de novo or somatic mutation in P2. Patient P2 was lost to follow-up in the absence of vascular endothelial growth factor C (VEGFC) and we were informed later that she died in 2017 from complica- (Fig. 2d). The MEK inhibitor trametinib rescued the increased tions of her lymphatic disease, five years after her diagnosis. sprouting in the mutant (Fig. 2d). We then performed a morpho- The Ser 214 residue, which is one of the 14-3-3 binding sites in logical analysis of the endothelial adherens junctions of primary conserved region 2 (CR2)14, in ARAF is highly conserved across HDLECs expressing ARAF-S214P. As shown by immunofluores- vertebrate species, as well as within the RAF proteins, suggesting cence microscopy, ARAF-S214P expression caused a significant that it may serve an essential role in the function of these kinases absence of VE-cadherin accumulation between adjacent cells sug- (Fig. 1g). The binding of 14-3-3 proteins to phosphorylated Ser 214 gesting increased VE-cadherin internalization (Fig. 2e, yellow of ARAF would prevent recruitment of ARAF protein to the plasma arrowheads, and Extended Data Fig. 2a). Additionally, expression membrane by activated Ras15. Previous studies showed that the of ARAF-S214P altered actin organization (Extended Data Fig. 2b), mutations in the ARAF-S214 paralogous residue Ser 259 in C-RAF with mutant-expressing cells possessing fewer discrete F-actin fila- impaired binding of 14-3-3 proteins, leading to plasma mem- ments within the cell body (Extended Data Fig. 2c). We then exam- brane localization and inducing ERK/MEK signaling16. As shown ined the ability of MEK1/2 inhibitors to reverse these abnormalities. in Fig. 2a, HEK293T cells transfected with ARAF-S214P showed The MEK inhibitor trametinib, at a concentration of 100 nM, res- reduced co-immunoprecipitation of 14-3-3 proteins, and in turn cued the loss of VE-cadherin from cell–cell junctions observed in

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HDLECs expressing ARAF-S214P with an almost complete restora- whether MEK signaling inhibitors can reverse the anomalies, we tion of the cell monolayer integrity and a recovery of the normal treated mrc1a:ARAFS214P larvae with cobimetinib from 3 d post appearance of VE-cadherin at junctions and actin filaments (Fig. 2e fertilization (dpf), when the lymphatic progenitor cells sprout to and Extended Data Fig. 2). Although ARAF-S214P clearly acti- form the TD17. We analyzed body segments (somites) with ARAF vates ERK in HDLECs, and ERK activation is typically associated expression in the TD at 7 dpf and found a significant rescue of duct with cell proliferation, we did not observe any measurable differ- morphology by cobimetinib (Fig. 2i,j). Meanwhile, we treated WT ences in proliferation between ARAF-WT- and -S214P-expressing Tg(mrc1a:egfp)y251 larvae with cobimetinib, and found that they tol- HDLECs across two independent retroviral transductions (Fig. 2f erated the drug well (Extended Data Fig. 6). and Extended Data Fig. 3). In view of our demonstration that the ARAF mutation led to a Analysis of lymphatic development in zebrafish was performed GoF effect in P1 that was unresponsive to sirolimus and that MEK in the Tg(mrc1a:egfp)y251 transgenic line17, where all lymphatic endo- inhibitors could rescue the lymphatic phenotype in both transduced thelial cells are labeled with EGFP. ARAF expression was targeted to endothelial cells and in a transgenic zebrafish model, we sought lymphatic vessels with the mrc1a promoter, and ARAF-expressing Institutional Review Board clearance to use MEK inhibitor therapy cells were marked by mCherry expression. ARAF-S214P expression in P1. Trametinib (Mekinist), a Food and Drug Administration induced dilated lymphatic vessels in different locations (Extended (FDA)-approved MEK inhibitor, was subsequently used off-label in Data Fig. 4), and most consistently we observed dilation of the trunk this 12-year-old patient following comprehensive baseline evalua- TD (Fig. 2g). Expression of ARAF-WT, in contrast, had no effect tion. We used a starting dose of 1 mg d−1 of trametinib and began on lymphatic morphology (Fig. 2h). Expression of ARAF-S214P observing improvement in pulmonary function testing within induces p-ERK in zebrafish (Extended Data Fig. 5). To determine 2 months of therapy (Fig. 3a). Moreover, there were significant

Fig. 2 | The ARAF-S214P mutation increases ERK1/2 activity, enhances lymphangiogenic capacity and alters actin skeleton and VE-cadherin junctions in HDLECs, and results in dilation of the thoracic duct (TD) in zebrafish that is reversed by cobimetinib. a, ARAF mutant transfection in HEK293T cells impairs association with 14-3-3 proteins and increases p-ERKs. The normalized 14-3-3/FLAG ratio is illustrated by the panel on the right, showing reduced co-immunoprecipitation of 14-3-3 proteins in the mutant. The data are shown as the mean ± s.e.m. of three independent experiments. Two-tailed unpaired t-test (with 4 degrees of freedom (df)), ****P = 8.6 × 10−6. The images were cropped for better presentation. b, ARAF mutant transfection in HEK293T cells induces increased expression of p-ERK1/2 compared with cells expressing the WT (**P = 0.0026; two-tailed unpaired t-test; df = 8). Phosphorylation of AKT, p70S6K, mTOR and p38 was not altered by ARAF-S214P. Normalized ratios are illustrated by the box and whisker plot on the right (minimum to maximum, showing all the points), where the center line represents the median, the box limits represent the interquartile range and the whiskers represent the minimum to maximum data range. Six independent experiments were performed. The images were cropped for better presentation. c, Primary HDLECs transduced with ARAF-WT or ARAF-S214P were cultured in increasing concentrations of trametinib. The results with cells from three independent transductions were quantified and graphed on a scatter dot plot with each individual value as a dot superimposed. The data are shown as the mean ± s.e.m. (error bars) of the three independent experiments. Transduction of ARAF-S214P significantly increased the level of p-ERKs (*P = 0.03; two-tailed unpaired t-test with 4 df). Trametinib treatment led to a significant reduction of p-ERKs (*P = 0.02 for 100 nM trametinib treatment and 300 nM trametinib treatment; *P = 0.01 for 1,000 nM trametinib treatment and 3,000 nM trametinib treatment; two-tailed unpaired t-test with 4 df); NS, not significant. The images were cropped for better presentation. d, Three-dimensional lymphatic spheroid sprouting assay shows the elevated sprouting activity in HDLECs expressing ARAF-S214P compared with ARAF-WT as measured by both number of sprouts (***P = 0.0002) and sprout length on the bottom (***P = 0.0005). Two-tailed unpaired t-test with 22 df. Spheroids were also cultured in increasing concentrations of trametinib, which significantly reduces both the number of sprouts at concentrations of 30 nM (****P = 4.68 × 10−5; df = 25), 100 nM (***P = 9.5 × 10−4; df = 23) and 300 nM (***P = 4.4 × 10−4; df = 24) and sprout length at concentrations of 30 nM (***P = 1.8 × 10−4; df = 25), 100 nM (**P = 0.001; df = 23) and 300 nM (***P = 3.2 × 10−4; df = 24). Two-tailed unpaired t-test. Three experiments performed with independent transductions of HDLECs were quantified, and points from all three experiments are plotted (15 points per condition) on the interleaved box and whisker plot (three to six spheroids per experiment), where the center line represents the median, the box limits represent the interquartile range and the whiskers represent the minimum to maximum data range. e, The ARAF mutant affects VE-cadherin localization (****P = 1.88 × 10−26), and treatment with trametinib results in increased cell surface localization of VE-cadherin (****P = 1.63 × 10−19). The red arrowheads point to staining referred to as plasma membrane staining, and the yellow arrowheads indicate intracellular staining. Three experiments performed with independent transductions of HDLECs were quantified for intracellular and plasma membrane staining, and points from all three experiments are plotted (75 points per condition) on the left box and whisker plot (minimum to maximum), where the center line represents the median, the box limits represent the interquartile range and the whiskers represent the minimum to maximum data range. Two-tailed unpaired t-test with 148 df; NS, not significant. The maximum length and width of cells from the experiment in e were measured, and the length-to-width ratios were calculated and plotted on the right box and whisker plot (minimum to maximum; bottom right), where the center line represents the median, the box limits represent the interquartile range and the whiskers represent the minimum to maximum data range. ARAF-S214P expression causes a significantly increased length/width ratio (****P = 9.57 × 10−15) and treatment with trametinib normalizes the ratio (****P = 7.51 × 10−17; two-tailed unpaired t-test with 148 df; NS, not significant). f, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay shows that ARAF-S214P leads to no increase in proliferation in transduced HDLECs. The metabolic activity is measured at two wavelengths (550 and 700 nm). The contents of triplicate wells (n = 3 independent wells) were collected at the indicated times, as described in the Methods. The trend lines connect the means for each transductant at each time point, and the points show the measured values for all data points. This experiment is representative of results from two independent retroviral transductions. The second experiment, performed independently, is shown in Extended Data Fig. 3. g–i, Top: an overview of the fish lymphatic system; the white frame indicates the area investigated in g–i, which shows an overlay (maximum intensity projection) of confocal scans. The TD and posterior cardinal vein (PCV) are labeled green (tg(mrc1a:EGFP)), and the TD is outlined by a dotted line in g–i. ARAF transgenic (mrc1a:araf) cells are marked red (green + red → yellow). g, ARAF-S214P expression leads to severe dilation of the TD. h, ARAF-WT (red) expression has no effect on the morphology of the TD and PCV (both green). i, Cobimetinib (1 uM) partially reverses dilation induced by the mutation. Three independent experiments were repeated with similar findings in g–i. j, Phenotype scoring categories of body segments for dilation with and without cobimetinib treatment: normal, moderate dilation (TD expanded but can be separated from the PCV) and severe dilation (TD and PCV overlapping). Cobimetinib treatment led to a significant reduction of severe dilation (****P = 1.33 × 10−5; one-tailed unpaired t-test; blue histograms) and rescue to normal morphology (***P = 0.00051; one-tailed unpaired t-test; green histogram). In three independent experiments, a total of 40 larvae and 120 body segments were analyzed. The data are shown as the mean ± s.e.m. of three independent experiments. Unprocessed blot images are available as source data.

