A conserved family of immune effectors cleaves cellular ATP upon viral infection.

During viral infection, cells can deploy immune strategies that deprive viruses of molecules essential for their replication. Here, we report a family of immune effectors in bacteria that, upon phage infection, degrade cellular adenosine triphosphate (ATP) and deoxyadenosine triphosphate (dATP) by cleaving the N-glycosidic bond between the adenine and sugar moieties. These ATP nucleosidase effectors are widely distributed within multiple bacterial defense systems, including cyclic oligonucleotide-based antiviral signaling systems (CBASS), prokaryotic argonautes, and nucleotide-binding leucine-rich repeat (NLR)-like proteins, and we show that ATP and dATP degradation during infection halts phage propagation. By analyzing homologs of the immune ATP nucleosidase domain, we discover and characterize Detocs, a family of bacterial defense systems with a two-component phosphotransfer-signaling architecture. The immune ATP nucleosidase domain is also encoded within diverse eukaryotic proteins with immune-like architectures, and we show biochemically that eukaryotic homologs preserve the ATP nucleosidase activity. Our findings suggest that ATP and dATP degradation is a cell-autonomous innate immune strategy conserved across the tree of life.

Personalized microbiome-driven effects of non-nutritive sweeteners on human glucose tolerance.

Non-nutritive sweeteners (NNS) are commonly integrated into human diet and presumed to be inert; however, animal studies suggest that they may impact the microbiome and downstream glycemic responses. We causally assessed NNS impacts in humans and their microbiomes in a randomized-controlled trial encompassing 120 healthy adults, administered saccharin, sucralose, aspartame, and stevia sachets for 2 weeks in doses lower than the acceptable daily intake, compared with controls receiving sachet-contained vehicle glucose or no supplement. As groups, each administered NNS distinctly altered stool and oral microbiome and plasma metabolome, whereas saccharin and sucralose significantly impaired glycemic responses. Importantly, gnotobiotic mice conventionalized with microbiomes from multiple top and bottom responders of each of the four NNS-supplemented groups featured glycemic responses largely reflecting those noted in respective human donors, which were preempted by distinct microbial signals, as exemplified by sucralose. Collectively, human NNS consumption may induce person-specific, microbiome-dependent glycemic alterations, necessitating future assessment of clinical implications.

The Identity of Human Tissue-Emigrant CD8+ T Cells.

Lymphocyte migration is essential for adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies have been restricted to immunological analyses of blood and various tissues. To address this knowledge gap, we used an integrated approach to characterize tissue-emigrant lineages in thoracic duct lymph (TDL). The most prevalent immune cells in human and non-human primate efferent lymph were T cells. Cytolytic CD8+ T cell subsets with effector-like epigenetic and transcriptional signatures were clonotypically skewed and selectively confined to the intravascular circulation, whereas non-cytolytic CD8+ T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Moreover, these anatomically distinct gene expression profiles were recapitulated within individual clonotypes, suggesting parallel differentiation programs independent of the expressed antigen receptor. Our collective dataset provides an atlas of the migratory immune system and defines the nature of tissue-emigrant CD8+ T cells that recirculate via TDL.

Urea Cycle Dysregulation Generates Clinically Relevant Genomic and Biochemical Signatures.

The urea cycle (UC) is the main pathway by which mammals dispose of waste nitrogen. We find that specific alterations in the expression of most UC enzymes occur in many tumors, leading to a general metabolic hallmark termed "UC dysregulation" (UCD). UCD elicits nitrogen diversion toward carbamoyl-phosphate synthetase2, aspartate transcarbamylase, and dihydrooratase (CAD) activation and enhances pyrimidine synthesis, resulting in detectable changes in nitrogen metabolites in both patient tumors and their bio-fluids. The accompanying excess of pyrimidine versus purine nucleotides results in a genomic signature consisting of transversion mutations at the DNA, RNA, and protein levels. This mutational bias is associated with increased numbers of hydrophobic tumor antigens and a better response to immune checkpoint inhibitors independent of mutational load. Taken together, our findings demonstrate that UCD is a common feature of tumors that profoundly affects carcinogenesis, mutagenesis, and immunotherapy response.

Clock proteins and training modify exercise capacity in a daytime-dependent manner.

