Jagged1 Research Articles

O-Fucose and Fringe-modified NOTCH1 extracellular domain fragments as decoys to release niche-lodged hematopoietic progenitor cells.

Successful hematopoietic progenitor cell (HPC) transplant therapy is improved by mobilizing HPCs from the bone marrow niche in donors. Notch receptor-ligand interactions are known to retain HPCs in the bone marrow, and neutralizing antibodies against Notch ligands, Jagged-1 or Delta-like ligand (DLL4), or NOTCH2 receptor potentiates HPC mobilization. Notch-ligand interactions are dependent on posttranslational modification of Notch receptors with O-fucose and are modulated by Fringe-mediated extension of O-fucose moieties. We previously reported that O-fucosylglycans on Notch are required for Notch receptor-ligand engagement controlling hematopoietic stem cell quiescence and retention in the marrow niche. Here, we generated recombinant fragments of NOTCH1 or NOTCH2 extracellular domain carrying the core ligand-binding regions (EGF11-13) either as unmodified forms or as O-fucosylglycan-modified forms. We found that the addition of O-fucose monosaccharide or the Fringe-extended forms of O-fucose to EGF11-13 showed substantial increases in binding to DLL4. Furthermore, the O-fucose and Fringe-extended NOTCH1 EGF11-13 protein displayed much stronger binding to DLL4 than the NOTCH2 counterpart. When assessed in an in vitro 3D osteoblastic niche model, we showed that the Fringe-extended NOTCH1 EGF11-13 fragment effectively released lodged HPC cells with a higher potency than the NOTCH2 blocking antibody. We concluded that O-fucose and Fringe-modified NOTCH1 EGF11-13 protein can be utilized as effective decoys for stem cell niche localized ligands to potentiate HPC egress and improve HPC collection for hematopoietic cell therapy.

PPARγ-p53-Mediated Vasculoregenerative Program to Reverse Pulmonary Hypertension.

In pulmonary arterial hypertension (PAH), endothelial dysfunction and obliterative vascular disease are associated with DNA damage and impaired signaling of BMPR2 (bone morphogenetic protein type 2 receptor) via two downstream transcription factors, PPARγ (peroxisome proliferator-activated receptor gamma), and p53. We investigated the vasculoprotective and regenerative potential of a newly identified PPARγ-p53 transcription factor complex in the pulmonary endothelium. In this study, we identified a pharmacologically inducible vasculoprotective mechanism in pulmonary arterial and lung MV (microvascular) endothelial cells in response to DNA damage and oxidant stress regulated in part by a BMPR2 dependent transcription factor complex between PPARγ and p53. Chromatin immunoprecipitation sequencing and RNA-sequencing established an inducible PPARγ-p53 mediated regenerative program regulating 19 genes involved in lung endothelial cell survival, angiogenesis and DNA repair including, EPHA2 (ephrin type-A receptor 2), FHL2 (four and a half LIM domains protein 2), JAG1 (jagged 1), SULF2 (extracellular sulfatase Sulf-2), and TIGAR (TP53-inducible glycolysis and apoptosis regulator). Expression of these genes was partially impaired when the PPARγ-p53 complex was pharmacologically disrupted or when BMPR2 was reduced in pulmonary artery endothelial cells (PAECs) subjected to oxidative stress. In endothelial cell-specific Bmpr2-knockout mice unable to stabilize p53 in endothelial cells under oxidative stress, Nutlin-3 rescued endothelial p53 and PPARγ-p53 complex formation and induced target genes, such as APLN (apelin) and JAG1, to regenerate pulmonary microvessels and reverse pulmonary hypertension. In PAECs from BMPR2 mutant PAH patients, pharmacological induction of p53 and PPARγ-p53 genes repaired damaged DNA utilizing genes from the nucleotide excision repair pathway without provoking PAEC apoptosis. We identified a novel therapeutic strategy that activates a vasculoprotective gene regulation program in PAECs downstream of dysfunctional BMPR2 to rehabilitate PAH PAECs, regenerate pulmonary microvessels, and reverse disease. Our studies pave the way for p53-based vasculoregenerative therapies for PAH by extending the therapeutic focus to PAEC dysfunction and to DNA damage associated with PAH progression.