1118 Nature Medicine | VOL 25 | JULY 2019 | 1116–1122 | www.nature.com/naturemedicine NATuRE MEDICInE Letters reductions in lymphatic fluid retention and supplemental oxygen doubling of his total lung capacity (TLC) and his forced expiratory requirements after three months of treatment, and he was able volume in 1 s (FEV1) improved from 23% to 42% predicted (Fig. 3a). to wean to room air with improved levels of physical activity and Electrolytes (low Na and K) normalized and his magnetic resonance without any adverse events being observed from trametinib. At imaging scan showed lymphatic remodeling with restructuring 12 months of therapy, his pulmonary function tests showed near of his lymphatic system (Fig. 3b–f), a remarkable recovery in an

a e ARAF-WTARAF-S214P

HEK293T Whole cell Anti-FLAG-IP lysate 1.5 DMSO FLAG (ARAF) 75 kDa ****

14-3-3 (Pan) 25 kDa 1.0

s.e. 100 µm 100 m

EV µ 37 kDa p-ERKs 0.5

l.e. 37 kDa ARAF-WT 50 kDa

ARAF-S214P 0.0 β-actin Normalized 14-3-3/FLAG ratio EV 100 nM EV trametinib ARAF-WT ARAF-S214P ARAF-WT ARAF-S214P

100 µm 100 µm b HEK293T

p-mTOR (S2448) *** 20 250 kDa ** *** **** DMSO EV 3 NS **** 8 DMSO 100 nM 75 kDa 15 FLAG (ARAF) ARAF-WT 100 nM trametinib 7 2 trametinib 10 NS 75 kDa 6 ARAF-S214P

p-p70s6K (T389) staining 1 5 5 4 0 Cell length/width 0 p-AKT (S473) 50 kDa 3 Membrane/intracellular l.e.

37 kDa Normalized ratio p-ERKs 2 ARAF-WT (T202/Y204) 1 ARAF-WT ARAF-WT ARAF-WT s.e. 37 kDa ARAF-S214P ARAF-S214P ARAF-S214P ARAF-S214P 0 p-p38 (T180)/Y182) 37 kDa 50 kDa f 1.0 -actin -actin -actin β-actin β β β-actin β β-actin ARAF-WT 0.8 ARAF-S214P FLAG/β-actin14-3-3/ p-p70/β-actinp-AKT/ p-p38/ p-mTOR/ p-ERKs/ Transfection: EV

700 nm 0.6 –A

ARAF-WT 0.4 nm

ARAF-S214P 550 0.2 A c 0.0 * * 0 24 48 72 96 120 * * Time (h) 8 * NS p-ERKs 37 KDa 6 ARAF-WT (T202/Y204) ARAF-S214P FLAG (ARAF) 75 KDa 4 300 m ERK 37 KDa µ Tg(mrc1a:EGFP) 2 [Trametinib] 30 nM 30 nM DMSO 100 nM300 nM DMSO 100 nM300 nM S214P 1,0003,000 nM nM 1,0003,000 nM nM mrc1a:ARAF WT 0 ghmrc1a:ARAF

Normalized p-ERKs/ERK ratio No treatment ARAF-WT ARAF-S214P 0 30 100 300 1,000 3,000 [Trametinib] (nM) TD TD d

ARAF-WT

TD 200 µm 200 µm 200 µm 200 µm TD PCV { 100 µm 100 µm

ARAF-S214P S214P ijmrc1a:ARAF Cobimetinib 200 µm 200 µm 200 µm 200 µm Trametinib 0 nM 30 nM 100 nM 300 nM *** 100 *** TD normal TD 80 *** TD moderate dilation

*** m)

50 µ *** 5,000 *** *** 60 TD severe dilation *** 40 40 *** 4,000 ** 20 30 ARAF-WT 3,000 ARAF-WT ARAF-S214P Percentage of segments 0 20 2,000 ARAF-S214P TD

No. of sprouts 10 1,000 PCV { Untreated Cobimetinib 0 0 100 µm

030 100 300 Cumulative sprout length ( 030 100 300 [Trametinib] (nM) [Trametinib] (nM)

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a d

Baseline Post therapy March 17, 2017 April 4, 2017 May 4, 2017 October 23, 2017 March 8, 2018 Parameter Unit Per % ref Per % ref Per % ref Per % ref Per % ref Weight kg 38 40 42 40 39 Height cm 142 142 143 145 148 FVC L 0.58 23 0.88 35 0.88 31 0.95 35 1.18 40

FEV1 L 0.52 23 0.77 34 0.72 31 0.85 35 1.09 42

FEV1/FVC % 89.8 105 87 102 89.3 105 89.0 105 92.0 108 FEF25–75 L/s 0.8 29 1.12 41 1.10 39 1.46 50 1.79 58 TLC L 0.93 29 1.23 38 1.28 39 1.51 45 1.98 56 RV L 0.27 31 0.35 40 0.48 55 0.56 62 0.80 86 e RV/TLC % 28.94 107 28 104 37.67 140 37 137 40 154 DLCO [Hb] ml min–1 mmHg–1 – – – – – – 9.99 54 9.9 52 DLCO/VA ml min–1 mmHg–1 – – – – – – 9.08 135 7.67 116

MIP cmH2O 51.6 71 – – 62.2 82 70.0 95 85.0 115 MEP cmH2O 69.4 66 – – 77.6 74 89.0 85 83.0 75 O2 Sat % 92 – 97 100 97

f 90 75 50 55 25

50 10 5 45 c 40 Weight (kg) 35

10 11 12 *13 14 15 Age (years)

Fig. 3 | Pulmonary function tests and clinical images in the lead proband before and after MEK inhibitor therapy. a, Results from pulmonary function tests before MEK inhibitor therapy (started on 17 March, 2017) and after therapy (April 2017–March 2018). Note the significant improvements in all spirometry measures, with FEV1 improving from 23% to 42% predicated value, TLC improving from 29% to 56% predicted value and maximal inspiratory pressure (MIP) improving from 71% to 115% predicted value (marked in red). FVC, forced vital capacity; FEF25–75, mid forced expiratory flow rates; RV, residual volume; RV/TLC, ratio of RV to TLC; DLCO [Hb], diffusing capacity of the lung for carbon monoxide corrected for hemoglobin; DLCO/VA,