Exercise and circadian biology are closely intertwined with physiology and metabolism, yet the functional interaction between circadian clocks and exercise capacity is only partially characterized. Here, we tested different clock mutant mouse models to examine the effect of the circadian clock and clock proteins, namely PERIODs and BMAL1, on exercise capacity. We found that daytime variance in endurance exercise capacity is circadian clock controlled. Unlike wild-type mice, which outperform in the late compared with the early part of their active phase, PERIODs- and BMAL1-null mice do not show daytime variance in exercise capacity. It appears that BMAL1 impairs and PERIODs enhance exercise capacity in a daytime-dependent manner. An analysis of liver and muscle glycogen stores as well as muscle lipid utilization suggested that these daytime effects mostly relate to liver glycogen levels and correspond to the animals' feeding behavior. Furthermore, given that exercise capacity responds to training, we tested the effect of training at different times of the day and found that training in the late compared with the early part of the active phase improves exercise performance. Overall, our findings suggest that clock proteins shape exercise capacity in a daytime-dependent manner through changes in liver glycogen levels, likely due to their effect on animals' feeding behavior.

Fatty acid transport protein 2 interacts with ceramide synthase 2 to promote ceramide synthesis.

Dihydroceramide is a lipid molecule generated via the action of (dihydro)ceramide synthases (CerSs), which use two substrates, namely sphinganine and fatty acyl-CoAs. Sphinganine is generated via the sequential activity of two integral membrane proteins located in the endoplasmic reticulum. Less is known about the source of the fatty acyl-CoAs, although a number of cytosolic proteins in the pathways of acyl-CoA generation modulate ceramide synthesis via direct or indirect interaction with the CerSs. In this study, we demonstrate, by proteomic analysis of immunoprecipitated proteins, that fatty acid transporter protein 2 (FATP2) (also known as very long-chain acyl-CoA synthetase) directly interacts with CerS2 in mouse liver. Studies in cultured cells demonstrated that other members of the FATP family can also interact with CerS2, with the interaction dependent on both proteins being catalytically active. In addition, transfection of cells with FATP1, FATP2, or FATP4 increased ceramide levels although only FATP2 and 4 increased dihydroceramide levels, consistent with their known intracellular locations. Finally, we show that lipofermata, an FATP2 inhibitor which is believed to directly impact tumor cell growth via modulation of FATP2, decreased de novo dihydroceramide synthesis, suggesting that some of the proposed therapeutic effects of lipofermata may be mediated via (dihydro)ceramide rather than directly via acyl-CoA generation. In summary, our study reinforces the idea that manipulating the pathway of fatty acyl-CoA generation will impact a wide variety of down-stream lipids, not least the sphingolipids, which utilize two acyl-CoA moieties in the initial steps of their synthesis.

On the Origins of Enzymes: Phosphate-Binding Polypeptides Mediate Phosphoryl Transfer to Synthesize Adenosine Triphosphate.

Reactions involving the transfer of a phosphoryl (-PO32-) group are fundamental to cellular metabolism. These reactions are catalyzed by enzymes, often large and complex, belonging to the phosphate-binding loop (P-loop) nucleoside triphosphatase (NTPase) superfamily. Due to their critical importance in life, it is reasonable to assume that phosphoryl-transfer reactions were also crucial in the pre-LUCA (last universal common ancestor) world and mediated by precursors that were simpler, in terms of their sequence and structure, relative to their modern-day enzyme counterparts. Here, we demonstrate that short phosphate-binding polypeptides (∼50 residues) comprising a single, ancestrally inferred, P-loop or Walker A motif mediate the reversible transfer of a phosphoryl group between two adenosine diphosphate molecules to synthesize adenosine triphosphate and adenosine monophosphate. This activity, although rudimentary, bears resemblance to that of adenylate kinase (a P-loop NTPase enzyme). The polypeptides, dubbed as "P-loop prototypes", thus relate to contemporary P-loop NTPases in terms of their sequence and function, and yet, given their simplicity, serve as plausible representatives of the early "founder enzymes" involved in proto-metabolic pathways.

Sulfamate Acetamides as Self-Immolative Electrophiles for Covalent Ligand-Directed Release Chemistry.

