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Wu, Guangyu, Svetlana Lyapina, Indranil Das, Jinhe Li, Mark Gurney, Adele Pauley, Inca Chui, Raymond J. Deshaies, and Jan Kitajewski. "SEL-10 Is an Inhibitor of Notch Signaling That Targets Notch for Ubiquitin-Mediated Protein Degradation." Molecular and Cellular Biology 21, no. 21 (November 1, 2001): 7403–15. http://dx.doi.org/10.1128/mcb.21.21.7403-7415.2001.
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ABSTRACT Notch receptors and their ligands play important roles in both normal animal development and pathogenesis. We show here that the F-box/WD40 repeat protein SEL-10 negatively regulates Notch receptor activity by targeting the intracellular domain of Notch receptors for ubiquitin-mediated protein degradation. Blocking of endogenous SEL-10 activity was done by expression of a dominant-negative form containing only the WD40 repeats. In the case of Notch1, this block leads to an increase in Notch signaling stimulated by either an activated form of the Notch1 receptor or Jagged1-induced signaling through Notch1. Expression of dominant-negative SEL-10 leads to stabilization of the intracellular domain of Notch1. The Notch4 intracellular domain bound to SEL-10, but its activity was not increased as a result of dominant-negative SEL-10 expression. SEL-10 bound Notch4 via the WD40 repeats and bound preferentially to a phosphorylated form of Notch4 in cells. We mapped the region of Notch4 essential for SEL-10 binding to the C-terminal region downstream of the ankyrin repeats. When this C-terminal fragment of Notch4 was expressed in cells, it was highly labile but could be stabilized by the expression of dominant-negative SEL-10. Ubiquitination of Notch1 and Notch4 intracellular domains in vitro was dependent on SEL-10. Although SEL-10 interacts with the intracellular domains of both Notch1 and Notch4, these proteins respond differently to interference with SEL-10 function. Thus, SEL-10 functions to promote the ubiquitination of Notch proteins; however, the fates of these proteins may differ.
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Takam Kamga, Paul, Federica Resci, Giada Dal Collo, Annalisa Adamo, Riccardo Bazzoni, Angela Mercuri, Massimiliano Bonifacio, and Mauro Krampera. "Prognostic Impact of Notch Signaling in Acute Myeloid Leukemia (AML)." Blood 132, Supplement 1 (November 29, 2018): 5242. http://dx.doi.org/10.1182/blood-2018-99-118701.
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Abstract Background: Notch signaling is a developmental pathway involved in normal and malignant hematopoiesis. Mutations in Notch genes lead to T-ALL and are associated with poor prognosis in B-CLL. However its role in AML is still under investigation, with controversial results. In addition, although the pathway consists of 4 receptors and 5 ligands, many studies are mostly based on single receptor or ligand. Aims: This study was conducted to determine if the expression level of each Notch receptor and ligand are associated with known prognostic factors and patient's survival. Methods: AML primary cells were collected from 79 AML patients after informed consent. The follow-up was for 36 months. Flow cytometry analysis was used to study the expression of each Notch receptor and ligand. Prognostics or risk factors considered were sex, white blood counts, FAB and cytogenetics. The Mann-Whitney test was used for analyzing differences in expression levels between two groups. Spearman test was used for correlation between Notch expression levels and prognostics factors. Differences in overall (OS) and progression free (PFS) survival were established through Gehan-Breslow-Wilcoxon test. Results: Consistently with previous findings, we found expression of Notch1, Notch2, Notch3, Notch4, Jagged1, Jagged2 and DLL3 in AML samples. There were no differences in expression levels between male and female. For each receptor or ligand, higher expression levels were found in more immature samples (M0, M1, and M2). Notch3, Notch4 and Jagged2 were enriched in adverse cytogenenetics risk groups compared to favorable cytogenetics risk patients. Then, for each receptor or ligand, patients were divided into two groups; patients with higher expression levels and patients with lower expression levels. Analyzing OS and PFS, we found that patients with lower expression levels of Notch4, Jagged2 and DLL3 displayed a longer survival compared to patients with higher expression levels. Conclusion: Given its oncogenic role in T-ALL and other malignancies Notch1 has been the more studied receptors in hematological malignancies. However, we have previously demonstrated that Notch4, DLL3 and Jagged2 are all involved in survival of AML and B-ALL cells. Studying the prognostic value of all Notch receptors and ligands, the current research clearly shows that higher levels of Notch4 and Jagged2 are found in poor cytogenetics risk groups and are associated with a shorter patient's survival. These demonstrations suggest that expression levels of Notch receptors and ligands in AML patient's samples at diagnostic could stand as prognostic marker for clinical care. Disclosures No relevant conflicts of interest to declare.
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Liu, Xiaoxia, Qingqing Luo, Yanfang Zheng, Xiaoping Liu, Ying Hu, Weifang Liu, Minglian Luo, Yin Zhao, and Li Zou. "NOTCH4 signaling controls EFNB2-induced endothelial progenitor cell dysfunction in preeclampsia." Reproduction 152, no. 1 (July 2016): 47–55. http://dx.doi.org/10.1530/rep-16-0132.
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Preeclampsia is a serious complication of pregnancy and is closely related to endothelial dysfunction, which can be repaired by endothelial progenitor cells (EPCs). The DLL4/NOTCH–EFNB2 (ephrinB2) cascade may be involved in the pathogenesis of preeclampsia by inhibiting the biological activity of EPCs. In addition, both NOTCH1 and NOTCH4, which are specific receptors for DLL4/NOTCH, play critical roles in the various steps of angiogenesis. However, it has not been determined which receptor (NOTCH1, NOTCH4, or both) is specific for the DLL4/NOTCH–EFNB2 cascade. Accordingly, we performed a series of investigations to evaluate it. EFNB2 expression was examined when NOTCH4 or NOTCH1 was downregulated, with or without DLL4 treatment. Then, the effects of NOTCH4 on EPC function were detected. Additionally, we analyzed NOTCH4 and EFNB2 expression in the EPCs from preeclampsia and normal pregnancies. Results showed that NOTCH4 downregulation led to decreased expression of EFNB2, which maintained the same level in the presence of DLL4/NOTCH activation. By contrast, NOTCH1 silencing resulted in a moderate increase in EFNB2 expression, which further increased in the presence of DLL4/NOTCH activation. The downregulation of NOTCH4 resulted in an increase of EPC biological activity, which was similar to EFNB2 silencing. NOTCH4 expression, consistent with the EFNB2 level, increased notably in preeclampsia EPCs compared with the controls. These findings suggest that NOTCH4, not NOTCH1, is the specific receptor for the DLL4/NOTCH–EFNB2 cascade. Blockade of this cascade may enhance the angiogenic property of EPCs, and act as a potential target to promote angiogenesis in patients with preeclampsia.
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Tu, Jian, Yang Li, Zhiqiang Hu, and Zhongbin Chen. "Radiosurgery inhibition of the Notch signaling pathway in a rat model of arteriovenous malformations." Journal of Neurosurgery 120, no. 6 (June 2014): 1385–96. http://dx.doi.org/10.3171/2013.12.jns131595.
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Object Notch signaling has been suggested to promote the development and maintenance of arteriovenous malformations (AVMs), but whether radiosurgery inhibits Notch signaling pathways in AVMs is unknown. The aim of this study was to examine molecular changes of Notch signaling pathways following radiosurgery and to explore mechanisms of radiosurgical obliteration of “nidus” vessels in a rat model of AVMs. Methods One hundred eleven rats received common carotid artery–to–external jugular vein anastomosis to form an arteriovenous fistula (AVF) model. Six weeks postoperatively, dilated small vessels and capillaries formed a nidus. The rats with AVFs received 25-Gy radiosurgery. The expression of Notch1 and Notch4 receptors and their ligands, Delta-like1 and Delta-like4, Jagged1, Notch downstream gene target HES1, and an apoptotic marker caspase-3 in nidus vessels in the AVF rats was examined immunohistochemically and was quantified using LAS-AF software at 7 time points over a period of 42 days postradiosurgery. The interaction events between Notch1 receptor and Jagged1, as well as Notch4 receptor and Jagged1, were quantified in nidus vessels in the AVF rats using proximity ligation assay at different time points over 42 days postradiosurgery. Results The expression of Notch1 and Notch4 receptors, Delta-like1, Delta-like4, Jagged1, and HES1 was observed in nidus vessels in the AVF rats pre- and postradiosurgery. Radiosurgery enhanced apoptotic activity (p < 0.05) and inhibited the expression of Notch1 and Notch4 receptors and Jagged1 in the endothelial cells of nidus vessels in the AVF rats at 1, 2, 3, 7, 21, 28, and 42 days postradiosurgery (p < 0.05). Radiosurgery suppressed the interaction events between Notch1 receptor and Jagged1 (p < 0.001) as well as Notch4 receptor and Jagged1 (p < 0.001) in the endothelial cells of nidus vessels in the AVF rats over a period of 42 days postradiosurgery. Radiosurgery induced thrombotic occlusion of nidus vessels in the AVF rats. There was a positive correlation between the percentage of fully obliterated nidus vessels and time after radiosurgery (r = 0.9324, p < 0.001). Conclusions Radiosurgery inhibits endothelial Notch1 and Notch4 signaling pathways in nidus vessels while inducing thrombotic occlusion of nidus vessels in a rat model of AVMs. The underlying mechanisms of radiosurgery-induced AVM shrinkage could be a combination of suppressing Notch receptor signaling in blood vessel endothelial cells, leading to a reduction in nidus vessel size and thrombotic occlusion of nidus vessels.
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Hadland, Brandon K., Barbara Varnum-Finney, Randall T. Moon, Michael Gustave Poulos, Jason M. Butler, Shahin Rafii, and Irwin D. Bernstein. "Notch Signaling By Either Notch1 or Notch2 Mediates Expansion of AGM-Derived Long-Term HSC Populations in Vitro." Blood 124, no. 21 (December 6, 2014): 2897. http://dx.doi.org/10.1182/blood.v124.21.2897.2897.
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Abstract Long-term, adult-engrafting hematopoietic stem cells (HSC) first emerge from hemogenic endothelial (HE) precursors in the context of embryonic arterial vessels such as the dorsal aorta of the AGM (Aorta-Gonad-Mesonephros region), a process which requires Notch1 receptor signaling. However, a possible subsequent role for Notch receptor activation during the unique period of substantial HSC expansion in embryonic development remains less well defined. Here, we show that endothelial cells derived from the murine embryonic AGM region (AGM-EC) or fetal liver (FL-EC) provide an in vitro substrate for generation/maturation of HSC from HE/HSC-precursor populations derived from early stage murine embryos, which lack adult-engraftment capacity prior to co-culture. Notably, these EC substrates, endogenously expressing Notch ligands of the Jagged and Delta families, also support subsequent numerical expansion of AGM-derived HSC, expressing Notch1 and Notch2 receptors, as determined by limit dilution transplantation analysis. Consistent with a requirement for Notch activation in this process, phenotypic HSC expansion during EC co-culture is blocked by gamma-secretase mediated Notch pathway inhibition. Furthermore, we show that in vitro Notch activation by immobilized ligand Delta1, together with cytokines and inhibition of the TGF-beta pathway, is sufficient to increase the number of AGM-derived HSC in the absence of EC stroma. Expansion of phenotypic hematopoietic stem/progenitor cells generated by culture on Delta1 is inhibited by antibody-mediated blockade of the combination of Notch1 and Notch2, but not by either Notch1 or Notch2 alone. Consistent with this, Notch receptor-specific activation by either immobilized Notch1 or Notch2 antibody is sufficient to support AGM-derived HSC in vitro in preliminary experiments. Altogether, these studies suggest a role for Notch pathway activation by either Notch1 or Notch2 in supporting embryonic-stage HSC expansion subsequent to initial Notch1-mediated HSC specification. Disclosures Moon: Fate Therapeutics: co-founder Other. Rafii:Angiocrine Biosciences: Founder Other.
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Urata, Yusuke, Wataru Saiki, Yohei Tsukamoto, Hiroaki Sago, Hideharu Hibi, Tetsuya Okajima, and Hideyuki Takeuchi. "Xylosyl Extension of O-Glucose Glycans on the Extracellular Domain of NOTCH1 and NOTCH2 Regulates Notch Cell Surface Trafficking." Cells 9, no. 5 (May 14, 2020): 1220. http://dx.doi.org/10.3390/cells9051220.
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Biochemical and genetic studies have indicated that O-linked glycosylation such as O-glucose (Glc), fucose (Fuc), and N-acetylglucosamine (GlcNAc) is critical for Notch signaling; however, it is not fully understood how O-glycans regulate the Notch receptor function. Notch receptors are type-I transmembrane proteins with large extracellular domains (ECD), containing 29–36 epidermal growth factor-like (EGF) repeats. Here, we analyzed O-Glc glycans on NOTCH1 and NOTCH2 expressed in HEK293T cells using an Orbitrap Fusion mass spectrometer and successfully revealed the structures and stoichiometries of all 17 EGF repeats of NOTCH1 with the O-Glc consensus sequence (C1-X-S-X-(P/A)-C2), and 16 out of 17 EGF repeats of NOTCH2 with the same consensus sequence. High levels of O-Glc attachment and xylosyl elongation were detected on most NOTCH1 and NOTCH2 EGF repeats. When both glucoside xylosyltransferases, GXYLT1 and GXYLT2, responsible for the xylosyl elongation of O-glucose, were genetically deleted, the expression of endogenous NOTCH1 and NOTCH2 on the surface of HEK293T cells did not change, but the cell surface expression of overexpressed NOTCH1 and NOTCH2 decreased compared with that in the wild type cells. In vitro secretion assays consistently showed a reduced secretion of both the NOTCH1 and NOTCH2 ECDs in GXYLT1 and GXYLT2 double knockout cells compared with the wild type cells, suggesting a significant role of the elongation of O-Glc glycans on the Notch ECDs in the quality control of Notch receptors.