DLCO divided by the alveolar volume (VA); MEP, maximal expiratory pressure; O2 Sat, oxygen saturation. b, Coronal maximal intensity projections of a T2-weighted non-contrast lymphangiogram just before initiation of medical therapy (left) and 12 months after MEK inhibitor therapy began (right) demonstrate near-complete resorption of massively dilated and beading subcutaneous lymphatic ducts. c, Coronal maximal intensity projections of contrast lymphangiograms of the pelvis and chest before treatment (left) demonstrate paucity of central lymphatic ducts, lack of central lymphatic flow above the diaphragm, and massively dilated and beading bilateral subcutaneous ducts coursing along the abdominal wall. Twelve months after the start of treatment (right) there is resorption of the dilated subcutaneous ducts, with formation of new, more normal-appearing lymphatic networks now extending along the abdominal wall and into the chest. d, Coronal maximal intensity projections of contrast lymphangiograms of the pelvis and thighs before treatment (left) also demonstrate paucity of ducts in the thighs and massive dilation and beading of the lymphatic ducts. After treatment (right), again there is resorption of the abnormal dilated ducts and formation of new and more normal appearing lymphatic networks. e, Chest X-rays before (left) and 12 months after treatment (right) showing reduced effusions and notably improved lung volumes. f, The patient’s growth chart (left). Treatment with trametinib was initiated just before age 13 (*) and improvement in lymphedema and clinical status was observed starting approximately 3–6 months after initiation of treatment. A picture of the patient’s lower extremities immediately after removal of compression stockings at his peak weight is shown at the top right. The bottom right image shows the corresponding picture of the lower extremities immediately after removal of compression stockings shortly after his most recent clinic visit. individual who was frequently hospitalized before initiation of this lymphatic disease unresponsive to sirolimus therapy. HDLECs genetically guided therapy (Fig. 3f). transduced with the mutant ARAF showed elevated ERK1/2 In sum, we performed WES for two unrelated patients with activity, enhanced lymphangiogenic capacity, and disassembly of lymphatic anomaly and identified a recurrent GoF mutation in actin skeleton and VE-cadherin junctions, which were rescued the ARAF gene, including in a 12-year-old male with an advanced using the MEK inhibitor trametinib. Sprouting was observed

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Table 1 | Mutations identified in individuals with lymphatic disorders Patient Gene Mutation Origin Gender Age Main phenotypic features 3 KRAS NM_004985.4:c.35G>A:p.(G12D) Somatic Male 26 years Lymphatic abdominal anomaly and protein-losing enteropathy 4 BRAF NM_004333.4:c.1403T>C:p.(F468S) Germline Male 2 months Lymphatic malformation with cardiofaciocutaneous syndrome and chylothorax 5 RASA1 NM_002890.2:c.475_476del:p.(L159Gfs*20) Germline Female 18 months Lymphatic disorder with chylous de novo pericardial effusion and non- immune hydrops 6 SOS1 NM_005633.3:c.2536G>A:p.(E846K) Germline Female 14 years Noonan syndrome with de novo lymphatic malformation and left pleural effusion 7 PTPN11 NM_002834.3:c.1504T>G:p.(S502A) Germline Male 10 months Noonan syndrome with de novo lymphatic disorder and chronic severe chylothorax 8 PTPN11 NM_002834.3:c.1510A>G:p.(M504V) Germline Male 5 years Noonan syndrome with lymphatic disorder and acute onset of chylous ascites 9 PTPN11 NM_002834.3:c.1530G>C:p.(Q510H) Germline Male 17 days LEOPARD syndrome with de novo lymphangiectasia and respiratory distress

in ARAF-S214P-expressing HDLECs in the absence of VEGFC of the ARAF mutation we uncovered, using the cBioPortal39 data- (a potent lymphangiogenic factor)18. Under the same condi- base (n = 71,857 subjects and queried on February 6, 2019), reveals tions, sprouting was absent in cells expressing ARAF-WT. This 2 patients with the same exact mutation in ARAF. Interestingly, suggests that the ARAF mutant is mimicking the stimulatory they both have concurrent TP53 mutations, which are considered behavior of VEGFC or inducing the expression of VEGFC by the as oncogenic drivers. Different mutations at this residue (S214T, HDLECs, which is necessary for endothelial cell sprouting, as seen S214A, S214Y, S214C and S214F), three of which have been shown in many stromal cell types19–21. Further experiments would be to result in elevated MEK/ERK phosphorylation40, were also required to distinguish these possibilities. We reproduced the observed in ten patients with different types of cancer. However, anomalous lymphatic phenotype, which is attributed to a GoF nine out of ten patients have co-occurring oncogenic mutations in mutation in ARAF, in a zebrafish model observing rescue of the TP53, GNAS, AKT2, APC, EGFR, ATM, CHEK2, KIT or U2AF1, phenotype using MEK inhibitor therapy. Remarkably, therapy of raising the possibility that these oncogenic drivers may be respon- the lead proband with the ARAF mutation using trametinib resulted sible for the excessive proliferation in cancer cells. The lead pro- in dramatic improvement in patient symptoms, with remodeling of band with the ARAF mutation has dilated lymphatic vessels but the his dilated and torturous lymphatic vasculature, resolution of the lesion shows no increase in size over years of follow-up. Thus, these lymphatic edema and resumption of regular daily activities within data are consistent with our observation that the ARAF mutation 12 months of therapy. we uncovered may not drive increased proliferation in lymphatic From ongoing patient recruitment, we investigated additional endothelial cells in vitro. lymphatic anomaly patients, including patients with Noonan Regarding the prevalence of mutation-positive lymphatic anom- (or Noonan-related) syndrome, Gorham–Stout disease, kaposi- alies, among 11 centers in the USA forming a lymphatic anomaly form lymphangiomatosis (KLA), lymphangiectasia and CCLA. consortium to facilitate multi-center clinical trials for this group of On sequencing 43 additional patients, we identified 7 additional lymphatic anomalies, including but not limited to GLA, Gorham– mutations in KRAS, BRAF, RASA1, PTPN11 and SOS1 (Table 1), Stout disease, CCLA, KLA, Klippel–Trenaunay syndrome and kapo- suggesting that the RAS–MAPK signaling is a common pathway siform hemangioendothelioma, there are more than 3,000 patients responsible for the various clinical lymphatic disease manifes- recruited with moderate to severe disease course, and the number of tations. Indeed, it has been increasingly acknowledged that the new patients per year is about 300 combined. Based on the current RAS–MAPK pathway plays a key role in the signaling of lym- molecular diagnostic yield (20%), we anticipate that about 20% of phangiogenesis21–23. Reviewing the literature, we identified more them will have defects in the RAS–MAPK pathway, suggesting that than 50 patients who have mutations in KRAS, HRAS, BRAF, a few thousand patients overall in the USA may benefit from MEK RAF1, PTPN11, SHOC2, CBL, RIT1 and SOS1, and present inhibitor therapy. Thus, our work exemplifies how genetic discover- clinical features of Noonan or Noonan-related syndromes with ies can impact disease classification and uncover novel biological lymphatic defects, including pleural effusion, pericardial effusions, and life-saving treatments as represented here in a patient with lym- chylothorax, hydrops, lymphangiectasis and lymphedema24–36. phatic anomaly of a previously unknown etiology, a realization of a While our work was in progress, a recurrent NRAS variant was precision medicine approach. implicated in GLA37 and also in KLA38, lending further support for the shared genetic etiology between these disease entities and Online content the importance of mutations in the RAS–MAPK pathway in lym- Any methods, additional references, Nature Research reporting phatic anomalies. summaries, source data, statements of code and data availability and The widespread prevalence of mutations in RASopathies in associated accession codes are available at https://doi.org/10.1038/ human cancer has been recognized for decades. A close scrutiny s41591-019-0479-2.