Electrophiles for covalent inhibitors that are suitable for in vivo administration are rare. While acrylamides are prevalent in FDA-approved covalent drugs, chloroacetamides are considered too reactive for such purposes. We report sulfamate-based electrophiles that maintain chloroacetamide-like geometry with tunable reactivity. In the context of the BTK inhibitor ibrutinib, sulfamate analogues showed low reactivity with comparable potency in protein labeling, in vitro, and cellular kinase activity assays and were effective in a mouse model of CLL. In a second example, we converted a chloroacetamide Pin1 inhibitor to a potent and selective sulfamate acetamide with improved buffer stability. Finally, we show that sulfamate acetamides can be used for covalent ligand-directed release (CoLDR) chemistry, both for the generation of "turn-on" probes as well as for traceless ligand-directed site-specific labeling of proteins. Taken together, this chemistry represents a promising addition to the list of electrophiles suitable for in vivo covalent targeting.

Imaging and Interventions for Lymphatic and Lymphatic-related Disorders.

The lymphatic system is critical in fluid balance homeostasis. Yet, until recently, lymphatic imaging has been outside of mainstream medicine due to a lack of robust imaging and interventional options. However, during the last 20 years, both clinical lymphatic imaging and interventions have shown dramatic advancement. The key to imaging advancement has been the interstitial delivery of contrast agents through lymphatic-rich tissues. These techniques include intranodal lymphangiography and dynamic contrast-enhanced MR lymphangiography. These methods provide the ability to image and recognize lymphatic anatomy and pathologic conditions. Percutaneous thoracic duct catheterization and embolization became the first widely accepted interventional technique for the management of chyle leaks. Advances in interstitial lymphatic embolization, as well as liver and mesenteric lymphatic interventions, have broadened the scope of possible lymphatic interventions. Also, recent techniques of lymphatic decompression allow for the treatment of a variety of lymphatic disorders. Finally, immunologic studies of central lymphatic fluid reveal the potential of lymphatic interventions on immunity. These advances herald an exciting new chapter for lymphatic imaging and interventions in the coming years.

Dynamic Contrast-enhanced CT Lymphangiography to Quantify Thoracic Duct Lymphatic Flow.

Background CT lymphangiography has been used to image the lymphatic anatomy and assess lymphatic abnormalities. There is, however, a need to develop a method for quantification of lymphatic flow rate in the thoracic duct (TD). Purpose To develop and validate a TD lymphatic flow measurement technique using dynamic contrast-enhanced CT lymphangiography. Materials and Methods Lymphatic flow rate was measured with two techniques: a first-pass analysis technique based on a single compartment model and a thresholding technique distinguishing between opacified and nonopacified voxels within the TD. The measurements were validated in a swine animal model between November 2021 and September 2022. CT images were acquired at 100 kV and 200 mA using a fast-pitched helical scan mode covering the entire TD following contrast material injection into the bilateral inguinal lymph nodes. Two helical CT scans, acquired at the base and peak contrast enhancement of the TD, were used to measure lymphatic flow rate. A US flow probe surgically placed around the TD provided the reference standard measurement. CT lymphatic flow measurements were compared with the reference US flow probe measurements using regression and Bland-Altman analysis. Repeatability was determined using repeated flow measurements within approximately 10 minutes of each other. Results Eleven swine (10 male; mean weight, 43.6 kg ± 2.6 [SD]) were evaluated with 71 dynamic CT acquisitions. The lymphatic flow rates measured using the first-pass analysis and thresholding techniques were highly correlated with the reference US flow probe measurements (r = 0.99 and 0.91, respectively) and showed good agreement with the reference standard, with Bland-Altman analysis showing small mean differences of 0.04 and 0.05 mL/min, respectively. The first-pass analysis and thresholding techniques also showed good agreement for repeated flow measurements (r = 0.94 and 0.90, respectively), with small mean differences of 0.09 and 0.03 mL/min, respectively. Conclusion The first-pass analysis and thresholding techniques could be used to accurately and noninvasively quantify TD lymphatic flow using dynamic contrast-enhanced CT lymphangiography. © RSNA, 2023 See also the editorial by Choyke in this issue.

Intranodal Glue Embolization for Postoperative Lymphatic Leaks in the Groin and Pelvis: Comparison with Sclerotherapy.