A phenome-wide association study of 26 mendelian genes reveals phenotypic expressivity of common and rare variants within the general population

The clinical evaluation of a genetic syndrome relies upon recognition of a characteristic pattern of signs or symptoms to guide targeted genetic testing for confirmation of the diagnosis. However, individuals displaying a single phenotype of a complex syndrome may not meet criteria for clinical diagnosis or genetic testing. Here, we present a phenome-wide association study (PheWAS) approach to systematically explore the phenotypic expressivity of common and rare alleles in genes associated with four well-described syndromic diseases (Alagille (AS), Marfan (MS), DiGeorge (DS), and Noonan (NS) syndromes) in the general population. Using human phenotype ontology (HPO) terms, we systematically mapped 60 phenotypes related to AS, MS, DS and NS in 337,198 unrelated white British from the UK Biobank (UKBB) based on their hospital admission records, self-administrated questionnaires, and physiological measurements. We performed logistic regression adjusting for age, sex, and the first 5 genetic principal components, for each phenotype and each variant in the target genes (JAG1, NOTCH2 FBN1, PTPN1 and RAS-opathy genes, and genes in the 22q11.2 locus) and performed a gene burden test. Overall, we observed multiple phenotype-genotype correlations, such as the association between variation in JAG1, FBN1, PTPN11 and SOS2 with diastolic and systolic blood pressure; and pleiotropy among multiple variants in syndromic genes. For example, rs11066309 in PTPN11 was significantly associated with a lower body mass index, an increased risk of hypothyroidism and a smaller size for gestational age, all in concordance with NS-related phenotypes. Similarly, rs589668 in FBN1 was associated with an increase in body height and blood pressure, and a reduced body fat percentage as observed in Marfan syndrome. Our findings suggest that the spectrum of associations of common and rare variants in genes involved in syndromic diseases can be extended to individual phenotypes within the general population.

Abstract 14382: Notch Ligand Delta-like Ligand 1 Promotes Macrophage Activation and Arteriosclerosis

Introduction: Macrophage-mediated inflammation promotes various vascular diseases, but how macrophage functions are regulated has not been fully understood. Notch signaling is a fundamental pathway regulating cell differentiation in developmental stages, but its role in adult vascular diseases are largely unknown. Aim: The aim of this study is to explore the role of Notch ligands in macrophage activation and subsequent arteriosclerosis. Methods: 1) To screen the Notch ligands, blocking antibodies of each Notch ligands (Jagged-1, Jagged-2, Dll1, and Dll4) were prepared. After pre-treatment with these antibodies, mouse peritoneal macrophages were stimulated with 10 ng/mL LPS + 10 ng/mL IFN-γ or 10 ng/mL IL-4 for 24 hours. mRNA expressions were quantified by real-time PCR. 2) C57BL/6J mice were subjected to femoral arterial wire injury and blocking antibodies (250 μg/injection) were intraperitoneally injected twice a week for 4 weeks, 3) Apolipoprotein E-deficient mice ( Apoe -/- ) at 8 weeks of age were fed a high-fat diet and blocking antibodies (250 μg/injection) were intraperitoneally injected twice a week for 12 weeks. Results: Blockade of Jagged 2, Dll1 and Dll4 decreased expression of iNOS and IL-1β in macrophages treated with LPS and IFN-γ. In contrast, blockade of Dll1 increased arginase-1 and Ym1 in IL-4 treated macrophages. Immobilized recombinant Dll1 increased prototypical Notch-target gene Hes-1 and induced subsequent IL-1β expression. Therefore, we have tested Dll1 blocking antibodies in in vivo models. After femoral arterial injury, administration of Dll1 blocking antibodies attenuated intimal hyperplasia, perivascular fibrosis, and negative remodeling of injured arteries. In Apoe -/- mice, a high-fat diet feeding increased Dll1 expression in atherosclerotic plaques, especially in Mac-3 positive macrophages. Blockade of Dll1 decreased atherosclerotic plaque area and lipid content determined by oil red O staining. In addition, blockade of Dll1 decreased accumulation of Mac-3 positive macrophages to the plaque. Conclusions: These results indicate that Dll1 promotes macrophage activation and arteriosclerosis. Dll1 may be a potential therapeutic target for vascular diseases promoted by macrophage-mediated inflammation.

Depletion of circ_0007841 inhibits multiple myeloma development and BTZ resistance via miR-129-5p/JAG1 axis

ABSTRACT Circular RNAs (circRNAs) possess important regulatory effects on multiple myeloma (MM) progression. Here, we aimed at exploring the function of circ_0007841 in MM and the underlying molecular mechanism. Expression of circ_0007841, microRNA (miR)-129-5p and Jagged1 (JAG1) was determined via qRT-PCR or western blot assay. Methyl thiazolyl tetrazolium (MTT) assay was applied to examine cell viability and IC50 value of MM cells to bortezomib (BTZ). Colony formation assay was performed to analyze cell proliferation. Moreover, cell apoptosis was assessed by flow cytometry and western blot analysis. Cell metastasis was evaluated by wound healing assay and Transwell assay. Function of circ_0007841 in vivo was determined by xenograft tumor assay. Target relationship between miR-129-5p and circ_0007841 or JAG1 was confirmed via dual-luciferase reporter, RNA immunoprecipitation (RIP) and pull-down assays. The up-regulation of circ_0007841 and JAG1, and the down-regulation of miR-129-5p were detected in MM bone marrow aspirates and cells. Circ_0007841 knockdown significantly repressed cell proliferation, chemoresistance, and metastasis, while contributed to apoptosis of MM cells in vitro, and reduced tumor growth in vivo. Circ_0007841 targeted miR-129-5p, and miR-129-5p inhibition reversed impact of silencing of circ_0007841 on MM cells. JAG1 was a mRNA target of miR-129-5p, whose overexpression could undermine the miR-129-5p-mediated effects on MM cells. Circ_0007841 positively regulated JAG1 expression via absorbing miR-129-5p. Circ_0007841 knockdown inhibited MM cell proliferation, metastasis and chemoresistance through modulating miR-129-5p/JAG1 axis, suggesting that circ_0007841 might serve as a potential therapeutic target of MM.