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Received: 8 May 2018; Accepted: 6 May 2019; 29. Koenighofer, M. et al. Mutations in RIT1 cause Noonan syndrome Published online: 1 July 2019 - additional functional evidence and expanding the clinical phenotype. Clin. Genet. 89, 359–366 (2016). 30. Lee, K. A. et al. PTPN11 analysis for the prenatal diagnosis of Noonan References syndrome in fetuses with abnormal ultrasound fndings. Clin. Genet. 75, 1. Trenor, C. C. 3rd & Chaudry, G. Complex lymphatic anomalies. Semin. 190–194 (2009). Pediatr. Surg. 23, 186–190 (2014). 31. Croonen, E. A. et al. Prenatal diagnostic testing of the Noonan syndrome 2. Collins, F. S. & Varmus, H. A new initiative on precision medicine. genes in fetuses with abnormal ultrasound fndings. Eur. J. Hum. Genet. 21, N. Engl. J. Med. 372, 793–795 (2015). 936–942 (2013). 3. Adams, D. M. et al. Efcacy and safety of sirolimus in the treatment of 32. Joyce, S. et al. Te lymphatic phenotype in Noonan and cardiofaciocutaneous complicated vascular anomalies. Pediatrics 137, e20153257 (2016). syndrome. Eur. J. Hum. Genet. 24, 690–696 (2016). 4. Hammill, A. M. et al. Sirolimus for the treatment of complicated vascular 33. Yaoita, M. et al. Spectrum of mutations and genotype–phenotype analysis anomalies in children. Pediatr. Blood Cancer 57, 1018–1024 (2011). in Noonan syndrome patients with RIT1 mutations. Hum. Genet. 135, 5. McCormick, A., Rosenberg, S., Trier, K. & Balest, A. A case of a central 209–222 (2016). conducting lymphatic anomaly responsive to sirolimus. Pediatrics 137, 34. Lo, I. F. et al. Severe neonatal manifestations of Costello syndrome. e20152694 (2016). J. Med. Genet. 45, 167–171 (2008). 6. Hilliard, R. I., McKendry, J. B. & Phillips, M. J. Congenital abnormalities 35. Ebrahimi-Fakhari, D. et al. Congenital chylothorax as the initial of the lymphatic system: a new clinical classifcation. Pediatrics 86, presentation of PTPN11-associated Noonan syndrome. J. Pediatr. 185, 988–994 (1990). 248–248.e1 (2017). 7. Levine, C. Primary disorders of the lymphatic vessels—a unifed concept. 36. Morcaldi, G. et al. Lymphodysplasia and Kras mutation: a case report and J. Pediatr. Surg. 24, 233–240 (1989). literature review. Lymphology 48, 121–127 (2015). 8. Smeltzer, D. M., Stickler, G. B. & Fleming, R. E. Primary lymphatic dysplasia 37. Manevitz-Mendelson, E. et al. Somatic NRAS mutation in patient with in children: chylothorax, chylous ascites, and generalized lymphatic dysplasia. generalized lymphatic anomaly. Angiogenesis 21, 287–298 (2018). Eur. J. Pediatr. 145, 286–292 (1986). 38. Barclay S. F. et al. A somatic activating NRAS variant associated with 9. Wassef, M. et al. Vascular anomalies classifcation: recommendations from kaposiform lymphangiomatosis. Genet. Med. https://doi.org/10.1038/ the International Society for the Study of Vascular Anomalies. Pediatrics 136, s41436-018-0390-0 (2018). e203–e214 (2015). 39. Gao, J. et al. Integrative analysis of complex cancer genomics and clinical 10. Chen, W., Adams, D., Patel, M., Gupta, A. & Dasgupta, R. Generalized profles using the cBioPortal. Sci. Signal. 6, pl1 (2013). lymphatic malformation with chylothorax: long-term management of a highly 40. Imielinski, M. et al. Oncogenic and sorafenib-sensitive ARAF mutations in morbid condition in a pediatric patient. J. Pediatr. Surg. 48, e9–e12 (2013). lung adenocarcinoma. J. Clin. Invest. 124, 1582–1586 (2014). 11. Lala, S. et al. Gorham–Stout disease and generalized lymphatic anomaly— clinical, radiologic, and histologic diferentiation. Skeletal Radiol. 42, 917–924 (2013). Acknowledgements 12. Clemens, R. K., Pfammatter, T., Meier, T. O., Alomari, A. I. & Amann-Vesti, B. We thank all of the families involved in this study for their participation. We gratefully R. Combined and complex vascular malformations. Vasa 44, 92–105 (2015). acknowledge L. Klepper, T. Ferry and J. Kelly, who helped with the collection of the 13. Li, D. et al. Pathogenic variant in EPHB4 results in central conducting DNA samples and clinical data on patient P2. Research reported in this publication lymphatic anomaly. Hum. Mol. Genet. 27, 3233–3245 (2018). was supported in part by the Roberts Collaborative Functional Genomics Rapid 14. Wellbrock, C., Karasarides, M. & Marais, R. Te RAF proteins take centre Grant (to D.L.) from CHOP, Institutional Development Funds (to H.H.) from CHOP, stage. Nat. Rev. Mol. Cell Biol. 5, 875–885 (2004). CHOP’s Endowed Chair in Genomic Research (H.H) and donation from the Adele and 15. Lavoie, H. & Terrien, M. Regulation of RAF protein kinases in ERK Daniel Kubert family (to H.H. and CAG). The study was also funded in part through a signalling. Nat. Rev. Mol. Cell Biol. 16, 281–298 (2015). sponsored research agreement from Aevi Genomic Medicine Inc., funding discovery and 16. Molzan, M. et al. Impaired binding of 14-3-3 to C-RAF in Noonan syndrome translation of rare and orphan disease genes at the CAG. suggests new approaches in diseases with increased Ras signaling. Mol. Cell Biol. 30, 4698–4711 (2010). Author contributions 17. Jung, H. M. et al. Development of the larval lymphatic system in zebrafsh. H.H. designed and supervised all aspects of the study. D.L. conducted the analysis and Development 144, 2070–2081 (2017). writing of the study. D.L., L.T., T.W., C.N.K., P.M.A.S. and H.H. arranged and performed 18. Karkkainen, M. J. et al. Vascular endothelial growth factor C is required for genomic testing/analysis. M.E.M., A.G.-U., C.K., C.S., L.S.M. and M.R.B contributed sprouting of the frst lymphatic vessels from embryonic veins. Nat. Immunol. the functional investigations. E.P., E.J.B., T.L.W., J.A.P., M.A.L., P.J.H., J.S., J.B.B., Y.D. 5, 74–80 (2004). and H.H. contributed the clinical phenotyping and treatment. B.M.W., M.S. and H.M.J. 19. Carmeliet, P. & Jain, R. K. Molecular mechanisms and clinical applications of provided the zebrafish line. N.R. and R.C. coordinated research study subject enrollment. angiogenesis. Nature 473, 298–307 (2011). D.L., M.E.M., A.G.-U., C.S., C.N.K, R.C., J.A.P., M.A.L., P.M.A.S., Y.D. and H.H. read, 20. Karaman, S., Leppanen, V. M. & Alitalo, K. Vascular endothelial growth factor edited and approved of the manuscript, along with all other authors. signaling in development and disease. Development 145, dev151019 (2018). 21. Potente, M. & Makinen, T. Vascular heterogeneity and specialization in development and disease. Nat. Rev. Mol. Cell Biol. 18, 477–494 (2017). Competing interests 22. Coso, S., Bovay, E. & Petrova, T. V. Pressing the right buttons: signaling in H.H. is a scientific advisor to Aevi Genomic Medicine Inc. and he and CHOP own shares lymphangiogenesis. Blood 123, 2614–2624 (2014). in the company. The other authors declare no competing interests. 23. Brouillard, P., Boon, L. & Vikkula, M. Genetics of lymphatic anomalies. J. Clin. Invest. 124, 898–904 (2014). 24. Bulow, L. et al. Hydrops, fetal pleural efusions and chylothorax in three Additional information patients with CBL mutations. Am. J. Med. Genet. A 167A, 394–399 (2015). Extended data is available for this paper at https://doi.org/10.1038/s41591-019-0479-2. 25. Gargano, G. et al. Hydrops fetalis in a preterm newborn heterozygous for the Supplementary information is available for this paper at https://doi.org/10.1038/ c.4A>G SHOC2 mutation. Am. J. Med. Genet. A 164A, 1015–1020 (2014). s41591-019-0479-2. 26. Gos, M. et al. Contribution of RIT1 mutations to the pathogenesis of Noonan syndrome: four new cases and further evidence of heterogeneity. Am. J. Med. Reprints and permissions information is available at www.nature.com/reprints. Genet. A 164A, 2310–2316 (2014). Correspondence and requests for materials should be addressed to H.H. 27. Hanson, H. L. et al. Germline CBL mutation associated with a Noonan-like Peer review information: Kate Gao and Brett Benedetti were the primary editors on this syndrome with primary lymphedema and teratoma associated with acquired article and managed its editorial process and peer review in collaboration with the rest of uniparental isodisomy of chromosome 11q23. Am. J. Med. Genet. A 164A, the editorial team. 1003–1009 (2014). 28. Milosavljevic, D. et al. Two cases of RIT1 associated Noonan syndrome: Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in further delineation of the clinical phenotype and review of the literature. published maps and institutional affiliations. Am. J. Med. Genet. A 170, 1874–1880 (2016). © The Author(s), under exclusive licence to Springer Nature America, Inc. 2019