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Demitrack, Elise S., Gail B. Gifford, Theresa M. Keeley, Nobukatsu Horita, Andrea Todisco, D. Kim Turgeon, Christian W. Siebel, and Linda C. Samuelson. "NOTCH1 and NOTCH2 regulate epithelial cell proliferation in mouse and human gastric corpus." American Journal of Physiology-Gastrointestinal and Liver Physiology 312, no. 2 (February 1, 2017): G133—G144. http://dx.doi.org/10.1152/ajpgi.00325.2016.
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The Notch signaling pathway is known to regulate stem cells and epithelial cell homeostasis in gastrointestinal tissues; however, Notch function in the corpus region of the stomach is poorly understood. In this study we examined the consequences of Notch inhibition and activation on cellular proliferation and differentiation and defined the specific Notch receptors functioning in the mouse and human corpus. Notch pathway activity was observed in the mouse corpus epithelium, and gene expression analysis revealed NOTCH1 and NOTCH2 to be the predominant Notch receptors in both mouse and human. Global Notch inhibition for 5 days reduced progenitor cell proliferation in the mouse corpus, as well as in organoids derived from mouse and human corpus tissue. Proliferation effects were mediated through both NOTCH1 and NOTCH2 receptors, as demonstrated by targeting each receptor alone or in combination with Notch receptor inhibitory antibodies. Analysis of differentiation by marker expression showed no change to the major cell lineages; however, there was a modest increase in the number of transitional cells coexpressing markers of mucous neck and chief cells. In contrast to reduced proliferation after pathway inhibition, Notch activation in the adult stomach resulted in increased proliferation coupled with reduced differentiation. These findings suggest that NOTCH1 and NOTCH2 signaling promotes progenitor cell proliferation in the mouse and human gastric corpus, which is consistent with previously defined roles for Notch in promoting stem and progenitor cell proliferation in the intestine and antral stomach. NEW & NOTEWORTHY Here we demonstrate that the Notch signaling pathway is essential for proliferation of stem cells in the mouse and human gastric corpus. We identify NOTCH1 and NOTCH2 as the predominant Notch receptors expressed in both mouse and human corpus and show that both receptors are required for corpus stem cell proliferation. We show that chronic Notch activation in corpus stem cells induces hyperproliferation and tissue hypertrophy, suggesting that Notch may drive gastric tumorigenesis.
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Pancewicz, J., and P. L. Bernatowicz. "Differential Notch1 and Notch2 expression in non-small cell lung cancer." Progress in Health Sciences 1 (June 11, 2019): 8–12. http://dx.doi.org/10.5604/01.3001.0013.3687.
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<b>Introduction:</b> NOTCH signaling can be deregulated in non-small cell lung cancer and can have oncogenic or tumor suppressive functions. NOTCH1 is regulated equally with NOTCH2, and the stability of the expression of those Notch receptors could determine the biological functions of lung cancer cells. <br/><b>Purpose:</b> To estimate expression level of NOTCH2 receptor in diverse NSCLC cell lines. Furthermore, we compared the mRNA level of NOTCH2 expression to the level of NOTCH1 expression. <br/><b>Materials and methods:</b> We have evaluated the mRNA manifestation of NOTCH1 and NOTCH2 genes by using quantitative real time method (RT-PCR). Moreover, we associated the results from NSCLC cells with results achieved in non-cancerous human bronchial epithelial cells (HBEpC). <br/><b>Results:</b> The expression level of NOTCH1 and NOTCH2 was downregulated in NSCLC cell lines, when related to HBEpC. Nevertheless, the decrease in NOTCH1 expression was significant, whereas NOTCH2 was not much different from the expression in control cells. <br/><b>Conclusions:</b> We conclude that NOTCH1 and NOTCH2 most likely have different biological function in NSCLC. They are active in NSCLC cell lines; nonetheless both are downregulated in lung cancer cells used in this study. Moreover, NOTCH2 expression is comparatively higher than NOTCH1 expression.
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Shi, Jianjun, Mohammad Fallahi, Jun-Li Luo, and Howard T. Petrie. "Nonoverlapping functions for Notch1 and Notch3 during murine steady-state thymic lymphopoiesis." Blood 118, no. 9 (September 1, 2011): 2511–19. http://dx.doi.org/10.1182/blood-2011-04-346726.
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Abstract Notch1 signaling is absolutely essential for steady-state thymic lymphopoiesis, but the role of other Notch receptors, and their potential overlap with the function of Notch1, remains unclear. Here we show that like Notch1, Notch3 is differentially expressed by progenitor thymocytes, peaking at the DN3 progenitor stage. Using mice carrying a gene-trapped allele, we show that thymic cellularity is slightly reduced in the absence of Notch3, although progression through the defined sequence of TCR-αβ development is normal, as are NKT and TCRγδ cell production. The absence of a profound effect from Notch3 deletion is not explained by residual function of the gene-trapped allele because insertion mapping suggests that the targeted allele would not encode functional signaling domains. We also show that although Notch1 and Notch3 are coexpressed on some early intrathymic progenitors, the relatively mild phenotype seen after Notch3 deletion does not result from the compensatory function of Notch1, nor does Notch3 function explain the likewise mild phenotype seen after conditional (intrathymic) deletion of Notch1. Our studies indicate that Notch1 and Notch3 carry out nonoverlapping functions during thymocyte differentiation, and that while Notch1 is absolutely required early in the lymphopoietic process, neither receptor is essential at later stages.
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Watanabe, Kazuhide, Tadahiro Nagaoka, Joseph M. Lee, Caterina Bianco, Monica Gonzales, Nadia P. Castro, Maria Cristina Rangel, et al. "Enhancement of Notch receptor maturation and signaling sensitivity by Cripto-1." Journal of Cell Biology 187, no. 3 (November 2, 2009): 343–53. http://dx.doi.org/10.1083/jcb.200905105.
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Nodal and Notch signaling pathways play essential roles in vertebrate development. Through a yeast two-hybrid screening, we identified Notch3 as a candidate binding partner of the Nodal coreceptor Cripto-1. Coimmunoprecipitation analysis confirmed the binding of Cripto-1 with all four mammalian Notch receptors. Deletion analyses revealed that the binding of Cripto-1 and Notch1 is mediated by the Cripto-1/FRL-1/Cryptic domain of Cripto-1 and the C-terminal region of epidermal growth factor–like repeats of Notch1. Binding of Cripto-1 to Notch1 occurred mainly in the endoplasmic reticulum–Golgi network. Cripto-1 expression resulted in the recruitment of Notch1 protein into lipid raft microdomains and enhancement of the furin-like protein convertase-mediated proteolytic maturation of Notch1 (S1 cleavage). Enhanced S1 cleavage resulted in the sensitization to ligand-induced activation of Notch signaling. In addition, knockdown of Cripto-1 expression in human and mouse embryonal carcinoma cells desensitized the ligand-induced Notch signaling activation. These results suggest a novel role of Cripto-1 in facilitating the posttranslational maturation of Notch receptors.
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Luo, B., J. C. Aster, R. P. Hasserjian, F. Kuo, and J. Sklar. "Isolation and functional analysis of a cDNA for human Jagged2, a gene encoding a ligand for the Notch1 receptor." Molecular and Cellular Biology 17, no. 10 (October 1997): 6057–67. http://dx.doi.org/10.1128/mcb.17.10.6057.
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Signaling through Notch receptors has been implicated in the control of cellular differentiation in animals ranging from nematodes to humans. Starting from a human expressed sequence tag-containing sequence resembling that of Serrate, the gene for a ligand of Drosophila melanogaster Notch, we assembled a full-length cDNA, now called human Jagged2, from overlapping cDNA clones. The full-length cDNA encodes a polypeptide having extensive sequence homology to Serrate (40.6% identity and 58.7% similarity) and even greater homology to several putative mammalian Notch ligands that have subsequently been described. When in situ hybridization was performed, expression of the murine Jagged2 homolog was found to be highest in fetal thymus, epidermis, foregut, dorsal root ganglia, and inner ear. In Northern blot analysis of RNA from tissues of 2-week-old mice, the 5.0-kb Jagged2 transcript was most abundant in heart, lung, thymus, skeletal muscle, brain, and testis. Immunohistochemistry revealed coexpression of Jagged2 and Notch1 within thymus and other fetal murine tissues, consistent with interaction of the two proteins in vivo. Coculture of fibroblasts expressing human Jagged2 with murine C2C12 myoblasts inhibited myogenic differentiation, accompanied by increased Notch1 and the appearance of a novel 115-kDa Notch1 fragment. Exposure of C2C12 cells to Jagged2 led to increased amounts of Notch mRNA as well as mRNAs for a second Notch receptor, Notch3, and a second Notch ligand, Jagged1. Constitutively active forms of Notchl in C2C12 cells also induced increased levels of the same set of mRNAs, suggesting positive feedback control of these genes initiated by binding of Jagged2 to Notch1. This feedback control may function in vivo to coordinate differentiation across certain groups of progenitor cells adopting identical cell fates.
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Van de Walle, Inge, Els Waegemans, Jelle De Medts, Greet De Smet, Magda De Smedt, Sylvia Snauwaert, Bart Vandekerckhove, et al. "Specific Notch receptor–ligand interactions control human TCR-αβ/γδ development by inducing differential Notch signal strength." Journal of Experimental Medicine 210, no. 4 (March 25, 2013): 683–97. http://dx.doi.org/10.1084/jem.20121798.
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In humans, high Notch activation promotes γδ T cell development, whereas lower levels promote αβ-lineage differentiation. How these different Notch signals are generated has remained unclear. We show that differential Notch receptor–ligand interactions mediate this process. Whereas Delta-like 4 supports both TCR-αβ and -γδ development, Jagged1 induces mainly αβ-lineage differentiation. In contrast, Jagged2-mediated Notch activation primarily results in γδ T cell development and represses αβ-lineage differentiation by inhibiting TCR-β formation. Consistently, TCR-αβ T cell development is rescued through transduction of a TCR-β transgene. Jagged2 induces the strongest Notch signal through interactions with both Notch1 and Notch3, whereas Delta-like 4 primarily binds Notch1. In agreement, Notch3 is a stronger Notch activator and only supports γδ T cell development, whereas Notch1 is a weaker activator supporting both TCR-αβ and -γδ development. Fetal thymus organ cultures in JAG2-deficient thymic lobes or with Notch3-blocking antibodies confirm the importance of Jagged2/Notch3 signaling in human TCR-γδ differentiation. Our findings reveal that differential Notch receptor–ligand interactions mediate human TCR-αβ and -γδ T cell differentiation and provide a mechanistic insight into the high Notch dependency of human γδ T cell development.
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Merrill, Marsha J., Nancy A. Edwards, and Russell R. Lonser. "Notch receptor and effector expression in von Hippel-Lindau disease–associated central nervous system hemangioblastomas." Journal of Neurosurgery 115, no. 3 (September 2011): 512–17. http://dx.doi.org/10.3171/2011.5.jns11271.
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Object Central nervous system hemangioblastomas are the most common manifestation of von Hippel-Lindau (VHL) disease, an autosomal dominant tumor suppressor syndrome that results in loss of VHL protein function and continuous upregulation of hypoxia-inducible factors. These tumors are composed of neoplastic stromal cells and abundant vasculature. Stromal cells express markers consistent with multipotent embryonically arrested hemangioblasts, which are precursors for hematopoietic and vascular lineages. Notch receptors are transmembrane signaling molecules that regulate multiple developmental processes including hematopoiesis and vasculogenesis. To investigate the importance of notch signaling in the development of VHL disease–associated CNS hemangioblastomas, the authors examined the presence of the four notch receptors and downstream notch effectors in this setting. Methods The authors used surgical specimens obtained from confirmed VHL-associated hemangioblastomas. Immunohistochemical analysis for the four notch receptors and the downstream effectors was performed on formalin-fixed paraffin-embedded sections. Western blot analysis for HES1 was performed on frozen specimens. Results All four notch receptors are present in hemangioblastomas. NOTCH1 and NOTCH4 receptors were widely and prominently expressed in both the stromal and vascular cells, NOTCH2 receptor expression was limited to primarily stromal cells, and NOTCH3 receptor expression was limited to vascular cells. All 4 receptors displayed a nuclear presence. Immunohistochemical analysis also demonstrated that downstream notch effectors, HES1 and HES5, were uniformly expressed in tumor stromal and vascular cells, but HES3, HEY1, and HEY2 were not. Strong HES1 expression was confirmed by Western blot analysis. Conclusions The presence of all four notch receptors and downstream effector molecules suggests that the notch signaling pathway plays a critical role in the maintenance of the undifferentiated pluripotent phenotype of these tumors and in the associated vascular response. Moreover, the prominent expression of notch receptors in VHL-associated CNS hemangioblastomas reveals a new and possibly potent therapeutic target.
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Giuli, M. V., E. Giuliani, I. Screpanti, D. Bellavia, and S. Checquolo. "Notch Signaling Activation as a Hallmark for Triple-Negative Breast Cancer Subtype." Journal of Oncology 2019 (July 11, 2019): 1–15. http://dx.doi.org/10.1155/2019/8707053.