The Notch Ligand Jagged1 Is Required for the Formation, Maintenance, and Survival of Hensen's Cells in the Mouse Cochlea.

During cochlear development, the Notch ligand JAGGED 1 (JAG1) plays an important role in the specification of the prosensory region, which gives rise to sound-sensing hair cells and neighboring supporting cells (SCs). While JAG1's expression is maintained in SCs through adulthood, the function of JAG1 in SC development is unknown. Here, we demonstrate that JAG1 is essential for the formation and maintenance of Hensen's cells, a highly specialized SC subtype located at the edge of the auditory epithelium. Using Sox2CreERT2/+::Jag1loxP/loxP mice of both genders, we show that Jag1 deletion at the onset of differentiation, at embryonic day 14.5, disrupted Hensen's cell formation. Similar loss of Hensen's cells was observed when Jag1 was deleted after Hensen's cell formation at postnatal day (P) 0/P1 and fate-mapping analysis revealed that in the absence of Jag1, some Hensen's cells die, but others convert into neighboring Claudius cells. In support of a role for JAG1 in cell survival, genes involved in mitochondrial function and protein synthesis were downregulated in the sensory epithelium of P0 cochlea lacking Jag1 Finally, using Fgfr3-iCreERT2 ::Jag1loxP/loxP mice to delete Jag1 at P0, we observed a similar loss of Hensen's cells and found that adult Jag1 mutant mice have hearing deficits at the low-frequency range.SIGNIFICANCE STATEMENT Hensen's cells play an essential role in the development and homeostasis of the cochlea. Defects in the biophysical or functional properties of Hensen's cells have been linked to auditory dysfunction and hearing loss. Despite their importance, surprisingly little is known about the molecular mechanisms that guide their development. Morphologic and fate-mapping analyses in our study revealed that, in the absence of the Notch ligand JAGGED1, Hensen's cells died or converted into Claudius cells, which are specialized epithelium-like cells outside the sensory epithelium. Confirming a link between JAGGED1 and cell survival, transcriptional profiling showed that JAGGED1 maintains genes critical for mitochondrial function and tissue homeostasis. Finally, auditory phenotyping revealed that JAGGED1's function in supporting cells is necessary for low-frequency hearing.

Identifying specific Notch1 target proteins in lung carcinoma cells.

The Notch signaling pathway has different roles in many human neoplasms, being either tumor-promoting or anti-proliferative. In addition, Notch signaling in carcinogenesis can be tissue dependent. The aim of the current study is to elucidate the relation between Notch1 protein expression in lung cancer cells and the following Notch related proteins: Hes1, c-Myc, Jagged1 and Jagged2. Notch1 and its related proteins were detected in human lung cancer cell lines and in 54 surgically resected different lung carcinoma tissues. Then, we used small interfering RNA (siRNA) technology, to down-regulate the expression of Notch1 in H69AR and SBC3 small cell lung carcinoma (SCLC) cells. Also, we transfected venus Notch1 intracellular domain (v.NICD) plasmid into human SCLC lines; H69. The expression of Hes1, c-Myc and Jagged2 is affected by Notch1 in SCLC. There is a strong association between the expression of Notch1 protein and the expression of Hes1, c-Myc and Jagged2 proteins, which could aid in better understanding tumorigenesis in SCLC.

Endothelial Notch1 Regulates High-dose Radiation Therapy (HDRT)-induced Endothelial-to-mesenchymal Transition (EbdMT)

