Journal articles on the topic 'Notch'
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1
Yuan, Xiyu, Xue Hu, Yating Zheng, and Mengli Huang. "Abstract 5095: Investigation of NOTCH mutation and correlation with immunotherapy biomarker in Chinese colorectal cancer patients." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5095. http://dx.doi.org/10.1158/1538-7445.am2022-5095.
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Abstract Background: NOTCH pathway, a highly conserved signaling system, is regulated by short-range cell-cell interaction between NOTCH receptor (NOTCH1-4) and “canonical” ligand, or non-canonically through activation of other pathways. Previous research on the NOTCH gene revealed that NOTCH signaling is associated with tumorigenesis, mutagenesis, and immune tolerance in several tumors, indicating its association with the clinical benefit of immune checkpoint inhibitors (ICIs). However, the NOTCH characteristics, its correlation with immunogenic marker, and the predictive value of immunotherapy in colorectal cancer (CRC) was unknown. Methods: An independent cohort (the MSKCC study cohort) with next-generation sequencing (NGS) data from 109 patients with CRC of pan-cancer, were used to analyze the prognostic effect of NOTCH1-4 on immunotherapy. Tumor tissue samples from Chinese head and neck cancer were analyzed using NGS (panel on 381/733-gene). TMB was defined as the total number of somatic non-synonymous mutations in coding region. MSI was evaluated by NGS of 500 known MSI loci. PD-L1 expression was evaluated using immunohistochemistry (Dako 22C3). Result: In the MSKCC cohort, there were 49 (45.0%) patients harbored NOTCH mutation(NOTCHmut), including 16 (14.7%) patients of NOTCH1, 7 (6.4%) patients of NOTCH2, 16 (14.7%) patients of NOTCH3, and 10 (10.9%) patients of NOTCH4. NOTCH1 mutation (median, 65.4 Muts/Mb) was associated with higher TMB (P<0.001) than NOTCH1 wild-type (NOTCHwt) (median, 6.8 Muts/Mb), same as NOTCH2 (median, 61.4 Muts/Mb vs 7.8 Muts/Mb; P<0.001), NOTCH3 (median, 64.9 Muts/Mb vs 6.8 Muts/Mb; P<0.001), and NOTCH4 (median, 76.7 Muts/Mb vs 7.8 Muts/Mb; P<0.001). In terms of prognostic effect, there was significant difference on overall survival (OS) (HR = 0.17, P=0.008) between NOTCH3mut (median, 11.5 months) and NOTCH3wt (median, 8 months), but there was no difference in others of NOTCH. In Chinese patients, genetic mutation of 1166 CRC patients were analyzed using NGS, of which 157 (13.5%) harbored NOTCH mutation, including NOTCH1 (123, 10.5%), NOTCH2 (221, 18.9%), NOTCH3 (135, 11.6%), and NOTCH4 (55, 4.7%). NOTCH3mut (median, 47,5 Muts/Mb) was associated with higher TMB (P<0.001) than NOTCH3wt (median, 7.3 Muts/Mb), which was significant difference, same as the other three. For the TMB, there was also significant difference in MSI between NOTCH1-4mut and NOTCH1-4wt (all of the four P<0.001). There was significant difference in PD-L1 expression between NOTCHwt with NOTCH1mut (P =0.005) and NOTCH2mut (P<0.001), but not with NOTCH3mut and NOTCH4mut. Conclusions: The results showed that the NOTCH genes had a high correlation with TMB and MSI in CRC, and the NOTCH3 might a potential biomarker for immune checkpoint therapy. Citation Format: Xiyu Yuan, Xue Hu, Yating Zheng, Mengli Huang. Investigation of NOTCH mutation and correlation with immunotherapy biomarker in Chinese colorectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5095.
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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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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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Shah, Heer, Mittal Mistry, Nupur Patel, and Hemangini Vora. "Clinical significance of Notch receptors in triple negative breast cancer." Breast Disease 42, no. 1 (March 21, 2023): 85–100. http://dx.doi.org/10.3233/bd-220041.
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BACKGROUND: The Notch signaling pathway is an evolutionary conserved cell signaling pathway that plays an indispensable role in essential developmental processes. Aberrant activation of Notch pathway is known to initiate wide array of diseases and cancers. OBJECTIVE: To evaluate the clinical significance of Notch receptors in Triple Negative Breast Cancer. METHODS: We evaluated the association between Notch receptors and clinicopathological parameters including disease-free survival and overall survival of one hundred TNBC patients by immunohistochemistry. RESULTS: Positive expression of nuclear Notch1 receptor (18%) was found be significantly correlated with positive lymph node (p = 0.009), high BR score (p = 0.02) and necrosis (p = 0.004) while cytoplasmic expression of Notch2 receptor (26%) was significantly correlated with metastasis (p = 0.05), worse DFS (p = 0.05) and poor OS (p = 0.02) in TNBC patients. Membrane (18%) and cytonuclear (3%) Notch3 expression were significantly associated with poorly differentiated tumors (p = 0.007), high BR score (p = 0.002) and necrosis (p = 0.03) respectively. However, cytoplasmic Notch3 and Notch4 expression were negatively correlated with poor prognostic factors. CONCLUSIONS: Our data indicated that Notch receptors play a key role in promoting TNBC and mainly, Notch2 may contribute to poor prognosis of the disease. Hence, it is implicated that Notch2 may serve as a potential biomarker and therapeutic target for TNBC.
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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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Ito, T., N. Udaka, T. Yazawa, K. Okudela, H. Hayashi, T. Sudo, F. Guillemot, R. Kageyama, and H. Kitamura. "Basic helix-loop-helix transcription factors regulate the neuroendocrine differentiation of fetal mouse pulmonary epithelium." Development 127, no. 18 (September 15, 2000): 3913–21. http://dx.doi.org/10.1242/dev.127.18.3913.
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To clarify the mechanisms that regulate neuroendocrine differentiation of fetal lung epithelia, we have studied the expression of the mammalian homologs of achaete-scute complex (Mash1) (Ascl1 - Mouse Genome Informatics); hairy and enhancer of split1 (Hes1); and the expression of Notch/Notch-ligand system in the fetal and adult mouse lungs, and in the lungs of Mash1- or Hes1-deficient mice. Immunohistochemical studies revealed that Mash1-positive cells seemed to belong to pulmonary neuroendocrine cells (PNEC) and their precursors. In mice deficient for Mash1, no PNEC were detected. Hes1-positive cells belong to non-neuroendocrine cells. In the mice deficient in Hes1, in which Mash1 mRNA was upregulated, PNEC appeared precociously, and the number of PNEC was markedly increased. NeuroD (Neurod1 - Mouse Genome Informatics) expression in the lung was detected in the adult, and was enhanced in the fetal lungs of Hes1-null mice. Expression of Notch1, Notch2, Notch3 and Notch4 mRNAs in the mouse lung increased with age, and Notch1 mRNA was expressed in a Hes1-dependent manner. Notch1, Notch2 and Notch3 were immunohistochemically detected in non-neuroendocrine cells. Moreover, analyses of the lungs from the gene-targeted mice suggested that expression of Delta-like 1 (Dll1 - Mouse Genome Informatics) mRNA depends on Mash1. Thus, the neuroendocrine differentiation depends on basic helix-loop-helix factors, and Notch/Notch-ligand pathways may be involved in determining the cell differentiation fate in fetal airway epithelium.
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Mizuta, Ikuko, Yumiko Nakao-Azuma, Hideki Yoshida, Masamitsu Yamaguchi, and Toshiki Mizuno. "Progress to Clarify How NOTCH3 Mutations Lead to CADASIL, a Hereditary Cerebral Small Vessel Disease." Biomolecules 14, no. 1 (January 18, 2024): 127. http://dx.doi.org/10.3390/biom14010127.
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Notch signaling is conserved in C. elegans, Drosophila, and mammals. Among the four NOTCH genes in humans, NOTCH1, NOTCH2, and NOTCH3 are known to cause monogenic hereditary disorders. Most NOTCH-related disorders are congenital and caused by a gain or loss of Notch signaling activity. In contrast, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) caused by NOTCH3 is adult-onset and considered to be caused by accumulation of the mutant NOTCH3 extracellular domain (N3ECD) and, possibly, by an impairment in Notch signaling. Pathophysiological processes following mutant N3ECD accumulation have been intensively investigated; however, the process leading to N3ECD accumulation and its association with canonical NOTCH3 signaling remain unknown. We reviewed the progress in clarifying the pathophysiological process involving mutant NOTCH3.
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Zanotti, Stefano, and Ernesto Canalis. "Notch Suppresses Nuclear Factor of Activated T Cells (Nfat) Transactivation and Nfatc1 Expression in Chondrocytes." Endocrinology 154, no. 2 (December 21, 2012): 762–72. http://dx.doi.org/10.1210/en.2012-1925.
