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1
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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Kramer, Jan, Ralf Schwanbeck, Horst Pagel, Figen Cakiroglu, Jürgen Rohwedel, and Ursula Just. "Inhibition of Notch Signaling Ameliorates Acute Kidney Failure and Downregulates Platelet-Derived Growth Factor Receptor β in the Mouse Model." Cells Tissues Organs 201, no. 2 (2016): 109–17. http://dx.doi.org/10.1159/000442463.
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Ischemic acute kidney injury (AKI) is associated with high morbidity and frequent complications. Repeated episodes of AKI may lead to end-stage renal failure. The pathobiology of regeneration in AKI is not well understood and there is no effective clinical therapy that improves regeneration. The Notch signaling pathway plays an essential role in kidney development and has been implicated in tissue repair in the adult kidney. Here, we found that kidneys after experimental AKI in mice showed increased expression of Notch receptors, specifically Notch1-3, of the Notch ligands Jagged-1 (Jag1), Jag2 and Delta-like-4 (Dll4) and of the Notch target genes Hes1, Hey2, HeyL, Sox9 and platelet-derived growth factor receptor β (Pdgfrb). Treatment of ischemic mice with the γ-secretase inhibitor DBZ blocked Notch signaling and specifically downregulated the expression of Notch3 and the Notch target genes Hes1, Hey2, HeyL and Pdgfrb. After DBZ treatment, the mice developed less interstitial edema and displayed altered interstitial inflammation patterns. Furthermore, serum urea and creatinine levels were significantly decreased from 6 h onwards when compared to control mice treated with DMSO only. Our data are consistent with an amelioration of the severity of kidney injury by blocking Notch activation following AKI, and suggest an involvement of Notch-regulated Pdgfrb in AKI pathogenesis.
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Wu, Lizi, Ivan Maillard, Makoto Nakamura, Warren S. Pear, and James D. Griffin. "The MAML1 Transcriptional Co-Activator Is Required for the Development of Marginal Zone B Cells." Blood 108, no. 11 (November 16, 2006): 777. http://dx.doi.org/10.1182/blood.v108.11.777.777.
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Abstract Notch1 and Notch2 receptor-mediated signaling appear to have important and unique roles in lymphoid lineage commitment. Notch1 is required for T cell development, while Notch2 is essential for marginal zone B cell development. This specificity is not completely explained by differential expression patterns of Notch1 and 2 or Notch ligands, suggesting that there are other genes that contribute to specifying Notch receptor functions. We have previously shown that the MAML family of transcriptional co-activators is essential for Notch-induced transcriptional events, and functions by forming ternary complexes with Notch and the transcription factor CSL in the nucleus. This MAML family currently consists of three members, MAML1-3, all of which can function as co-activators for Notch receptors in vitro . In this study, we investigated the possibility that MAML1 co-activator contributes to determining Notch receptor function by generating mice deficient in the Maml1 gene. Maml1 -deficient mice fail to thrive and die within 10 days of birth. The morphology of marrow, nodes, and spleen was grossly intact. The ability of Maml1-deficient stem cells to generate different T and B lineages of lymphoid cells was determined by transplanting fetal liver cells isolated from E14.5 embryos into lethally irradiated wild-type recipient mice and analyzing donor-derived lymphoid cells 12 weeks post-transplantation. We found that the deletion of Maml1 results in complete loss of marginal zone B cell lineage (MZB, defined by B220+CD21hiCD23lo). Moreover, the number of MZB cells was reduced to about 50% in Maml1 -heterozygous fetal liver chimeras as compared to wild type controls. However, T cell development was largely unaffected, with only a modest but significant increase in the number of γδ T cells (about 2 fold) in both the thymus and the spleen. Therefore, these results suggest the unexpected finding that targeted deletion of Maml1 in hematopoietic cells is similar to the targeted deletion of either Notch2 or the Notch ligand, Delta-like 1 (Dll1) resulting in the loss of marginal zone B cells and minimal effects on T cell development. Moreover, the number of marginal zone B cells is correlated with Maml1 gene dosage, indicating haploinsufficiency. These data suggest that the Notch ligand Dll1 activates Notch2 signaling resulting in a Notch2/MAML1/CSL complex that is essential for marginal zone B cell development. Further studies with respect to relative expression levels of various MAML genes and interactions of MAML co-activators and Notch receptors may shed additional light into understanding how different Notch receptors regulate cell fate decisions in hematopoiesis.
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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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Wang, Qing, Ran Yan, Nancy Pinnell, Yiran Liu, Amparo Serna Alarcon, Jason Qin, Yitong Chen, et al. "The Direct Notch1 Cofactor Zmiz1 Differentially Regulates Notch1 Signals in a Stage-Specific Manner to Preserve Early T-Cell Precursors and Expand Committed T Cells." Blood 128, no. 22 (December 2, 2016): 426. http://dx.doi.org/10.1182/blood.v128.22.426.426.
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Abstract When stem cells first enter the thymus and become early T-cell precursor (ETP) cells, they are exposed to high levels of Notch1 ligand. Notch1 signal strength must be tightly regulated because on one hand, excessive Notch1 signals drive premature T-cell commitment, resulting in loss of ETP cells and alternative cell fates. On the other hand, complete loss of Notch1 signals impairs ETP proliferation, also resulting in loss of ETP cells. Thus, keeping Notch signals finely balanced in ETP cells preserves "stemness". However, after ETP cells commit to the T-cell lineage by the DN3 cell stage, Notch1 signals ramp up dramatically to drive proliferation. It is unclear how Notch1 signals are initially restrained and then amplified. We previously showed that the PIAS-like coregulator Zmiz1 is a direct, context-dependent cofactor of Notch1 in T-cell leukemia. In contrast to drosophila Zmiz1, mammalian Zmiz1 evolved a tetratricopeptide repeat (TPR) domain that binds directly to Notch1 and selectively induces oncogenic target genes such as Myc through its transactivation domain (TAD). To understand the role of Zmiz1 in T-cell development, we bred conditional Zmiz1 and Notch1 knockout mice to Cre strains that delete floxed genes in hematopoietic cells (VavCre/MxCre), in early T cells (LckCre), and in late T-cells (CD4Cre). Like deletion of Notch1, deletion of Zmiz1 caused an early defect at the ETP stage and a late defect at the DN3 stage. The defect with Zmiz1 deletion was less severe than with Notch1 deletion (~4-fold reduction for Zmiz1 deletion