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Triple-negative breast cancer (TNBC) is a subgroup of 15%-20% of diagnosed breast cancer patients. It is generally considered to be the most difficult breast cancer subtype to deal with, due to the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2), which usually direct targeted therapies. In this scenario, the current treatments of TNBC-affected patients rely on tumor excision and conventional chemotherapy. As a result, the prognosis is overall poor. Thus, the identification and characterization of targets for novel therapies are urgently required. The Notch signaling pathway has emerged to act in the pathogenesis and tumor progression of TNBCs. Firstly, Notch receptors are associated with the regulation of tumor-initiating cells (TICs) behavior, as well as with the aetiology of TNBCs. Secondly, there is a strong evidence that Notch pathway is a relevant player in mammary cancer stem cells maintenance and expansion. Finally, Notch receptors expression and activation strongly correlate with the aggressive clinicopathological and biological phenotypes of breast cancer (e.g., invasiveness and chemoresistance), which are relevant characteristics of TNBC subtype. The purpose of this up-to-date review is to provide a detailed overview of the specific role of all four Notch receptors (Notch1, Notch2, Notch3, and Notch4) in TNBCs, thus identifying the Notch signaling pathway deregulation/activation as a pathognomonic feature of this breast cancer subtype. Furthermore, this review will also discuss recent information associated with different therapeutic options related to the four Notch receptors, which may be useful to evaluate prognostic or predictive indicators as well as to develop new therapies aimed at improving the clinical outcome of TNBC patients.
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Tran, Ivy T., Ashley R. Sandy, Alexis Carulli, Gloria T. Shan, Vedran Radojcic, Ann Friedman, Amy Shelton, et al. "In Vivo Blockade of Individual Notch Ligands and Receptors Provides a New Targeted Therapeutic Approach In Graft-Versus-Host Disease." Blood 118, no. 21 (November 18, 2011): 819. http://dx.doi.org/10.1182/blood.v118.21.819.819.
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Abstract Abstract 819 Notch signaling is a cell-cell communication pathway with multiple functions in health and disease. Notch ligands of the Delta-like (Dll1, 3, 4) or Jagged (Jagged1, 2) family interact with one of four mammalian Notch receptors (Notch1-4), leading to proteolytic activation of the receptors by gamma-secretase. We have discovered a critical role for Notch signaling in the differentiation of pathogenic host-reactive T cells during graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (allo-BMT). Expression of the pan-Notch inhibitor DNMAML in donor T cells led to markedly reduced GVHD severity, without causing global immunosuppression (Blood 2011, 117(1): 299–308). These findings identify Notch signaling in alloreactive T cells as an attractive therapeutic target after allo-BMT. To explore preclinical strategies of Notch blockade in GVHD, we first assessed the effects of systemic pan-Notch inhibition with gamma-secretase inhibitors. In the B6 anti-BALB/c MHC-mismatched model of allo-BMT, administration of the gamma-secretase inhibitor dibenzazepine was as efficient as genetic strategies at blocking Notch target gene expression and production of inflammatory cytokines in donor T cells (IFN-γ, TNF-α, IL-2). However, dibenzazepine induced severe gastrointestinal toxicity after total body irradiation due to inhibition of both Notch1 and Notch2 in the gut epithelium. To avoid these side effects, we hypothesized that targeting individual Notch receptors or ligands could provide safe therapeutic Notch blockade after allo-BMT. Among the four mammalian Notch receptors (Notch1-4), donor alloreactive T cells expressed Notch1 and Notch2. Host dendritic cells expressed Notch ligands of the Jagged and Delta-like (Dll) families, with markedly increased Dll4 but not Jagged1/2 transcripts after total body irradiation. This suggested that blockade of Notch1 and/or Notch2 in T cells or Delta-like Notch ligands in dendritic cells could abrogate GVHD. To explore this possibility, we used specific monoclonal antibodies to neutralize Notch receptors and ligands in vivo after allo-BMT (Nature 2006, 444(7122):1083–7; Nature 2010, 464(7291): 1052–7). Combined blockade of Notch1 and Notch2 in vivo reduced the production of key inflammatory cytokines by alloreactive CD4+ and CD8+ T cells to a similar extent as DNMAML-mediated pan-Notch inhibition. Inhibition of Notch1 alone led to a large decrease in cytokine secretion, indicating that Notch1 is a dominant non-redundant Notch receptor in alloreactive T cells. Consistently, transplantation of Notch1-deficient but not Notch2-deficient B6 T cells allowed for decreased GVHD and improved survival in BALB/c recipients, similarly to global Notch inhibition by DNMAML. We then studied the consequences of inhibiting Dll1, Dll4 or both Dll1/Dll4 Notch ligands during acute GVHD. Combined Dll1/Dll4 blockade was as potent as DNMAML expression in decreasing cytokine production by alloreactive T cells, demonstrating that Delta-like and not Jagged ligands are the key Notch agonists at the alloimmune synapse. Dll4 inhibition was superior to Dll1 blockade in reducing cytokine production, abrogating GVHD, and prolonging recipient survival. Importantly, combined Dll1/Dll4 inhibition provided long-term protection against GVHD morbidity and mortality, while avoiding severe gastrointestinal side effects from Notch inhibition. Protection was observed even upon transient Dll1/Dll4 blockade during 1–2 weeks after transplantation. Altogether, our data suggest that Notch1 and Dll4 preferentially interact during alloreactive T cell priming and identify novel strategies to safely and efficiently target individual elements of the Notch pathway after allo-BMT. Humanized antibodies against Notch receptors and ligands were designed to block both mouse and human proteins, thus our preclinical work could lead to new strategies for GVHD control in human patients. Disclosures: Shelton: Genentech Inc.: Employment. Yan:Genentech Inc.: Employment. Siebel:Genentech Inc.: Employment.
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Jin, Yuting, Changyong Li, Dongwei Xu, Jianjun Zhu, Song Wei, Andrew Zhong, Mingwei Sheng, et al. "Jagged1-mediated myeloid Notch1 signaling activates HSF1/Snail and controls NLRP3 inflammasome activation in liver inflammatory injury." Cellular & Molecular Immunology 17, no. 12 (October 31, 2019): 1245–56. http://dx.doi.org/10.1038/s41423-019-0318-x.
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AbstractNotch signaling plays important roles in the regulation of immune cell functioning during the inflammatory response. Activation of the innate immune signaling receptor NLRP3 promotes inflammation in injured tissue. However, it remains unknown whether Jagged1 (JAG1)-mediated myeloid Notch1 signaling regulates NLRP3 function in acute liver injury. Here, we report that myeloid Notch1 signaling regulates the NLRP3-driven inflammatory response in ischemia/reperfusion (IR)-induced liver injury. In a mouse model of liver IR injury, Notch1-proficient (Notch1FL/FL) mice receiving recombinant JAG1 showed a reduction in IR-induced liver injury and increased Notch intracellular domain (NICD) and heat shock transcription factor 1 (HSF1) expression, whereas myeloid-specific Notch1 knockout (Notch1M-KO) aggravated hepatocellular damage even with concomitant JAG1 treatment. Compared to JAG1-treated Notch1FL/FL controls, Notch1M-KO mice showed diminished HSF1 and Snail activity but augmented NLRP3/caspase-1 activity in ischemic liver. The disruption of HSF1 reduced Snail activation and enhanced NLRP3 activation, while the adoptive transfer of HSF1-expressing macrophages to Notch1M-KO mice augmented Snail activation and mitigated IR-triggered liver inflammation. Moreover, the knockdown of Snail in JAG1-treated Notch1FL/FL livers worsened hepatocellular functioning, reduced TRX1 expression and increased TXNIP/NLRP3 expression. Ablation of myeloid Notch1 or Snail increased ASK1 activation and hepatocellular apoptosis, whereas the activation of Snail increased TRX1 expression and reduced TXNIP, NLRP3/caspase-1, and ROS production. Our findings demonstrated that JAG1-mediated myeloid Notch1 signaling promotes HSF1 and Snail activation, which in turn inhibits NLRP3 function and hepatocellular apoptosis leading to the alleviation of IR-induced liver injury. Hence, the Notch1/HSF1/Snail signaling axis represents a novel regulator of and a potential therapeutic target for liver inflammatory injury.
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Wu, Lizi, Ivan Maillard, Makoto Nakamura, Warren S. Pear, and James D. Griffin. "The MAML1 Transcriptional Co-Activator Is Required for the Development of Marginal Zone B Cells." Blood 108, no. 11 (November 16, 2006): 777. http://dx.doi.org/10.1182/blood.v108.11.777.777.
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Abstract Notch1 and Notch2 receptor-mediated signaling appear to have important and unique roles in lymphoid lineage commitment. Notch1 is required for T cell development, while Notch2 is essential for marginal zone B cell development. This specificity is not completely explained by differential expression patterns of Notch1 and 2 or Notch ligands, suggesting that there are other genes that contribute to specifying Notch receptor functions. We have previously shown that the MAML family of transcriptional co-activators is essential for Notch-induced transcriptional events, and functions by forming ternary complexes with Notch and the transcription factor CSL in the nucleus. This MAML family currently consists of three members, MAML1-3, all of which can function as co-activators for Notch receptors in vitro . In this study, we investigated the possibility that MAML1 co-activator contributes to determining Notch receptor function by generating mice deficient in the Maml1 gene. Maml1 -deficient mice fail to thrive and die within 10 days of birth. The morphology of marrow, nodes, and spleen was grossly intact. The ability of Maml1-deficient stem cells to generate different T and B lineages of lymphoid cells was determined by transplanting fetal liver cells isolated from E14.5 embryos into lethally irradiated wild-type recipient mice and analyzing donor-derived lymphoid cells 12 weeks post-transplantation. We found that the deletion of Maml1 results in complete loss of marginal zone B cell lineage (MZB, defined by B220+CD21hiCD23lo). Moreover, the number of MZB cells was reduced to about 50% in Maml1 -heterozygous fetal liver chimeras as compared to wild type controls. However, T cell development was largely unaffected, with only a modest but significant increase in the number of γδ T cells (about 2 fold) in both the thymus and the spleen. Therefore, these results suggest the unexpected finding that targeted deletion of Maml1 in hematopoietic cells is similar to the targeted deletion of either Notch2 or the Notch ligand, Delta-like 1 (Dll1) resulting in the loss of marginal zone B cells and minimal effects on T cell development. Moreover, the number of marginal zone B cells is correlated with Maml1 gene dosage, indicating haploinsufficiency. These data suggest that the Notch ligand Dll1 activates Notch2 signaling resulting in a Notch2/MAML1/CSL complex that is essential for marginal zone B cell development. Further studies with respect to relative expression levels of various MAML genes and interactions of MAML co-activators and Notch receptors may shed additional light into understanding how different Notch receptors regulate cell fate decisions in hematopoiesis.
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Tyagi, Ashish, Arun K. Sharma, and Chendil Damodaran. "A Review on Notch Signaling and Colorectal Cancer." Cells 9, no. 6 (June 25, 2020): 1549. http://dx.doi.org/10.3390/cells9061549.
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Colorectal cancer (CRC) has one of the highest mortality rates despite the advancement of treatment options. Aggressive CRC remains difficult to treat owing to the activation of oncogenic signaling pathways such as the Notch signaling pathway. The role of Notch receptors varies according to the difference in their structures; in particular, aberrant activation of Notch1 has been attributed to the severity of CRC. Notch1 activation in CRC is inhibited by small molecule inhibitors that target γ-secretase, an enzyme responsible for the third and last cleavage step of Notch receptors. γ-Secretase also produces the intracellular domain that finally carries out cellular functions by activating downstream effectors. However, most inhibitors block γ-secretase non-selectively and cause severe toxicity. Plant-source-derived small molecules, monoclonal antibodies, biological molecules (such as SiRNAs), and compounds targeting the Notch1 receptor itself or the downstream molecules such as HES1 are some of the options that are in advanced stages of clinical trials. The Negative Regulatory Region (NRR), which plays a central role in the transduction of Notch1 signaling in the event of ligand-dependent and ligand-independent Notch1 processing is also being targeted specifically by monoclonal antibodies (mAbs) to prevent aberrant Notch1 activation. In this review, we discuss the role of Notch1 in CRC, particularly its metastatic phenotype, and how mutations in Notch1, specifically in its NRR region, contribute to the aberrant activation of Notch1 signaling, which, in turn, contributes to CRC pathogenesis. We also discuss prevailing and emerging therapies that target the Notch1 receptor and the NRR region, and we highlight the potential of these therapies in abrogating Notch signaling and, thus, CRC development and progression.
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Zhou, Lan, Quanjian Yan, David Yao, Lebing W. Li, Stanton L. Gerson, and John B. Lowe. "Notch-Dependent Control of Blood Lineage Development is Modified by Fucosylation." Blood 112, no. 11 (November 16, 2008): 2448. http://dx.doi.org/10.1182/blood.v112.11.2448.2448.
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Abstract Notch receptors are conserved cell surface molecules essential for hematopoietic cell fate determination. Activated Notch enhances self-renewal of hematopoietic stem cells and promotes T lymphopoiesis. O-linked fucose moieties attached to the EGF domains of Notch receptors and its modification by Fringe can strongly modulate Notch signaling. Our recently published results indicate that Notch-dependent signaling controls myelopoiesis both in vitro and in vivo, and identify a requirement for Notch fucosylation in the expression of Notch ligand binding activity and Notch signaling efficiency in hematopoietic progenitor cells. In the current study, we tested the hypothesis that fucosylation controlled Notch signaling regulates hematopoietic lineage homeostasis. Genetically-modified mouse embryonic stem (ES) cells deficient in Notch1 receptor (NOTCH1−/−) or pofut1 (POFUT1−/−) that controls O-fucose modification of Notch receptor EGF repeats are studied in an in vitro co-culture assay with Notch ligand-expressing OP9 cells. Activation of Notch in wild type ES cells promotes T lymphopoiesis, while exposure of NOTCH1−/− or POFUT1−/− ES cells to Notch ligand failed to generate T lymphocytes but sustained granulocytic production. When probed with recombinant Notch ligand Dll1 or Dll4, hematopoietic cells derived from wild type ES line displayed robust Notch ligand binding, but cells from NOTCH1−/− or POFUT1−/− ES lines showed completely absent or reduced Notch ligand interaction, respectively. In comparison, ES cells deficient in pofut2 (POFUT2−/−) that controls O-fucose modification on thrombospondin repeats (TSR) displayed a wild type lineage development phenotype and normal Notch ligand binding ability. When examined for their in vivo hematopoietic reconstitution, blood cells derived from NOTCH1−/− or POFUT1−/− ES lines, but not POFUT2−/− ES line, showed enhanced granulocytic but suppressed T and B lymphoid lineage development. These results are consistent with our bone marrow transplantation findings that hematopoietic reconstitution by fucosylation-deficient marrow progenitor cells exhibited increased granulocytopoiesis while wild type or fucosylation-intact marrow cells have normal lineage distribution. Our observations indicate that Notch signaling maintains blood lineage homeostasis by promoting lymphoid lineage development and suppressing overt myeloid development. O-fucose modification of EGF repeats on Notch receptor is essential for this Notch-dependent control of blood lineage homeostasis as deficiency of fucose on Notch receptor results in enhanced myeloid development.