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Notch1 to Notch4 transmembrane receptors determine cell fate, and release of the Notch intracellular domain (NICD) in the cytoplasm induces gene expression. Notch regulates endochondral ossification, but it is not clear whether Notch interacts with signals controlling chondrocyte differentiation. Nuclear factor of activated T cells (Nfatc) transcription factors regulate chondrogenesis, and we asked whether Notch modifies Nfat signaling in chondrocytes. Notch was induced in teratocarcinoma ATDC5 chondrogenic cells infected with a retroviral vector, where the cytomegalovirus (CMV) promoter directs NICD expression. NICD suppressed chondrocyte differentiation and inhibited Nfat transactivation and Nfatc1 expression. Notch was activated in chondrocytes from RosaNotch mice, where the Rosa26 promoter is upstream of a loxP-flanked STOP cassette and NICD. To excise the STOP cassette and express NICD, RosaNotch chondrocytes were infected with an adenoviral vector where the CMV promoter directs Cre expression (Ad-CMV-Cre). Notch1 and Notch2 mediate the effects of Notch in skeletal cells, and to inhibit Notch signaling, chondrocytes from mice homozygous for Notch1 and Notch2 alleles targeted with loxP sites were infected with Ad-CMV-Cre. NICD suppressed chondrogenic nodules formation and expression of selected chondrocyte gene markers, induced Col10a1 and Mmp13, and suppressed Nfat transactivation and Nfatc1 expression, whereas inactivation of Notch1 and Notch2 did not affect chondrocyte differentiation. To investigate Nfatc1 function in chondrocytes, Nfatc1 was induced in RosaNotch chondrocytes overexpressing NICD or controls. Nfatc1 suppressed chondrocyte differentiation and opposed Col10a1 induction by Notch. In conclusion, Notch suppresses Nfat transactivation in chondrocytes and Notch and Nfatc1 regulate chondrocyte differentiation.
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Takam Kamga, Paul, Giada Dal Collo, Federica Resci, Riccardo Bazzoni, Angela Mercuri, Francesca Maria Quaglia, Ilaria Tanasi, et al. "Notch Signaling Molecules as Prognostic Biomarkers for Acute Myeloid Leukemia." Cancers 11, no. 12 (December 6, 2019): 1958. http://dx.doi.org/10.3390/cancers11121958.
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The role of Notch signaling in acute myeloid leukemia (AML) is still under investigation. We have previously shown that high levels of Notch receptors and ligands could interfere with drug response. In this study, the protein expression of 79 AML blast samples collected from newly diagnosed patients was examined through flow cytometry. Gamma-secretase inhibitors were used in AML mouse xenograft models to evaluate the contribution of Notch pharmacological inhibition to mouse survival. We used univariate analysis for testing the correlation and/or association between protein expression and well-known prognostics markers. All the four receptors (Notch1–4) and some ligands (Jagged2, DLL-3) were highly expressed in less mature subtypes (M0–M1). Notch3, Notch4, and Jagged2 were overexpressed in an adverse cytogenetic risk group compared to good cytogenetic risk patients. Chi-square analysis revealed a positive association between the complete remission rate after induction therapy and weak expression of Notch2 and Notch3. We also found an association between low levels of Notch4 and Jagged2 and three-year remission following allogeneic stem cell transplantation (HSCT). Accordingly, Kaplan–Meier analysis showed improved OS for patients lacking significant expression of Notch4, Jagged2, and DLL3. In vivo experiments in an AML mouse model highlighted both improved survival and a significant reduction of leukemia cell burden in the bone marrow of mice treated with the combination of Notch pan-inhibitors (GSIs) plus chemotherapy (Ara-C). Our results suggest that Notch can be useful as a prognostic marker and therapeutic target in AML.
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Mao, Yongzhong, Shaotao Tang, Li Yang, and Kang Li. "Inhibition of the Notch Signaling Pathway Reduces the Differentiation of Hepatic Progenitor Cells into Cholangiocytes in Biliary Atresia." Cellular Physiology and Biochemistry 49, no. 3 (2018): 1115–23. http://dx.doi.org/10.1159/000493290.
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Background/Aims: Viral infections, especially with rotavirus, are often considered an initiator of the pathogenesis of biliary atresia (BA). However, the mechanism by which rotavirus induces BA is still unclear. Methods: A BA mouse model was induced in newborn mice by i.p. inoculation with rhesus rotavirus within 6 h of birth. The expression of Notch pathway-associated molecules (JAG1, JAG2, Notch1, Notch2, Notch3, Notch4, DII1, DII3, and DII4) was measured by quantitative PCR and western blot analysis. Bile duct obstruction was detected by hematoxylin and eosin staining and CK-19 immunohistochemical staining. DAPT was used to inhibit the Notch pathway in vivo and in vitro. Results: In the livers of patients with BA and rotavirus-induced BA mice, the expression of JAG1 and Notch2 was significantly increased. Inhibition of the Notch pathway by DAPT in vivo ameliorated bile duct obstruction and delayed BA-induced mortality. The serum levels of inflammation cytokines (TNF-α, IL-2, IL-8, and IL-18) were reduced by inhibiting the Notch pathway. The expression of CK19, Sox9, and EpCAM was significantly increased in BA liver, while DAPT treatment decreased the expression of CK19, Sox9, and EpCAM. Conclusion: Notch activation is involved in the pathogenesis of BA by promoting the differentiation of hepatic progenitor cells into cholangiocytes.
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Broner, Esther Channah, Genia Alpert, Udi Gluschnaider, Adi Mondshine, Oz Solomon, Ido Sloma, Rami Rauch, Evgeny Izumchenko, Jon Christopher Aster, and Matti Davis. "AL101 mediated tumor inhibition in notch-altered TNBC PDX models." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): 1064. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.1064.
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1064 Background: The Notch pathway is activated during mammary gland development and has been implicated as a key driver in breast cancer. There is an urgent need to identify new therapeutic strategies for triple-negative breast cancer (TNBC), a sub-type associated with poor prognosis and no available targeted therapies. Notch gain of function (GOF) genetic alterations are potential tumor drivers found in ~10% of TNBC. This motivated the development of Notch inhibitors, including AL101 a pan-Notch, gamma secretase inhibitor (J Clin Oncol 36, 2018 abstract 2515). AL101 is currently being evaluated in Adenoid Cystic Carcinoma patients with activating Notch mutations (NCT03691207, ACCURACY trial). Here, we aim to test the activity of AL101 in TNBC patient derived xenograft (PDX) models with Notch activating genetic alterations. Methods: Gene expression cluster analysis was performed for 38 TNBC PDX tumors using a list of 21 Notch target genes. Seven tumors, bearing a “Notch-on” signature, were enriched with mutated/fusion (M/F) Notch genes and clustered separately from all other tumors. Of 9 models selected for study, 4 had a Notch-on signature and were expected to respond to AL101. Tumors were implanted into female athymic nude mice. Once tumors reached an average size of 150-300 mm3, mice (n = 5/group) were randomized to Vehicle or AL101 treatment arms (3 mg/kg, PO, 4on/3off) until tumors reached 1500 mm3 or day 60. Results: As measured by tumor growth inhibition (TGI), AL101 was more potent in tumors with a putative Notch-on signature. Within these 4 models, M/F genes were present in Notch1-NRR GOF (103% TGI p = 0.0004); Notch2-fusion (62%TGI p = 0.036); Notch3-fusion (75% TGI p = 0.032); or Notch4-fusion (147% TGI p < 0.00001). Tumors lacking the Notch signature did not respond significantly to AL101: WT Notch (43% TGI p = 0.0104; 64% TGI p = 0.13); Notch1 with a predicted loss of function mutation (12% TGI p = 0.53), Notch1 Variant of Unknown Significance (VUS) (30% TGI p = 0.44), Notch2 VUS (41% TGI p = 0.44). Conclusions: We demonstrate that in TNBC PDX models, the presence of a Notch-on signature and Notch GOF mutations/fusions correlates with potent response to AL101. These data support the clinical development of AL101 as a targeted therapy for TNBC with Notch GOF alterations.
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Lachej, Nadežda, Violeta Jonušienė, Augustina Mažeikė, Aušra Sasnauskienė, Daiva Dabkevičienė, Julija Šimienė, Kęstutis Sužiedėlis, and Janina Didžiapetrienė. "Changes in the expression of Notch and Wnt signalling molecules in human endometrial cancer." Acta medica Lituanica 26, no. 3 (January 11, 2020): 181–90. http://dx.doi.org/10.6001/actamedica.v26i3.4148.
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Background. Endometrial cancer is the sixth most frequent type of cancer among women worldwide. Type I adenocarcinomas account for 80–85% of endometrial cancer cases and sometimes require more aggressive treatment than the remaining part of this group. Therefore, molecular markers to stratify adenocarcinomas are needed. Materials and methods. In this study, we analysed Notch and Wnt signalling in human endometrial cancer cases to evaluate these pathway elements as potential biomarkers for type I endometrial cancer. Endometrial samples were obtained from 47 women undergoing surgery for stage I–IV endometrial cancer in the National Cancer Institute (Vilnius, Lithuania) in 2015–2016. The expression at the mRNA level of signalling molecules genes (NOTCH1, NOTCH2, NOTCH3, NOTCH4, JAG1, JAG2, DLL1, HES1, AXIN2 and CTNNB1) was analysed by the quantitative real-time polymerase chain reaction. Relative expression of NOTCH1, NOTCH4, HES1 and β-catenin proteins in endometrioid adenocarcinoma was evaluated by the Western blot method. Results. The expression level of Notch receptors, ligands, and the target gene, as well as CTNNB1 and AXIN2, was reduced in stage I endometrioid adenocarcinoma if compared to the adjacent non-tumour tissue. The expression of all receptors, ligands, and target molecules was reduced in adenocarcinomas of later stages. The statistically significant correlations between transcript amounts of Notch receptors and ligands were found. There was a statistically significant difference in the gene expression of Notch signalling pathway components between different tumour differentiation grade samples. A positive correlation between mRNA and protein the expression level of NOTCH1, NOTCH4, HES1 was determined in stage I samples. Conclusions. Analysis of 47 human endometrial cancer samples revealed a reduction in the transcript levels of Notch and Wnt signalling molecule compared to the adjacent non-tumour tissue. These results suggest tumour suppressor function of Notch and Wnt signalling in human endometrial cancer. More detailed research on these signalling pathways should reveal their importance as potential biomarkers.