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Methods MV 2 (Promocell) according to the manufacturer’s directions. The full-length 41 Patients. Afer obtaining approval from the Institutional Review Board at Te ARAF cDNA obtained from Addgene (plasmid no. 23725) was amplified from Children’s Hospital of Philadelphia (CHOP) and written informed consent, the original vector and cloned as a BamHI/XhoI fragment into the pcDNA3.1 blood specimens from the lead proband (P1) and his parents were obtained for vector that contains two copies of the FLAG tag (DYKDDDDK), followed by two sequencing analysis. Te proband had severe accumulation of lymphatic fuid in STREP tags (WSHPQFEK). The S214P mutation was introduced by site-directed his chest, pericardium, abdomen, lower extremities and genitalia and was being mutagenesis using the Q5 mutagenesis kit from NEB following the manufacturer’s followed and treated at the Center for Lymphatic Imaging and Interventions at instructions. Transfections in HEK293T and HeLa were performed using Fugene CHOP. An unrelated second adult patient (P2) was recruited through the Patient HD (Promega), with 3 μg DNA (empty vector, WT ARAF (ARAF-WT) or ARAF Registry of the Lymphangiomatosis & Gorham’s Disease Alliance (LGDA), together mutant (ARAF-S214P)) and 9 μl of the transfection reagent, according to the with available family members. manufacturer’s protocols. At 36–48 h after transfection, cells were washed twice Birth and family history for P1 were unremarkable except for a capillary with ice-cold phosphate-buffered saline (PBS) and lysed on ice using a freshly malformation on the left side of his abdomen and his childhood growth and prepared ice-cold cell lysis buffer containing 50 mM Tris-HCl, pH 7.4, 100 mM development milestones were normal. At age 10 years, he developed swelling of his NaCl, 50 mM β-glycerophosphate, 10% glycerol (w/v), 1% NP-40 (w/v), 1 mM lower abdomen, thighs, scrotum and penis. Two months later, he presented to a EDTA, 2 mM NaVO4 and a complete, EDTA-free protease inhibitor cocktail local hospital with shortness of breath and exercise intolerance. A chest radiograph (Roche Applied Science) at 20 μl per millilitre of lysis buffer. After clearing the cell demonstrated cardiomegaly and echocardiogram revealed a large pericardial lysates by centrifugation, the supernatants were collected and used for western effusion. Pericardiocentesis was performed with drainage of 1 l of chylous fluid. blotting or immunoprecipitation with Anti-FLAG M2 Affinity Gel (cat. no. A2220, Despite institution of total parenteral nutrition, the drainage continued and he Sigma) followed by western blotting. Immunoprecipitates and lysates were run was transferred to CHOP for further management. At CHOP, his initial evaluation on NuPAGE 4–12% Bis-Tris gels (Thermo Fisher Scientific) and blotted with included dynamic contrast-enhanced magnetic resonance lymphangiography primary antibodies including anti-phospho-p70S6K-Thr389 (cat. no. 9205S, Cell that demonstrated large pericardial effusion and antegrade flow in dilated lumbar Signaling Technology; 1:1,000), anti-phospho-mTOR Ser2448 (cat. no. 5536P, Cell and retroperitoneal networks into a dilated and tortuous TD coursing towards Signaling Technology; 1:1,000), anti-FLAG (cat. no. F3165, Sigma; 1:4,000), anti- the innominate vein on the left (Fig. 1a,c,d). An image of an unaffected person is phospho-p38 Thr180/Tyr182 (cat. no. 4511, Cell Signaling Technology; 1:1,000), shown in Fig. 1b as a reference. In addition, there was retrograde lymphatic flow anti-PAN-14-3-3 (cat. no. sc-629, Santa Cruz Biotechnology; 1:500), anti-phospho- into the liver, mesentery, penis and scrotum, and from the distal TD there was Akt-Ser473 (cat. no. 4060, Cell Signaling Technology; 1:1,000), anti-phospho- retrograde flow into the mediastinum and pericardium (Fig. 1c–e). He underwent p44/42-(Erk1/2)-Thr202/Tyr204 (cat. no. 4376, Cell Signaling Technology; 1:1,000) placement of a stent in the distal TD and Lipiodol embolization with the aim of or anti-β-actin (cat. no. sc-69879, Santa Cruz Biotechnology; 1:1,000) antibodies. stopping the abnormal mediastinal and pericardial lymphatic effusion. He was The ARAF sequence, from pCDNA3.1-F2S2-ARAF-WT or -S214P constructs as discharged after a month with a stable pericardial effusion but then presented previously indicated, was cut with BamHI/XhoI and introduced into the BglII/ shortly thereafter in respiratory distress due to large fluid re-accumulation that XhoI sites of a modified version of the pMSCV plasmid that contains amino- necessitated an increasing requirement for supplemental oxygen (up to 5 l by terminal FLAG and HA tags. Viral production was performed using Fugene, with nasal cannula). He was started on sirolimus and was dosed based on trough levels 8 μg of total DNA (pMSCV-ARAF-WT or -S214P together with envelope and tolerated well on a dose of 2.5 mg per day, which resulted in a median trough packaging plasmids) and 18 μl of the transfection reagent in HEK293T. After 72 h, level of 11.8 between May and November 2016 (range 6.8–16.2 μg dl−1). Over viral supernatant was collected and filtered. HDLECs were infected by replacing 1 the course of 1.5 years, he underwent multiple percutaneous interventional and the cell culture medium with the viral supernatant, supplemented with 8 μg ml− surgical lymphatic procedures, including repetitive thoracentesis and pleural Polybrene and filtered through a 0.45 μm filter. Cells were spinfected at 650g for drains, multiple percutaneous lymphatic embolizations, bilateral surgical 90 min, and subsequently cultured for 6 h at which point the viral supernatant was pleurodesis twice, surgical lymphovenous anastomosis in his thighs, abdomen and replaced by standard culture medium. Transduced HDLECs were cultured for 48 h retroperitoneum and, due to worsening penile and scrotal edema, surgical ligation before use in experiments. Transduction efficiencies observed by HA staining were and embolization of groin lymph channels. Despite multiple attempts to control between 40% and 60%. his pericardial effusions, his penile, scrotal, lower extremity and lower abdominal lymphedema worsened and his condition continued to deteriorate to the point that Immunofluorescence staining and western blotting of HDLECs. Round (12 mm) consideration of palliative care was discussed. The last procedure was performed coverslips (VWR) were coated with 0.1% gelatin in water for 10 min in 24-well and sirolimus was discontinued five months before trametinib began in March plates (Corning), and then air-dried for 15 min. Transduced HDLECs were plated 2017 (Fig. 3a). at 100,000 cells per well in 0.5 ml of culture medium in the presence or absence of Patient P2, an unrelated adult female, was diagnosed with lymphangiomatosis trametinib for 48 h. Cells were washed in warm serum-free Dulbecco’s modified at the age of 31 in 2012 before the International Society for the Study of Vascular Eagle’s medium and fixed in 4% paraformaldehyde. Fixed cells were washed twice Anomalies classification was established in 2015. She had extensive symptoms with PBS and twice with 0.1% BSA in PBS. Cells were permeablized and blocked for many years before her diagnosis with prominent pulmonary involvement by incubation with 10% normal donkey serum (Jackson Immunoresearch) and and required multiple pleurocentesis procedures before pleurodesis. She had 0.3% Triton X-100 (Sigma Aldrich) in PBS. VE-cadherin antibody (Thermo Fisher widespread involvement of her gastrointestinal tract, requiring a specialized Scientific) was diluted (final concentration: 2 μg ml−1) in 0.01% normal donkey fat-restricted diet and medium-chain triglyceride oil supplementation with serum, 0.1% BSA and 0.3% Triton X-100 in PBS, and staining was performed intermittent total parenteral nutrition. She underwent computed tomography and for 1 h. Coverslips were washed twice with 0.1% BSA in PBS. Goat-anti-rabbit magnetic resonance imaging after persistent unexplained symptoms, which were Alexa546 (Thermo Fisher Scientific; final concentration: 8 μg ml−1) and phalloidin consistent with lymphangiomatosis affecting her kidneys, liver, spleen and lungs. Alexa350 (Thermo Fisher Scientific; final concentration: 5 units ml−1) were diluted A liver biopsy confirmed the diagnosis of lymphangiomatosis. She was additionally in 0.01% normal donkey serum, 0.1% BSA and 0.3% Triton X-100 in PBS, and treated with albuterol and diuretics and used a motorized scooter because of staining was performed for 1 h. When used, HA-Tag (6E2) mouse antibody fatigue and dyspnea. She was never confirmed to have bone involvement. As the (cat. no. 2367, Cell Signaling Technology) was diluted 1:100 in 0.1% BSA and patient was recruited from the Lymphangiomatosis & Gorham’s Disease Alliance 0.3% Triton X-100 in PBS, and staining was performed for 1 h. Coverslips were and was not local, she was lost to follow-up and was not available for a trial of other washed twice with 0.1% BSA in PBS and twice with PBS. Coverslips were dipped in therapies (we later learned she had died in 2017 from complications related to her water to remove residual salts, and mounted to slides using Prolong Gold antifade underlying lymphatic disorder). reagent (Thermo Fisher Scientific). Image acquisition was performed on a Leica DM6000 motorized upright microscope with a Photometrics HQ2 high-resolution WES and bioinformatics analysis. We examined missense, nonsense, splice- monochrome CCD (charge-coupled device) camera using LAS AF software altering and coding indels matching either the dominant or recessive inheritance (Leica Microsystems). Z-stacks were acquired at ×10 magnification. Images models in the exome data. Results were filtered to exclude variants with the were further processed in the Fiji software package42. Brightness and contrast following factors: synonymous variants; variants in known pseudogenes; variants adjustments were made. Identical brightness and contrast settings were applied with a minor allele frequency (MAF) greater than 0.5% in either the 1000 Genomes to all images. Fluorescence values were measured in regions of interest (ROIs) Project or the 6,503 exomes from the National Heart, Lung, and Blood Institute drawn to contain entire individual cells, or in ROIs drawn to contain the entire Exome Sequencing Project (ESP6500SI); variants previously identified in controls cell body but exclude the cell–cell junction. From those measured values, a value by our in-house exome variant database. Subsequent gene prioritization was for the plasma membrane was derived (total cell − intracellular), and the ratio of performed on the basis of deleterious prediction and biological relevance by plasma membrane to intracellular values was derived and plotted. Additionally, the referring to the Online Mendelian Inheritance in Man database. length and width of cells were measured with the line tool and ROI manager. For both analyses, five clearly ARAF-expressing cells, as determined by HA staining, Expression and characterization of ARAF mutation in mammalian cell were analyzed per ×10 field. Five ×10 fields were acquired per condition per lines. HEK293T and HeLa cells were obtained from the American Type experiment. Experiments were conducted with cells from 3 independent thaws and Culture Collection and grown at 37 °C in Dulbecco’s modified Eagle’s medium transductions of HDLECs, for a total of 75 cells per condition. For western blotting supplemented with 10% fetal bovine serum. Primary adult HDLECs were of HDLECs with trametinib, 20,000 transduced HDLECs cells were plated into obtained from Promocell, and were cultured in Endothelial Cell Growth Medium 96-well plates in the presence of increasing amounts of trametinib. Cells were