versus ~8-fold reduction for Notch1 deletion). Unexpectedly, the ETP defect in Zmiz1-deficient mice partially phenocopied excessive Notch1 activation with increased differentiation to DN2 cells and loss of ETP cells and alternative cell fates (myeloid and NK). To confirm this effect, we plated Zmiz1-deficient hematopoietic stem and progenitor cells (HSPCs) directly on OP9-DL stromal cells. Accordingly, these cultures recapitulated the in vivo phenotype, including suppression of myeloid cells. Reducing Notch signals slightly with modest doses of Notch inhibitors restored myeloid differentiation. In contrast to the ETP defect, the DN3 defect resembled Notch1 loss-of function. Accordingly, overexpression of activated Notch1 or a fusion protein containing only the Notch-interacting domain (TPR) and the TAD was sufficient to rescue the DN3 block. To determine mechanism, we performed RNA-Seq in sorted ETP and DN3 cells from Zmiz1-deficient mice and mice treated with the anti-NRR Notch1 antibody. Zmiz1 coregulated ~16% of Notch1 target genes in ETP cells and ~24% in DN3 cells. In ETP cells, Zmiz1 primarily acted as a repressor of Notch1 target genes. Enrichment analyses showed that Notch1 promoted changes associated with T-lineage commitment, such as induction of Dtx1, Notch3, and Ptcra. In contrast, Zmiz1 reversed these changes. Although generally antagonistic with each other, Zmiz1 and Notch1 concordantly activated a minority of target genes important for proliferation, such as Myc. Upon differentiation to the DN3 cell stage, Zmiz1 switched from primarily a repressor of Notch1 target genes to primarily an activator, inducing Myc and Wnt pathway genes. Accordingly, overexpression of Myc in Zmiz1-deficient DN3 cells was sufficient to rescue the DN3 block in OP9-DL culture. To determine whether Zmiz1 needed to bind Notch1 in order activate or repress Notch1 target genes, we used HSQC NMR to identify amino acids in the TPR that were required for Notch1 binding. Two amino acids, R14 and E34, were confirmed by reporter and co-IP assays to be critical for the Zmiz1-Notch1 interaction. The Zmiz1(R14A+E34A) mutant, which was incapable of binding Notch1, failed to induce Myc. In contrast, this mutant retained full ability to repress Notch target genes associated with T-cell commitment. These data suggest that in ETP cells, Zmiz1 preferentially restrains Notch1 T-cell commitment genes. However, after T-lineage commitment, Zmiz1 switches primarily into a Notch1-interaction mode that preferentially promotes Notch1 signals. It has been puzzling how Notch1 can drive seemingly conflicting biological processes of self-renewal and commitment. Zmiz1 appears to be one solution that evolved to differentially regulate Notch1 signals at a given Notch dosage strength in order to preserve ETP "stemness" while at the same time expanding committed T cells. Disclosures No relevant conflicts of interest to declare.
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Wu, Lizi, Ivan Maillard, Makoto Nakamura, Warren S. Pear, and James D. Griffin. "The transcriptional coactivator Maml1 is required for Notch2-mediated marginal zone B-cell development." Blood 110, no. 10 (November 15, 2007): 3618–23. http://dx.doi.org/10.1182/blood-2007-06-097030.
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Abstract Signaling mediated by various Notch receptors and their ligands regulates diverse biological processes, including lymphoid cell fate decisions. Notch1 is required during T-cell development, while Notch2 and the Notch ligand Delta-like1 control marginal zone B (MZB) cell development. We previously determined that Mastermind-like (MAML) transcriptional coactivators are required for Notchinduced transcription by forming ternary nuclear complexes with Notch and the transcription factor CSL. The 3 MAML family members (MAML1-MAML3) are collectively essential for Notch activity in vivo, but whether individual MAMLs contribute to the specificity of Notch functions is unknown. Here, we addressed this question by studying lymphopoiesis in the absence of the Maml1 gene. Since Maml1−/− mice suffered perinatal lethality, hematopoietic chimeras were generated with Maml1−/−, Maml1+/−, or wild-type fetal liver progenitors. Maml1 deficiency minimally affected T-cell development, but was required for the development of MZB cells, similar to the phenotype of Notch2 deficiency. Moreover, the number of MZB cells correlated with Maml1 gene dosage. Since all 3 Maml genes were expressed in MZB cells and their precursors, these results suggest that Maml1 is specifically required for Notch2 signaling in MZB cells.
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Hamada, Y., Y. Kadokawa, M. Okabe, M. Ikawa, J. R. Coleman, and Y. Tsujimoto. "Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality." Development 126, no. 15 (August 1, 1999): 3415–24. http://dx.doi.org/10.1242/dev.126.15.3415.
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Notch family genes encode transmembrane proteins involved in cell-fate determination. Using gene targeting procedures, we disrupted the mouse Notch2 gene by replacing all but one of the ankyrin repeat sequences in the cytoplasmic domain with the E. coli (beta)-galactosidase gene. The mutant Notch2 gene encodes a 380 kDa Notch2-(beta)-gal fusion protein with (beta)-galactosidase activity. Notch2 homozygous mutant mice die prior to embryonic day 11.5, whereas heterozygotes show no apparent abnormalities and are fully viable. Analysis of Notch2 expression patterns, revealed by X-gal staining, demonstrated that the Notch2 gene is expressed in a wide variety of tissues including neuroepithelia, somites, optic vesicles, otic vesicles, and branchial arches, but not heart. Histological studies, including in situ nick end labeling procedures, showed earlier onset and higher incidence of apoptosis in homozygous mutant mice than in heterozygotes or wild type mice. Dying cells were particularly evident in neural tissues, where they were seen as early as embryonic day 9.5 in Notch2-deficient mice. Cells from Notch2 mutant mice attach and grow normally in culture, demonstrating that Notch2 deficiency does not interfere with cell proliferation and that expression of the Notch2-(beta)-gal fusion protein is not toxic per se. In contrast to Notch1-deficient mice, Notch2 mutant mice did not show disorganized somitogenesis, nor did they fail to properly regulate the expression of neurogenic genes such as Hes-5 or Mash1. In situ hybridization studies show no indication of altered Notch1 expression patterns in Notch2 mutant mice. The results indicate that Notch2 plays an essential role in postimplantation development in mice, probably in some aspect of cell specification and/or differentiation, and that the ankyrin repeats are indispensable for its function.
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Du, Wei, Jared Sipple, Jonathan Schick, and Qishen Pang. "Enhanced Notch Signaling Skews Hematopoietic Stem Cell Differentiation in Fanconi Anemia Murine Models." Blood 120, no. 21 (November 16, 2012): 1191. http://dx.doi.org/10.1182/blood.v120.21.1191.1191.