There is increasing evidence that high-dose radiation therapy (HDRT) induces endothelial-to-mesenchymal-transition (EdnMT) which promotes abnormal tumor vascularity and radioresistance. We have previously shown that HDRT induces Notch1 signaling in tumor endothelial cells. Here, we investigate if endothelial Notch1 regulates HDRT induced EdnMT. HUVEC monolayers, in vitro, were also treated with 0, 2, 8, and 12Gy and immunoblot was performed for Notch1, Jagged1, VE-Cadherin. HUVEC monolayers were also treated with a soluble Notch inhibitor, Notch1 decoy, to assess the effect of Notch inhibition on EdnMT. Human neuroblastoma cells NGP were engineered to express Fc(control) and Notch1 decoy. These cells were intrarenally injected to develop xenograft tumors. Tumors were treated with 0 Gy and 12 Gy single fraction doses of HDRT. Mice were sacked at 72 hr after HDRT. Tumor sections were subjected to double immunofluorescence staining for aSMA (mesenchymal marker) and endomucin (endothelial marker). We also assessed the effect of combining HDRT with Notch inhibition on macrophage infiltration into the tumor microenvironment by performing immunofluorescence staining for F480 (a marker for tumor associated macrophage), INOS (marker for M1 macrophage) and MRC1 (a marker for M2 macrophage). In HUVEC monolayers, HDRT doses of 8 Gy and 12 Gy profoundly increased Notch1 and Jagged1 protein levels after 72 hr. The increased level of Notch1 and Jagged1 was associated with the reduction of endothelial marker VE-Cadherin level. Notch1 decoy treatment inhibited reduction of VE-cadherin indicating Notch inhibition blocked EdnMT. In vivo in NGP xenograft mice model, increased colocalization of endomucin and αSMA was observed at 72 hr 12 Gy post-irradiation, when compared to nonirradiated tumors, indicating transition of ECs. Interestingly, Notch inhibition reduced colocalizing αSMA (+) endomucin (+) staining in Notch1decoy+12 Gy tumors. Quantification showed that while ∼4% and ∼3% of endomucin (+) vessels expressed αSMA, following Fc+0 Gy and Notch1decoy + 0 Gy treatments, respectively, ∼30% of the surviving vessels were αSMA (+) after Fc+12 Gy. Notch inhibition reduced αSMA staining in surviving vessels as ∼4% of the vessels were αSMA (+) in N11-24-decoy+12 Gy tumors. Increased F480 and MRC1 staining was observed in tumor treated with 12 Gy, compared to untreated tumors, while no change in INOS staining was noted. Notch1 decoy treatment significantly decreased F480 and MRC1 suggesting Notch blockade decreased HDRT induced M2 macrophage infiltration. Our data shows that Notch blockade inhibits HDRT-induced EdnMT. Our data further shows that HDRT increased tumor-promoting immune suppressive M2 macrophage infiltration. Combing HDRT with Notch blockade inhibits M2 macrophage infiltration into the tumor microenvironment and can be a potential therapeutic approach to minimize immunosuppressive immune response.

Effect of Notch pathway on lipid accumulation induced by mono-2-ethylhexyl phthalate on 3T3-L1 cells

BackgroundMono-2-ethylhexyl phthalate (MEHP) is a major metabolite of di (2-ethylhexyl) phthalate (DEHP). Our previous researches have shown that MEHP can induce lipid accumulation in preadipocytes, while, the underlying mechanism is unclear. The present study was undertaken to clarify the effect of Notch pathway on lipid accumulation induced by MEHP. Methods3T3-L1 preadipocytes were exposed to MEHP (0, 10, 50, 250 µM and 0.1%DMSO) for the whole differentiation phase. Then the level of TG and cell cycle were detected. RT-PCR was used to detect the mRNA expression and Western blot was used to detect the expression of protein by Notch pathway genes and lipid metabolic related genes. ResultsIn this study, the level of TG in the 250 µM and 250 µM MEHP groups was significantly higher than that in the control, DMSO and 10 µM MEHP groups (P < 0.05). The relative mRNA level of Notch-1, Notch-3, Notch-4, Jagged-2 and Dll-4 in 250 µM group was higher than other groups (P < 0.05). The expression of Notch signal pathway proteins increased in MEHP treated groups, and the expression of Notch-2, Jagged-1, Jagged-2, Dll-1 and Dll-4 in 250 µM group were significantly higher than control group (P < 0.05). The expression of lipid metabolic related gene mRNA and protein increased in MEHP treated groups, and 250 µM MEHP group was higher than other groups (P < 0.05). The intracellular TG content was significantly correlated with the expression levels of Notch-1 and Jagged-2 mRNA (P < 0.05). ConclusionIn this study, we have found that MEHP exposure could increase the TG content in 3T3-L1 cells. The expression of Notch pathway mRNA and proteins were disturbed by the MEHP. Notch-1 and its ligand Jagged-2 play a critical role in the abnormal lipid metabolism in 3T3-L1 cells caused by MEHP.

Jagged1-Notch1-deployed tumor perivascular niche promotes breast cancer stem cell phenotype through Zeb1

Zinc finger E-box binding homeobox 1 (Zeb1) has been demonstrated to participate in the acquisition of the properties of cancer stem cells (CSCs). However, it is largely unknown how signals from the tumor microenvironment (TME) contribute to aberrant Zeb1 expression. Here, we show that Zeb1 depletion suppresses stemness, colonization and the phenotypic plasticity of breast cancer. Moreover, we demonstrate that, with direct cell-cell contact, TME-derived endothelial cells provide the Notch ligand Jagged1 (Jag1) to neighboring breast CSCs, leading to Notch1-dependent upregulation of Zeb1. In turn, ectopic Zeb1 in tumor cells increases VEGFA production and reciprocally induces endothelial Jag1 in a paracrine manner. Depletion of Zeb1 disrupts this positive feedback loop in the tumor perivascular niche, which eventually lessens tumor initiation and progression in vivo and in vitro. In this work, we highlight that targeting the angiocrine Jag1-Notch1-Zeb1-VEGFA loop decreases breast cancer aggressiveness and thus enhances the efficacy of antiangiogenic therapy.