Nature Medicine | www.nature.com/naturemedicine Letters NATuRE MEDICInE cultured for 24 h in the presence of the drug, and then lysed with 40 mM HEPES Inhibitory drug treatment in zebrafish. Drug treatments were performed in pH 7.5, 120 mM NaCl, 0.3% CHAPS, 50 mM NaF, 1.5 mM NaVO3 and a protease 6-well plates with up to 20 larvae per group. Cobimetinib was diluted in embryo inhibitor cocktail. Lysates were cleared by centrifugation at 20,000g for 5 min medium containing 0.01 M Tris pH 7.2 and 0.1% DMSO. Cobimetinib was used at 4 °C. Proteins were separated on 4–12% NuPAGE Bis-Tris gels. Blotting was at 1 μM. performed using the antibodies described above. p-ERK antibody staining in zebrafish. Fish were injected as described above and Three-dimensional lymphatic spheroid sprouting assay. Multicellular spheroids larvae with prominent WT or mutant ARAF/mcherry expression were selected for the lymphatic sprouting assay were initiated by seeding 7,500 HDLECs for analysis. Larvae were fixed overnight in a 4% paraformaldehyde solution in expressing ARAF-WT or ARAF-S214P into wells of a 96-well plate that were PBS with Tween-20 (PBST). Larvae were washed with PBST and incubated in 2% precoated with 1.5% agarose. Under these conditions, all of the HDLECs would Triton X-100 for 24 h at 4 °C. Then, larvae were blocked in 10% bovine serum and aggregate into a single spheroid by 24 h. After formation, each spheroid was stained with phospho-ERK T202/Y204 antibody (cat. no. 9101, Cell Signaling transferred into a gelling solution comprised of type I collagen (cat. no. 354236, Technology, 1:200) overnight at 4 °C, washed with PBST and stained with Alexa Corning; final concentration = 1.5 mg ml−1; pH neutralized with NaOH) and Fluor 488 goat anti-rabbit secondary antibody (cat. no. A11008, Thermo Fisher trametinib at the indicated concentrations, which was then allowed to polymerize Scientific, 1:400). at 37 °C. Once solidified, Endothelial Cell Growth Medium MV 2 (without VEGFC) containing trametinib at the appropriate concentration was added onto Statistics. For all of the cell-based assays, significance was assessed by unpaired, the collagen gels. After 2 days of incubation, z-stack images with a step size of two-tailed Student’s t-tests for comparison of two groups. Statistical analysis ~8.5 μm were taken of the embedded spheroids using an EVOS FL Auto Imaging was performed with GraphPad Prism 7.0d software. The data are represented System (Thermo Fisher Scientific). The numbers and lengths of capillary-like as box-and-whisker plots with boxes ranging from the 25th to 75th percentile, sprouts growing from each spheroid were measured using the software ImageJ whiskers from the minimum to maximum and the median as the center, or as (https://imagej.nih.gov/ij/). dot plots with bar graphs for mean ± s.e.m., as indicated. For all of the assays performed on HDLECs, three independent experiments were performed with MTT proliferation assay with transduced HDLECs. Proliferation of transduced independent transductions of HDLECs, except for the proliferation study, where HDLECs was measured using Cell Proliferation Kit I (MTT) from Roche Applied no statistical analysis was performed. For the 14-3-3 protein association assay, Science. Briefly, at 2 d post retroviral transduction, ARAF-WT- and -S214P- three independent experiments were performed with independent transfection of expressing HDLECs were collected, counted and replated into flat-bottom 96-well HEK293T cells, while other results for HEK293T cells represent six independent plates at 10,000 cells per well in 100 μl of medium. At the indicated times after experiments. All of the zebrafish-related assays were performed in three plating, 10 μl of the MTT was added to the appropriate wells, and incubated for independent experiments and tested by unpaired, one-tailed Student’s t-tests for 4 h at 37 °C. A 100 μl volume of the solubilization reagent was added followed by comparison of two groups. overnight incubation at 37 °C. Absorbance at 550 nm and 700 nm was measured on a Spectramax i3 Multi Mode plate reader (Molecular Devices), and A550 nm–A700 nm Reporting Summary. Further information on research design is available in the was calculated. A time point of 4 h after plating was included as an approximate Nature Research Reporting Summary linked to this article. measure of cells loaded into the experiment with minimal proliferation. Data availability Transgenic expression of human ARAF in zebrafish. All procedures using WES data have been deposited in dbGaP with the accession number zebrafish were approved by the Institutional Animal Care and Use Committee of phs001802.v1.p1. CHOP (IAC 001154) and were in accordance with the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. Human mutant and WT ARAF cDNAs were cloned without stop codons into the pDONR221 vector; References a zebrafish-adapted kozak sequence (GCAAACATGG) was used43. Expression 41. Johannessen, C. M. et al. COT drives resistance to RAF inhibition through constructs were assembled using a Tol2 backbone vector including a gateway MAP kinase pathway reactivation. Nature 468, 968–972 (2010). cloning cassette44,45. Constructs were co-injected with Tol2 messenger RNA46. 42. Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. ARAF was expressed in vein and lymphatic vessels using the zebrafish mrc1a Nat. Methods 9, 676–682 (2012). promoter, and expression was visualized by mCherry linked to ARAF by an 43. Grzegorski, S. J., Chiari, E. F., Robbins, A., Kish, P. E. & Kahana, A. Natural autocatalytic V2a protein cleavage site. For imaging, larvae were mounted in low- variability of Kozak sequences correlates with function in a zebrafsh model. melting agarose, and multiple Z-images were taken with a Zeiss LSM710 confocal PLoS One 9, e108475 (2014). microscope using a ×20 lens. Confocal z-stacks of images were superimposed 44. Kwan, K. M. et al. Te Tol2kit: a multisite gateway-based construction using Zeiss Zen software’s maximum intensity projection function. To analyze kit for Tol2 transposon transgenesis constructs. Dev. Dyn. 236, dilation of the TD, body segments separated by intersegmental lymphatic vessels 3088–3099 (2007). with expression of the transgene in the TD were selected. Morphology was scored 45. Villefranc, J. A., Amigo, J. & Lawson, N. D. Gateway compatible vectors for as normal (WT), moderate dilation (TD expanded but separate from the PCV) or analysis of gene function in the zebrafsh. Dev. Dyn. 236, 3077–3087 (2007). severe dilation (TD and PCV not distinguishable in Z-projections). Images were 46. Kawakami, K. & Shima, A. Identifcation of the Tol2 transposase of the compiled in ImageJ (Fiji). Each experiment was performed 3 times, and a total of medaka fsh Oryzias latipes that catalyzes excision of a nonautonomous Tol2 40 animals were analyzed. element in zebrafsh Danio rerio. Gene 240, 239–244 (1999).