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Abstract Abstract 1191 Objective: Hematopoietic stem cells (HSCs) can either self-renew or differentiate into various types of cells of the blood lineage. Little is known about the signaling pathways that regulate this choice of self-renewal versus differentiation. We studied the effect of altered Notch signaling on HSC differentiation in mouse models of Fanconi anemia (FA), a genetic disorder associated with bone marrow failure and progression to leukemia and other cancers. Methods: The study used a Notch reporter mouse, in which Notch-driven GFP expression acts as a sensor for HSC differentiation. Long-term hematopoietic stem cell (LT-HSC) and multipotential progenitor (MPP) cell compartments, as well as GFP expression in different cell populations were detected by Flow Cytometry analysis using primary bone marrow cells from Notch-eGFP-WT, Notch-eGFP-Fanca−/− or Notch-eGFP-Fancc−/− mice. Cell Cycle analysis was performed to distinguish the difference of quiescent state in GFP-gated LSK cells from these Notch-eGFP reporter mice. Colony forming units (CFU) assay and bone marrow transplantation (BMT) were utilized to determine HSC self-renew capacity. Gene arrays for pathways involved in DNA repair, cell cycle control, anti-oxidant defense, inflammatory response and apoptotic signaling were employed to define the gene expression signatures of the MPP population. Results and conclusions: In mice expressing a transgenic Notch reporter, deletion of the Fanca or Fancc gene enhanced Notch signaling in MPPs, which was correlated with decreased phenotypic long-term HSCs and increased formation of MPP1 progenitors. Furthermore, we found a functional correlation between Notch signaling and self-renewal capacity in FA hematopoietic stem and progenitor cells (HSPCs). Significantly, we show that FA deficiency in MPPs deregulates a complex network of genes in the Notch and canonical NF-kB pathways. Specifically, enhanced Notch signaling in FA MPPs was associated with the unregulation of genes involved in inflammatory and stress responses (including Rela, Tnfrsf1b, Gadd45b, Sod2, Stat1, Irf1 and Xiap), cell-cycle regulation (including Ccnd1, Cdc16, Cdkn1a, Gsk3b, Notch2 and Nr4a2), and transcription regulation (including Rela, Stat1, Hes1, Hey1, Hoxb4, Notch1 and Notch2). Consequently, TNF-a stimulation enhanced Notch signaling of FA LSK cells, leading to decreased HSC quiescence and compromised HSC self-renewal. Finally, genetic ablation of NF-kB reduced Notch signaling in FA MPPs to nearly wide-type level, and blocking either NF-kB or Notch signaling partially restored FA HSC quiescence and self-renewal capacity. Translational Applicability: The study identifies a functional interaction between the FA pathway and Notch signaling in HSC differentiation and establishes a role of FA proteins in the control of balance between renewal and lineage commitment, hence contributing to hematopoiesis. These findings indicate that the Notch signaling pathway may represent a novel and therapeutically accessible pathway in FA. Disclosures: No relevant conflicts of interest to declare.
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Ziouti, Fani, Regina Ebert, Maximilian Rummler, Melanie Krug, Sigrid Müller-Deubert, Martin Lüdemann, Franz Jakob, Bettina M. Willie, and Franziska Jundt. "NOTCH Signaling Is Activated through Mechanical Strain in Human Bone Marrow-Derived Mesenchymal Stromal Cells." Stem Cells International 2019 (February 26, 2019): 1–13. http://dx.doi.org/10.1155/2019/5150634.
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Skeletal development and remodeling of adult bone are critically controlled by activated NOTCH signaling in genetically modified mice. It is yet unclear whether NOTCH signaling is activated by mechanical strain sensed by bone cells. We found that expression of specific NOTCH target genes is induced after in vivo tibial mechanical loading in wild-type mice. We further applied mechanical strain through cyclic stretching in human bone marrow-derived mesenchymal stromal cells (BMSCs) in vitro by using a bioreactor system and detected upregulation of NOTCH target gene expression. Inhibition of the NOTCH pathway in primary BMSCs as well as telomerase-immortalized human BMSCs (hMSC-TERT) through the gamma-secretase inhibitor GSI XII blocked mechanotransduction and modulated actin cytoskeleton organization. Short-hairpin RNA gene silencing identified NOTCH2 as the key receptor mediating NOTCH effects on hMSC-TERT cells. Our data indicate a functional link between NOTCH activation and mechanotransduction in human BMSCs. We suggest that NOTCH signaling is an important contributor to molecular mechanisms that mediate the bone formation response to mechanical strain.
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Vanorny, Dallas A., Rexxi D. Prasasya, Abha J. Chalpe, Signe M. Kilen, and Kelly E. Mayo. "Notch Signaling Regulates Ovarian Follicle Formation and Coordinates Follicular Growth." Molecular Endocrinology 28, no. 4 (April 1, 2014): 499–511. http://dx.doi.org/10.1210/me.2013-1288.
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Abstract Ovarian follicles form through a process in which somatic pregranulosa cells encapsulate individual germ cells from germ cell syncytia. Complementary expression of the Notch ligand, Jagged1, in germ cells and the Notch receptor, Notch2, in pregranulosa cells suggests a role for Notch signaling in mediating cellular interactions during follicle assembly. Using a Notch reporter mouse, we demonstrate that Notch signaling is active within somatic cells of the embryonic ovary, and these cells undergo dramatic reorganization during follicle histogenesis. This coincides with a significant increase in the expression of the ligands, Jagged1 and Jagged2; the receptor, Notch2; and the target genes, Hes1 and Hey2. Histological examination of ovaries from mice with conditional deletion of Jagged1 within germ cells (J1 knockout [J1KO]) or Notch2 within granulosa cells (N2 knockout [N2KO]) reveals changes in follicle dynamics, including perturbations in the primordial follicle pool and antral follicle development. J1KO and N2KO ovaries also contain multi-oocytic follicles, which represent a failure to resolve germ cell syncytia, and follicles with enlarged oocytes but lacking somatic cell growth, signifying a potential role of Notch signaling in follicle activation and the coordination of follicle development. We also observed decreased cell proliferation and increased apoptosis in the somatic cells of both conditional knockout lines. As a consequence of these defects, J1KO female mice are subfertile; however, N2KO female mice remain fertile. This study demonstrates important functions for Jagged1 and Notch2 in the resolution of germ cell syncytia and the coordination of somatic and germ cell growth within follicles of the mouse ovary.
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Murta, D., M. Batista, E. Silva, A. Trindade, L. Mateus, A. Duarte, and L. Lopes-da-Costa. "Differential expression of Notch component and effector genes during ovarian follicle and corpus luteum development during the oestrous cycle." Reproduction, Fertility and Development 27, no. 7 (2015): 1038. http://dx.doi.org/10.1071/rd13399.
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Ovarian dynamics throughout the female oestrous cycle (EC) are characterised by cyclical follicle and corpus luteum (CL) development. These events are tightly regulated, involving extensive cell-to-cell communication. Notch is an evolutionarily well conserved cell-signalling pathway implicated in cell-fate decisions in several tissues. Here, we evaluated the extra-vascular expression patterns of Notch component and effector genes during follicle and CL development throughout the EC. Five mature CD1 female mice were killed at each EC stage. Blood samples were collected for progesterone measurement, ovaries were processed for immunohistochemistry and expression patterns of Notch components (Notch1, 2 and 3, Jagged1 and Delta-like1 and 4) and effectors (Hes1, Hes2 and Hes5) were characterised. Nuclear detection of Notch effectors indicates that Notch signalling is active in the ovary. Notch components and effectors are differentially expressed during follicle and CL development throughout the EC. The spatial and temporal specific expression patterns are associated with follicle growth, selection and ovulation or atresia and CL development and regression.
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Zhou, Lan, Lebing Wei Li, Quanjian Yan, Bronislawa Petryniak, Yunfang Man, Charles Su, Jeongsup Shim, Stephanie Chervin, and John B. Lowe. "Notch-dependent control of myelopoiesis is regulated by fucosylation." Blood 112, no. 2 (July 15, 2008): 308–19. http://dx.doi.org/10.1182/blood-2007-11-115204.