SNHG16 aggravates chronic constriction injury-induced neuropathic pain in rats via binding with miR-124-3p and miR-141-3p to upregulate JAG1

Neuropathic pain is caused by damage to the nervous system. Increasing studies have confirmed that jagged 1 (JAG1) plays a significant role in nervous system diseases. However, the regulatory mechanisms of JAG1 in neuropathic pain remain vague. In this study, a chronic constriction injury (CCI) rat model was performed. JAG1 was found to be upregulated in CCI rats. The recombinant lentiviruses containing sh-JAG1 were injected to the CCI rats for knockdown of JAG1 in rats. JAG1 knockdown improved the mechanical allodynia and thermal hyperalgesia in CCI rats, and decreased the concentrations and mRNA expression of inflammatory cytokines (IL‐6, TNF-α and IL-1β) in spinal cord dorsal horn of CCI rats, suggesting that JAG1 knockdown attenuated neuropathic pain. In addition, we explored for the upstream mechanism of JAG1. Through RNA pull down assay and luciferase reporter assay, we confirmed that miR-124-3p and miR-141-3p bound with JAG1. Long non-coding RNA (lncRNA) small nucleolar RNA host gene 6 (SNHG16) was verified to be the upstream molecule of miR-124-3p and miR-141-3p to negatively regulate miR-124-3p and miR-141-3p. SNHG16 positively regulated JAG1 expression through competitively binding with miR-124-3p and miR-141-3p. Moreover, SNHG16 was found to be upregulated in CCI rats. SNHG16 knockdown improved the mechanical allodynia and thermal hyperalgesia as well as reduced the concentrations and mRNA expression of inflammatory cytokines in CCI rats. Finally, SNHG16 was confirmed to aggravate neuropathic pain in CCI rats via upregulating JAG1. In conclusion, this study verified that SNHG16 aggravated neuropathic pain in CCI rats via binding with miR-124-3p and miR-141-3p to upregulate JAG1, which may provide new insights into the development of gene therapy for neuropathic pain.

Notch Signaling and Liver Cancer.

Interactions between liver cells are closely regulated by Notch signaling. Notch signaling has been reported clinically related to bile duct hypogenesis in Alagille syndrome, which is caused by mutations in the Jagged1 gene. Notch activation and hepatocarcinogenesis are closely associated since cancer signaling is affected by the development of liver cells and cancer stem cells. Gene expression and genomic analysis using a microarray revealed that abnormalities in Notch-related genes were associated with the aggressiveness of liver cancer. This pattern was also accompanied with α-fetoprotein- and EpCAM-expressing phenotypes in vitro, in vivo, and in clinical tissues. Hepatitis B or C virus chronic infection or alcohol- or steatosis-related liver fibrosis induces liver cancer. Previous reports demonstrated that HBx, a hepatitis B virus protein, was associated with Jagged1 expression. We found that the Jagged1 and Notch1 signaling pathways were closely associated with the transcription of covalently closed circular hepatitis B virus DNA, which regulated cAMP response element-binding protein, thereby affecting Notch1 regulation by the E3 ubiquitin ligase ITCH. This viral pathogenesis in hepatocytes induces liver cancer. In conclusion, Notch signaling exerts various actions and is a clinical signature associated with hepatocarcinogenesis and liver context-related developmental function.

Peritoneal dialysis in an adult patient with tetralogy of Fallot diagnosed with incomplete Alagille syndrome

BackgroundAlagille syndrome is an autosomal dominant disorder usually caused by pathogenic variants of the JAG1 gene. In the past, cholestasis was a condition sine qua non for diagnosis of the syndrome. However, recent advancements in genetic testing have revealed that clinical presentations vary from lack of symptoms, to multiorgan involvement. Tetralogy of Fallot, the most frequent complex congenital heart defect in Alagille Syndrome, very rarely leads to renal failure requiring dialysis – there are only single reports of such cases in the literature, with none of them in Alagille Syndrome.Case presentationA 41-year-old woman suffering from cyanosis, dyspnea and plethora was admitted to the hospital. The patient suffered from chronic kidney disease and tetralogy of Fallot and had been treated palliatively with Blalock-Taussig shunts in the past; at admission, only minimal flow through the left shunt was preserved. These symptoms, together with impaired mental status and dysmorphic facial features, led to extensive clinical and genetic testing including whole exome sequencing. A previously unknown missense variant c.587G > A within the JAG1 gene was identified. As there were no signs of cholestasis, and subclinical liver involvement was only suggested by elevated alkaline phosphatase levels, the patient was diagnosed with incomplete Alagille Syndrome. End-stage renal disease required introduction of renal replacement therapy. Continuous ambulatory peritoneal dialysis was chosen and the patient’s quality of life significantly increased. However, after refusal of further treatment, the patient died at the age of 45.ConclusionsTetralogy of Fallot should always urge clinicians to evaluate for Alagille Syndrome and offer patients early nephrological care. Although tetralogy of Fallot rarely leads to end-stage renal disease requiring dialysis, if treated palliatively and combined with renal dysplasia (typical of Alagille Syndrome), it can result in severe renal failure as in the presented case. There is no standard treatment for such cases, but based on our experience, peritoneal dialysis is worth consideration. Finally, clinical criteria for the diagnosis of Alagille Syndrome require revision. Previously, diagnosis was based on cholestasis – however, cardiovascular anomalies are found to be more prevalent. Furthermore, the criteria do not include renal impairment, which is also common.