PURPOSE: To compare the effectiveness of and adverse events related to intranodal glue embolization (IGE) with those of intracavitary sclerotherapy for the treatment of postoperative groin and pelvic lymphatic leaks. MATERIALS AND METHODS: From November 2015 to July 2021, IGE for postoperative pelvic or groin lymphocele or lymphorrhea was performed in 33 patients. From January 2010 to July 2021, 28 patients with postoperative pelvic or groin lymphocele were treated with sclerosis alone. Clinical success was defined as resolution of drainage within 3 weeks of the last intervention performed without recurrence. Patients presenting >1 year after surgery or with <30 days of follow-up were excluded. Patients with lymphorrhea treated with IGE were not statistically compared with those in the sclerosis group because they were not eligible for sclerosis. RESULTS: Clinical success was similar between the groups (lymphocele IGE, 15/18, 83.3%, vs sclerosis, 15/23, 65.2% [P = .29]; lymphorrhea IGE, 8/9, 88.9%). The mean number of interventions performed to successfully treat a lymphocele was significantly higher in the sclerosis group (2.5 for sclerosis vs 1.3 for IGE; P = .003; lymphorrhea IGE, 1.0). The mean time to resolution was significantly longer for sclerosis than for IGE (27 vs 7 days; P = .002; 4 days for lymphorrhea IGE). There were no sclerosis-related adverse events and 2 IGE-related adverse events: (a) 1 case of mild lymphedema and (b) 1 case of nontarget embolization resulting in deep vein thrombosis. CONCLUSIONS: For treatment of postoperative pelvic and groin lymphoceles, IGE results in faster resolution with fewer interventions compared with sclerosis. IGE is also an effective treatment for postoperative groin lymphorrhea.

The cisterna chyli: a systematic review of definition, prevalence, and anatomy.

The cisterna chyli is a lymphatic structure found at the caudal end of the thoracic duct that receives lymph draining from the abdominal and pelvic viscera and lower limbs. In addition to being an important landmark in retroperitoneal surgery, it is the key gateway for interventional radiology procedures targeting the thoracic duct. A detailed understanding of its anatomy is required to facilitate more accurate intervention, but an exhaustive summary is lacking. A systematic review was conducted, and 49 published human studies met the inclusion criteria. Studies included both healthy volunteers and patients and were not restricted by language or date. The detectability of the cisterna chyli is highly variable, ranging from 1.7 to 98%, depending on the study method and criteria used. Its anatomy is variable in terms of location (vertebral level of T10 to L3), size (ranging 2-32 mm in maximum diameter and 13-80 mm in maximum length), morphology, and tributaries. The size of the cisterna chyli increases in some disease states, though its utility as a marker of disease is uncertain. The anatomy of the cisterna chyli is highly variable, and it appears to increase in size in some disease states. The lack of well-defined criteria for the structure and the wide variation in reported detection rates prevent accurate estimation of its natural prevalence in humans.

Intranodal CT Lymphangiography with Water-soluble Iodinated Contrast Medium for Imaging of the Central Lymphatic System.

Background Dynamic contrast-enhanced MR lymphangiography (DCMRL) is the reference standard used to diagnose various thoracic lymphatic disorders, such as traumatic chylothorax and plastic bronchitis. However, accessibility and logistical challenges have prevented the wide dissemination of this technology. Purpose To evaluate the feasibility of intranodal CT lymphangiography (ICTL) in the diagnosis and planning of subsequent intervention in patients with thoracic lymphatic disorders. Materials and Methods In this retrospective review, five women suspected of having lymphatic abnormalities (ranging from traumatic chylothorax to plastic bronchitis) and with contraindications to MRI underwent ICTL from September 2019 to May 2020. Needles (25 gauge) were placed in the bilateral inguinal lymph nodes with US guidance, and water-soluble iodinated contrast material was injected. CT fluoroscopy was used to monitor the opacification of the cisterna chyli to determine the timing of CT. After ICTL, the thoracic duct was catheterized, and lymphangiography was performed through the thoracic duct catheter. The ICTL and subsequent lymphangiographic findings were then visually compared by using three-dimensional reconstructions. Results Intranodal injection of water-soluble contrast medium was successful in all patients evaluated (five women; mean age, 68 years ± 11 [standard deviation]; range, 53-83 years). The central lymphatics were opacified in four of the five women, demonstrating abnormal pulmonary lymphatic flow from the thoracic duct into the lung parenchyma. In one of the five women, thoracic duct injection showed successful ligation of the thoracic duct. The time elapsed from injection of contrast medium to visualization of the thoracic duct ranged from 2 to 27 minutes. ICTL and lymphangiographic findings matched well. Conclusion Intranodal CT lymphangiography sufficiently depicted central lymphatic anatomy in patients with lymphatic abnormalities, thereby demonstrating its use as a feasible alternative to more technically challenging methods, such as dynamic contrast-enhanced MR lymphangiography. © RSNA, 2021.