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Abstract Cell-cell contact–dependent mechanisms that modulate proliferation and/or differentiation in the context of hematopoiesis include mechanisms characteristic of the interactions between members of the Notch family of signal transduction molecules and their ligands. Whereas Notch family members and their ligands clearly modulate T lymphopoietic decisions, evidence for their participation in modulating myelopoiesis is much less clear, and roles for posttranslational control of Notch-dependent signal transduction in myelopoiesis are unexplored. We report here that a myeloproliferative phenotype in FX−/− mice, which are conditionally deficient in cellular fucosylation, is consequent to loss of Notch-dependent signal transduction on myeloid progenitor cells. In the context of a wild-type fucosylation phenotype, we find that the Notch ligands suppress myeloid differentiation of progenitor cells and enhance expression of Notch target genes. By contrast, fucosylation-deficient myeloid progenitors are insensitive to the suppressive effects of Notch ligands on myelopoiesis, do not transcribe Notch1 target genes when cocultured with Notch ligands, and have lost the wild-type Notch ligand-binding phenotype. Considered together, these observations indicate that Notch-dependent signaling controls myelopoiesis in vivo and in vitro and identifies a requirement for Notch fucosylation in the expression of Notch ligand binding activity and Notch signaling efficiency in myeloid progenitors.
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Lin, Neng-Yu, Alfiya Distler, Christian Beyer, Ariella Philipi-Schöbinger, Silvia Breda, Clara Dees, Michael Stock, et al. "Inhibition of Notch1 promotes hedgehog signalling in a HES1-dependent manner in chondrocytes and exacerbates experimental osteoarthritis." Annals of the Rheumatic Diseases 75, no. 11 (February 5, 2016): 2037–44. http://dx.doi.org/10.1136/annrheumdis-2015-208420.
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ObjectivesNotch ligands and receptors have recently been shown to be differentially expressed in osteoarthritis (OA). We aim to further elucidate the functional role of Notch signalling in OA using Notch1 antisense transgenic (Notch1 AS) mice.MethodsNotch and hedgehog signalling were analysed by real-time PCR and immunohistochemistry. Notch-1 AS mice were employed as a model of impaired Notch signalling in vivo. Experimental OA was induced by destabilisation of the medial meniscus (DMM). The extent of cartilage destruction and osteophyte formation was analysed by safranin-O staining with subsequent assessment of the Osteoarthritis Research Society International (OARSI) and Mankin scores and µCT scanning. Collagen X staining was used as a marker of chondrocyte hypertrophy. The role of hairy/enhancer of split 1 (Hes-1) was investigated with knockdown and overexpression experiments.ResultsNotch signalling was activated in human and murine OA with increased expression of Jagged1, Notch-1, accumulation of the Notch intracellular domain 1 and increased transcription of Hes-1. Notch1 AS mice showed exacerbated OA with increases in OARSI scores, osteophyte formation, increased subchondral bone plate density, collagen X and osteocalcin expression and elevated levels of Epas1 and ADAM-TS5 mRNA. Inhibition of the Notch pathway induced activation of hedgehog signalling with induction of Gli-1 and Gli-2 and increased transcription of hedgehog target genes. The regulatory effects of Notch signalling on Gli-expression were mimicked by Hes-1.ConclusionsInhibition of Notch signalling activates hedgehog signalling, enhances chondrocyte hypertrophy and exacerbates experimental OA including osteophyte formation. These data suggest that the activation of the Notch pathway may limit aberrant hedgehog signalling in OA.
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Canalis, Ernesto, Tamar R. Grossman, Michele Carrer, Lauren Schilling, and Jungeun Yu. "Antisense oligonucleotides targeting Notch2 ameliorate the osteopenic phenotype in a mouse model of Hajdu-Cheney syndrome." Journal of Biological Chemistry 295, no. 12 (January 28, 2020): 3952–64. http://dx.doi.org/10.1074/jbc.ra119.011440.
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Notch receptors play critical roles in cell-fate decisions and in the regulation of skeletal development and bone remodeling. Gain–of–function NOTCH2 mutations can cause Hajdu-Cheney syndrome, an untreatable disease characterized by osteoporosis and fractures, craniofacial developmental abnormalities, and acro-osteolysis. We have previously created a mouse model harboring a point 6955C→T mutation in the Notch2 locus upstream of the PEST domain, and we termed this model Notch2tm1.1Ecan. Heterozygous Notch2tm1.1Ecan mutant mice exhibit severe cancellous and cortical bone osteopenia due to increased bone resorption. In this work, we demonstrate that the subcutaneous administration of Notch2 antisense oligonucleotides (ASO) down-regulates Notch2 and the Notch target genes Hes-related family basic helix–loop–helix transcription factor with YRPW motif 1 (Hey1), Hey2, and HeyL in skeletal tissue from Notch2tm1.1Ecan mice. Results of microcomputed tomography experiments indicated that the administration of Notch2 ASOs ameliorates the cancellous osteopenia of Notch2tm1.1Ecan mice, and bone histomorphometry analysis revealed decreased osteoclast numbers in Notch2 ASO-treated Notch2tm1.1Ecan mice. Notch2 ASOs decreased the induction of mRNA levels of TNF superfamily member 11 (Tnfsf11, encoding the osteoclastogenic protein RANKL) in cultured osteoblasts and osteocytes from Notch2tm1.1Ecan mice. Bone marrow-derived macrophage cultures from the Notch2tm1.1Ecan mice displayed enhanced osteoclastogenesis, which was suppressed by Notch2 ASOs. In conclusion, Notch2tm1.1Ecan mice exhibit cancellous bone osteopenia that can be ameliorated by systemic administration of Notch2 ASOs.
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Kindler, Thomas, Melanie G. Cornejo, Claudia Scholl, Jianing Liu, Dena S. Leeman, J. Erika Haydu, Stefan Fröhling, Benjamin H. Lee, and D. Gary Gilliland. "K-RasG12D–induced T-cell lymphoblastic lymphoma/leukemias harbor Notch1 mutations and are sensitive to γ-secretase inhibitors." Blood 112, no. 8 (October 15, 2008): 3373–82. http://dx.doi.org/10.1182/blood-2008-03-147587.
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Abstract To study the impact of oncogenic K-Ras on T-cell leukemia/lymphoma development and progression, we made use of a conditional K-RasG12D murine knockin model, in which oncogenic K-Ras is expressed from its endogenous promoter. Transplantation of whole bone marrow cells that express oncogenic K-Ras into wild-type recipient mice resulted in a highly penetrant, aggressive T-cell leukemia/lymphoma. The lymphoblasts were composed of a CD4/CD8 double-positive population that aberrantly expressed CD44. Thymi of primary donor mice showed reduced cellularity, and immunophenotypic analysis demonstrated a block in differentiation at the double-negative 1 stage. With progression of disease, approximately 50% of mice acquired Notch1 mutations within the PEST domain. Of note, primary lymphoblasts were hypersensitive to γ-secretase inhibitor treatment, which is known to impair Notch signaling. This inhibition was Notch-specific as assessed by down-regulation of Notch1 target genes and intracellular cleaved Notch. We also observed that the oncogenic K-Ras-induced T-cell disease was responsive to rapamycin and inhibitors of the RAS/MAPK pathway. These data indicate that patients with T-cell leukemia with K-Ras mutations may benefit from therapies that target the NOTCH pathway alone or in combination with inhibition of the PI3K/AKT/MTOR and RAS/MAPK pathways.