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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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Haider, Sandra, Gudrun Meinhardt, Philipp Velicky, Gerlinde R. Otti, Guy Whitley, Christian Fiala, Jürgen Pollheimer, and Martin Knöfler. "Notch Signaling Plays a Critical Role in Motility and Differentiation of Human First-Trimester Cytotrophoblasts." Endocrinology 155, no. 1 (January 1, 2014): 263–74. http://dx.doi.org/10.1210/en.2013-1455.
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Failures in human extravillous trophoblast (EVT) development could be involved in the pathogenesis of pregnancy diseases. However, the underlying mechanisms have been poorly characterized. Here, we provide evidence that Notch signaling could represent a key regulatory pathway controlling trophoblast proliferation, motility, and differentiation. Immunofluorescence of first-trimester placental tissues revealed expression of Notch receptors (Notch2 and Notch3) and membrane-anchored ligands (delta-like ligand [DLL] 1 and -4 and Jagged [JAG] 1 and -2) in villous cytotrophoblasts (vCTBs), cell column trophoblasts (CCTs), and EVTs. Notch4 and Notch1 were exclusively expressed in vCTBs and in CCTs, respectively. Both proteins decreased in Western blot analyses of first-trimester, primary cytotrophoblasts (CTBs) differentiating on fibronectin. Luciferase reporter analyses suggested basal, canonical Notch activity in SGHPL-5 cells and primary cells that was increased upon seeding on DLL4-coated dishes and diminished in the presence of the Notch/γ-secretase inhibitors N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) or L-685,458. Bromodeoxyuridine labeling, cyclin D1 mRNA expression, and cell counting indicated that chemical inhibition of Notch signaling elevated proliferation in the different primary trophoblast model systems. Notch inhibition also increased motility of SGHPL-5 cells through uncoated and fibronectin-coated Transwells, motility of primary CTBs, as well as migration in villous explant cultures on collagen I. Accordingly, small interfering RNA-mediated gene silencing of Notch1 also elevated SGHPL-5 cell migration. In contrast, motility of primary cultures and SGHPL-5 cells was diminished in the presence of DLL4. Moreover, DAPT increased markers of differentiated EVT, ie, human leukocyte antigen G1, integrin α5, and T-cell factor 4, whereas DLL4 provoked the opposite. In summary, the data suggest that canonical Notch signaling impairs motility and differentiation of first-trimester CTBs.
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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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Okuhashi, Yuki, Mai Itoh, Nobuo Nara, and Shuji Tohda. "Effects of NOTCH Knockdown on the Proliferation and mTOR Signaling of T-ALL and AML Cell Lines." Blood 122, no. 21 (November 15, 2013): 1396. http://dx.doi.org/10.1182/blood.v122.21.1396.1396.
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Abstract Background The activation of Notch is crucial for the growth of T-ALL cells with NOTCH1 mutations. The precise roles of Notch signaling in AML cells are not fully understood, although the growth of AML cells is diversely affected by Notch ligand stimulation as we previously reported. We also reported the effects of γ-secretase inhibitors (GSIs), which block Notch activation, on the growth of leukemia cells. However, these effects might not necessarily be due to Notch inhibition as GSIs also exhibit some off-target effects. To elucidate the roles of Notch signaling in leukemia cells, we examined the effects of small interfering RNA (siRNA)-mediated knockdown of NOTCH1 and NOTCH2on cell proliferation and down-stream signaling pathways such as mTOR signaling in T-ALL and AML cell lines. Methods Two T-ALL cell lines (DND-41 and KOPT-K1) and 2 AML cell lines (THP-1 and TMD7) were used in this study. The cells were transfected with siRNAs targeting NOTCH1 (siN1), NOTCH2 (siN2), or control siRNA by using the pipette tip chamber-based electroporation system. The effects of siN1 and siN2 transfection on cell proliferation and induction of apoptosis were examined using a colorimetric WST-8 assay and by observing cytospin preparations of the harvested cells, respectively. The effects of the siRNA-transfection on the mRNA and protein expression were examined by quantitative RT-PCR and immunoblotting, respectively. Results Transfection with siN1 and siN2 selectively suppressed the expression of Notch1 and Notch2 mRNA and protein, respectively. In T-ALL cell lines, NOTCH1 knockdown as well as NOTCH2 knockdown suppressed cell proliferation and induced apoptosis. Immunoblot analysis showed that Myc expression was downregulated in NOTCH1-knockdown cells but not affected in NOTCH2-knockdown cells. In AML cell lines, cell proliferation was not significantly affected by NOTCH siRNAs. NOTCH2 knockdown increased the level of cleaved Notch1 fragment without increasing Notch1 expression. The knockdown of NOTCH1 and NOTCH2 reduced the expression and phosphorylation of mTOR protein in THP-1 cells. To confirm this finding, we examined the effects of activation of Notch by the recombinant Notch ligands, Jagged1 and Delta1, on the expression of mTOR protein. The activation of Notch resulted in an increase in the level of the mTOR protein and its phosphorylation in THP-1 cells. Thus, siRNA-transfection and ligand stimulation of Notch showed contrasting effects. Phosphorylation of Akt, 4E-BP1, and S6K was also induced after the stimulation. Discussion Using siRNA-mediated knockdown experiments, we found that Notch2 signaling plays a role in the growth of T-ALL cells, independent of Myc expression. The autonomous activation of Notch signaling in AML cells had little effect on the proliferation of these cells. Notch2 protein seemed to be involved in the activation of Notch1 in AML cells. Regarding the crosstalk between Notch and mTOR signaling, it is known that the Notch-induced Hes1 protein suppresses PTEN transcription, resulting in the promotion of Akt phosphorylation. We found an alternative pathway linking Notch and mTOR signaling in THP-1 cells in which the PTEN gene is homozygously deleted. In THP-1 cells, Notch activation promotes the expression and phosphorylation of the mTOR protein and the activation of mTOR signaling. These findings would contribute to the development of effective Notch-targeted therapy against leukemia. Disclosures: No relevant conflicts of interest to declare.
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Mikheil, Dareen, Kirthana Prabhakar, Tun Lee Ng, Sireesh Teertam, B. Jack Longley, Michael A. Newton, and Vijayasaradhi Setaluri. "Notch Signaling Suppresses Melanoma Tumor Development in BRAF/Pten Mice." Cancers 15, no. 2 (January 14, 2023): 519. http://dx.doi.org/10.3390/cancers15020519.
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Both oncogenic and tumor suppressor roles have been assigned to Notch signaling in melanoma. In clinical trials, Notch inhibitors proved to be ineffective for melanoma treatment. Notch signaling has also been implicated in melanoma transdifferentiation, a prognostic feature in primary melanoma. In this study, we investigated the role of Notch signaling in melanoma tumor development and growth using the genetic model of mouse melanoma by crossing BRAFCA/+/Pten+/+/Tyr-CreER+ (B) and BRAFCA/+/Pten-/-/Tyr-CreER + (BP) mice with Notch1 or Notch2 floxed allele mice. The topical application of tamoxifen induced tumors in BP mice but not in B mice with or without the deletion of either Notch1 or Notch2. These data show that the loss of either Notch1 nor Notch2 can substitute the tumor suppressor function of Pten in BRAFV600E-induced melanomagenesis. However, in Pten-null background, the loss of either Notch1 or Notch2 appeared to accelerate BRAFV600E-induced tumor development, suggesting a tumor suppressor role for Notch1 and Notch2 in BRAFV600E/Pten-null driven melanomagenesis. Quantitative immunochemical analysis of a human cutaneous melanoma tissue microarray that consists of >100 primary tumors with complete clinical history showed a weak to moderate correlation between NOTCH protein levels and clinical and pathological parameters. Our data show that Notch signaling is involved during melanomagenesis and suggest that the identification of genes and signaling pathways downstream of Notch could help devise strategies for melanoma prevention.
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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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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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Severson, Eric Allan, Shakti Ramkissoon, Sugganth Daniel, Jo-Anne Vergilio, Laurie M. Gay, Julia Andrea Elvin, James Suh, et al. "Association of tumor mutational burden in cutaneous squamous cell carcinoma with genomic alterations in Notch family receptors." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): e13031-e13031. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.e13031.