Lipoxygenase functions in 1O2 production during root responses to osmotic stress.

Drought induces osmotic stress in roots, a condition simulated by the application of high-molecular-weight polyethylene glycol. Osmotic stress results in the reduction of Arabidopsis thaliana root growth and production of 1O2 from an unknown non-photosynthetic source. Reduced root growth can be alleviated by application of the 1O2 scavenger histidine (HIS). Here, we examined the possibility that 1O2 production involves Russell reactions occurring among the enzymatic products of lipoxygenases (LOXs), the fatty acid hydroperoxides. LOX activity was measured for purified soybean (Glycine max) LOX1 and in crude Arabidopsis root extracts using linoleic acid as substrate. Formation of the 13(S)-Hydroperoxy-9(Z),11(E)-octadecadienoic acid product was inhibited by salicylhdroxamic acid, which is a LOX inhibitor, but not by HIS, whereas 1O2 production was inhibited by both. D2O, which specifically extends the half-life of 1O2, augmented the LOX-dependent generation of 1O2, as expected from a Russell-type reaction. The addition of linoleic acid to roots stimulated 1O2 production and inhibited growth, suggesting that the availability of LOX substrate is a rate-limiting step. Indeed, water stress rapidly increased linoleic and linolenic acids by 2.5-fold in roots. Mutants with root-specific microRNA repression of LOXs showed downregulation of LOX protein and activity. The lines with downregulated LOX displayed significantly less 1O2 formation, improved root growth in osmotic stress, and an altered transcriptome response compared with wild type. The results show that LOXs can serve as an enzymatic source of "dark" 1O2 during osmotic stress and demonstrate a role for 1O2 in defining the physiological response.

Enhanced proteostasis, lipid remodeling, and nitrogen remobilization define barley flag leaf senescence.

Leaf senescence is a developmental process allowing nutrient remobilization to sink organs. We characterized flag leaf senescence at 7, 14, and 21 d past anthesis in two near-isogenic barley lines varying in the allelic state of the HvNAM1 transcription factor gene, which influences senescence timing. Metabolomics and microscopy indicated that, as senescence progressed, thylakoid lipids were transiently converted to neutral lipids accumulating in lipid droplets. Senescing leaves also exhibited an accumulation of sugars including glucose, while nitrogen compounds (nucleobases, nucleotides, and amino acids) decreased. RNA-Seq analysis suggested lipid catabolism via ß-oxidation and the glyoxylate cycle, producing carbon skeletons and feeding respiration as a replacement of the diminished carbon supply from photosynthesis. Comparison of the two barley lines highlighted a more prominent up-regulation of heat stress transcription factor- and chaperone-encoding genes in the late-senescing line, suggesting a role for these genes in the control of leaf longevity. While numerous genes with putative roles in nitrogen remobilization were up-regulated in both lines, several peptidases, nucleases, and nitrogen transporters were more highly induced in the early-senescing line; this finding identifies processes and specific candidates which may affect nitrogen remobilization from senescing barley leaves, downstream of the HvNAM1 transcription factor.

The seventh international RASopathies symposium: Pathways to a cure-expanding knowledge, enhancing research, and therapeutic discovery.

RASopathies are a group of genetic disorders that are caused by genes that affect the canonical Ras/mitogen-activated protein kinase (MAPK) signaling pathway. Despite tremendous progress in understanding the molecular consequences of these genetic anomalies, little movement has been made in translating these findings to the clinic. This year, the seventh International RASopathies Symposium focused on expanding the research knowledge that we have gained over the years to enhance new discoveries in the field, ones that we hope can lead to effective therapeutic treatments. Indeed, for the first time, research efforts are finally being translated to the clinic, with compassionate use of Ras/MAPK pathway inhibitors for the treatment of RASopathies. This biannual meeting, organized by the RASopathies Network, brought together basic scientists, clinicians, clinician scientists, patients, advocates, and their families, as well as representatives from pharmaceutical companies and the National Institutes of Health. A history of RASopathy gene discovery, identification of new disease genes, and the latest research, both at the bench and in the clinic, were discussed.

Neonatal lymphatic flow disorders: central lymphatic flow disorder and isolated chylothorax, diagnosis and treatment using novel lymphatic imaging and interventions technique.