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McCarter, Anna, Ran Yan, Amparo Serna Alarcon, Catherine Chang, Erin Kim, Cher Sha, Yiran Liu, et al. "Ets1 Enhances Context-Dependent Notch1 Activity in T-Cell Leukemia." Blood 132, Supplement 1 (November 29, 2018): 2595. http://dx.doi.org/10.1182/blood-2018-99-111376.
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Abstract The discovery of NOTCH1 as the most frequently mutated oncogene in T-ALL patients raised hopes for targeted therapy in this cancer. Unfortunately, in clinical trials, the pan-Notch inhibitor GSI caused excessive GI toxicity. Mice treated continuously with GSI die from intestinal stem cell loss and severe intestinal secretory cell metaplasia. Intermittent dosing of GSI is tolerable, but has weak anti-cancer effects. Thus, the challenge has been to find ways to selectively disable Notch in T-ALL. Our idea to meet this challenge stems from work by others showing that Notch cannot activate enhancers by itself. Notch requires a favorable "chromatin context" at its enhancers that is created by cooperating transcription factors. In theory, one could target cell-specific factors at these enhancers in order to avoid the intolerable effects of pan-Notch inhibition. In support of this, others showed that ubiquitous deletion of the T-cell specific Notch-dependent Myc enhancer in mice impairs T-ALL proliferation and thymopoiesis, but has no effect on other tissues. We previously showed that the transcriptional coactivator Zmiz1 is a direct cofactor of Notch1 that selectively promotes Notch activity at the T-cell Myc enhancer. However, it was unclear what other factors promote context-dependent Notch activity. Ets1 is an attractive candidate. It can bind nucleosome-occupied regions in T-cell precursors and most Notch response elements in T-ALL cells, including the T-cell MYC enhancer. To investigate its importance, we generated conditional Ets1 knockout mice. Deletion of Ets1 in hematopoietic cells using the VavCre transgene caused a 21-fold loss of thymocytes starting at the earliest stage. This was 4-fold more severe than the loss of thymocytes in Notch-deficient mice. Deletion of Ets1 using a ubiquitous tamoxifen-inducible Cre caused a Notch loss-of-function phenotype in the intestine with a 1.4 to 2.3-fold increase in goblet cells. This was milder than the effects of GSI (3.3 to 4.2-fold increase). ~64% of the Ets1-deleted mice died from unclear causes. In vivo deletion of Ets1 in Notch1-induced murine T-ALLs reduced blast counts by 30-fold and prolonged survival. In a panel of human T-ALL cell lines, on average, knockdown with two different shEts1 reduced proliferation by 2 and 9-fold respectively over ~1.5 weeks of culture. This was superior to the effects of GSI (up to 2-fold inhibition). A small molecule inhibitor of Usp9x, the deubiquitinase of Ets1, induced Ets1 protein degradation and impaired T-ALL cell proliferation with submicromolar GI50. In PDX models, shEts1 reduced circulating blasts by 44-fold and prolonged survival. To identify the mechanism by which Ets1 promotes T-ALL, we performed endogenous co-IP assays, which showed that Ets1 interacts with Notch1 and its cofactor Zmiz1. Further, Ets1 binding by ChIP correlated with Zmiz1 binding (R2=0.93). Knockdown of Ets1 reduced Zmiz1, Ets1, and Notch1 binding to enhancers of major T-ALL oncogenes, MYC and IL7R. RNA-Seq showed that Ets1 co-regulates the expression of ~30% of Notch1 target genes. Multiple MSigDB enrichment analyses of both Ets1 and Notch-regulated genes showed that the MYC and MTORC pathways were the #1 or #2 most enriched list. Enforced expression of Myc partially rescued the proliferation of human T-ALL cell lines deprived of Ets1. Based on these data, we predicted that Ets1 inhibition would sensitize enhancers to Notch inhibition. Accordingly, Ets1 withdrawal promoted the effects of GSI in repressing Myc expression and cell proliferation. Further, in our mouse model of Notch-induced T-ALL, Ets1 deletion in combination with intermittent doses of GSI reduced blast counts and prolonged survival more effectively than either treatment alone. Our data support an emerging model in which cofactors like Ets1 create a favorable chromatin context for Notch1 to activate a subset of response elements. The context dependence of Ets1 action, which promotes certain oncogenic signals of Notch1 in T cells, might be clinically relevant. Ets1 deprivation inhibited thymopoiesis and leukemic proliferation more effectively and with less intestinal toxicity than Notch deprivation. Our data suggest that inhibiting Ets1, possibly through targeted protein degradation, would combat important drivers of the Notch pathway with reduced adverse effects linked to pan-Notch inhibition. Disclosures No relevant conflicts of interest to declare.
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De Decker, Matthias, Marieke Lavaert, Juliette Roels, Laurentijn Tilleman, Bart Vandekerckhove, Georges Leclercq, Filip Van Nieuwerburgh, Pieter Van Vlierberghe, and Tom Taghon. "HES1 and HES4 have non-redundant roles downstream of Notch during early human T-cell development." Haematologica 106, no. 1 (January 9, 2020): 130–41. http://dx.doi.org/10.3324/haematol.2019.226126.
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In both mouse and human, Notch1 activation is the main initial driver to induce T-cell development in hematopoietic progenitor cells. The initiation of this developmental process coincides with Notch1-dependent repression of differentiation towards other hematopoietic lineages. Although well described in mice, the role of the individual Notch1 target genes during these hematopoietic developmental choices is still unclear in human, particularly for HES4 since no orthologous gene is present in the mouse. Here, we investigated the functional capacity of the Notch1 target genes HES1 and HES4 to modulate human Notch1-dependent hematopoietic lineage decisions and their requirement during early T-cell development. We show that both genes are upregulated in a Notch-dependent manner during early T-cell development and that HES1 acts as a repressor of differentiation by maintaining a quiescent stem cell signature in CD34+ hematopoietic progenitor cells. While HES4 can also inhibit natural killer and myeloid cell development like HES1, it acts differently on the T- versus B-cell lineage choice. Surprisingly, HES4 is not capable of repressing B-cell development, the most sensitive hematopoietic lineage with respect to Notch-mediated repression. In contrast to HES1, HES4 promotes initiation of early T-cell development, but ectopic expression of HES4, or HES1 and HES4 combined, is not sufficient to induce T-lineage differentiation. Importantly, knockdown of HES1 or HES4 significantly reduces human T-cell development. Overall, we show that the Notch1 target genes HES1 and HES4 have non-redundant roles during early human T-cell development which may relate to differences in mediating Notch-dependent human hematopoietic lineage decisions.
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Jing, Yaxun, Joao Antonio Gimenes, Rahul Mishra, Duc Pham, Adam T. Comstock, Daohai Yu, and Umadevi Sajjan. "NOTCH3 contributes to rhinovirus-induced goblet cell hyperplasia in COPD airway epithelial cells." Thorax 74, no. 1 (July 10, 2018): 18–32. http://dx.doi.org/10.1136/thoraxjnl-2017-210593.