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e13031 Background: In Cutaneous Squamous Cell Carcinoma (cSqCC), Notch1, Notch2, and Notch3 have been identified as tumor suppressors with a high rate of inactivating mutations early in cSqCC pathogenesis. Despite the high frequency of alterations, the biologic and therapeutic implications of Notch genomic alterations (GAs) in cSqCC are incompletely understood. Methods: 232 FFPE samples of cSqCC were evaluated by comprehensive genomic profiling (CGP) of 315 genes and analyzed for all classes of GAs, with diagnoses confirmed by central pathology review. Notch GAs with unknown functional impact were excluded. Tumor mutational burden (TMB) was calculated from 1.11 Mb of sequenced DNA and reported as mutations/Mb. Results: The cohort of 232 samples was 78% male and 22% female, aged from 17 to 88, with confirmed metastatic disease in 32% of cases. 115 (50%) samples had a Notch family member GA with a known or predicted functional impact. 96 cases had Notch1 GAs, 43 cases had Notch2 GAs, and 14 cases had Notch3 GAs with 40 cases having GAs in multiple Notch family members. These mutations were 41% missense, 34% nonsense, 15% splice site, 9% frameshift indels, and 1% non-frameshift indels alterations. Patients with Notch GAs were significantly older (median 70 years old vs median 64, p < 0.01). TMB was increased among samples with Notch GAs (median TMB of 63 mutations/mb versus 20 mutations/mb, p < 1x10-6), with similar differences for primary and metastatic samples. Cases with Notch GAs had more total GAs per case (mean 10.4 vs 7.4, p < 1.5x10-10); however, the top co-mutated genes were the same (TP53, CDKN2A, FAT1, MLL2). Both groups had a high proportion of C- > T/G- > A transitions, consistent with UV damage; however, the proportion was higher in cases with Notch GAs (median 83% vs 80%) - a difference seen in primary and metastatic samples. Conclusions: 50% of cSqCC cases had a Notch family member loss of function GA, which was associated with increased TMB in the primary and metastatic setting. With pre-clinical models and case studies showing responses to PD-1 inhibitors in cSqCC, further investigations are warranted into the associations of Notch GAs with mutational burden and response to immunotherapy.
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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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Shaik, Jilani Purusottapatnam, Ibrahim O. Alanazi, Akbar Ali Khan Pathan, Narasimha Reddy Parine, Majid A. Almadi, Nahla A. Azzam, Abdulrahman M. Aljebreen, Othman Alharbi, Mohammad Saud Alanazi, and Zahid Khan. "Frequent Activation of Notch Signaling Pathway in Colorectal Cancers and Its Implication in Patient Survival Outcome." Journal of Oncology 2020 (March 9, 2020): 1–8. http://dx.doi.org/10.1155/2020/6768942.
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Colorectal cancer is a major health concern as it ranks third in incidence and second major cause of cancer-related deaths worldwide. A leading cause of treatment failure has been attributed to cancer stem cells that can invariably resist existing chemotherapeutic regimens. Notch signaling pathway has been involved in the maintenance of stem cells besides being crucial in cell fate decision and embryonic development. This pathway has also been implicated in several human malignancies including colorectal cancer. We investigated mRNA expression of four Notch receptors (Notch1–4), five ligands (Jag1, Jag2, Dll1, Dll3, and Dll4), and four target genes (Hes1, Hes5, Hey1, and Hey2) using highly specific TaqMan gene expression assays in colorectal adenomas and cancers. Upregulated expression of Notch receptors ranged between 29 and 73% in colorectal cancers and between 11 and 56% in adenomas. Expression of Notch3 and Notch4 receptors was significantly higher in colorectal cancers compared to normal and adenoma tissues. The Jagged and Delta-like ligands were overexpressed between 25 and 52% in colorectal cancers, while in adenomas, it ranged between 0 and 33%. Combining the data for upregulation of receptors and ligands suggests that 86% colorectal cancers and 56% adenomas exhibited overexpression of Notch pathway genes in our cohort. Notch target genes were upregulated between 24 and 33% in colorectal cancers and between 11 and 22% in adenomas. Collating upregulation of Notch receptors and ligands with the target genes showed concordance in 58% colorectal tumors. Additionally, we evaluated expression of Notch receptors, ligands, and target genes with prognosis using the TCGA mRNA expression dataset. Patients overexpressing Notch3, Notch4, and Hey1 had significantly poorer overall survival relative to those having lower levels of these genes. Taken together, Notch signaling components are aberrantly overexpressed in colorectal tumors, and development of therapeutics targeting the Notch pathway may prove to be beneficial in the management of colorectal cancers.
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Kakuda, Shinako, Rachel K. LoPilato, Atsuko Ito, and Robert S. Haltiwanger. "Canonical Notch ligands and Fringes have distinct effects on NOTCH1 and NOTCH2." Journal of Biological Chemistry 295, no. 43 (August 19, 2020): 14710–22. http://dx.doi.org/10.1074/jbc.ra120.014407.
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Notch signaling is a cellular pathway regulating cell-fate determination and adult tissue homeostasis. Little is known about how canonical Notch ligands or Fringe enzymes differentially affect NOTCH1 and NOTCH2. Using cell-based Notch signaling and ligand-binding assays, we evaluated differences in NOTCH1 and NOTCH2 responses to Delta-like (DLL) and Jagged (JAG) family members and the extent to which Fringe enzymes modulate their activity. In the absence of Fringes, DLL4–NOTCH1 activation was more than twice that of DLL4–NOTCH2, whereas all other ligands activated NOTCH2 similarly or slightly more than NOTCH1. However, NOTCH2 showed less sensitivity to the Fringes. Lunatic fringe (LFNG) enhanced NOTCH2 activation by DLL1 and -4, and Manic fringe (MFNG) inhibited NOTCH2 activation by JAG1 and -2. Mass spectral analysis showed that O-fucose occurred at high stoichiometry at most consensus sequences of NOTCH2 and that the Fringe enzymes modified more O-fucose sites of NOTCH2 compared with NOTCH1. Mutagenesis studies showed that LFNG modification of O-fucose on EGF8 and -12 of NOTCH2 was responsible for enhancement of DLL1–NOTCH2 activation, similar to previous reports for NOTCH1. In contrast to NOTCH1, a single O-fucose site mutant that substantially blocked the ability of MFNG to inhibit NOTCH2 activation by JAG1 could not be identified. Interestingly, elimination of the O-fucose site on EGF12 allowed LFNG to inhibit JAG1-NOTCH2 activation, and O-fucosylation on EGF9 was important for trafficking of both NOTCH1 and NOTCH2. Together, these studies provide new insights into the differential regulation of NOTCH1 and NOTCH2 by Notch ligands and Fringe enzymes.
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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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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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Hubmann, Rainer, Susanne Schnabl, Mohammad Araghi, Christian Schmidl, André F. Rendeiro, Martin Hilgarth, Dita Demirtas, et al. "Targeting Nuclear NOTCH2 by Gliotoxin Recovers a Tumor-Suppressor NOTCH3 Activity in CLL." Cells 9, no. 6 (June 18, 2020): 1484. http://dx.doi.org/10.3390/cells9061484.
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NOTCH signaling represents a promising therapeutic target in chronic lymphocytic leukemia (CLL). We compared the anti-neoplastic effects of the nuclear NOTCH2 inhibitor gliotoxin and the pan-NOTCH γ-secretase inhibitor RO4929097 in primary CLL cells with special emphasis on the individual roles of the different NOTCH receptors. Gliotoxin rapidly induced apoptosis in all CLL cases tested, whereas RO4929097 exerted a variable and delayed effect on CLL cell viability. Gliotoxin-induced apoptosis was associated with inhibition of the NOTCH2/FCER2 (CD23) axis together with concomitant upregulation of the NOTCH3/NR4A1 axis. In contrast, RO4929097 downregulated the NOTCH3/NR4A1 axis and counteracted the spontaneous and gliotoxin-induced apoptosis. On the cell surface, NOTCH3 and CD23 expression were mutually exclusive, suggesting that downregulation of NOTCH2 signaling is a prerequisite for NOTCH3 expression in CLL cells. ATAC-seq confirmed that gliotoxin targeted the canonical NOTCH signaling, as indicated by the loss of chromatin accessibility at the potential NOTCH/CSL site containing the gene regulatory elements. This was accompanied by a gain in accessibility at the NR4A1, NFκB, and ATF3 motifs close to the genes involved in B-cell activation, differentiation, and apoptosis. In summary, these data show that gliotoxin recovers a non-canonical tumor-suppressing NOTCH3 activity, indicating that nuclear NOTCH2 inhibitors might be beneficial compared to pan-NOTCH inhibitors in the treatment of CLL.
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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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Liu, Ren, Xiuqing Li, Anil Tulpule, Yue Zhou, Jeffrey S. Scehnet, Shaobing Zhang, Jong-Soo Lee, Preet M. Chaudhary, Jae Jung, and Parkash S. Gill. "KSHV-induced notch components render endothelial and mural cell characteristics and cell survival." Blood 115, no. 4 (January 28, 2010): 887–95. http://dx.doi.org/10.1182/blood-2009-08-236745.