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Zheng, Li, and Sean D. Conner. "PI5P4Kγ functions in DTX1-mediated Notch signaling." Proceedings of the National Academy of Sciences 115, no. 9 (February 12, 2018): E1983—E1990. http://dx.doi.org/10.1073/pnas.1712142115.
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Notch signaling is an evolutionarily conserved pathway that is essential for development, where it controls processes ranging from cell differentiation to survival. Transport through endosomes is a critical step in regulating Notch signaling capacity, where the E3 ubiquitin ligase DTX1 is thought to control Notch1 intracellular transport decisions by direct receptor ubiquitination. However, how DTX1 regulates Notch1 transport within endosomes and the consequence of Notch1 ubiquitination by DTX1 remain unresolved. Here we demonstrate that DTX1 colocalizes with Notch1 on tubulovesicular recycling endosomes. We find that DTX1 silencing leads to enhanced Notch1 recycling from this compartment to the cell surface via a rab4a-mediated transport route. This, in turn, increases Notch1 cell-surface levels and enhances signaling. Surprisingly, we discovered that DTX1 depletion also elevates Notch1 activity mediated by a mutant form of the receptor that lacks lysine residues for ubiquitination, suggesting that DTX1 targets additional factors. Using an activity-based screen for ubiquitination targets, we identified multiple DTX1 substrates including PI5P4Kγ, a lipid kinase involved in PI(4,5)P2 production. Immunolocalization analysis reveals that PI5P4Kγ, like DTX1 and Notch1, is present on tubulovesicular recycling endosomes. However, in contrast to DTX1, Notch1 signaling is inhibited by pharmacological inactivation or siRNA depletion of PI5P4Kγ. Moreover, loss of PI5P4Kγ activity decreases Notch1 recycling rates and reduces receptor cell-surface levels. Collectively, these findings argue that PI5P4Kγ positively regulates the Notch pathway by promoting receptor recycling. Additionally, they support a model where DTX1 controls Notch1 endosomal sorting decisions by controlling PI5P4Kγ-mediated production of PI(4,5)P2.
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Kannan, Sankaranarayanan, Robert M. Sutphin, Mandy G. Hall, Leonard S. Golfman, Wendy Fang, Riitta M. Nolo, Lauren J. Akers, et al. "Notch activation inhibits AML growth and survival: a potential therapeutic approach." Journal of Experimental Medicine 210, no. 2 (January 28, 2013): 321–37. http://dx.doi.org/10.1084/jem.20121527.
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Although aberrant Notch activation contributes to leukemogenesis in T cells, its role in acute myelogenous leukemia (AML) remains unclear. Here, we report that human AML samples have robust expression of Notch receptors; however, Notch receptor activation and expression of downstream Notch targets are remarkably low, suggesting that Notch is present but not constitutively activated in human AML. The functional role of these Notch receptors in AML is not known. Induced activation through any of the Notch receptors (Notch1–4), or through the Notch target Hairy/Enhancer of Split 1 (HES1), consistently leads to AML growth arrest and caspase-dependent apoptosis, which are associated with B cell lymphoma 2 (BCL2) loss and enhanced p53/p21 expression. These effects were dependent on the HES1 repressor domain and were rescued through reexpression of BCL2. Importantly, activated Notch1, Notch2, and HES1 all led to inhibited AML growth in vivo, and Notch inhibition via dnMAML enhanced proliferation in vivo, thus revealing the physiological inhibition of AML growth in vivo in response to Notch signaling. As a novel therapeutic approach, we used a Notch agonist peptide that led to significant apoptosis in AML patient samples. In conclusion, we report consistent Notch-mediated growth arrest and apoptosis in human AML, and propose the development of Notch agonists as a potential therapeutic approach in AML.
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Pettersson, Susanne, Matylda Sczaniecka, Lorna McLaren, Fiona Russell, Karen Gladstone, Ted Hupp, and Maura Wallace. "Non-degradative ubiquitination of the Notch1 receptor by the E3 ligase MDM2 activates the Notch signalling pathway." Biochemical Journal 450, no. 3 (February 28, 2013): 523–36. http://dx.doi.org/10.1042/bj20121249.
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The Notch receptor is necessary for modulating cell fate decisions throughout development, and aberrant activation of Notch signalling has been associated with many diseases, including tumorigenesis. The E3 ligase MDM2 (murine double minute 2) plays a role in regulating the Notch signalling pathway through its interaction with NUMB. In the present study we report that MDM2 can also exert its oncogenic effects on the Notch signalling pathway by directly interacting with the Notch 1 receptor through dual-site binding. This involves both the N-terminal and acidic domains of MDM2 and the RAM [RBP-Jκ (recombination signal-binding protein 1 for Jκ)-associated molecule] and ANK (ankyrin) domains of Notch 1. Although the interaction between Notch1 and MDM2 results in ubiquitination of Notch1, this does not result in degradation of Notch1, but instead leads to activation of the intracellular domain of Notch1. Furthermore, MDM2 can synergize with Notch1 to inhibit apoptosis and promote proliferation. This highlights yet another target for MDM2-mediated ubiquitination that results in activation of the protein rather than degradation and makes MDM2 an attractive target for drug discovery for both the p53 and Notch signalling pathways.
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Zheng, Li, and Sean D. Conner. "Glycogen synthase kinase 3β inhibition enhances Notch1 recycling." Molecular Biology of the Cell 29, no. 4 (February 15, 2018): 389–95. http://dx.doi.org/10.1091/mbc.e17-07-0474.
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The Notch signaling pathway is essential throughout development and remains active into adulthood, where it performs a critical role in tissue homeostasis. The fact that defects in signaling can lead to malignancy illustrates the need to control Notch activity tightly. GSK3β is an established regulator of the Notch signaling pathway, although its mechanism of action remains unclear. Given the emerging role for GSK3β in receptor trafficking, we tested the idea that GSK3β controls signaling by regulating Notch transport. Consistent with published reports, we find that GSK3β inhibition enhances Notch1 signaling activity. Immunolocalization analysis reveals that Notch1 localization within a tubulovesicular compartment is altered when GSK3β activity is disrupted. We also find that receptor cell surface levels increase following acute GSK3β inhibition. This is followed by elevated Notch intracellular domain (NICD) production and a corresponding increase in signaling activity. Moreover, Notch transport assays reveal that receptor recycling rates increase when GSK3β activity is inhibited. Collectively, results presented here support a model where GSK3β regulates signaling by controlling postendocytic transport of Notch1. Given that GSK3β activity is suppressed following stimulation by multiple signal transduction pathways, our findings also suggest that cells can modulate Notch1 activity in response to extracellular signals by mobilizing Notch1 from endosomal stores.
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Yao, David, Yuanshuai Huang, Xiaoran Huang, Weihuan Wang, Quanjian Yan, Lebing Wei, Wei Xin, et al. "Protein O-fucosyltransferase 1 (Pofut1) regulates lymphoid and myeloid homeostasis through modulation of Notch receptor ligand interactions." Blood 117, no. 21 (May 26, 2011): 5652–62. http://dx.doi.org/10.1182/blood-2010-12-326074.
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Abstract Notch signaling is essential for lymphocyte development and is also implicated in myelopoiesis. Notch receptors are modified by O-fucosylation catalyzed by protein O-fucosyltransferase 1 (Pofut1). Fringe enzymes add N-acetylglucosamine to O-fucose and modify Notch signaling by altering the sensitivity of Notch receptors to Notch ligands. To address physiologic functions in hematopoiesis of Notch modified by O-fucose glycans, we examined mice with inducible inactivation of Pofut1 using Mx-Cre. These mice exhibited a reduction in T lymphopoiesis and in the production of marginal-zone B cells, in addition to myeloid hyperplasia. Restoration of Notch1 signaling rescued T lymphopoiesis and the marrow myeloid hyperplasia. After marrow transfer, both cell-autonomous and environmental cues were found to contribute to lymphoid developmental defects and myeloid hyperplasia in Pofut1-deleted mice. Although Pofut1 deficiency slightly decreased cell surface expression of Notch1 and Notch2, it completely abrogated the binding of Notch receptors with Delta-like Notch ligands and suppressed downstream Notch target activation, indicating that O-fucose glycans are critical for efficient Notch-ligand binding that transduce Notch signals. The combined data support a key role for the O-fucose glycans generated by Pofut1 in Notch regulation of hematopoietic homeostasis through modulation of Notch-ligand interactions.
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Afshar, Yalda, Lucio Miele, and Asgerally T. Fazleabas. "Notch1 Is Regulated by Chorionic Gonadotropin and Progesterone in Endometrial Stromal Cells and Modulates Decidualization in Primates." Endocrinology 153, no. 6 (April 24, 2012): 2884–96. http://dx.doi.org/10.1210/en.2011-2122.
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No other tissue in the body undergoes such a vast and extensive growth and remodeling in a relatively short period of time as the primate endometrium. Endometrial integrity is coordinated by ovarian hormones, namely, estrogens, progesterone, and the embryonic hormone chorionic gonadotropin (CG). These regulated events modulate the menstrual cycle and decidualization. The Notch family of transmembrane receptors regulate cellular proliferation, differentiation, and apoptosis, cellular processes required to maintain endometrial integrity. In two primate models, the human and the simulated pregnant baboon model, we demonstrated that Notch1 is increased during the window of uterine receptivity, concomitant with CG. Furthermore, CG combined with estrogens and progesterone up-regulate the level of Notch1, whereas progesterone increases the intracellular transcriptionally competent Notch1, which binds in a complex with progesterone receptor. Inhibition of Notch1 prevented decidualization, and alternatively, when decidualization is biochemically recapitulated in vitro, Notch1 is down-regulated. A focused microarray demonstrated that the Notch inhibitor, Numb, dramatically increased when Notch1 decreased during decidualization. We propose that in the endometrium, Notch has a dual role during the window of uterine receptivity. Initially, Notch1 mediates a survival signal in the uterine endometrium in response to CG from the implanting blastocyst and progesterone, so that menstrual sloughing is averted. Subsequently, Notch1 down-regulation may be critical for the transition of stromal fibroblast to decidual cells, which is essential for the establishment of a successful pregnancy.
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Boni, Alessandro, Konrad Urbanek, Angelo Nascimbene, Toru Hosoda, Hanqiao Zheng, Francesca Delucchi, Katsuya Amano, et al. "Notch1 regulates the fate of cardiac progenitor cells." Proceedings of the National Academy of Sciences 105, no. 40 (October 1, 2008): 15529–34. http://dx.doi.org/10.1073/pnas.0808357105.
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The Notch receptor mediates cell fate decision in multiple organs. In the current work we tested the hypothesis that Nkx2.5 is a target gene of Notch1 and raised the possibility that Notch1 regulates myocyte commitment in the adult heart. Cardiac progenitor cells (CPCs) in the niches express Notch1 receptor, and the supporting cells exhibit the Notch ligand Jagged1. The nuclear translocation of Notch1 intracellular domain (N1ICD) up-regulates Nkx2.5 in CPCs and promotes the formation of cycling myocytes in vitro. N1ICD and RBP-Jk form a protein complex, which in turn binds to the Nkx2.5 promoter initiating transcription and myocyte differentiation. In contrast, transcription factors of vascular cells are down-regulated by Jagged1 activation of the Notch1 pathway. Importantly, inhibition of Notch1 in infarcted mice impairs the commitment of resident CPCs to the myocyte lineage opposing cardiomyogenesis. These observations indicate that Notch1 favors the early specification of CPCs to the myocyte phenotype but maintains the newly formed cells in a highly proliferative state. Dividing Nkx2.5-positive myocytes correspond to transit amplifying cells, which condition the replicative capacity of the heart. In conclusion, Notch1 may have critical implications in the control of heart homeostasis and its adaptation to pathologic states.
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Kamińska, Alicja, Sylwia Marek, Laura Pardyak, Małgorzata Brzoskwinia, Barbara Bilinska, and Anna Hejmej. "Crosstalk between Androgen-ZIP9 Signaling and Notch Pathway in Rodent Sertoli Cells." International Journal of Molecular Sciences 21, no. 21 (November 5, 2020): 8275. http://dx.doi.org/10.3390/ijms21218275.