Cholera toxin induces food allergy through Th2 cell differentiation which is unaffected by Jagged2

AimsCholera toxin is often used to induce food allergies. However, its exact mode of action and effect remain ambiguous. In this study, we established a BALB/c mouse cholera toxin/ovalbumin-induced food allergy model to determine the molecular basis and signaling mechanisms of the immune regulation of cholera toxin during food allergy. Materials and methodsThe adjuvant activity of cholera toxin was analyzed by establishing mouse allergy model, and the allergic reaction of each group of mice was evaluated. The effect of cholera toxin on Th1/Th2 cell differentiation was analyzed to further explore the role of cholera toxin in allergen immune response. We stimulated bone marrow-derived dendritic cells (BMDCs) with cholera toxin in vitro to investigate the effect of cholera toxin on Notch ligand expression. BMDCs and naive CD4+T cells were co-cultured in vitro, and their cytokine levels were examined to investigate whether cholera toxin regulates Th cell differentiation via the Jagged2 Notch signaling pathway. Key findingsThe results showed that in the presence of allergens, cholera toxin promotes Th2 cell differentiation and enhances the body's immune response. Cholera toxin induces expression of the Notch ligand Jagged2, but Jagged2 Notch signaling pathway is not required to promote BMDCs-mediated differentiation of Th2 cells. SignificanceThis study initially revealed the mechanism by which cholera toxin plays an adjuvant role in food allergy, and provides reference for future related research.

JAGGED1 stimulates cranial neural crest cell osteoblast commitment pathways and bone regeneration independent of canonical NOTCH signaling.

Craniofacial bone loss is a complex clinical problem with limited regenerative solutions. Currently, BMP2 is used as a bone-regenerative therapy in adults, but in pediatric cases of bone loss, it is not FDA-approved due to concerns of life-threatening inflammation and cancer. Development of a bone-regenerative therapy for children will transform our ability to reduce the morbidity associated with current autologous bone grafting techniques. We discovered that JAGGED1 (JAG1) induces cranial neural crest (CNC) cell osteoblast commitment during craniofacial intramembranous ossification, suggesting that exogenous JAG1 delivery is a potential craniofacial bone-regenerative approach. In this study, we found that JAG1 delivery using synthetic hydrogels containing O9-1 cells, a CNC cell line, into critical-sized calvarial defects in C57BL/6 mice provided robust bone-regeneration. Since JAG1 signals through canonical (Hes1/Hey1) and non-canonical (JAK2) NOTCH pathways in CNC cells, we used RNAseq to analyze transcriptional pathways activated in CNC cells treated with JAG1±DAPT, a NOTCH-canonical pathway inhibitor. JAG1 upregulated expression of multiple NOTCH canonical pathway genes (Hes1), which were downregulated in the presence of DAPT. JAG1 also induced bone chemokines (Cxcl1), regulators of cytoskeletal organization and cell migration (Rhou), signaling targets (STAT5), promoters of early osteoblast cell proliferation (Prl2c2, Smurf1 and Esrra), and, inhibitors of osteoclasts (Id1). In the presence of DAPT, expression levels of Hes1 and Cxcl1 were decreased, whereas, Prl2c2, Smurf1, Esrra, Rhou and Id1 remain elevated, suggesting that JAG1 induces osteoblast proliferation through these non-canonical genes. Pathway analysis of JAG1+DAPT-treated CNC cells revealed significant upregulation of multiple non-canonical pathways, including the cell cycle, tubulin pathway, regulators of Runx2 initiation and phosphorylation of STAT5 pathway. In total, our data show that JAG1 upregulates multiple pathways involved in osteogenesis, independent of the NOTCH canonical pathway. Moreover, our findings suggest that JAG1 delivery using a synthetic hydrogel, is a bone-regenerative approach with powerful translational potential.

Jagged Ligands Enhance the Pro-Angiogenic Activity of Multiple Myeloma Cells.