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AbstractKaposi sarcoma–associated herpesvirus (KSHV) infection is essential to the development of Kaposi sarcoma (KS). Notch signaling is also known to play a pivotal role in KS cell survival and lytic phase entrance of KSHV. In the current study, we sought to determine whether KSHV regulates Notch components. KSHV-infected lymphatic endothelial cells showed induction of receptors Notch3 and Notch4, Notch ligands Dll4 and Jagged1, and activated Notch receptors in contrast to uninfected lymphatic endothelial cells. In addition, KSHV induced the expression of endothelial precursor cell marker (CD133) and mural cell markers (calponin, desmin, and smooth muscle alpha actin), suggesting dedifferentiation and trans-differentiation. Overexpression of latency proteins (LANA, vFLIP) and lytic phase proteins (RTA, vGPCR, viral interleukin-6) further supported the direct regulatory capacity of KSHV viral proteins to induce Notch receptors (Notch2, Notch3), ligands (Dll1, Dll4, Jagged1), downstream targets (Hey, Hes), and endothelial precursor CD133. Targeting Notch pathway with γ-secretase inhibitor and a decoy protein in the form of soluble Dll4 inhibited growth of KSHV-transformed endothelial cell line. Soluble Dll4 was also highly active in vivo against KS tumor xenograft. It inhibited tumor cell growth, induced tumor cell death, and reduced vessel perfusion. Soluble Dll4 is thus a candidate for clinical investigation.
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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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Kamstrup, Maria R., Lise Mette Rahbek Gjerdrum, Edyta Biskup, Britt Thyssing Lauenborg, Elisabeth Ralfkiaer, Anders Woetmann, Niels Ødum, and Robert Gniadecki. "Notch1 as a potential therapeutic target in cutaneous T-cell lymphoma." Blood 116, no. 14 (October 7, 2010): 2504–12. http://dx.doi.org/10.1182/blood-2009-12-260216.
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AbstractDeregulation of Notch signaling has been linked to the development of T-cell leukemias and several solid malignancies. Yet, it is unknown whether Notch signaling is involved in the pathogenesis of mycosis fungoides and Sézary syndrome, the most common subtypes of cutaneous T-cell lymphoma. By immunohistochemistry of 40 biopsies taken from skin lesions of mycosis fungoides and Sézary syndrome, we demonstrated prominent expression of Notch1 on tumor cells, especially in the more advanced stages. The γ-secretase inhibitor I blocked Notch signaling and potently induced apoptosis in cell lines derived from mycosis fungoides (MyLa) and Sézary syndrome (SeAx, HuT-78) and in primary leukemic Sézary cells. Specific down-regulation of Notch1 (but not Notch2 and Notch3) by siRNA induced apoptosis in SeAx. The mechanism of apoptosis involved the inhibition of nuclear factor-κB, which is the most important prosurvival pathway in cutaneous T-cell lymphoma. Our data show that Notch is present in cutaneous T-cell lymphoma and that its inhibition may provide a new way to treat cutaneous T-cell lymphoma.
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Shanmugam, Vignesh, Jeffrey W. Craig, Laura K. Hilton, Matthew H. Nguyen, Christopher K. Rushton, Kian Fahimdanesh, Scott Lovitch, Ben Ferland, David W. Scott, and Jon C. Aster. "Notch activation is pervasive in SMZL and uncommon in DLBCL: implications for Notch signaling in B-cell tumors." Blood Advances 5, no. 1 (January 5, 2021): 71–83. http://dx.doi.org/10.1182/bloodadvances.2020002995.
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Abstract Notch receptors participate in a signaling pathway in which ligand-induced proteolysis frees the Notch intracellular domain (NICD), allowing it to translocate to the nucleus, form a transcription complex, and induce target gene expression. Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), splenic marginal zone B-cell lymphoma (SMZL), and distinct subsets of diffuse large B-cell lymphoma (DLBCL) are strongly associated with mutations in the 3′ end of NOTCH1 or NOTCH2 that disrupt a proline, glutamic acid, serine, and threonine (PEST) degron domain and stabilize NICD1 and NICD2. By contrast, mutations leading to constitutive Notch activation are rare in primary B-cell neoplasms, suggesting that Notch activation is confined to ligand-rich tumor microenvironments, or that cryptic strong gain-of-function mutations have been missed in prior analyses. To test these ideas, we used immunohistochemical stains to screen a broad range of B-cell tumors for Notch activation. Our analyses reveal that among small B-cell neoplasms, NICD2 is primarily detected in SMZL and is a common feature of both NOTCH2 wild-type and NOTCH2-mutated SMZLs, similar to prior findings with NOTCH1 in CLL/SLL. The greatest NOTCH2 activation was observed in NOTCH2-mutated SMZLs, particularly within splenic marginal zones. By contrast, little evidence of NOTCH2 activation was observed in DLBCL, even in NOTCH2-mutated tumors, suggesting that selective pressure for NOTCH2 activation is mainly confined to low-grade B-cell neoplasms, whereas DLBCLs with NOTCH1 mutations frequently showed evidence of ongoing NOTCH1 activation. These observations have important implications for the pathogenic role of Notch and its therapeutic targeting in B-cell lymphomas.
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Shanmugam, Vignesh, Jeffrey W. Craig, Laura K. Hilton, Matthew H. Nguyen, Christopher K. Rushton, Kian Fahimdanesh, Scott Lovitch, Ben Ferland, David W. Scott, and Jon C. Aster. "Notch activation is pervasive in SMZL and uncommon in DLBCL: implications for Notch signaling in B-cell tumors." Blood Advances 5, no. 1 (January 5, 2021): 71–83. http://dx.doi.org/10.1182/bloodadvances.2020002995.
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Abstract Notch receptors participate in a signaling pathway in which ligand-induced proteolysis frees the Notch intracellular domain (NICD), allowing it to translocate to the nucleus, form a transcription complex, and induce target gene expression. Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL), splenic marginal zone B-cell lymphoma (SMZL), and distinct subsets of diffuse large B-cell lymphoma (DLBCL) are strongly associated with mutations in the 3′ end of NOTCH1 or NOTCH2 that disrupt a proline, glutamic acid, serine, and threonine (PEST) degron domain and stabilize NICD1 and NICD2. By contrast, mutations leading to constitutive Notch activation are rare in primary B-cell neoplasms, suggesting that Notch activation is confined to ligand-rich tumor microenvironments, or that cryptic strong gain-of-function mutations have been missed in prior analyses. To test these ideas, we used immunohistochemical stains to screen a broad range of B-cell tumors for Notch activation. Our analyses reveal that among small B-cell neoplasms, NICD2 is primarily detected in SMZL and is a common feature of both NOTCH2 wild-type and NOTCH2-mutated SMZLs, similar to prior findings with NOTCH1 in CLL/SLL. The greatest NOTCH2 activation was observed in NOTCH2-mutated SMZLs, particularly within splenic marginal zones. By contrast, little evidence of NOTCH2 activation was observed in DLBCL, even in NOTCH2-mutated tumors, suggesting that selective pressure for NOTCH2 activation is mainly confined to low-grade B-cell neoplasms, whereas DLBCLs with NOTCH1 mutations frequently showed evidence of ongoing NOTCH1 activation. These observations have important implications for the pathogenic role of Notch and its therapeutic targeting in B-cell lymphomas.
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Taghon, Tom, Inge Van de Walle, Els Waegemans, Jelle De Medts, Greet De Smet, Magda De Smedt, Bart Vandekerckhove, et al. "Notch ligands control the human αβ/γδ lineage choice through receptor-specific induction of differential Notch signal strength. (111.29)." Journal of Immunology 188, no. 1_Supplement (May 1, 2012): 111.29. http://dx.doi.org/10.4049/jimmunol.188.supp.111.29.
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Abstract Differential Notch signal strength influences the αβ versus γδ T-cell lineage decision in both mouse and human. Here, we show that the Notch ligands Delta-like-4, Jagged1 and Jagged2, differentially impact this lineage decision in human. Whereas Delta-like-4 supports both αβ and γδ T-cell development, Jagged1 induces αβ-lineage differentiation and Jagged2 primarily γδ T-cell development. Jagged2 induces the strongest Notch signal in human thymocytes as a result of interactions with both Notch1 and Notch3, whereas Delta-like-4 only binds Notch1. In agreement, Notch3 is a stronger activator of Notch target genes and only supports γδ-lineage development, whereas Notch1 is a weaker activator supporting both αβ and γδ T cell development. Blockage of Notch3 activation in Jagged2 cocultures rescues αβ-lineage differentiation, showing the importance of this interaction for driving γδ-lineage differentiation. Further analysis revealed that Jagged2-mediated Notch3 activation blocks αβ-lineage differentiation as a result of inhibition of TCR-β chain expression. Consistently, TCR-αβ T cell development is restored in Jagged2 cocultures by providing immature human T cell progenitors with a TCR-β chain. Our results show that Notch ligands control the human αβ/γδ lineage choice through receptor-specific induction of differential Notch signal strength and that Jagged2-mediated Notch3 activation drives human γδ-lineage differentiation as a result of inhibition of αβ-lineage differentiation.
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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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Bigas, Anna, David I. K. Martin, and Laurie A. Milner. "Notch1 and Notch2 Inhibit Myeloid Differentiation in Response to Different Cytokines." Molecular and Cellular Biology 18, no. 4 (April 1, 1998): 2324–33. http://dx.doi.org/10.1128/mcb.18.4.2324.
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ABSTRACT We have compared the ability of two mammalian Notch homologs, mouse Notch1 and Notch2, to inhibit the granulocytic differentiation of 32D myeloid progenitor cells. 32D cells undergo granulocytic differentiation when stimulated with either granulocyte colony-stimulating factor (G-CSF) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Expression of the activated intracellular domain of Notch1 inhibits the differentiation induced by G-CSF but not by GM-CSF; conversely, the corresponding domain of Notch2 inhibits differentiation in response to GM-CSF but not to G-CSF. The region immediately C-terminal to the cdc10 domain of Notch confers cytokine specificity on the cdc10 domain. The cytokine response patterns of Notch1 and Notch2 are transferred with this region, which we have termed the Notch cytokine response (NCR) region. The NCR region is also associated with differences in posttranslational modification and subcellular localization of the different Notch molecules. These findings suggest that the multiple forms of Notch found in mammals have structural differences that allow their function to be modulated by specific differentiation signals.