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RationaleGoblet cell hyperplasia (GCH) is one of the cardinal features of chronic obstructive pulmonary disease (COPD) and contributes to airways obstruction. Rhinovirus (RV), which causes acute exacerbations in patients with COPD, also causes prolonged airways obstruction. Previously, we showed that RV enhances mucin gene expression and increases goblet cell number in a COPD mouse model. This study examines whether RV causes sustained GCH in relevant models of COPD.MethodsMucociliary-differentiated COPD and normal airway epithelial cell cultures and mice with normal or COPD phenotype were infected with RV or sham and examined for GCH by immunofluorescence and/or mucin gene expression. In some experiments, RV-infected COPD cells and mice with COPD phenotype were treated with γ-secretase inhibitor or interleukin-13 neutralising antibody and assessed for GCH. To determine the contribution of NOTCH1/3 in RV-induced GCH, COPD cells transduced with NOTCH1/3 shRNA were used.ResultsRV-infected COPD, but not normal cell cultures, showed sustained GCH and increased mucin genes expression. Microarray analysis indicated increased expression of NOTCH1, NOTCH3 and HEY1 only in RV-infected COPD cells. Blocking NOTCH3, but not NOTCH1, attenuated RV-induced GCH in vitro. Inhibition of NOTCH signalling by γ-secretase inhibitor, but not neutralising antibody to IL-13, abrogated RV-induced GCH and mucin gene expression.ConclusionsRV induces sustained GCH via NOTCH3 particularly in COPD cells or mice with COPD phenotype. This may be one of the mechanisms that may contribute to RV-induced prolonged airways obstruction in COPD.
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Lee, Sung-UK, Min Li, Manami Maeda, Nagisa Sakurai, Yuichi Ishikawa, Freddy Radtke, Minhong Yan, Hugh Robson Macdonald, and Takahiro Maeda. "Notch Repression by LRF Is Necessary for the Maintenance of Adult Hematopoietic Stem Cell Pool." Blood 116, no. 21 (November 19, 2010): 2633. http://dx.doi.org/10.1182/blood.v116.21.2633.2633.
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Abstract Abstract 2633 Among the different stem cells, hematopoietic stem cells (HSCs) are one of the best studied and characterized stem cells. To maintain life-long hematopoiesis in the bone marrow (BM), signals governing the balance between self-renewal and differentiation are tightly regulated in HSC compartment. Notch signals are critical regulators of the lymphoid lineage fate, but their role in adult HSC function in the BM is currently under debate. LRF (Leukemia/Lymphoma Related Factor, also known as Zbtb7a/pokemon) is a transcription factor that acts as a proto-oncogene and plays a key role in lymphoid and erythroid development. Previously we reported that the pool of LT-HSCs, CD150+CD48−Flt3−Vcam-1+/−IL7Rα−LSK (Lin−Sca-1+c-Kit+), was significantly reduced, while lymphoid-biased multi-potential progenitors (LMPPs: CD150−CD48+Flt3+Vcam-1+/−IL7Rα−LSK) and common lymphoid progenitors (CLPs: Lin−CD150−CD48+Flt3+Vcam-1−IL7Rα+) were barely detectable in LRF deficient mice. This was due to excessive differentiation of HSC into aberrant CD4/CD8 DP (double positive) T cell development in the BM caused by high Notch activity, implicating LRF role on HSC maintenance. Both gene expression profile (GSEA and DAVID analysis) and Q-PCR results indicated that LRF deficient LT-HSCs had loss of stem cell signature; but gain of T cell signature and up-regulated Notch-target gene, Hes-1, without affecting mRNA expression of Notch (1-4) or related (DLL1, DLL4, Jagged-1) genes. To determine LRF function in HSCs, we performed in vivo and in vitro experiments: 1) 5-FU (5-fluorouracil, the chemotherapy agent) treated LRF deficient mice were not able to compensate for their loss of LT-HSCs; 2) multi-lineage defects were shown in second recipient mice transplanted with 1 million of LRF deficient bone marrow cells in serial bone marrow transplantation assays, suggesting that LRF deficient LT-HSCs had defect in self-renewal and 3) LRF deficient FL-HSCs (CD150+CD48−LSK cells) were cultured on OP9 cells expressing delta-like ligand (DLL1, DLL4 and Jagged1), and enhanced T cell differentiation was only observed when they were co-cultured with delta-expressing OP9 cells. Among the Notch family, these phenotypes were Notch1-dependent. In fact, Notch1flox/floxLRFflox/floxMx1-Cre+ mice demonstrated normal LT-HSC numbers and restored B cell development, and prolonged survival over LRFflox/floxMx1-Cre+ mice in sequential 5-FU treatment in vivo. To explore which Notch-ligand(s) in BM niche is responsible for aberrant T-cell development in LRF deficient mice as well, we treated wild-type and LRFflox/floxMx1-Cre+ with anti-DLL4 antibody twice per week for 3 weeks. DLL4 blockage in LRF deficient mice rescued B cell development and prevented the development of aberrant DP T-cell development in LRF deficient mice. To further elucidate the relationship between LRF and Notch in adult HSC function, we analyzed Notch protein expression levels in HSCs and performed in-depth analysis of HSC/progenitor (HSC, LMPP and CLP) compartments in wild-type and LRF knockout (KO). Interestingly, Notch1 proteins were differentially expressed in LT-HSCs and ~50 % of them were positive for Notch1, while Notch2 was abundantly expressed in LT-HSCs. Notch1 expressing LT-HSCs were in more active cell-cycle (S phase) and absent in LRF conditional knockout mice. It is most likely that Notch1 expressing LT-HSCs were continually differentiating toward T cells in the absence of LRF, as CD4+CD8+ T cells were evident in the BM 10 months after pIpC injection. Taken together, our data strongly indicate that LRF is indispensable for hematopoietic homeostasis by preventing the lymphoid-primed HSCs from Notch/Delta-mediated T-instructive signal in the BM niche. Currently we're investigating the functional significances of Notch1 expressing LT-HSCs in detail. Our studies help us to better understand the underlying mechanism for HSC fate decision (self-renewal v.s. differentiation) in stem cell biology and its therapeutic approach in regenerative medicine. Disclosures: No relevant conflicts of interest to declare.
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Phelan, James D., Ingrid Saba, Chinavenmeni S. Velu, Tarik Moroy, and H. Leighton Grimes. "Notch Signaling Requires Gfi1 for T Cell Development." Blood 118, no. 21 (November 18, 2011): 2174. http://dx.doi.org/10.1182/blood.v118.21.2174.2174.