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Our recent study demonstrated altered expression of Notch ligands, receptors, and effector genes in testes of pubertal rats following reduced androgen production or signaling. Herein we aimed to explore the role of nuclear androgen receptor (AR) and membrane androgen receptor (Zrt- and Irt-like protein 9; ZIP9) in the regulation of Notch pathway activation in rodent Sertoli cells. Experiments were performed using TM4 and 15P-1 Sertoli cell lines and rat primary Sertoli cells (PSC). We found that testosterone (10−8 M–10−6 M) increased the expression of Notch1 receptor, its active form Notch1 intracellular domain (N1ICD) (p < 0.05, p < 0.01, p < 0.001), and the effector genes Hey1 (p < 0.05, p < 0.01, p < 0.001) and Hes1 (p < 0.05, p < 0.001) in Sertoli cells. Knockdown of AR or ZIP9 as well as antiandrogen exposure experiments revealed that (i) action of androgens via both AR and ZIP9 controls Notch1/N1ICD expression and transcriptional activity of recombination signal binding protein (RBP-J), (ii) AR-dependent signaling regulates Hey1 expression, (iii) ZIP9-dependent pathway regulates Hes1 expression. Our findings indicate a crosstalk between androgen and Notch signaling in Sertoli cells and point to cooperation of classical and non-classical androgen signaling pathways in controlling Sertoli cell function.
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Guidos, Cynthia J. "Synergy between the pre–T cell receptor and Notch: cementing the αβ lineage choice." Journal of Experimental Medicine 203, no. 10 (September 25, 2006): 2233–37. http://dx.doi.org/10.1084/jem.20060998.
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Notch1 signaling suppresses B cell development and promotes T lineage commitment in thymus-seeding hematopoietic progenitors. Notch1 is also activated in early T cell progenitors, but the functions of these later Notch signals have not been clearly defined. Recent studies reveal that Notch signaling is not essential for pre–T cell receptor (TCR) expression or γδ lineage choice. Rather, pre-TCR signaling enhances progenitor competitiveness for limiting Notch ligands, leading to preferential expansion of TCRβ-bearing progenitors.
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Sanchez-Irizarry, Cheryll, Michael Malecki, Woojoong Lee, Mina Xu, Stephen C. Blacklow, and Jon C. Aster. "Functional Analysis of Leukemia-Associated Mutations Involving the Heterodimerization Domain of NOTCH1." Blood 106, no. 11 (November 16, 2005): 843. http://dx.doi.org/10.1182/blood.v106.11.843.843.
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Abstract NOTCH1 is a type I transmembrane receptor that regulates T cell development via a signaling pathway that relies on regulated proteolysis. During its maturation, most NOTCH1 is cleaved at a position 70 amino acids external to the transmembrane domain by a furin-like protease, creating extracellular (NEC) and transmembrane (NTM) subunits that are held together non-covalently by a juxtamembranous heterodimerization (HD) domain. Ligand-binding to NEC promotes cleavage by i) metalloproteases at a site in the ectodomain of NTM, followed by ii) gamma-secretase within the transmembrane domain. This releases the NOTCH1 intracellular domain (ICN1), allowing it to translocate to the nucleus and activate target genes. Normally, proteolysis is constrained prior to ligand-binding by an extracellular negative regulatory region consisting of 3 iterated LNR repeats and the N- and C-terminal portions of the HD domain, which flank the furin cleavage site. Recent work has shown that human T-ALL is frequently associated with gain of function mutations that map to the HD domain of NOTCH1. These mutations are distributed in both parts of the HD domain and include point mutations, short insertions, and deletions, suggesting that there might be variation in their relative strength and the mechanisms by which they act. To investigate these issues, we introduced 16 of the HD domain mutations found in primary T-ALLs or T-ALL cell lines into a full-length NOTCH1 cDNA, and tested their ability to activate a NOTCH sensitive luciferase reporter gene. Except for the "mutation" R1609S, which was found in only one primary T-ALL sample, all of the mutations stimulated NOTCH1 signaling. These increases in signaling were abolished by a gamma-secretase inhibitor and were associated with increased rates of metalloprotease-mediated cleavage, indicating that activation proceeds through the normal series of proteolytic events. The mutations also caused gains in function when introduced into NOTCH1 polypeptides lacking the ligand-binding region of NEC, indicating that the HD domain mutations can cause ligand-independent receptor activation. Since NEC dissociation can lead to activation of NOTCH signaling (and is a proposed mechanism for normal ligand-mediated NOTCH activation), one simple way for HD domain mutations to act is through the destabilization of NOTCH1 heterodimers. To test this model, each mutation was introduced into soluble NOTCH1 mini-receptors bearing N-terminal FLAG and C-terminal HA tags. When expressed transiently, the normal NOTCH1 mini-receptor was secreted into conditioned media as a furin-processed heterodimer. Certain activating HD domain mutations, such as L1601P, resulted in complete dissociation of the furin-processed mini-receptor subunits under native conditions, and all other HD domain mutations save one were more sensitive to urea-induced dissociation than normal NOTCH1. The exception was an unusual insertional mutation (identified in the P12-Ichikawa cell line) that introduces a 14 amino acid direct repeat sequence at a position immediately N-terminal of the metalloprotease cleavage site. We hypothesize that this mutation, which was associated with the greatest increases in signaling in NOTCH1 reporter gene assays, displaces protective HD and LNR domain residues and thereby unveils the metalloprotease cleavage site. We conclude that most T-ALL-associated HD domain mutations confer ligand-independent gain-of-function on NOTCH1 receptors, but vary in strength and are likely to act through several distinct mechanisms.
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Croquelois, Adrien, Andrea A. Domenighetti, Mohamed Nemir, Mario Lepore, Nathalie Rosenblatt-Velin, Freddy Radtke, and Thierry Pedrazzini. "Control of the adaptive response of the heart to stress via the Notch1 receptor pathway." Journal of Experimental Medicine 205, no. 13 (December 8, 2008): 3173–85. http://dx.doi.org/10.1084/jem.20081427.
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In the damaged heart, cardiac adaptation relies primarily on cardiomyocyte hypertrophy. The recent discovery of cardiac stem cells in the postnatal heart, however, suggests that these cells could participate in the response to stress via their capacity to regenerate cardiac tissues. Using models of cardiac hypertrophy and failure, we demonstrate that components of the Notch pathway are up-regulated in the hypertrophic heart. The Notch pathway is an evolutionarily conserved cell-to-cell communication system, which is crucial in many developmental processes. Notch also plays key roles in the regenerative capacity of self-renewing organs. In the heart, Notch1 signaling takes place in cardiomyocytes and in mesenchymal cardiac precursors and is activated secondary to stimulated Jagged1 expression on the surface of cardiomyocytes. Using mice lacking Notch1 expression specifically in the heart, we show that the Notch1 pathway controls pathophysiological cardiac remodeling. In the absence of Notch1, cardiac hypertrophy is exacerbated, fibrosis develops, function is altered, and the mortality rate increases. Therefore, in cardiomyocytes, Notch controls maturation, limits the extent of the hypertrophic response, and may thereby contribute to cell survival. In cardiac precursors, Notch prevents cardiogenic differentiation, favors proliferation, and may facilitate the expansion of a transient amplifying cell compartment.
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Yuan, Julie S., Joanne B. Tan, Ioana Visan, Irina R. Matei, Peter Urbanellis, Keli Xu, Jayne S. Danska, Sean E. Egan, and Cynthia J. Guidos. "Lunatic Fringe prolongs Delta/Notch-induced self-renewal of committed αβ T-cell progenitors." Blood 117, no. 4 (January 27, 2011): 1184–95. http://dx.doi.org/10.1182/blood-2010-07-296616.
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Abstract Lunatic Fringe (Lfng) enhances Notch1 activation by Delta-like 4 (DL4) to promote Notch1-dependent T-lineage commitment of thymus-seeding progenitors. Subsequently, Notch1 and T-cell receptor-β (TCRβ)–containing pre-TCR complexes signal CD4/CD8 double-negative 3 (DN3) committed T-cell progenitors to survive, proliferate, and differentiate into CD4/CD8 double-positive (DP) αβ T-cell precursors. Few DP thymocytes develop without Notch1 or pre-TCR signals, whereas ectopic Notch1 activation causes T-cell leukemia. However, mechanisms of a Notch-pre-TCR collaboration during this “β-selection” process are poorly understood. We genetically manipulated Lfng to attenuate or enhance Notch1 activation in DN3 thymocytes without inducing leukemogenesis. We show that Lfng temporally sustains DL-induced Notch1 signaling to prolong proliferative self-renewal of pre-DP thymocytes. Pre-TCR signaling greatly augmented Notch trophic functions to promote robust proliferation of pre-DP progenitors. In contrast, in the absence of DL/Notch signaling, pre-TCR-expressing progenitors rapidly atrophied and differentiated into DP thymocytes. Thus, Lfng prolongs Notch1 signaling to promote self-renewal more than differentiation during the early stages of β-selection. Our data provide novel insights into the Notch-pre-TCR collaboration, and suggest that decreasing Lfng expression during the DN3-DP transition minimizes the potent leukemogenic potential of Notch1 signaling.
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Sharma, Ankur, Annapoorni Rangarajan, and Rajan R. Dighe. "Antibodies against the extracellular domain of human Notch1 receptor reveal the critical role of epidermal-growth-factor-like repeats 25–26in ligand binding and receptor activation." Biochemical Journal 449, no. 2 (December 14, 2012): 519–30. http://dx.doi.org/10.1042/bj20121153.
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The Notch signalling pathway is implicated in a wide variety of cellular processes throughout metazoan development. Although the downstream mechanism of Notch signalling has been extensively studied, the details of its ligand-mediated receptor activation are not clearly understood. Although the role of Notch ELRs [EGF (epidermal growth factor)-like-repeats] 11–12 in ligand binding is known, recent studies have suggested interactions within different ELRs of the Notch receptor whose significance remains to be understood. Here, we report critical inter-domain interactions between human Notch1 ELRs 21–30 and the ELRs 11–15 that are modulated by calcium. Surface plasmon resonance analysis revealed that the interaction between ELRs 21–30 and ELRs 11–15 is ~10-fold stronger than that between ELRs 11–15 and the ligands. Although there was no interaction between Notch1 ELRs 21–30 and the ligands in vitro, addition of pre-clustered Jagged1Fc resulted in the dissociation of the preformed complex between ELRs 21–30 and 11–15, suggesting that inter-domain interactions compete for ligand binding. Furthermore, the antibodies against ELRs 21–30 inhibited ligand binding to the full-length Notch1 and subsequent receptor activation, with the antibodies against ELRs 25–26 being the most effective. These results suggest that the ELRs 25–26 represent a cryptic ligand-binding site which becomes exposed only upon the presence of the ligand. Thus, using specific antibodies against various domains of the Notch1 receptor, we demonstrate that, although ELRs 11–12 are the principal ligand-binding site, the ELRs 25–26 serve as a secondary binding site and play an important role in receptor activation.
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Pancewicz, J., P. L. Bernatowicz, and P. Golec. "Human bronchial epithelial cells as a good control for evaluation potential therapeutic Notch signaling in non-small cell lung cancer." Progress in Health Sciences 8, no. 1 (June 18, 2018): 7–13. http://dx.doi.org/10.5604/01.3001.0012.1111.
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Purpose: Notch signaling is often deregulated in non-small cell lung cancer (NSCLC), but little is known about the initial endogenous mRNA status of Notch ligands and receptors. Therefore, the aim of this study was to evaluate expression level of NOTCH1 receptor and Notch ligands, such as delta like ligands (DLL1, DLL3, DLL4), and jagged ligands (JAG1, JAG2), as well as target gene-hes family bHLH transcription factor 1 (HES1) in diverse NSCLC cell lines. Materials and Methods: We have investigated the mRNA expression of chosen genes by using quantitative real time method (RT-PCR). We compared the results from NSCLC cells with results obtained in non-cancerous human bronchial epithelial cells (HBEpC). We also measured NOTCH1 expression in A549 cells, before and after treatment with γ-secretase inhibitor (GSI). Results: The expression level of NOTCH1, HES1, JAG1 and JAG2 was downregulated when compared to HBEpC. The expression of Notch ligand DLL1 was lower in all cancer cell lines, but mRNA level of DLL3 was elevated in H1299 and A549 cells when related to HBEpC. The mRNA level of DLL4 was higher in H520 and in A549 cell lines. Moreover, the mRNA level of NOTCH1 dropped down after GSI treatment, in addition A549 cells proliferated slower after drug implementation. Conclusions: We conclude that non-cancerous HBEpC cells could serve as a good control for Notch mRNAs expression analysis in NSCLC. Moreover, GSI-treated cells could inhibit proliferation through suppressing NOTCH1 in A549 cells.
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Yang, Liang-Tung, James T. Nichols, Christine Yao, Jennifer O. Manilay, Ellen A. Robey, and Gerry Weinmaster. "Fringe Glycosyltransferases Differentially Modulate Notch1 Proteolysis Induced by Delta1 and Jagged1." Molecular Biology of the Cell 16, no. 2 (February 2005): 927–42. http://dx.doi.org/10.1091/mbc.e04-07-0614.
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Fringe O-fucose-β1,3-N-acetylglucosaminyltransferases modulate Notch signaling by potentiating signaling induced by Delta-like ligands, while inhibiting signaling induced by Serrate/Jagged1 ligands. Based on binding studies, the differential effects of Drosophila fringe (DFng) on Notch signaling are thought to result from alterations in Notch glycosylation that enhance binding of Delta to Notch but reduce Serrate binding. Here, we report that expression of mammalian fringe proteins (Lunatic [LFng], Manic [MFng], or Radical [RFng] Fringe) increased Delta1 binding and activation of Notch1 signaling in 293T and NIH 3T3 cells. Although Jagged1-induced signaling was suppressed by LFng and MFng, RFng enhanced signaling induced by either Delta1 or Jagged1, underscoring the diversity of mammalian fringe glycosyltransferases in regulating signaling downstream of different ligand-receptor combinations. Interestingly, suppression of Jagged1-induced Notch1 signaling did not correlate with changes in Jagged1 binding as found for Delta1. Our data support the idea that fringe glycosylation increases Delta1 binding to potentiate signaling, but we propose that although fringe glycosylation does not reduce Jagged1 binding to Notch1, the resultant ligand–receptor interactions do not effectively promote Notch1 proteolysis required for activation of downstream signaling events.