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Xue, Dongyun, Dong Li, Cong Dou, and Junshan Li. "A Comprehensive Bioinformatic Analysis of NOTCH Pathway Involvement in Stomach Adenocarcinoma." Disease Markers 2021 (December 8, 2021): 1–19. http://dx.doi.org/10.1155/2021/4739868.
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Background. Activation of NOTCH signaling pathways, which are key regulators of multiple cellular functions, has been frequently implicated in cancer pathogenesis, and NOTCH inhibitors have received much recent focus in the context of cancer therapeutics. However, the role and possible involvement of NOTCH pathways in stomach adenocarcinoma (STAD) are unclear. Here, putative regulatory mechanisms and functions of NOTCH pathways in STAD were investigated. Methods. Publicly available data from the TCGA-STAD database were utilized to explore the involvement of canonical NOTCH pathways in STAD by analyzing RNA expression levels of NOTCH receptors, ligands, and downstream genes. Statistical analysis of the data pertaining to cancer and noncancerous samples was performed using R software packages and public databases/webservers. Results. Significant differential gene expression between control and STAD samples was noted for all NOTCH receptors (NOTCH1, 2, 3, and 4), the delta-like NOTCH ligands (DLL-3 and 4), and typical downstream genes (HES1 and HEY1). Four genes (NOTCH1, NOTCH2, NOTCH3, and HEY1) presented prognostic values for the STAD outcome in terms of overall survival. Functional enrichment analysis indicated that NOTCH family genes-strongly correlated genes were mainly enriched in several KEGG signaling pathways such as the PI3K-Akt signaling pathway, human papillomavirus infection, focal adhesion, Rap1 signaling pathway, and ECM-receptor interaction. Gene set enrichment analysis (GSEA) results showed that NOTCH family genes-significantly correlated genes were mainly enriched in four signaling pathways, ECM (extracellular matrix), tumor angiogenesis, inflammatory response, and immune regulation. Conclusions. NOTCH family genes may play an essential role in the progression of STAD by modulating immune cells and mediating ECM synthesis, angiogenesis, focal adhesion, and PI3K-Akt signaling. Multiple NOTCH family genes are valuable candidate biomarkers or therapeutic targets for the management of STAD.
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Canalis, Ernesto, Michele Carrer, Tabitha Eller, Lauren Schilling, and Jungeun Yu. "Use of antisense oligonucleotides to target Notch3 in skeletal cells." PLOS ONE 17, no. 5 (May 10, 2022): e0268225. http://dx.doi.org/10.1371/journal.pone.0268225.
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Notch receptors are determinants of cell fate and function, and play an important role in the regulation of bone development and skeletal remodeling. Lateral Meningocele Syndrome (LMS) is a monogenic disorder associated with NOTCH3 pathogenic variants that result in the stabilization of NOTCH3 and a gain-of-function. LMS presents with neurological developmental abnormalities and bone loss. We created a mouse model (Notch3em1Ecan) harboring a 6691TAATGA mutation in the Notch3 locus, and heterozygous Notch3em1Ecan mice exhibit cancellous and cortical bone osteopenia. In the present work, we explored whether Notch3 antisense oligonucleotides (ASO) downregulate Notch3 and have the potential to ameliorate the osteopenia of Notch3em1Ecan mice. Notch3 ASOs decreased the expression of Notch3 wild type and Notch36691-TAATGA mutant mRNA expressed by Notch3em1Ecan mice in osteoblast cultures without evidence of cellular toxicity. The effect was specific since ASOs did not downregulate Notch1, Notch2 or Notch4. The expression of Notch3 wild type and Notch36691-TAATGA mutant transcripts also was decreased in bone marrow stromal cells and osteocytes following exposure to Notch3 ASOs. In vivo, the subcutaneous administration of Notch3 ASOs at 25 to 50 mg/Kg decreased Notch3 mRNA in the liver, heart and bone. Microcomputed tomography demonstrated that the administration of Notch3 ASOs ameliorates the cortical osteopenia of Notch3em1Ecan mice, and ASOs decreased femoral cortical porosity and increased cortical thickness and bone volume. However, the administration of Notch3 ASOs did not ameliorate the cancellous bone osteopenia of Notchem1Ecan mice. In conclusion, Notch3 ASOs downregulate Notch3 expression in skeletal cells and their systemic administration ameliorates cortical osteopenia in Notch3em1Ecan mice; as such ASOs may become useful strategies in the management of skeletal diseases affected by Notch gain-of-function.
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Liu, Qiaoyuan, Chuxi Chen, Yunxuan He, Wenhao Mai, Shipeng Ruan, Yunshan Ning, and Yan Li. "Notch Signaling Regulates the Function and Phenotype of Dendritic Cells in Helicobacter pylori Infection." Microorganisms 11, no. 11 (November 20, 2023): 2818. http://dx.doi.org/10.3390/microorganisms11112818.
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Notch signaling manipulates the function and phenotype of dendritic cells (DCs), as well as the interaction between DCs and CD4+ T cells. However, the role of Notch signaling in Helicobacter pylori (H. pylori) infection remains elusive. Murine bone marrow-derived dendritic cells (BMDCs) were pretreated in the absence or presence of Notch signaling inhibitor DAPT prior to H. pylori stimulation and the levels of Notch components, cytokines and surface markers as well as the differentiation of CD4+ T cells in co-culture were measured using quantitative real-time PCR (qRT-PCR), Western blot, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. Compared with the control, the mRNA expression of all Notch receptors and Notch ligands Dll4 and Jagged1 was up-regulated in H. pylori-stimulated BMDCs. The blockade of Notch signaling by DAPT influenced the production of IL-1β and IL-10 in H. pylori-pulsed BMDCs, and reduced the expression of Notch1, Notch3, Notch4, Dll1, Dll3 and Jagged2. In addition, DAPT pretreatment decreased the expression of maturation markers CD80, CD83, CD86, and major histocompatibility complex class II (MHC-II) of BMDCs, and further skewed Th17/Treg balance toward Treg. Notch signaling regulates the function and phenotype of DCs, thus mediating the differentiation of CD4+ T cells during H. pylori infection.
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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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Takeuchi, Hideyuki, Michael Schneider, Daniel B. Williamson, Atsuko Ito, Megumi Takeuchi, Penny A. Handford, and Robert S. Haltiwanger. "Two novel protein O-glucosyltransferases that modify sites distinct from POGLUT1 and affect Notch trafficking and signaling." Proceedings of the National Academy of Sciences 115, no. 36 (August 20, 2018): E8395—E8402. http://dx.doi.org/10.1073/pnas.1804005115.
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The Notch-signaling pathway is normally activated by Notch–ligand interactions. A recent structural analysis suggested that a novel O-linked hexose modification on serine 435 of the mammalian NOTCH1 core ligand-binding domain lies at the interface with its ligands. This serine occurs between conserved cysteines 3 and 4 of Epidermal Growth Factor-like (EGF) repeat 11 of NOTCH1, a site distinct from those modified by protein O-glucosyltransferase 1 (POGLUT1), suggesting that a different enzyme is responsible. Here, we identify two novel protein O-glucosyltransferases, POGLUT2 and POGLUT3 (formerly KDELC1 and KDELC2, respectively), which transfer O-glucose (O-Glc) from UDP-Glc to serine 435. Mass spectrometric analysis of NOTCH1 produced in HEK293T cells lacking POGLUT2, POGLUT3, or both genes showed that either POGLUT2 or POGLUT3 can add this novel O-Glc modification. EGF11 of NOTCH2 does not have a serine residue in the same location for this O-glucosylation, but EGF10 of NOTCH3 (homologous to EGF11 in NOTCH1 and -2) is also modified at the same position. Comparison of the sites suggests a consensus sequence for modification. In vitro assays with POGLUT2 and POGLUT3 showed that both enzymes modified only properly folded EGF repeats and displayed distinct acceptor specificities toward NOTCH1 EGF11 and NOTCH3 EGF10. Mutation of the O-Glc modification site on EGF11 (serine 435) in combination with sensitizing O-fucose mutations in EGF8 or EGF12 affected cell-surface presentation of NOTCH1 or reduced activation of NOTCH1 by Delta-like1, respectively. This study identifies a previously undescribed mechanism for fine-tuning the Notch-signaling pathway in mammals.
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Sun, Yuan, Xue Gao, Jia Liu, Qing-You Kong, Xiao-Wei Wang, Xiao-Yan Chen, Qian Wang, Yi-Fan Cheng, Xiao-Xia Qu, and Hong Li. "Differential Notch1 and Notch2 Expression and Frequent Activation of Notch Signaling in Gastric Cancers." Archives of Pathology & Laboratory Medicine 135, no. 4 (April 1, 2011): 451–58. http://dx.doi.org/10.5858/2009-0665-oa.1.