Simple SummaryThe Jagged family of ligands are aberrantly expressed during multiple myeloma progression and contributes to activate Notch signaling both in myeloma cells and in the nearby bone marrow cell populations activating several pro-tumor effects. This work elucidates, in vitro, in vivo as well as in patients’ bone marrow biopsies, different mechanisms by which tumor cell-derived Jagged1 and 2 contribute to myeloma-associated angiogenesis. These include the ability to induce myeloma and bone marrow stromal cell secretion of VEGF along with a direct activation of the pro-angiogenic Notch signaling pathway in endothelial cells. This research provides a rational for a Jagged-directed therapy in multiple myeloma.Multiple myeloma (MM) is an incurable plasma cell malignancy arising primarily within the bone marrow (BM). During MM progression, different modifications occur in the tumor cells and BM microenvironment, including the angiogenic shift characterized by the increased capability of endothelial cells to organize a network, migrate and express angiogenic factors, including vascular endothelial growth factor (VEGF). Here, we studied the functional outcome of the dysregulation of Notch ligands, Jagged1 and Jagged2, occurring during disease progression, on the angiogenic potential of MM cells and BM stromal cells (BMSCs). Jagged1–2 expression was modulated by RNA interference or soluble peptide administration, and the effects on the MM cell lines’ ability to induce human pulmonary artery cells (HPAECs) angiogenesis or to indirectly increase the BMSC angiogenic potential was analyzed in vitro; in vivo validation was performed on a zebrafish model and MM patients’ BM biopsies. Overall, our results indicate that the MM-derived Jagged ligands (1) increase the tumor cell angiogenic potential by directly triggering Notch activation in the HPAECs or stimulating the release of angiogenic factors, i.e., VEGF; and (2) stimulate the BMSCs to promote angiogenesis through VEGF secretion. The observed pro-angiogenic effect of Notch activation in the BM during MM progression provides further evidence of the potential of a therapy targeting the Jagged ligands.

Identification of Genetic Factors Underlying the Association between Sodium Intake Habits and Hypertension Risk.

The role of sodium in hypertension remains unresolved. Although genetic factors have a significant impact on high blood pressure, studies comparing genetic susceptibility between people with low and high sodium diets are lacking. We aimed to investigate the genetic variations related to hypertension according to sodium intake habits in a large Korean population-based study. Data for a total of 57,363 participants in the Korean Genome and Epidemiology Study Health Examination were analyzed. Sodium intake was measured by a semi-quantitative food frequency questionnaire. We classified participants according to sodium intake being less than or greater than 2 g/day. We used logistic regression to test single-marker variants for genetic association with a diagnosis of hypertension, adjusting for age, sex, body mass index, exercise, alcohol, smoking, potassium intake, principal components 1, and principal components 2. Significant associations were defined as p < 5 × 10−8. In participants whose sodium intake was greater than 2 g/day, chromosome 6 open reading frame 10 (C6orf10)-human leukocyte antigen (HLA)-DQB1 rs6913309, ring finger protein (RNF)213 rs112735431, glycosylphosphatidylinositol anchored molecule-like (GML)- cytochrome P450 family 11 subfamily B member 1(CYP11B1) rs3819496, myosin light chain 2 (MYL2)-cut like homeobox 2 (CUX2) rs12229654, and jagged1 (JAG1) rs1887320 were significantly associated with hypertension. In participants whose intake was less than 2 g/day, echinoderm microtubule-associated protein-like 6(EML6) rs67617923 was significantly associated with hypertension. Genetic susceptibility associated with hypertension differed according to sodium intake. Identifying gene variants that contribute to the dependence of hypertension on sodium intake status could make possible more individualized nutritional recommendations for preventing cardiovascular diseases.

Abstract 6691: Delivering intra-tumoral immune modulators and targeting cancer stem cells using recombinant- AAVs