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Weng, Andrew P., Yunsun Nam, Michael S. Wolfe, Warren S. Pear, James D. Griffin, Stephen C. Blacklow, and Jon C. Aster. "Growth Suppression of Pre-T Acute Lymphoblastic Leukemia Cells by Inhibition of Notch Signaling." Molecular and Cellular Biology 23, no. 2 (January 15, 2003): 655–64. http://dx.doi.org/10.1128/mcb.23.2.655-664.2003.
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ABSTRACT Constitutive NOTCH signaling in lymphoid progenitors promotes the development of immature T-cell lymphoblastic neoplasms (T-ALLs). Although it is clear that Notch signaling can initiate leukemogenesis, it has not previously been established whether continued NOTCH signaling is required to maintain T-ALL growth. We demonstrate here that the blockade of Notch signaling at two independent steps suppresses the growth and survival of NOTCH1-transformed T-ALL cells. First, inhibitors of presenilin specifically induce growth suppression and apoptosis of a murine T-ALL cell line that requires presenilin-dependent proteolysis of the Notch receptor in order for its intracellular domain to translocate to the nucleus. Second, a 62-amino-acid peptide derived from a NOTCH coactivator, Mastermind-like-1 (MAML1), forms a transcriptionally inert nuclear complex with NOTCH1 and CSL and specifically inhibits the growth of both murine and human NOTCH1-transformed T-ALLs. These studies show that continued growth and survival of NOTCH1-transformed lymphoid cell lines require nuclear access and transcriptional coactivator recruitment by NOTCH1 and identify at least two steps in the Notch signaling pathway as potential targets for chemotherapeutic intervention.
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Roti, Giovanni, Jun Qi, Angela Su, Antony Varca, Jon C. Aster, James E. Bradner, and Kimberly Stegmaier. "Selectively Targeting Mutated NOTCH1 with a Folate-Thapsigargin Derivative." Blood 124, no. 21 (December 6, 2014): 2158. http://dx.doi.org/10.1182/blood.v124.21.2158.2158.
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Abstract Gain-of-function mutations in NOTCH1, encoding a trans-membrane receptor involved in a transcriptional activation complex, are the most common genetic abnormality in human T-cell acute lymphoblastic leukemia (T-ALL). Thus, targeting NOTCH1 represents one promising therapeutic opportunity in T-ALL. The implementation of NOTCH1 pathway antagonists, however, poses several challenges secondary to concerns about inhibiting normal Notch receptors, leading to gut toxicity and secondary malignancies. We recently reported the results of a gene-expression based, NOTCH1 chemical genomic loss-of-function screen and a cell-based screen for cDNA enhancers of a NOTCH1 allele bearing a leukemia-associated mutation. SERCA (Sarco-endoplasmic reticulum Ca2+ ATPases) emerged at the intersection of these complementary approaches. We discovered that SERCA inhibition impairs the maturation of mutated NOTCH1 receptors and induces a G0/G1 arrest in NOTCH1-mutated human T-ALL cells. A small-molecule SERCA inhibitor, thapsigargin, a sesquiterpene-g-lactone, had on-target activity in mouse models of human T-ALL and also interfered with Notch signaling in Drosophila. Remarkably, thapsigargin preferentially inhibited mutated NOTCH1 receptors. This selectivity provides a therapeutic window not observed with other Notch pathway inhibitors, such as gamma-secretase inhibitors, that are equipotent inhibitors of mutated and wild-type receptors. While thapsigargin is a highly potent tool compound, in its native form, it is unlikely to have direct clinical application because of its global effects on intracellular Ca2+. Aberrant expression of folate receptors (FR) on the surface of cancer cells, including leukemia, has previously been reported, and efforts have been undertaken to leverage folate receptor-mediated endocytosis to deliver cancer therapeutics in vivo. We therefore developed a thapsigargin derivative conjugated with folic acid (JQ-FT) for preclinical testing in T-ALL. The FR gene family includes four members: FOLR1-4. The high affinity of FOLR1 and FOLR2 for folate binding, their endocytic capacity and their restricted expression pattern have prompted evaluation of folate-drug conjugates as a therapeutic approach in cancer. Analysis of gene-expression profiling suggested that FOLR2 is more highly and consistently expressed compared to FOLR1 in human T-ALL cell lines. We confirmed this finding by qRT-PCR across a panel of T-ALL cell lines and three primary patient samples. Surface expression of FOLR1 and FOLR2 measured by flow cytometry paralleled the transcriptional findings. We next validated the dependency of the uptake of a folate-FITC probe in T-ALL cells on active endocytosis via FR. First, we demonstrated that treatment of T-ALL cells with increasing concentrations of folate-FITC corresponds to an increase in measurable fluorescent signal by flow cytometry. The uptake is specific for FR as overexpression of FOLR1 or FOLR2 leads to increased, and folic acid competition decreased, fluorescent signal. Moreover, active endocytosis is supported by data demonstrating that folate-FITC fluorescence is not eliminated with an acidic wash step and that cold temperature does diminish internalization of the chemical probe. Next we demonstrated that increasing concentrations of JQ-FT impede the growth of T-ALL cells and induce apoptosis as measured by the activation of caspase 2. Moreover, the overexpression of ICN1, the intracellular active form of NOTCH1, partially rescues the small-molecule anti-proliferative effect indicating that the JQ-FT retains the on target activity similar to the underivatized thapsigargin. As observed with thapsigargin treatment of T-ALL cells, JQ-FT treatment decreased the levels of the intracellular active form of NOTCH1, ICN1, and the furin-processed transmembrane form of NOTCH1, while the full length precursor accumulated. In summary, these pre-clinical studies confirm the selective uptake of a folate-conjugated thapsigargin by an FR-mediated mechanism with the suppression of mutated NOTCH1 maturation and support the ongoing testing of this approach in mouse models of NOTCH1-mutated T-ALL. If successful, this approach would enhance the therapeutic window of thapsigargin as a NOTCH1 inhibitor providing dual selectivity: leukemia over normal cell and NOTCH1 mutated over wild-type receptors. Disclosures No relevant conflicts of interest to declare.
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Kox, Corinne, Svenja Leible, Stephen Breit, Martin Stanulla, Martin Schrappe, Martina Ulrike Muckenthaler, and Andreas Kulozik. "Notch Pathway Activation by a Combination of Activating NOTCH1-Receptor and Inactivating FBXW7 Mutations Robustly Predicts Excellent Early Treatment Response in Pediatric T-ALL." Blood 112, no. 11 (November 16, 2008): 1493. http://dx.doi.org/10.1182/blood.v112.11.1493.1493.
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Abstract In contrast to pre B-ALL, robust DNA-based biomarkers with prognostic and predictive value are not available in T-ALL. We have previously shown that common activating mutations of the NOTCH1 receptor predict good early treatment response and excellent long term outcome in T-ALL (Breit et al., Blood 2006). We now aimed at analyzing if recently described inactivating mutations of FBXW7, the E3 ubiquitin ligase of NOTCH, contribute to the predictive power of the Notch1 pathway activation in pediatric T-ALL. Figure Figure We genotyped FBXW7 in 137 T-ALL and found inactivating mutations in 22 patients. We next analyzed the influence of this mutation on treatment response and found that most patients with FBXW7 mutations showed a prednisone good response (PGR; 19/22), whereas PGR was observed in only 58/115 with no FBWX7 mutation (p=0.0006). This beneficial effect of FBXW7 inactivating mutations is enhanced when the NOTCH1 mutational status was considered. Among 17 patients with NOTCH1 and FBXW7 mutations, only 1 showed a prednisone poor response (PPR; p=0.0003). These results suggest a synergistic effect of NOTCH1 and FBXW7 mutations in early treatment response in pediatric T-ALL. Notably, FBXW7 mutations occur in the group of leukemias with NOTCH1 heterodimerisation (HD) domain mutations only and do not occur in those with truncations of the PEST domain, where FBXW7 physiologically targets NOTCH for degradation. One might thus predict that patients with NOTCH1-HD/PEST double mutations would be biologically similar to those with NOTCH1-HD/FBXW7 double mutations. A comparison of early treatment response between these groups indeed showed PGR in 10/13 patients with NOTCH1-HD/PEST and 16/17 with NOTCH1-HD/FBXW7 status. Furthermore, these groups taken together exhibit a significantly better prednisone response than both, the patients with single mutations in either NOTCH1 or in FBXW7 or those with detectable mutations in neither NOTCH1 nor FBWX7 (see bar diagram). These data indicate that activation of the Notch1 pathway by a combination of receptor gain of function mutations and inactivating mutations of its E3 ubiquitin ligase has a strong favorable effect on in-vivo prednisone sensitivity. Notch pathway activation may thus define a novel group of biomarkers that predict early treatment response and thus help to stratify risk of children and adolescents with T-ALL.
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Roti, Giovanni, Kenneth N. Ross, Adolfo A. Ferrando, Stephen C. Blacklow, Jon Aster, and Kimberly Stegmaier. "Expression-Based Screen Identifies the Calcium Channel Antagonist Bepridil as a Notch1 Modulator in T-ALL." Blood 114, no. 22 (November 20, 2009): 366. http://dx.doi.org/10.1182/blood.v114.22.366.366.
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Abstract Abstract 366 Gain of function mutations in Notch1, which encodes a signaling protein that is converted into a transcription factor upon activation, are the most common genetic abnormality in human T-cell acute lymphoblastic leukemia (T-ALL). Although inhibiting Notch1 activity represents a potential therapeutic opportunity, the discovery of new Notch1 pathway antagonists poses a difficult challenge. Traditional small molecule library screening approaches have not been amenable to modulating transcription factor abnormalities. In order to overcome this challenge, we applied Gene Expression-based High-throughput Screening (GE-HTS) to discover new Notch1 modulators. GE-HTS uses gene expression signatures as surrogates for different biological states. We derived a 32-gene Notch1 expression signature from genome-wide microarray expression profiling of 7 different Notch1 mutant T-ALL cell lines treated with vehicle (Notch1 on) versus a Notch1 inactivating γ-secretase inhibitor (GSI; Notch off) and screened a small molecule library for compounds inducing the Notch1 off state in DND41 mutant Notch1 T-ALL cells. Among numerous ion flux modulators validated to induce the Notch1 off signature, one of the top hits was the FDA-approved calcium channel blocker, bepridil, used to treat patients with cardiac disease. In multiple mutant Notch1 T-ALL cell lines, bepridil induced the Notch1 off signature. We next confirmed that bepridil indeed targets Notch signaling by demonstrating its inhibitory effects on a Notch-sensitive luciferase reporter gene in heterologous U2OS cells expressing a mutated form of Notch1. Similar to the phenotypic effects of GSI, bepridil induced a G0/G1 cell cycle arrest, inhibited cellular viability, and decreased cell size in multiple T-ALL cell lines, including the GSI-resistant cell line PF382. Next, in order to confirm dependency of the induced phenotype on inhibition of Notch, we utilized the 8946 T-ALL cell line. This murine line depends on a doxycycline-repressible human c-myc transgene for growth and can be rescued from transgene withdrawal with activated Notch1, which upregulates the endogenous c-myc gene. In these cells, the phenotypic effect of bepridil on viability is also dependent on Notch1 inhibition because cells rescued from transgene withdrawal with activated Notch1 were more sensitive to the effects of the drug than were those cells still dependent on the c-myc transgene. Finally, we asked whether bepridil altered the level of active Notch1 protein in T-ALL cell lines. As with GSI, bepridil treatment results in decreased levels of intracellular Notch (ICN1). In contrast to GSI, however, bepridil treatment decreased levels of the furin-processed extracellular and transmembrane forms of Notch1 while the full length Notch1 precursor form accumulated upon bepridil treatment. One hypothesis is that by altering ER/Golgi compartment calcium, bepridil prevents the folding of newly synthesized Notch1 polypeptides, leading to its retention in the ER/Golgi and a failure to traffic to cellular compartments where receptor activation occurs. Consistent with this hypothesis co-localization studies in U2OS cell lines expressing the L1601P mutant Notch1 suggest retention of Notch1 in the ER/Golgi. An alternative hypothesis under investigation is that bepridil affects the activity of furin, a calcium-dependent protease that is required for processing of Notch receptors. In summary, we have identified an FDA-approved drug with Notch1 modulating activity in T-ALL by a mechanism unique from GSI. These studies have potential for rapid translation to clinical testing. Disclosures: Ferrando: Merck, Pfizer: Research Funding.
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Arruga, Francesca, Branimir Gizdic, Sara Serra, Tiziana Vaisitti, Davide Rossi, Marta Coscia, Luca Laurenti, et al. "Functional Effects Of NOTCH1 Mutations In Chronic Lymphocytic Leukemia Patients." Blood 122, no. 21 (November 15, 2013): 4117. http://dx.doi.org/10.1182/blood.v122.21.4117.4117.