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Abstract Abstract 2174 Growth factor independent-1 (Gfi1) is a zinc finger transcriptional repressor protein originally identified in a rodent model of T-cell leukemia. Gfi1 deficient mice have defects in T cell development and a moderate loss of thymic cellularity. In Drosophila, orthologs of Notch1 and Gfi1 cooperate to specify embryo sensory organ precursors. Given the established requirement for Notch1 in T cell specification and development as well as the functional relationship of Notch and Gfi1 orthologs in Drosophila genetics, we investigated the ability of Gfi1 and Notch to cooperate in T-cell development. Utilizing transgenic mice in which the expression of Cre recombinase is controlled by the proximal Lck promoter (LckCre) to both activate intracellular Notch1 (ICN) while simultaneously deleting Gfi1, we demonstrate that T cells overexpressing ICN require Gfi1 for their survival and proper integration of ICN signaling. First, we validated our approach by showing that Lck-Cre-mediated deletion of Gfi1 alleles (Gfi1flox/-) or activation of ICN expression (Rosa26lox-stop-loxICN ires eGFP, “RosaICN”) lead to expected phenotypes. We next examined the consequences of ICN activation with simultaneous deletion of Gfi1. Whereas inducible deletion of Gfi1 alone decreases thymic cellularity by ∼4-fold, Gfi1 deletion coupled with ICN activation leads to complete thymic involution with a 14-fold reduction in total T cell numbers (p<0.0001). To determine whether developmental context controlled this interaction, we used a series of temporally regulated T cell promoters to drive Cre expression. In addition to targeting thymocytes before TCRβ-selection with Lck-Cre, we also examined CD4-Cre (deleting after TCRβ-selection), as well as the distal Lck promoter-Cre (deleting after negative selection). Notably, CD4-Cre mediated activation of ICN and deletion of Gfi1 results in an ∼9-fold reduction in thymocyte numbers, similar to proximal Lck-Cre. However, the requirement for Gfi1 in ICN-expressing cells is not global, in that distal Lck-Cre mediated deletion in post-negative selection thymocytes revealed normal cell numbers. Variation in Notch signaling defects may explain the profound differences in cellularity observed between deleting Gfi1 early verses late in T cell development. We limited one allele of Gfi1 and examined the transcriptional effect upon ICN target genes. First, FACS sorted DN3 thymocytes (CD4−, CD8−, CD44−, CD25+) from proximal LckCre+RosaICNGfi1f/+ transgenic mice, showed that a full one-third of all ICN-activated genes are differentially regulated upon the loss of a single copy of Gfi1. In contrast, splenic T cells from distal Lck-iCre+RosaICNGfi1f/+, display an equivalent expression level of many Notch1 target genes as their Gfi1+/+ littermate controls (dLck-iCre+RosaICNGfi1+/+). Moreover, these Notch signaling defects do not appear to require supraphysiological levels of activated ICN as evidenced by dysregulated endogenous Notch1 target gene activation in Gfi1−/− mice, including FACS sorted DN1 thymocytes and early bone marrow progenitors. Finally, this defect is cell autonomous in that Gfi1−/− early thymic progenitors do not develop on OP9-DL1 stroma cells whereas their WT littermates develop into DN3 T cells within 6 days. Therefore, our data both confirms and extends a functional genetic relationship between Notch1 and Gfi1 from fruit fly to mammalian lymphocyte development. Furthermore, our data suggests that Gfi1−/− developing thymocytes are incapable of correctly interpreting Notch signals, which ultimately leads to their death. Disclosures: No relevant conflicts of interest to declare.
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Ungerbäck, Jonas, Josefine Åhsberg, Tobias Strid, Rajesh Somasundaram, and Mikael Sigvardsson. "Combined heterozygous loss of Ebf1 and Pax5 allows for T-lineage conversion of B cell progenitors." Journal of Experimental Medicine 212, no. 7 (June 8, 2015): 1109–23. http://dx.doi.org/10.1084/jem.20132100.
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To investigate how transcription factor levels impact B-lymphocyte development, we generated mice carrying transheterozygous mutations in the Pax5 and Ebf1 genes. Whereas combined reduction of Pax5 and Ebf1 had minimal impact on the development of the earliest CD19+ progenitors, these cells displayed an increased T cell potential in vivo and in vitro. The alteration in lineage fate depended on a Notch1-mediated conversion process, whereas no signs of de-differentiation could be detected. The differences in functional response to Notch signaling in Wt and Pax5+/−Ebf1+/− pro–B cells were reflected in the transcriptional response. Both genotypes responded by the generation of intracellular Notch1 and activation of a set of target genes, but only the Pax5+/−Ebf1+/− pro–B cells down-regulated genes central for the preservation of stable B cell identity. This report stresses the importance of the levels of transcription factor expression during lymphocyte development, and suggests that Pax5 and Ebf1 collaborate to modulate the transcriptional response to Notch signaling. This provides an insight on how transcription factors like Ebf1 and Pax5 preserve cellular identity during differentiation.
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Kalinichenko, Vladimir V., Galina A. Gusarova, Il-Man Kim, Brian Shin, Helena M. Yoder, Jean Clark, Alexander M. Sapozhnikov, Jeffrey A. Whitsett, and Robert H. Costa. "Foxf1 haploinsufficiency reduces Notch-2 signaling during mouse lung development." American Journal of Physiology-Lung Cellular and Molecular Physiology 286, no. 3 (March 2004): L521—L530. http://dx.doi.org/10.1152/ajplung.00212.2003.
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The forkhead box (Fox) f1 transcription factor is expressed in the mouse splanchnic (visceral) mesoderm, which contributes to development of the liver, gallbladder, lung, and intestinal tract. Pulmonary hemorrhage and peripheral microvascular defects were found in approximately half of the newborn Foxf1(+/-) mice, which expressed low levels of lung Foxf1 mRNA [low- Foxf1(+/-) mice]. Microvascular development was normal in the surviving newborn high- Foxf1(+/-) mice, which compensated for pulmonary Foxf1 haploinsufficiency and expressed wild-type Foxf1 levels. To identify expression of genes regulated by Foxf1, we used Affymetrix microarrays to determine embryonic lung RNAs influenced by Foxf1 haploinsufficiency. Embryonic Foxf1(+/-) lungs exhibited diminished expression of hepatocyte growth factor receptor c-Met, myosin VI, the transcription factors SP-3, BMI-1, ATF-2, and glucocorticoid receptor, and cell cycle inhibitors p53, p21Cip1, retinoblastoma, and p107. Furthermore, Notch-2 signaling was decreased in embryonic Foxf1(+/-) lungs, as evidenced by significantly reduced levels of the Notch-2 receptor and the Notch-2 downstream target hairy enhancer of split-1. The severity of the Notch-2-signaling defect in 18-day postcoitus Foxf1(+/-) lungs correlated with Foxf1 mRNA levels. Disruption of pulmonary Notch-2 signaling continued in newborn low- Foxf1(+/-) mice, which died of lung hemorrhage and failed to compensate for Foxf1 haploinsufficiency. In contrast, in newborn high- Foxf1(+/-) lungs, Notch-2 signaling was restored to the level found in wild-type mice, which was associated with normal microvascular formation and survival. Foxf1 haploinsufficiency disrupted pulmonary expression of genes in the Notch-2-signaling pathway and resulted in abnormal development of lung microvasculature.
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Loganathan, Sampath K., Krista Schleicher, Ahmad Malik, Rene Quevedo, Ellen Langille, Katie Teng, Robin H. Oh, et al. "Rare driver mutations in head and neck squamous cell carcinomas converge on NOTCH signaling." Science 367, no. 6483 (March 12, 2020): 1264–69. http://dx.doi.org/10.1126/science.aax0902.