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Extended Data Fig. 1 | Western blot analysis of ARAF overexpression in HeLa cells. It demonstrates the phosphorylation status of ERK1/2, AKT, p70S6K, mTOR and p38 in HeLa cells after serum deprivation or serum deprivation followed by a short stimulation with 10% fetal bovine serum (FBS). Normalized ratios are illustrated by the panel on the bottom. The data are shown as the mean ± s.e.m. (error bars) of five independent experiments. Two-tailed unpaired t-test with 8 df. *P = 014; ****P = 7.7 × 10−5. The images were cropped for better presentation.

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Extended Data Fig. 2 | Cell morphology study in primary HDLECs. Primary HDLECs transduced with ARAF-WT or ARAF-S214P were plated in the presence or absence of 100 nM trametinib. Cells were fixed and stained for VE-cadherin and actin. Here are the full ×10 magnification fields. a, VE-cadherin staining. b, Actin staining of the same ×10 field shown in a. c, Zoomed-in views of the regions with the red boxes in b. Zoomed-in views are presented for improved visualization of the actin filaments.

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Extended Data Fig. 3 | MTT assay demonstrating that the ARAF-S214P mutation does not enhance proliferation. There is no increase in proliferation observed in transduced HDLECs from an independent experiment. The contents of triplicate wells (n = 3 independent wells) were collected at the indicated times as described in the Methods. The trend lines connect the means for each transductant at each time point, and the points show the measured values for all data points. This experiment is the second of two representative experiments, as described in Fig. 2f.

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Extended Data Fig. 4 | Examples of dilated lymphatic vessels induced by ARAF-S214P. a, The construct used for transgene expression. b,c, ARAF-S214P expression (red) leads to dilation of the intersegmental lymphatic vessel (ISLV, b) and the parachordal line (PL, c). Scale bar, 200 μm.

Nature Medicine | www.nature.com/naturemedicine NATuRE MEDICInE Letters

Extended Data Fig. 5 | ARAF-S214P expression in zebrafish induces phosphorylation of ERK. Staining with p-ERK antibody is labeled green, and transgene expression is labeled red; the bottom panel shows an overlay of the staining. a, For ARAF-WT the area of the TD is outlined by dotted lines; that of the PCV is outlined by dashed lines. Red ARAF-WT transgenic cells (arrows in a) and cells without transgene expression do not show significantly different p-ERK levels. b, Expression of ARAF-S214P causes expansion and fusion of the TD and PCV (outlined by dotted lines). (We observed comparable findings in n = 10 larvae.) Scale bar, 100 μm.

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Extended Data Fig. 6 | WT larvae show good tolerance following treatment with 1 μM cobimetinib from 3 to 7 dpf. a,b, The treatment does not affect the overall morphology and development (b) in WT zebrafish compared to control (DMSO)-treated larvae (a). The morphologies of the TD at 7 dpf (dotted outline) and the PCV are also comparable in cobimetinib-treated (d) and DMSO-treated larvae (c). The boxes indicate the area investigated in c and d. Scale bar, 100 μm.

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Corresponding author(s): DBPR NMED-L91762C

Reporting Summary Nature Research wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist.

Statistical parameters When statistical analyses are reported, confirm that the following items are present in the relevant location (e.g. figure legend, table legend, main text, or Methods section). n/a Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement An indication of whether measurements were taken from distinct samples or whether the same sample was measured repeatedly The statistical test(s) used AND whether they are one- or two-sided Only common tests should be described solely by name; describe more complex techniques in the Methods section. A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistics including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)

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Software and code Policy information about availability of computer code Data collection Illumina CASAVA software (version 1.8.2); Leica LAS AF v3.0; FIJI v1.51; Zeiss Zen software

Data analysis BWA, v.0.7.12; Picard, v.1.97; GATK, v.2.6-5; ANNOVAR, 2012May25; SnpEff v.2.0.5; Graphpad prism 7.0d

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Data

Policy information about availability of data April 2018 All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: - Accession codes, unique identifiers, or web links for publicly available datasets - A list of figures that have associated raw data - A description of any restrictions on data availability

Whole exome sequencing data have been deposited in dbGaP with accession number phs001802.v1.p1

1 nature research | reporting summary Field-specific reporting Please select the best fit for your research. If you are not sure, read the appropriate sections before making your selection. Life sciences Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/authors/policies/ReportingSummary-flat.pdf

Life sciences study design All studies must disclose on these points even when the disclosure is negative. Sample size No sample-size calculation was performed. 45 patients with very rare lymphatic diseases were enrolled in the study through Center for Lymphatic Imaging and Interventions at CHOP and Patient Registry at Lymphangiomatosis & Gorham’s Disease Alliance (LGDA). Given WES- based testing is being used for rare congenital anomaly patients with a molecular diagnostic yield of 20-30%, we anticipated a few individuals would obtain a molecular diagnosis.

Data exclusions No data exclusion

Replication The experimental findings were reliably reproduced through at least three repeated experiments

Randomization For in vitro studies cells with the same condition were randomly distributed into studied groups

Blinding To eliminate operator bias, the researchers performing the procedures and analyses were blinded to zebrafish genotype identity as well as the identity of the experimental groups.

Reporting for specific materials, systems and methods

Materials & experimental systems Methods n/a Involved in the study n/a Involved in the study Unique biological materials ChIP-seq Antibodies Flow cytometry Eukaryotic cell lines MRI-based neuroimaging Palaeontology Animals and other organisms Human research participants

Unique biological materials Policy information about availability of materials Obtaining unique materials All unique materials used are available from standard commercial sources or from the authors upon request.