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Abstract Chronic lymphocytic leukemia (CLL), is characterized by the expansion of mature B lymphocytes present in blood, bone marrow and lymphoid organs. Clinical behavior is highly heterogeneous, thus requiring timely identification of high-risk patients. NOTCH1 encodes a trans-membrane receptor acting as a ligand-activated transcription factor. NOTCH signaling initiates when the ligand, from either the Jagged or Delta families, binds to the receptor and induces successive proteolytic cleavages, resulting in the release and nuclear translocation of the NOTCH intra-cellular domain (NICD). Signaling is terminated by phosphorylation of the PEST domain of NOTCH1, triggering its ubiquitination and proteasomal degradation. Whole exome sequencing approaches have revealed NOTCH1 mutations in 5-10% newly diagnosed CLL cases, with their prevalence increasing to 15-20% in progressive or relapsed patients. The most frequent mutation is 7544-7545delCT frameshift deletion in exon 34, resulting in disruption of the C-terminal PEST domain. Truncation of the PEST domain is predicted to result in NOTCH1 impaired degradation, stabilization of the active NOTCH1, and deregulated signaling. The present study was undertaken with the aim to compare NOTCH1 expression and functional role in CLL patients harboring wild type (WT) or mutated (M) NOTCH1 gene. NOCTH1 mRNA and surface protein were expressed at comparable high levels in peripheral blood (PB) CLL cells obtained from NOTCH1 M and WT patients, consistent with a more general requirement of NOTCH1 signaling in this leukemia. However, at a variance of NOTCH1 WT cases, NOCTH1 M patients displayed remarkable accumulation of both the intermediate molecular species of the activated NOCTH1 receptor, as well as of the active NICD. Consistently, by gene expression profiling NOCTH1 M patients displayed significantly higher levels of HES1 and DTX1, the main NOTCH1 target genes. Overall, these data suggest a more active signaling pathway in NOTCH1 M CLL than in NOTCH1 WT cases. Expression of NOTCH1 and of its target gene (DTX1) varied across disease compartments, being higher in CLL cells obtained from the lymph nodes (LN), as compared to paired samples derived from the PB or the bone marrow (BM). By immunohistochemical analyses of primary LN tissue samples, NOTCH1 M CLL showed an intense nuclear staining as opposed to the more cytoplasmic distribution observed in NOTCH1 WT samples. These data suggest a more active NOTCH1 signaling in CLL residing in the LN microenvironment and confirm the functional effect of NOTCH1 mutations in vivo. When PB CLL cells were cultured in vitro in the absence of any supporting layer or stimulation, they showed a rapid down regulation of the NOTCH pathway, with complete loss of NICD after 24 hours paralleled by a sharp decrease in HES1 and DTX1 transcription. Consistently, levels of presenilin-1 (PSEN1), the catalytic subunit of the g-secretase complex, were also down-regulated offering a partial mechanistic explanation for the NICD loss. NOTCH1 mRNA levels remained unchanged, with accumulation of the receptor at the plasma membrane. These effects were independent of NOTCH1 mutation status and suggested the dependence of NOTCH1 signaling activation upon in vivo microenvironmental interaction, even in NOTCH1 M CLL. Within primary LN biopsies from CLL patients, the NOTCH1 ligand, was highly expressed on CD68+ elements of myeloid origin. This observation prompted the in vitro recreation of a lymphoid niche by co-culturing Jagged1+ nurse-like cells (NLC) with autologous CLL cells. Under these conditions, NOTCH1 activity in CLL cells was sustained over time, as shown by Q-PCR analyses of DTX1 and PSEN1. Moreover, NLCs protect NOTCH1 M CLL cells from fludarabine-induced apoptosis. This microenvironment-induced chemoresistance was prevented by pre-treatment of NOTCH1 M CLL cells with specific g-secretase inhibitors, to block NOTCH1 activation. Taken together, these results show that the 7544-7545delCT mutation in the PEST domain of NOTCH1 has a stabilizing effect on NOTCH1 signaling pathway. They also show that micro-environmental interactions are critical in activating NOTCH1 pathway both in the M and WT patients. Lastly, these results show that NOTCH1 signals micht create local conditions that favour drug resistance, thus making NOTCH1 a potential molecular target in CLL. Disclosures: No relevant conflicts of interest to declare.
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Karsan, Aly. "The role of notch in modeling and maintaining the vasculature." Canadian Journal of Physiology and Pharmacology 83, no. 1 (January 1, 2005): 14–23. http://dx.doi.org/10.1139/y04-125.
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The Notch proteins encompass a family of transmembrane receptors that have been highly conserved through evolution as mediators of cell fate, and are comprised of 4 members in mammals (Notch1 to Notch4). Following intra cellular processing of the full-length protein, Notch is expressed at the cell surface as a heterodimeric receptor. Engagement by ligand results in a 2-step cleavage of the Notch heterodimer, releasing the intracellular domain of Notch and allowing translocation to the nucleus. The intracellular domain of Notch interacts with the DNA-binding factor, CSL, resulting in transactivation at various promoters, in particular those of various basic helix-loop-helix factors of the HES (Hairy and Enhancer of Split) and HRT families (Hairy-Related Transcription factor). Recent findings implicate Notch as playing a critical and non-redundant role in vascular development and maintenance. This article briefly reviews vessel development and Notch signaling and highlights studies that examine Notch functions such as proliferation, cell survival, migration, adhesion, and mesenchymal transformation in the vasculature. Human diseases caused by Notch pathway members are also discussed.Key words: vascular, endothelial, Notch, angiogenesis, mesenchymal transformation.
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Rojas, Katerin Ingrid, M. Rocio Martín, Federico Rojo, Angelo Gámez-Pozo, Francisco J. De Castro, Elena Filipovich, Jaime Ceballos, M. Rosario Hernández, and Jose Ales-Martinez. "Transcriptomic mapping on Notch signaling in A luminal phenotype breast cancer." Journal of Clinical Oncology 39, no. 15_suppl (May 20, 2021): e12539-e12539. http://dx.doi.org/10.1200/jco.2021.39.15_suppl.e12539.
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e12539 Background: The Notch signaling pathway plays an important role in cell-differentiation, survival, proliferation, stem-cell renewal, and in determining cell fate during development and morphogenesis. The dysregulation of the Notch pathways contributes to carcinogenesis, cancer stem cell renewal, angiogenesis, and chemo-resistance. Elevated levels of Notch receptors and ligands have been associated with cancer-progression and poor survival. A less explored function of Notch pathways in cancer is their role in leukocyte homing and activation. Understanding their role and relationship with immune infiltrates is an area of interest in cancer research. Methods: The expression of four different Notch genes (Notch1, Notch2, Notch3 and Notch4) was explored in relation with A luminal breast cancer patient outcome using transcriptomic data (Affymetrix dataset, exploratory cohort) and the METABRIC study (validation cohort). The TIMER online tool was used to explore the association of the identified notch and immune infiltration, and the TCGA and METABRIC studies to analyze the correlation between notch1 - 4 expression and genomic signatures of immune activation. Results: We identified 2 individual genes called Notch1 and Notch2, which predict favorable prognosis in luminal A breast cancer. Their expression positively correlated with the presence of immune infiltrates within the tumor (dendritic cells, CD4+ T cells, neutrophils, CD8+ T cells and B cells), with markers of T cell activation and antigen presentation, and with gene signatures of immune surveillance (cytotoxic T lymphocyte activation and IFN gamma signature). By contrast Notch3 and Notch4 which predicted for detrimental outcome were not associated with any of these parameters. Conclusions: Our analysis identifies a Notch signature composed of 2 genes with potential to recognize immune infiltrated and activated A luminal phenotype breast cancers with favorable prognosis.
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Hubmann, Rainer, Martin Hilgarth, Susanne Schnabl, Elena Ponath, Dita Demirtas, Marlies Reiter, Ulrich Jäger, and Medhat Shehata. "Differential Expression and Functions of NOTCH Family Members and Involvement of NR4A1 in the Regulation of Apoptosis in CLL." Blood 120, no. 21 (November 16, 2012): 3919. http://dx.doi.org/10.1182/blood.v120.21.3919.3919.
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Abstract Abstract 3919 Chronic lymphocytic leukemia (CLL) cells express constitutively activated NOTCH2 in a protein kinase C (PKC) dependent manner linking NOTCH2 to the activated state of the leukemic cells. The transcriptional activity of NOTCH2 is associated with the expression of CD23 and enhanced CLL cell viability. However, the regulation and possible functions of the individual NOTCH family members (NOTCH1–4) in CLL cells remain to be clarified. We took advantage of targeting nuclear NOTCH2 using the recently identified NOTCH2 transactivation inhibitor gliotoxin (WO 2006/135949). We also analysed the regulation and possible function of NOTCH1–4 in PKC stimulated CLL cells using a PMA model (Hubmann et al., BJH 2010) and a microenvironment model where CLL lymphocytes were co-cultured with primary bone marrow stromal cells (BMSC) (Shehata et al., BLOOD 2010). Electrophoretic mobility shift assays (EMSA) demonstrated that gliotoxin inhibited DNA-bound NOTCH2 complexes in PMA stimulated CLL cells in parallel to increasing the rate of apoptosis (mean±SD: 67±31% in gliotoxin treated cells versus 13±14% in the untreated controls, n=21). This was associated with downregulation of CD23A mRNA expression and CD23 surface expression (mean±SD: 42±32% versus 83±17%, n=21) as assessed by RT-PCR and FACS analysis. Exceptionally, one CLL case with a recently described NOTCH1 gain of function mutation appeared to be less sensitive to gliotoxin and had a persistent high expression of CD23. We next tested whether NOTCH2 inhibition by gliotoxin is a selective process or indirectly mediated by effects on proteasome regulated apoptosis. Proteasome assays showed that gliotoxin had a minimal or no effect on the chymotrypsin like activity of the proteasome in CLL cells. In addition, the activity of the proteasome regulated transcription factor NFκB and the expression of its target genes like BCL2 and MCL1 were also not influenced by gliotoxin. These data point to the selectivity of targeting NOTCH2 signaling by gliotoxin rather than indirectly through the regulation of proteasome activity. Short term (4 hours) exposure of CLL cells revealed that NOTCH1 was equally transcribed in unstimulated and in PMA activated CLL cells. NOTCH2 was upregulated in PMA activated CLL lymphocytes whereas NOTCH4 was only weakly detectable in unstimulated CLL cells. Gliotoxin treatment resulted in the downregulation of NOTCH1, NOTCH2 and NOTCH4 mRNA expression. Interestingly, the inhibition of NOTCH2 activity by gliotoxin was associated with the concomitant induction of NOTCH3 signaling especially in the presence of PMA. This was indicated by the induced mRNA expression of NOTCH3 and its preferred target gene HEY1. Moreover, the induced transcription of HEY1 correlated with the upregulation of NR4A1, a key regulator of apoptosis in activated lymphocytes. These data may thus point to a pro-apoptotic role for NOTCH3/HEY1/NR4A1 signaling in CLL cells. The data also suggest that gliotoxin induced apoptosis is associated with differential regulation of the anti-apoptotic and pro-apoptotic arms of NOTCH signalling in CLL cells. RT-PCR revealed that NOTCH1 and NOTCH2 are the main NOTCH family members which are expressed in CLL cells under co-culture conditions with BMSC and in freshly isolated CLL cells. Exposure to gliotoxin in co-culture selectively induced apoptosis in CLL cells and led to downregulation of NOTCH1 and NOTCH2 together with upregulation of NOTCH3 mRNA expression. In summary, the data suggest that nuclear NOTCH2 activity might protect activated CLL cells from apoptosis by modulating the expression of NR4A1. The induced expression of NOTCH3 and its target gene HEY1 by gliotoxin reveals the complex role of different NOTCH family members in the regulation of apoptosis in CLL cells. Therefore, the individual NOTCH receptors may have opposite effects on CLL cell viability which should be considered in therapeutic approaches aimed to target NOTCH signaling in CLL. Disclosures: No relevant conflicts of interest to declare.
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Gordon-Thomson, Clare, Steven A. Botto, Graham R. Cam, and G. Philip M. Moore. "Notch pathway gene expression and wool follicle cell fates." Australian Journal of Experimental Agriculture 48, no. 5 (2008): 648. http://dx.doi.org/10.1071/ea07315.
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The Notch family of genes has been implicated in specifying cell fates during hair follicle morphogenesis. We examined Notch gene expression during wool follicle formation, as an understanding of genes that influence cell distributions in the developing follicle is a prerequisite for devising molecular strategies to manipulate fibre characters and follicle density. We identified transcripts for the Notch1 receptor and one of its ligands, Jagged1, in fetal sheep skin by reverse transcriptase polymerase chain reaction. The sheep-specific cDNA sequences were used as templates to produce probes to investigate the expression patterns of Notch1 and Jagged1 in developing ovine fetal skin by in situ hybridisation. Notch1 and Jagged1 were detected in the epidermis and in a subpopulation of mesenchymal cells before follicle initiation. At day 70 during follicle initiation, transcripts were also detected in cells at the tip of the epidermal plug and in dermal condensates. By day 86, Notch1 and Jagged1 were detected in the distal cells of the epidermal downgrowths and epidermis and Notch1 was no longer detected in the mesenchyme and dermal condensates. After day 96, transcripts were absent from the epidermis, but localised to differentiating outer root sheath (ORS) cells. The distributions of transcripts implicate a Notch1–Jagged1 signal pathway in the fates of prospective ORS cells. The transient appearance of Notch1 in cells at the epidermal–mesenchymal junction during early follicle morphogenesis suggests that the receptor may be responsible for the specification of a cell subpopulation committed to a prepapilla fate at initiation.
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Engel, Michael E., Hong N. Nguyen, Jolene Mariotti, Aubrey Hunt, and Scott W. Hiebert. "Myeloid Translocation Gene 16 (MTG16) Interacts with Notch Transcription Complex Components To Integrate Notch Signaling in Hematopoietic Cell Fate Specification." Molecular and Cellular Biology 30, no. 7 (February 1, 2010): 1852–63. http://dx.doi.org/10.1128/mcb.01342-09.