PURPOSE OF REVIEW: Neonatal lymphatic disorders (NLDs) are conditions that are relatively rare and difficult to treat. The recent development of lymphatic imaging, such as Dynamic Contrast-Enhanced MR Lymphangiography and Intranodal Lymphangiography has led to a new, better understanding of the anatomical substrate and pathophysiological mechanisms of the diseases. Consequently, this has allowed the development of new targeted therapeutic interventions as well as prognostication for this population with lymphatic flow disorders. RECENT FINDINGS: The underlying causes of all NLD is an obstruction or altered flow of the central lymphatic flow. Two types of NLD have been described: isolated neonatal chylothorax and central lymphatic flow disorder (CLFD). Isolated neonatal chylothorax can be treated successfully with oil-based contrast (lipiodol) embolization. CLFD secondary to obstruction of the thoraco-venous junction can be successfully treated with surgical thoracic duct-venous anastomosis. CLFD caused by elevated central pressure and/or thoracic duct dysplasia can be treated medically, including with new systemic therapies such as mammalian target of rapamycin inhibitors. SUMMARY: New diagnostic and interventional tools have recently allowed for classification, prognostication, and targeted interventions for neonatal patients with lymphatic flow disorders. Further research will build on these discoveries.

Lymphatic Interventional Treatment for Chyluria via Retrograde Thoracic Duct Access.

Chyluria is the leakage of intestinal lymph (chyle) into the urine. Novel lymphatic intervention techniques, such as interstitial lymphatic embolization, proved to be a useful treatment option for chyluria. However, one of the challenges of this approach is the difficulty in identifying connections between the lymphatic system and kidney collecting system. Here, embolization of the abnormal lymphatic connection through retrograde thoracic duct access in 3 chyluria patients is introduced.

Research Priorities in Lymphatic Interventions: Recommendations from a Multidisciplinary Research Consensus Panel.

Recognizing the increasing importance of lymphatic interventions, the Society of Interventional Radiology Foundation brought together a multidisciplinary group of key opinion leaders in lymphatic medicine to define the priorities in lymphatic research. On February 21, 2020, SIRF convened a multidisciplinary Research Consensus Panel (RCP) of experts in the lymphatic field. During the meeting, the panel and audience discussed potential future research priorities. The panelists ranked the discussed research priorities based on clinical relevance, overall impact, and technical feasibility. The following research topics were prioritized by RCP: lymphatic decompression in patients with congestive heart failure, detoxification of thoracic duct lymph in acute illness, development of newer agents for lymphatic imaging, characterization of organ-based lymph composition, and development of lymphatic interventions to treat ascites in liver cirrhosis. The RCP priorities underscored that the lymphatic system plays an important role not only in the intrinsic lymphatic diseases but in conditions that traditionally are not considered to be lymphatic such as congestive heart failure, liver cirrhosis, and critical illness. The advancement of the research in these areas will lead the field of lymphatic interventions to the next level.

Pathogenic variant in EPHB4 results in central conducting lymphatic anomaly.

Central conducting lymphatic anomaly (CCLA) is one of the complex lymphatic anomalies characterized by dilated lymphatic channels, lymphatic channel dysmotility and distal obstruction affecting lymphatic drainage. We performed whole exome sequencing (WES) of DNA from a four-generation pedigree and examined the consequences of the variant by transfection of mammalian cells and morpholino and rescue studies in zebrafish. WES revealed a heterozygous mutation in EPHB4 (RefSeq NM_004444.4; c.2334 + 1G>C) and RNA-Seq demonstrated that the EPHB4 mutation destroys the normal donor site, which leads to the use of a cryptic splice donor that results in retention of the intervening 12-bp intron sequence. Transient co-expression of the wild-type and mutant EPHB4 proteins showed reduced phosphorylation of tyrosine, consistent with a loss-of-function effect. Zebrafish ephb4a morpholino resulted in vessel misbranching and deformities in the lymphatic vessel development, indicative of possible differentiation defects in lymphatic vessels, mimicking the lymphatic presentations of the patients. Immunoblot analysis using zebrafish lysates demonstrated over-activation of mTORC1 as a consequence of reduced EPHB4 signaling. Strikingly, drugs that inhibit mTOR signaling or RAS-MAPK signaling effectively rescued the misbranching phenotype in a comparable manner. Moreover, knock-in of EPHB4 mutation in HEK293T cells also induced mTORC1 activity. Our data demonstrate the pathogenicity of the identified EPHB4 mutation as a novel cause of CCLA and suggesting that ERK inhibitors may have therapeutic benefits in such patients with complex lymphatic anomalies.