Antibodies Antibodies used Antibody Company Calalog # Clone Lot Number Dilution Mouse anti-FLAG M2 affinity Gel Sigma, St Louis, MO A2220 M2 SLBS2543V N/A Rabbit anti-phospho-p70S6K-Thr389 Cell Signaling Technology, Danvers, MA 9205 polyclonal Ref:04/2016 1:1000 anti-phospho-mTOR Ser2448 Cell Signaling Technology, Danvers, MA 5536 polyclonal Ref:04/2016 1:1000 Mouse anti-FLAG Sigma, St Louis, MO F3165 M2 SLBT6752 1:4000 Rabbit anti-phospho-p38 Thr180/Tyr182 Cell Signaling Technology, Danvers, MA 4511 polyclonal Ref:02/2016 1:1000 April 2018 Rabbit anti-PAN-14-3-3 Santa cruz biotechnology, Dallas, TX sc-629 polyclonal A2216 1:500 anti-phospho-Akt-Ser473 Cell Signaling Technology, Danvers, MA 4060 polyclonal Ref:01/2017 1:1000 Rabbit anti-phospho-p44/42-(Erk1/2)-Thr202/Tyr204 Cell Signaling Technology, Danvers, MA 4376 polyclonal Ref:03/2016 1:1000 Mouse anti-β-actin Santa cruz biotechnology, Dallas, TX sc-69879 AC-15 B1517 1:1000 HA-Tag (6E2) mouse antibody Cell Signaling Technology, Danvers, MA 2367 6E2 Ref:04/2016 1:100 Rabbit phospho-ERK T202/Y204 antibody Cell Signaling Technology, Danvers, MA 9101 polyclonal 30 1:200 anti-rabbit secondary antibody Thermo Fisher Scientific; Waltham, MA A11008 polyclonal 1622775 1:400

2 Antibody Validation: Validation nature research | reporting summary For western blots, proper performance of antibodies was determined experimentally through observation of a single specific band of the appropriate molecular weight in whole cell lysates, as can be seen for each antibody in the uncropped gel images provided with the manuscript. The FLAG antibody used to detect transfected or transduced FLAG-tag ARAF gave no signal of the appropriate size in untransfected/untransduced cells, and readily visualized ARAF when present. Additionally, M2 is the most heavily described clone of anti-FLAG monoclonal antibody. The same antibody was used for immunofluorescence, and a large increase in signal was confirmed between untransduced cells and ARAF-expressing cells, confirming specific staining. All antibodies used for western blotting of phosphorylated substrates were raised against human epitopes, and as stated above generated a single band of the expected molecular weight, with only considerably weaker minor/nonspecific bands in some cases. (Nonspecific bands observed in most cases are residual signals left after stripping of membranes. Particularly strong signals are not always completely removed by stripping.) Obviously, ERK and phospho-ERK westerns show two bands, corresponding to both p42 and p44 ERK. VE-cadherin antibody used for staining of human primary endothelial cells was raised against a human epitope, and staining was confirmed on untransduced primary cells, yielding the expected cobblestone pattern commonly described for endothelial cell VE-cadherin staining (see PMCID: PMC2948582 for one of many examples of the expected staining pattern). Additionally, each antibody was described by its manufacturer as raised against human epitopes and reacting with endogenous levels of human protein. The phospho-ERK T202/Y204 antibody used for zebrafish staining was previously described to react with zebrafish p-ERK1/2 (see PMID: 30794326).

Manufacturers statements about antibody validation, taken verbatim from websites. Further information, including manufacturer provided references, available at manufacturers’ websites.

Cell Signaling Technology: #9205: Phospho-p70 S6 Kinase (Thr389) Antibody detects endogenous levels of p70 S6 kinase only when phosphorylated at threonine 389. This antibody also detects p85 S6 kinase when phosphorylated at the analogous site (Thr412), and possibly S6KII phosphorylated at Thr388. Species Reactivity: Human, Mouse, Rat, Monkey

#5536: Phospho-mTOR (Ser2448) (D9C2) XP® Rabbit mAb detects endogenous levels of mTOR protein only when phosphorylated at Ser2448. Species Reactivity:Human, Mouse, Rat, Monkey Species predicted to react based on 100% sequence homology: Rat, Chicken, Pig, Horse

#4511: Phospho-p38 MAPK (Thr180/Tyr182) (D3F9) XP® Rabbit mAb detects endogenous levels of p38 MAPK only when phosphorylated at Thr180 and Tyr182. This antibody does not cross-react with the phosphorylated forms of either p42/44 MAPK or SAPK/JNK. Species Reactivity: Human, Mouse, Rat, Monkey, Mink, Pig, S. cerevisiae Species predicted to react based on 100% sequence homology:Hamster, Chicken, Zebrafish, Bovine, Pig

#4060: Phospho-Akt (Ser473) (D9E) XP® Rabbit mAb detects endogenous levels of Akt only when phosphorylated at Ser473. Species Reactivity: Human, Mouse, Rat, Hamster, Monkey, D. melanogaster, Zebrafish, Bovine Species predicted to react based on 100% sequence homology: Chicken, Xenopus, Dog, Pig

#4376: Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11) Rabbit mAb detects endogenous levels of p44 and p42 MAP Kinase (Erk1 and Erk2) when dually phosphorylated at Thr202 and Tyr204 of Erk1 (Thr185 and Tyr187 of Erk2), and singly phosphorylated at Thr202. The antibody does not cross-react with the corresponding phosphorylated residues of either JNK/ SAPK or p38 MAP kinase. Species Reactivity: Human, Mouse, Rat, Hamster, Monkey, Mink, D. melanogaster, Zebrafish, Pig, S. cerevisiae

#2367: HA-Tag (6E2) Mouse mAb detects recombinant proteins containing the HA epitope tag. The antibody recognizes the HA- tag fused to either the amino or carboxy terminus of targeted proteins in transfected cells.

#9101: Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody detects endogenous levels of p44 and p42 MAP Kinase (Erk1 and Erk2) when phosphorylated either individually or dually at Thr202 and Tyr204 of Erk1 (Thr185 and Tyr187 of Erk2). The antibody does not cross-react with the corresponding phosphorylated residues of either JNK/SAPK or p38 MAP Kinase, and does not cross-react with non-phosphorylated Erk1/2. Species Reactivity: Human, Mouse, Rat, Hamster, Monkey, Mink, D. melanogaster, Zebrafish, Bovine, Pig, C. elegans Species predicted to react based on 100% sequence homology: Chicken

Sigma

#A2220: Anti-FLAG M2 Affinity gel is a mouse monoclonal antibody that is covalently attached to agarose. The antibody binds FLAG at the N-terminal, Met-N-terminal, C-terminal and internal locations of fusion proteins. Binding is calcium-independent. Anti-FLAG® M2 affinity gel has been used for western blotting, immunoprecipitation and for the purification of FLAG fusion proteins.

#F3165: Anti Flag M2 antibody is used for the detection of Flag fusion proteins.This monoclonal antibody is produced in mouse April 2018 and recognizes the FLAG sequence at the N-terminus, Met N-terminus, and C-terminus. The antibody is also able to recognize FLAG at an internal site. M2, unlike M1 antibody is not Calcium dependent.

Santa Cruz Biotechnologies

#4511: epitope mapping at the N-terminus of 14-3-3 β of human origin recommended for detection of pan 14-3-3 of mouse, rat, human and avian origin by WB, IP, IF, IHC(P), FCM and ELISA; also reactive with additional species, including and equine, canine, bovine, porcine and avian

#sc-69879: raised against a slightly modified synthetic peptide corresponding to β-cytoplasmic actin nature research | reporting summary recommended for detection of β-Actin of broad species origin by WB, IP and IF; not recommended for detection of adult cardiac and skeletal muscle actins or detection in Dictyostelium discoideum.

Eukaryotic cell lines Policy information about cell lines Cell line source(s) HEK293T and HeLa cells were obtained from ATCC. Primary human dermal lymphatic endothelial cells (HDLECs) were obtained from Promocell

Authentication STR Analysis and morphology check by microscope

Mycoplasma contamination All cell lines tested mycoplasma negative.

Commonly misidentified lines No commonly misidentified cell lines were used. (See ICLAC register)

Animals and other organisms Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal research Laboratory animals Zebrafish (Tg(mrc1a:egfp)y251 transgenic line) between 0-7 days post fertilization were used in the study. Zebrafish at the analyzed stage were gender neutral, there was no bias towards male or female animals in the sample. Additional detailed information is provided in methods and supplementary method section.

Wild animals The study did not involve wild animals

Field-collected samples The study did not involve samples collected from the field

Human research participants Policy information about studies involving human research participants Population characteristics 42 pediatric and 3 adult patients with age ranging from 2 months to 41 years old with uncommon Noonan (or Noonan-related) syndrome with lymphatic anomaly, Gorham Stout disease (GSD), kaposiform lymphangiomatosis (KLA), lymphangiectasia, and central conducting lymphatic anomaly (CCLA) were enrolled for WES study, among which 26 are females and 19 are males.

Recruitment After obtaining approval from the Institutional Review Board (IRB) at The Children’s Hospital of Philadelphia (CHOP) and written informed consent, blood specimens from the lead proband (P1) with ARAF mutation and his parents were obtained for sequencing analysis. An unrelated second adult patient (P2) with ARAF mutation was recruited through the Patient Registry of the Lymphangiomatosis & Gorham’s Disease Alliance (LGDA), together with available family members. 43 additional patients and their family members were recruited similarly through CHOP or LGDA. April 2018