Abstract Solid tumors are composed of heterogeneous cell populations that support rapid growth of cancer cells and suppress immune cell responses. Unleashing the body's own immune response to eradicate cancer cells is one of the most promising ways to treat cancer patients. However, cancer immunotherapy is not without challenges. Some of these include: 1) Phenotypic plasticity and clonal selection in a highly dynamic, heterogeneous and mutable cell population, which leads to de novo and acquired resistance; 2) Immune editing by cancer cells; 3) Expression of immune check point ligands and receptors to suppress immune response locally within the tumor microenvironment, and systemically using exosomes as immune checkpoint carriers; 4) Cross-talk between cancer stem cells and the immune microenvironment; 5) Feasibility of developing personalized treatment strategies, such as cost vs benefit of CAR-T-cell therapy and time constraint, and humanized antibody production; 6) Systemic toxicity of current immunotherapy strategies; To address these challenges, we propose to use a recombinant adeno-associated virus (rAAV) platform to simultaneously enhance tumor immunity and target cancer stem cells by intra-tumoral administration. We express programmed cell death ligand-1 (PD-L1), and B7-H3 (CD276), in combination with a Notch1 decoy. The latter was chosen because: 1) Notch is a crucial signaling component of cancer stem cells (CSCs) maintenance and resistance and 2) Studies have shown that within the tumor, regulatory T-cells (Treg) are activated by OX40 and Notch ligand Jagged1. Blocking Notch signaling would have dual advantages: blocking Notch signaling in CSCs, and preventing activation of immunosuppressive Tregs. Based on current clinical trials and our experimental results, we hypothesized that intra-tumoral injection of rAAV viral particles engineered to express soluble PD-1, B7-H3 and Notch1 decoys in combination with lipopolysaccharide (LPS) is a promising strategy. To test our hypothesis, we used two mouse triple negative breast (TNBC) cancer models: C0321 in the FVB background and M-Wnt in the C57/Bl6 background; both are aggressive, highly metastatic, and similar to human TNBC. Using tumor spheroids in vitro, in the presence of soluble decoys of PD-1, B7-H3 and Notch1 synergistically induced the most effective tumor cell killing, compared to single decoy application. Importantly, intra-tumoral injection of PD-1, B7-H3 and Notch1 decoys in combination with LPS induced necrosis of tumors in vivo in our preliminary findings. Citation Format: Deniz A. Ucar, Giulia Monticone, Fokhrul Hossain, Samarpan Majumder, Dorota Wyczechowska, Matthew J. Dean, Luis Del Valle, Jovanny Zabaleta, Yong Ran, Sudarvili Shanthalingam, Abraham Bert Chabot, Bridgette M. Collins-Burow, Matthew E. Burrow, Barbara Osborne, Todd Eliot Golde, Lucio Miele. Delivering intra-tumoral immune modulators and targeting cancer stem cells using recombinant- AAVs [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6691.

Tumour-associated neutrophils orchestrate intratumoural IL-8-driven immune evasion through Jagged2 activation in ovarian cancer

BackgroundTumour associated neutrophils (TANs) play a controversial role in regulating immune surveillance and immune evasion in various malignancies. Here, we investigated the relevance of TANs with the prognosis and immune microenvironment of epithelial ovarian cancer (EOC).MethodsWe characterised TANs using flow cytometric analysis and immunofluorescence analysis. The prognostic merit of TANs in EOC was evaluated using cox regression analysis. Furthermore, we explored the therapeutic merit of targeting Notch signalling in EOC and determined its involvement in the immune microenvironment.ResultsHigh level of TANs is associated with a dismal prognosis and immune tolerance in EOC. TANs impaired cytotoxic effects of CD8+ T cells partly through Jagged2 (JAG2). Notch pathway blocked using γ-secretase inhibitor LY3039478 and anti-JAG2 antibody led to retarded tumour growth and augmented cytotoxic effects of CD8+ T cells. IL-8 contributes to the recruitment of TANs and the induction of JAG2 expression in TANs. Blockade of CXCR2 signalling reduces tumour growth rate, accompanied by a decreasing amount of TANs and increasing activity of CD8+ T cells. JAG2+TANs is an independent predictor of clinical outcomes.ConclusionJAG2+TANs are closely linked to IL-8-driven immune evasion microenvironment and may serve as a promising therapeutic target for the reinvigoration of anti-tumour immunity.

Antisense Oligonucleotides Targeting Jagged 1 Reduce House Dust Mite-induced Goblet Cell Metaplasia in the Adult Murine Lung.

Goblet cell metaplasia, excessive mucus production, and inadequate mucus clearance accompany and exacerbate multiple chronic respiratory disorders, such as asthma and chronic obstructive pulmonary disease. Notch signaling plays a central role in controlling the fate of multiple cell types in the lung, including goblet cells. In the present study, we explored the therapeutic potential of modulating the Notch pathway in the adult murine lung using chemically modified antisense oligonucleotides (ASOs). To this end, we designed and characterized ASOs targeting the Notch receptors Notch1, Notch2, and Notch3 and the Notch ligands Jag1 (Jagged 1) and Jag2 (Jagged 2). Pulmonary delivery of ASOs in healthy mice or mice exposed to house dust mite, a commonly used mouse model of asthma, resulted in a significant reduction of the respective mRNAs in the lung. Furthermore, ASO-mediated knockdown of Jag1 or Notch2 in the lungs of healthy adult mice led to the downregulation of the club cell marker Scgb1a1 and the concomitant upregulation of the ciliated cell marker FoxJ1 (forkhead box J1). Similarly, ASO-mediated knockdown of Jag1 or Notch2 in the house dust mite disease model led to reduced goblet cell metaplasia and decreased mucus production. Because goblet cell metaplasia and excessive mucus secretion are a common basis for many lung pathologies, we propose that ASO-mediated inhibition of JAG1 could provide a novel therapeutic path for the treatment of multiple chronic respiratory diseases.