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Abstract Context.—The biologic effects of Notch1 and Notch2 vary with cancer types and their potential role(s) in gastric cancers (GCs) remains largely unknown. Objectives.—This study aimed to address the previously mentioned issue by checking the expression of Notch1, Notch2, and Notch target gene Hes1 in GCs, premalignant gastric lesions, and noncancerous endoscopic gastric mucosa and by inhibiting Notch signal transduction in GC cells. Design.—The status of Notch1, Notch2, and Hes1 expression in 74 GC surgical specimens, 10 endoscopic samples, and 4 human GC cell lines was evaluated by tissue microarray–based immunohistochemical staining, Western blotting, and reverse transcription-polymerase chain reaction, and the importance of Notch signaling was elucidated by treating 2 GC cell lines with 2 γ-secretase inhibitors. Results.—Notch1 was undetectable in noncancerous gastric mucosa but was expressed with nuclear translocation in 16.7% (4 of 24) of chronic gastritis, 50.0% (9 of 18) of intestinal metaplasia, 54.2% (26 of 48) of intestinal GC, and 23.1% (6 of 26) of diffuse GC, showing distinct differences of Notch1 detection rates between either intestinal metaplasia and chronic gastritis or intestinal GCs and diffuse GCs (P = .03; P = .005, respectively). Notch2 nuclear translocation frequencies were 10.0% (1 of 10) in noncancerous endoscopic mucosa, 71.4% (30 of 42) in premalignant lesions, and 97.3% (72 of 74) in GC tissues, demonstrating a correlation of Notch2 expression with both intestinal GC and diffuse GC formation (P < .001). The rates of nuclear-Hes1 labeling were 1 of 10 among noncancerous, 42.9% premalignant, and 81.1% cancer tissues, which were closely correlated with Notch2 (P < .001) rather than Notch1 (P = .42) nuclear translocation. Only Notch2 was expressed accompanied with Hes1 nuclear labeling in the 4 GC cell lines established from diffuse GC cases. Inhibition of Notch signaling with γ-secretase inhibitors, L-685,458 and DAPT, prevented Hes1 nuclear translocation but neither suppressed growth nor induced cell death. Conclusions.—This study demonstrated a close correlation of Notch2 expression with GC formation and the potential link of Notch1 upregulation with intestinal-like phenotypes of gastric lesions. Although inhibition of Notch activity failed to achieve anti-GC effects, the activated Notch signaling may reflect a potential GC risk.
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Vercauteren, Suzanne M., and Heather J. Sutherland. "Constitutively active Notch4 promotes early human hematopoietic progenitor cell maintenance while inhibiting differentiation and causes lymphoid abnormalities in vivo." Blood 104, no. 8 (October 15, 2004): 2315–22. http://dx.doi.org/10.1182/blood-2004-01-0204.
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Abstract Notch transmembrane receptors are known to play a critical role in cell-fate decisions, with Notch1 shown to enhance self-renewal of hematopoietic stem cells and cause T-cell leukemia. Four Notch receptors exist, and the extent of redundancy and overlap in their function is unknown. Notch4 is structurally distinct from Notch1 through Notch3 and has not been extensively studied in hematopoiesis. By polymerase chain reaction (PCR) we find Notch4 transcript expression in human marrow cells and in both CD34+ and CD34– populations. When constitutively active Notch1 or Notch4 was overexpressed in normal human marrow or cord cells, we found reduced colony-forming and short-term proliferative ability while the primitive progenitor content of myeloid long-term cultures was significantly increased. Notch4–intracellular domain (Notch4-IC)–transduced cord cells transplanted into β2-microglobulin–/– nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in significantly higher levels of engraftment of both green fluorescent protein–positive (GFP+) and GFP– populations as compared with controls. GFP+ cells in bone marrow and spleen of animals that had received transplants gave rise to an immature CD4+CD8+ T-cell population, whereas B-cell development was blocked. These results indicate that activation of Notch4 results in enhanced stem cell activity, reduced differentiation, and altered lymphoid development, suggesting it may influence both stem cells and the fate of the common lymphoid progenitor.
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Jiang, Da, Zhaojian Niu, Jianli Zhang, Yanlei Wang, Liang Shang, Bixun Li, Jun Guo, et al. "Notch family gene mutations associate with high tumor mutational burden in diverse cancers." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e14616-e14616. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e14616.
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e14616 Background: The Notch pathway has an important role in tumorigenesis in many types of cancer. Clinical trials with Notch inhibitors are ongoing. Recently, Notch pathway has been reported to be part of tumor immunity and patients with NOTCH1 mutations (mut) showed better outcome to PD-1 inhibitors, however, the underlying mechanism is unknown. Methods: Next generation sequencing (NGS) panel of 450 cancer genes was performed on FFPE tissue and matched blood samples from 1341 solid tumors, including hepatocellular carcinoma (HCC, n = 644), esophageal carcinoma (EC, n = 255), breast cancer (BC, n = 175), small cell lung cancer (SCLC, n = 141), head and neck cancer (HNC, n = 77). Notch family gene (NOTCH1/2/3/4) muts were analyzed. Genomic alterations including single base substitution, short and long insertions/deletion, copy number variation, gene fusion and rearrangement were assessed. Tumor mutational burden (TMB) was calculated in all patients by NGS algorithms. TMB high (TMB-H) was defined as TMB values ≥75% tumors in each tumor type. Results: For all patients, Notch family gene mut (substitution/indel/truncation) were found in 27% EC, 19% SCLC, 12% HNC, 6% BC and 5% HCC. The median TMB was 6.1 muts/Mb. In general, tumors with Notch family gene mut had significantly higher median TMB in pan-cancer cohort when compared with tumors without Notch family gene mut (9.2 vs. 6.1 muts/Mb, p < 0.001). Specifically, tumors with NOTCH1 mut had higher TMB in HCC (8.5 vs 5.5, p = 0.034), in BC (23.2 vs. 4.6, p = 0.006) and in SCLC (14.0 vs. 9.4, p = 0.002), while NOTCH2 mutant tumors presented higher TMB in HCC (18.5 vs. 5.5, p = 0.026), in EC (14.7 vs. 6.9, p = 0.001) and in SCLC (13.2 vs. 4.6, p = 0.026). We found 65% of NOTCH1 mut were located in epidermal growth factor (EGF)-like repeats domain. Classes of the NOTCH1 mutation type were substitution/indel (non-frameshift) (58%) and truncation (42%), while NOTCH2 mut were 88% and 12%. In TMB-H tumors, substitution/indel is the predominant mutation type of Notch family gene (NOTCH1 61%, NOTCH2 100%). Conclusions: Tumors with Notch family gene mut presented higher TMB in multiple cancer types, indicating a potential strategy for targeted and immunotherapy in NOTCH mutant cancers.
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Shimizu, Kiyoshi, Shigeru Chiba, Noriko Hosoya, Keiki Kumano, Toshiki Saito, Mineo Kurokawa, Yoshinobu Kanda, Yoshio Hamada, and Hisamaru Hirai. "Binding of Delta1, Jagged1, and Jagged2 to Notch2 Rapidly Induces Cleavage, Nuclear Translocation, and Hyperphosphorylation of Notch2." Molecular and Cellular Biology 20, no. 18 (September 15, 2000): 6913–22. http://dx.doi.org/10.1128/mcb.20.18.6913-6922.2000.
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ABSTRACT Delta1, Jagged1, and Jagged2, commonly designated Delta/Serrate/LAG-2 (DSL) proteins, are known to be ligands for Notch1. However, it has been less understood whether they are ligands for Notch receptors other than Notch1. Meanwhile, ligand-induced cleavage and nuclear translocation of the Notch protein are considered to be fundamental for Notch signaling, yet direct observation of the behavior of the Notch molecule after ligand binding, including cleavage and nuclear translocation, has been lacking. In this report, we investigated these issues for Notch2. All of the three DSL proteins bound to endogenous Notch2 on the surface of BaF3 cells, although characteristics of Jagged2 for binding to Notch2 apparently differed from that of Delta1 and Jagged1. After binding, the three DSL proteins induced cleavage of the membrane-spanning subunit of Notch2 (Notch2TM), which occurred within 15 min. In a simultaneous time course, the cleaved fragment of Notch2TMwas translocated into the nucleus. Interestingly, the cleaved Notch2 fragment was hyperphosphorylated also in a time-dependent manner. Finally, binding of DSL proteins to Notch2 also activated the transcription of reporter genes driven by the RBP-Jκ-responsive promoter. Together, these data indicate that all of these DSL proteins function as ligands for Notch2. Moreover, the findings of rapid cleavage, nuclear translocation, and phosphorylation of Notch2 after ligand binding facilitate the understanding of the Notch signaling.
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Chabanais, Julien, François Labrousse, Alain Chaunavel, Agnès Germot, and Abderrahman Maftah. "POFUT1 as a Promising Novel Biomarker of Colorectal Cancer." Cancers 10, no. 11 (October 30, 2018): 411. http://dx.doi.org/10.3390/cancers10110411.