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ABSTRACT The Notch signaling pathway regulates gene expression programs to influence the specification of cell fate in diverse tissues. In response to ligand binding, the intracellular domain of the Notch receptor is cleaved by the γ-secretase complex and then translocates to the nucleus. There, it binds the transcriptional repressor CSL, triggering its conversion to an activator of Notch target gene expression. The events that control this conversion are poorly understood. We show that the transcriptional corepressor, MTG16, interacts with both CSL and the intracellular domains of Notch receptors, suggesting a pivotal role in regulation of the Notch transcription complex. The Notch1 intracellular domain disrupts the MTG16-CSL interaction. Ex vivo fate specification in response to Notch signal activation is impaired in Mtg16 −/− hematopoietic progenitors, and restored by MTG16 expression. An MTG16 derivative lacking the binding site for the intracellular domain of Notch1 fails to restore Notch-dependent cell fate. These data suggest that MTG16 interfaces with critical components of the Notch transcription complex to affect Notch-dependent lineage allocation in hematopoiesis.
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Weinmaster, G., V. J. Roberts, and G. Lemke. "Notch2: a second mammalian Notch gene." Development 116, no. 4 (December 1, 1992): 931–41. http://dx.doi.org/10.1242/dev.116.4.931.
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Notch is a cell surface receptor that mediates a wide variety of cellular interactions that specify cell fate during Drosophila development. Recently, homologs of Drosophila Notch have been isolated from Xenopus, human and rat, and the expression patterns of these vertebrate proteins suggest that they may be functionally analogous to their Drosophila counterpart. We have now identified a second rat gene that exhibits substantial nucleic and amino acid sequence identity to Drosophila Notch. This gene, designated Notch2, encodes a protein that contains all the structural motifs characteristic of a Notch protein. Thus, mammals differ from Drosophila in having more than one Notch gene. Northern and in situ hybridisation analyses in the developing and adult rat identify distinct spatial and temporal patterns of expression for Notch1 and Notch2, indicating that these genes are not redundant. These results suggest that the great diversity of cell-fate decisions regulated by Notch in Drosophila may be further expanded in vertebrates by the activation of distinct Notch proteins.
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Pross, Seth, John M. Millholland, Hong Sai, Andrew P. Weng, Jon C. Aster, Warren S. Pear, and Ivan Maillard. "Efficient Inhibition of Notch3 and Notch4 Family Members In Vivo by a Dominant Negative Mutant of Mastermind." Blood 104, no. 11 (November 16, 2004): 1617. http://dx.doi.org/10.1182/blood.v104.11.1617.1617.
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Abstract The four mammalian Notch receptors play multiple roles in the hematolymphoid system. Notch1 is critical for the generation of hematopoietic stem cells (HSCs) during embryonic development and for commitment to the T cell lineage, while Notch2 regulates marginal zone B cell development. In addition, important roles for Notch are starting to be identified in peripheral T cells, in which all four Notch family members are upregulated upon T cell activation. Canonical Notch signaling involves proteolytic cleavage of Notch receptors and translocation of intracellular Notch (ICN) to the nucleus where it interacts with the transcription factor CSL/RBP-J and recruits Mastermind-like proteins (MAMLs) for transcriptional activation. By inhibiting Notch1 and Notch2-dependent developmental decisions with a dominant negative mutant of MAML1 (DNMAML1), we have previously demonstrated that MAMLs critically regulate Notch signaling in vivo (Maillard et al., Blood 2004). However, it is unclear if Notch3 and Notch4 are equally dependent on the activity of MAMLs in vivo, and if DNMAML1 can thus be used as a true pan-Notch inhibitor. To address this question, we used a modified fetal thymic organ culture (FTOC) system and Notch-mediated T lineage commitment as a readout for Notch function. Fetal liver cells (FLCs) were transduced with retroviruses expressing ICN1, ICN3 or ICN4 and a retrovirus expressing DNMAML1. Irradiated fetal thymic lobes were reconstituted with transduced FLCs and cultured in the presence of gamma secretase inhibitors to inhibit the activation of endogenous Notch receptors. In this manner the effect of a specific ICN could be evaluated in the absence of endogenous Notch signals. Gamma secretase inhibitors resulted in a profound block in T cell development and in the generation of intrathymic B cells, consistent with Notch1 inhibition. ICN1, ICN3 and ICN4 were all able to rescue commitment to the T cell lineage and to block B cell development, indicating that each ICN can deliver appropriate signals for T lineage commitment in the absence of endogenous Notch1 signaling. In cells doubly transduced with specific ICNs and DNMAML1, the rescuing effect of ICN1, ICN3 or ICN4 was blocked, leading to the generation of B cells. In this competitive situation, DNMAML1 only partially inhibited ICN1, the most potent of all four Notch family members, but it was able to completely inhibit ICN3 and ICN4. We conclude that DNMAML1 can efficiently inhibit Notch3 and Notch4 in vivo. The results validate the use of DNMAML1 as a bona fide pan-Notch inhibitor in multiple settings, including studying the role of Notch in HSCs and peripheral T cells.
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Gordon, Wendy R., Monideepa Roy, Didem Vardar-Ulu, Megan Garfinkel, Marc R. Mansour, Jon C. Aster, and Stephen C. Blacklow. "Structure of the Notch1-negative regulatory region: implications for normal activation and pathogenic signaling in T-ALL." Blood 113, no. 18 (April 30, 2009): 4381–90. http://dx.doi.org/10.1182/blood-2008-08-174748.
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Abstract Proteolytic resistance of Notch prior to ligand binding depends on the structural integrity of a negative regulatory region (NRR) of the receptor that immediately precedes the transmembrane segment. The NRR includes the 3 Lin12/Notch repeats and the juxtamembrane heterodimerization domain, the region of Notch1 most frequently mutated in T-cell acute lymphoblastic leukemia lymphoma (T-ALL). Here, we report the x-ray structure of the Notch1 NRR in its autoinhibited conformation. A key feature of the Notch1 structure that maintains its closed conformation is a conserved hydrophobic plug that sterically occludes the metalloprotease cleavage site. Crystal packing interactions involving a highly conserved, exposed face on the third Lin12/Notch repeat suggest that this site may normally be engaged in intermolecular or intramolecular protein-protein interactions. The majority of known T-ALL–associated point mutations map to residues in the hydrophobic interior of the Notch1 NRR. A novel mutation (H1545P), which alters a residue at the crystal-packing interface, leads to ligand-independent increases in signaling in reporter gene assays despite only mild destabilization of the NRR, suggesting that it releases the autoinhibitory clamp on the heterodimerization domain imposed by the Lin12/Notch repeats. The Notch1 NRR structure should facilitate a search for antibodies or compounds that stabilize the autoinhibited conformation.
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Kramer, Jan, Ralf Schwanbeck, Horst Pagel, Figen Cakiroglu, Jürgen Rohwedel, and Ursula Just. "Inhibition of Notch Signaling Ameliorates Acute Kidney Failure and Downregulates Platelet-Derived Growth Factor Receptor β in the Mouse Model." Cells Tissues Organs 201, no. 2 (2016): 109–17. http://dx.doi.org/10.1159/000442463.
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Ischemic acute kidney injury (AKI) is associated with high morbidity and frequent complications. Repeated episodes of AKI may lead to end-stage renal failure. The pathobiology of regeneration in AKI is not well understood and there is no effective clinical therapy that improves regeneration. The Notch signaling pathway plays an essential role in kidney development and has been implicated in tissue repair in the adult kidney. Here, we found that kidneys after experimental AKI in mice showed increased expression of Notch receptors, specifically Notch1-3, of the Notch ligands Jagged-1 (Jag1), Jag2 and Delta-like-4 (Dll4) and of the Notch target genes Hes1, Hey2, HeyL, Sox9 and platelet-derived growth factor receptor β (Pdgfrb). Treatment of ischemic mice with the γ-secretase inhibitor DBZ blocked Notch signaling and specifically downregulated the expression of Notch3 and the Notch target genes Hes1, Hey2, HeyL and Pdgfrb. After DBZ treatment, the mice developed less interstitial edema and displayed altered interstitial inflammation patterns. Furthermore, serum urea and creatinine levels were significantly decreased from 6 h onwards when compared to control mice treated with DMSO only. Our data are consistent with an amelioration of the severity of kidney injury by blocking Notch activation following AKI, and suggest an involvement of Notch-regulated Pdgfrb in AKI pathogenesis.
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Zine, A., T. R. Van De Water, and F. de Ribaupierre. "Notch signaling regulates the pattern of auditory hair cell differentiation in mammals." Development 127, no. 15 (August 1, 2000): 3373–83. http://dx.doi.org/10.1242/dev.127.15.3373.
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The development of the mammalian cochlea is an example of patterning in the peripheral nervous system. Sensory hair cells and supporting cells in the cochlea differentiate via regional and cell fate specification. The Notch signaling components shows both distinct and overlapping expression patterns of Notch1 receptor and its ligands Jagged1 (Jag1) and Jagged2 (Jag2) in the developing auditory epithelium of the rat. On embryonic day 16 (E16), many precursor cells within the Kolliker's organ immunostained for the presence of both Notch1 and Jag1, while the area of hair cell precursors did not express either Notch1 and Jag1. During initial events of hair cell differentiation between E18 and birth, Notch1 and Jag1 expression predominated in supporting cells and Jag2 in nascent hair cells. Early after birth, Jag2 expression decreased in hair cells while the pattern of Notch1 expression now included both supporting cells and hair cells. We show that the normal pattern of hair cell differentiation is disrupted by alteration of Notch signaling. A decrease of either Notch1 or Jag1 expression by antisense oligonucleotides in cultures of the developing sensory epithelium resulted in an increase in the number of hair cells. Our data suggest that the Notch1 signaling pathway is involved in a complex interplay between the consequences of different ligand-Notch1 combinations during cochlear morphogenesis and the phases of hair cell differentiation.
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Rakowski, Lesley A., Derek D. Garagiola, Sarah C. Caruso, and Mark Y. Chiang. "ZMIZ1 Collaborates with NOTCH1 During Induction and Maintenance of T-Cell Acute Lymphoblastic Leukemia." Blood 118, no. 21 (November 18, 2011): 75. http://dx.doi.org/10.1182/blood.v118.21.75.75.
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Abstract Abstract 75 Activating NOTCH1 mutations are found in 40–60% of human T-cell acute lymphoblastic leukemia (T-ALL) samples. In mouse models, most leukemia-associated NOTCH1 mutations fail to induce leukemia. This observation suggests that cooperating oncogenes must be recruited by NOTCH1 to fully induce leukemia. In murine retroviral/transposon insertional mutagenesis screens, induction of the Zmiz1 gene was frequently associated with activation of the Notch1 receptor during leukemogenesis (Uren et al., Cell, 2008; Dupuy et al., Nature, 2005). ZMIZ1 is a transcriptional co-activator of the Protein Inhibitor of Activated STAT-like family that has been implicated for prostate cancer survival. It directly interacts with the androgen receptor to enhance its transcriptional activity. To investigate the role of ZMIZ1 during leukemogenesis, activating NOTCH1 mutations and ZMIZ1 were transduced into hematopoietic progenitor cells. These cells were then used to reconstitute lethally irradiated mice. ZMIZ1 or NOTCH1 alone failed to induce T-ALL after 1 year of observation. In contrast, ZMIZ1 and NOTCH1 in combination induced T-ALL with ∼50% penetrance by 100 days after transplantation. These data show that ZMIZ1 can promote leukemogenesis in cooperation with NOTCH1. To determine the relevance of ZMIZ1 to human leukemia, we screened 15 primary human adult T-ALL samples for ZMIZ1 mRNA and protein. 20% expressed ZMIZ1. In publically available data sets, ZMIZ1 gene expression was significantly enriched by ∼2-fold in early thymocyte precursor ALL (ETP-ALL) samples. The ETP-ALL subgroup comprises about 13% of all T-ALL and may have a highly unfavorable prognosis. We discovered expression of ZMIZ1 in two ETP-like cell lines. To investigate whether ZMIZ1 is a potential therapeutic target, we transduced these cell lines with shRNA directed against ZMIZ1. ZMIZ1 inhibition reduced cell size, increased apoptosis by ∼2-fold, and reduced growth by 75–94%. Furthermore, ZMIZ1 knockdown overcame resistance to NOTCH signaling blockade with g-secretase inhibitors. Since the glucocorticoid receptor is highly homologous to the androgen receptor, we considered the possibility that ZMIZ1 inhibition may promote glucocorticoid resistance. However, we treated ZMIZ1-inhibited T-ALL cell lines with increasing doses of dexamethasone. ZMIZ1-inhibited T-ALL cell lines were twice as sensitive to dexamethasone than uninhibited cells. These data suggest that ZMIZ1 is required for leukemia growth and survival. Inhibition of ZMIZ1 may potentially enhance targeting of T-ALL with NOTCH pathway inhibitors and glucocorticoids. To determine the mechanism underlying ZMIZ1 function, we performed gene expression profiling. We identified C-MYC as a potential downstream target of ZMIZ1. C-MYC is also a direct target of NOTCH1. Ectopic expression of ZMIZ1 or NOTCH1 had weak effects on endogenous C-MYC expression and failed to rescue a C-MYC-dependent T-ALL cell line after withdrawal of ectopic C-MYC. In contrast, ZMIZ1 in combination with NOTCH1 dramatically induced C-MYC expression by ∼7000 fold, induced C-MYC target gene expression, and rescued the C-MYC dependent cell line. ZMIZ1 inhibition lowered C-MYC levels by ∼93%. The interaction between ZMIZ1 and NOTCH appeared to be specific for C-MYC, as modulation of ZMIZ1 levels did not affect the NOTCH1 target genes Hes1, Dtx1, and Cd25. Downregulation of C-MYC partly phenocopied the effects of ZMIZ1 downregulation. However, ectopic expression of C-MYC failed to rescue the growth of ZMIZ1-inhibited cells. These data suggest that C-MYC is an essential but insufficient downstream effector of ZMIZ1 function. In conclusion, ZMIZ1 is new potential therapeutic target in a subset of T-ALL. It functionally interacts with NOTCH1 to promote C-MYC expression and activity. Disclosures: No relevant conflicts of interest to declare.