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Background: While protein O-fucosyltransferase 1 (POFUT1) overexpression has been recently proposed as a potential biomarker for different cancer types, no study was carried out on POFUT1 implication in colorectal cancer (CRC). Methods: Data from 626 tumors and 51 non-tumor adjacent tissues available in FireBrowse had been used in this study. Statistical analyses on POFUT1 expression and gene copy number, NOTCH receptors (main targets of POFUT1 enzymatic activity) expression and association of POFUT1 and NOTCH1 expressions with clinical parameters were investigated. Data were completed by POFUT1 histological labeling on six tumor tissues from patients with CRC. Results: We found that POFUT1 is overexpressed from the stage I (p < 0.001) and 76.02% of tumors have a 20q11.21 amplification, associated in 90.13% of cases with a POFUT1 overexpression, compared to non-tumor adjacent tissues. The POFUT1 copy number in tumors is mainly between 2 and 3. POFUT1 is positively correlated with NOTCH1 (rs = 0.34, p < 0.001), NOTCH3 (rs = 0.087, p = 0.0297), and NOTCH4 (rs = 0.097, p = 0.0148) expressions, while negatively correlated with NOTCH2 expression (rs = −0.098, p = 0.0142). POFUT1 overexpression is markedly associated with rectal location, non-mucinous adenocarcinoma and cancer stages IV and M1. NOTCH1 overexpression is only associated with rectal location and non-mucinous adenocarcinoma. Conclusion: We conclude that POFUT1 is overexpressed in CRC from stage I, and its high expression is associated with metastatic process, probably through NOTCH pathway activation. Then, POFUT1 could represent a potential novel biomarker for CRC diagnosis.
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Park, Sung-Gyoo, San-Heon Park, and Jung-Ah Kang. "Notch1 is an important secondary stimulator for activated B cells (IRM10P.606)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 131.4. http://dx.doi.org/10.4049/jimmunol.194.supp.131.4.
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Abstract The roles of Notch1 and Notch2 in T cell function have been well studied, but the functional roles of Notch in B cells have not been extensively investigated, except for Notch2 involvement in peripheral marginal zone B cell differentiation. This study examined the roles of Notch1 in murine primary B cells. We found that the intracellular domain of Notch1 could substitute for Notch2 in marginal zone B cell differentiation, and that Notch1 expression was important for further activation of B cells. During B cell activation by B cell receptor ligation, Notch1 was up-regulated while Notch2 was not. In addition, Notch1 up-regulation itself did not contribute to the further activation of B cells, but the Notch ligand was important for Notch1-mediated further B cell activation. Moreover, Notch1 increased antibody secretion from activated B cells. These data suggest that Notch1 is more important for B cell activation than Notch2, while Notch2 is more important for marginal zone B cell differentiation.
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Parmigiani, Elena, and Claudio Giachino. "Genetic Inactivation of Notch1 Synergizes with Loss of Trp53 to Induce Tumor Formation in the Adult Mouse Forebrain." Cancers 14, no. 21 (November 2, 2022): 5409. http://dx.doi.org/10.3390/cancers14215409.
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Simultaneous genetic inactivation of the key Notch signaling mediator RBP-Jk and p53 leads to the formation of forebrain tumors in mice, suggesting a tumor suppressor role of the Notch pathway in this context. However, the contribution of individual Notch receptors to the tumor-suppressive activity of Notch signaling in the brain remains elusive. Here, we show that simultaneous Notch1 and Notch2 deletion, similar to complete ablation of canonical Notch signaling by Rbpj inactivation, cooperates with Trp53 deletion to promote tumor growth in the adult forebrain. We also demonstrate that inactivation of Notch1 and Trp53 in cells with active Notch signaling is sufficient to induce brain tumor or hyperplasia formation. Analysis of tumor location suggests a multifocal origin and shows that ventral forebrain regions and olfactory bulbs are the most affected sites. Hence, Notch1 cooperates with p53 to repress malignant transformation in the adult mouse forebrain.
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Krebs, Luke T., Yingzi Xue, Christine R. Norton, John R. Shutter, Maureen Maguire, John P. Sundberg, Daniel Gallahan, et al. "Notch signaling is essential for vascular morphogenesis in mice." Genes & Development 14, no. 11 (June 1, 2000): 1343–52. http://dx.doi.org/10.1101/gad.14.11.1343.
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The Notch gene family encodes large transmembrane receptors that are components of an evolutionarily conserved intercellular signaling mechanism. To assess the role of the Notch4 gene, we generatedNotch4-deficient mice by gene targeting. Embryos homozygous for this mutation developed normally, and homozygous mutant adults were viable and fertile. However, the Notch4 mutation displayed genetic interactions with a targeted mutation of the relatedNotch1 gene. Embryos homozygous for mutations of both theNotch4 and Notch1 genes often displayed a more severe phenotype than Notch1 homozygous mutant embryos. BothNotch1 mutant and Notch1/Notch4 double mutant embryos displayed severe defects in angiogenic vascular remodeling. Analysis of the expression patterns of genes encoding ligands for Notch family receptors indicated that only the Dll4gene is expressed in a pattern consistent with that expected for a gene encoding a ligand for the Notch1 and Notch4 receptors in the early embryonic vasculature. These results reveal an essential role for the Notch signaling pathway in regulating embryonic vascular morphogenesis and remodeling, and indicate that whereas theNotch4 gene is not essential during embryonic development, theNotch4 and Notch1 genes have partially overlapping roles during embryogenesis in mice.
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Lee, Suk Young, Keiki Kumano, Kumi Nakazaki, Masashi Sanada, Go Yamamoto, Yasuhito Nannya, Ritsuro Suzuki, et al. "Gain-of-Function Mutations and Copy Number Increases of Notch2 in Diffuse Large B-Cell Lymphoma." Blood 110, no. 11 (November 16, 2007): 695. http://dx.doi.org/10.1182/blood.v110.11.695.695.
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Abstract [Background] Signaling through the Notch receptors, triggered by the binding of ligands expressed on neighboring cells, has a key role in determining cell fate in a variety of cell lineages, including lymphocytes. The physiologic roles of two of the four mammalian Notch genes, Notch1 and Notch2, have been clarified in mouse models. Notch1 is preferentially expressed in immature T cells and is essential for specification of early hematopoietic progenitors toward the T-cell fate and for early T-cell development in the thymus. In contrast, Notch2 is preferentially expressed in mature B cells and is required for the generation of a mature B-cell subset, known as splenic marginal zone B (MZB) cells. Deregulation of Notch1 function results in the development of T-cell acute lymphoblastic leukemia (T-ALL) in a number of mouse experimental models, and more importantly, more than 50% of childhood and 30% to 40% of adult human T-ALL cases carry Notch1 mutations that lead to deregulated activation of Notch signaling, indicating that accelerated Notch signaling contributes to the development of human neoplasms. [Results and Discussion] We screened Notch2 gene mutations at the heterodimerization and PEST domains in 109 B-cell lymphoma samples, and found mutations in 5 samples, all of which were diffuse large B-cell lymphomas (DLBCL; in DLBCL, 5 of 63 samples, ∼8%). These mutations lead to partial or complete deletion of the PEST domain, or a single amino acid substitution at the C-terminus of Notch2 protein. All five DLBCL cases with Notch2 mutations were uniformly immunohistochemically negative for CD10 and positive for BCL6 and MUM-1, whereas only 10 out of 24 Notch2 mutation-negative subjects showed this staining pattern. This observation might imply a link between Notch2 mutations and a fraction of non-germinal center B-cell-like DLBCL (non-GCB-DLBCL), according to the immunohistchemistry-based DLBCL subclassification. Interestingly, high-density oligonucleotide microarray analysis revealed that two of the five Notch2 mutation-carrying samples had an increased copy number of the mutated Notch2 allele, and in another sample of the five the total copy number of the Notch2 allele was increased. Furthermore, in the Notch activation-sensitive luciferase reporter assay in vitro, mutant Notch2 receptors showed increased activity compared with wild-type Notch2. These findings implicate Notch2 gain-of-function mutations in the pathogenesis of a subset of B-cell lymphomas, and suggest broader roles for Notch gene mutations in human cancers.
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Crow, Jacob J., and Allan R. Albig. "Notch family members follow stringent requirements for intracellular domain dimerization at sequence-paired sites." PLOS ONE 15, no. 11 (November 24, 2020): e0234101. http://dx.doi.org/10.1371/journal.pone.0234101.
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Notch signaling is essential for multicellular life, regulating core functions such as cellular identity, differentiation, and fate. These processes require highly sensitive systems to avoid going awry, and one such regulatory mechanism is through Notch intracellular domain dimerization. Select Notch target genes contain sequence-paired sites (SPS); motifs in which two Notch transcriptional activation complexes can bind and interact through Notch’s ankyrin domain, resulting in enhanced transcriptional activation. This mechanism has been mostly studied through Notch1, and to date, the abilities of the other Notch family members have been left unexplored. Through the utilization of minimalized, SPS-driven luciferase assays, we were able to test the functional capacity of Notch dimers. Here we show that the Notch 2 and 3 NICDs also exhibit dimerization-induced signaling, following the same stringent requirements as seen with Notch1. Furthermore, our data suggested that Notch4 may also exhibit dimerization-induced signaling, although the amino acids required for Notch4 NICD dimerization appear to be different than those required for Notch 1, 2, and 3 NICD dimerization. Interestingly, we identified a mechanical difference between canonical and cryptic SPSs, leading to differences in their dimerization-induced regulation. Finally, we profiled the Notch family members’ SPS gap distance preferences and found that they all prefer a 16-nucleotide gap, with little room for variation. In summary, this work highlights the potent and highly specific nature of Notch dimerization and refines the scope of this regulatory function.