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In most human cancers, only a few genes are mutated at high frequencies; most are mutated at low frequencies. The functional consequences of these recurrent but infrequent “long tail” mutations are often unknown. We focused on 484 long tail genes in head and neck squamous cell carcinoma (HNSCC) and used in vivo CRISPR to screen for genes that, upon mutation, trigger tumor development in mice. Of the 15 tumor-suppressor genes identified, ADAM10 and AJUBA suppressed HNSCC in a haploinsufficient manner by promoting NOTCH receptor signaling. ADAM10 and AJUBA mutations or monoallelic loss occur in 28% of human HNSCC cases and are mutually exclusive with NOTCH receptor mutations. Our results show that oncogenic mutations in 67% of human HNSCC cases converge onto the NOTCH signaling pathway, making NOTCH inactivation a hallmark of HNSCC.
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Wang, Jishi, Yingya Wu, Lila Mei, Yuan Yang, and Lu Shen. "Regulation of Differentiation and Proliferation of Marrow Hematogenesis Stem Cells by Notch1 Signaling System from Patients with Aplastic Anemia and Chronic Myelogenous Leukemia." Blood 108, no. 11 (November 16, 2006): 4215. http://dx.doi.org/10.1182/blood.v108.11.4215.4215.
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Abstract Notch genes encode evolutionarily conserved transmembrane receptors that regulate cell fate determination. Notch activation promotes proliferation and inhibits differentiation of bone marrow stem cells. Our research was to study the differential expression of Notch1 on bone marrow mononuclear cells (BMMNC) from chronic aplastic anemia (AA) and chronic myelogenous leukemia (CML) patients. We demonstrated that Notch signaling is inactivated in AA patients and activated in CML patients. In CML patients, Notch1 furthers stimulation by its ligand Jagged1, resulting in a strong increase of tumor cell growth. Therefore, we suggest that inactivated Notch signaling plays a pivotal role in the pathogenesis of AA and that activated Notch signaling plays a key role in the pathogenesis of CML. We used the antibody to inactivate Notch1 signaling in mice, and then to detect the hematopoietic state of these mice. Methods: 5ml marrow were abstracted from chronic AA patient s, CML patients in chronic phase and normal people and the BMMNC was separated from the marrow. RNA was abstracted from these cells. Real Time Quantitative PCR was used to investigate the expression of Notch1 on BMMNCs in 30 AA patients, 20 normal controls and 30 CML patients, and the changes of their expression after chemotherapy. Notch1 protein of these BMMNC was also detected through Western Blots. We injected monoclonal anti-Notch1 and isotonic Na chloride as controls into the tail vein of mice, to detect the hematopoietic state through bone marrow slides and flow cytometry. Results: Real Time PCR: Expression of Notch1 on BMMNCs from AA patients was lower than that of controls (P<0.05). The expression of Notch1 on BMMNCs from CML patients was higher than that of controls(P<0.05). Notch1 on the BMMNCs from AA patients was expressed higher after chemotherapy (P<0.05). Notch1 on the BMMNCs from CML patients was expressed lower after chemotherapy(P<0.05). Western Blots: Notch1 receptors were highly expressed in BMMNCs of CML patient. BMMNCs of AA patients expressed low amounts of Notch1. Jagged1 induces Notch signaling in cultured cells; We performed RT-PCR analysis of mRNA expression of Hes-1, a member of the hairy enhancer of split family of transcriptional repressors that are direct transcriptional targets of activated Notch. The result was that the expression of Hes-1 of CML was much higher than AA. The growth rate of CML BMMNCs was also much higher than AA BMMNCs. Compared with controls, the hematopoietic state of mice treated with anti-Notch1 had an obvious low hematopoietic ability. Conclusions: The expression of Notch1 on BMMNCs from AA patients and normal controls was different, which may account for the occurrence of AA. The expression of Notch1 of AA patients was improved after effective chemotherapy, which may be one of the reason of recovery of hematopoietic function. The expression of Notch1 on BMMNCs from CML patients was much higher than that of controls, which decreased after chemotherapy and may be one of the factors accounting for occurrence of CML. BMMNCs cocultured with Jagged-1 expressing cell lines can enhance the growth rate, which indicate that Notch1 can improve proliferation of leukemic cells. The mice treated with anti-Notch1 had lower hematopoietic ability manifesting that Notch1 signaling is essential to maintain normal hematopoietic function.
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Pinnell, Nancy, Ran Yan, Hyoje Cho, Paula Jeon, Theresa Keeley, Jordan McHugh, Yiran Liu, et al. "Direct Coregulation of Notch1 By Zmiz1 in T-Cell Development and Leukemia." Blood 124, no. 21 (December 6, 2014): 55. http://dx.doi.org/10.1182/blood.v124.21.55.55.
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Abstract The Notch1 receptor is required throughout normal T-cell development. NOTCH1 is also the most recurrently mutated oncogene in T-ALL, occurring in ~60% of human samples. However, Notch inhibitors cannot be used at full-strength because of intolerable on-target side effects, such as GI toxicity. Another concern is that these inhibitors would reverse the tumor suppressor functions of Notch. Lower doses of Notch inhibitors are better tolerated, but lead to residual Notch signaling. Moreover, collaborating pathways can reinforce these weak signals and drive resistance. Thus, it is important to identify the collaborative factors that selectively amplify the oncogenic functions of Notch1 as opposed to the physiological and tumor suppressor functions. We previously reported that the PIAS coactivator Zmiz1 and Notch1 alleles collaborated to induce T-ALL in mice. ZMIZ1 and activated NOTCH1 were co-expressed in 20-30% of patient samples and cell lines. To explore the significance of Zmiz1 in normal physiology and leukemia, we generated conditional Zmiz1 knockout mice. Global loss of Zmiz1 was previously shown to cause embryonic lethality. Thus, we used Mx-cre and pIpC injection to delete Zmiz1. Similar to Notch1 deletion, Zmiz1 deletion caused a cell autonomous five-fold loss of all T-cell subsets. Interestingly, RT-PCR of sorted Zmiz1-deficient T cells showed an unexpected, selective reduction of certain Notch targets such as Myc. In contrast to some mouse models of Notch deficiency, we did not observe myeloproliferative disease, effects on hematopoietic stem cells, or histological GI toxicity at the time points we examined. To determine if Zmiz1 was required for leukemia initiation, we transduced constitutively active Notch1 alleles into hematopoietic progenitors and transplanted them into recipient mice. All control mice developed circulating preleukemic cells with 20% progressing to T-ALL. In contrast, conditional deletion of Zmiz1 abolished preleukemic cells and T-ALL. We also deleted Zmiz1 in mice with established T-ALL. To date, 30% of control mice and none of the Zmiz1-deleted mice have succumbed to leukemia. To identify the mechanisms of Zmiz1 effects we first showed that the N-terminal domain (NTD) of Zmiz1 was important for enhancing Myc expression and Notch1 reporter activity. We solved the crystal structure of the NTD, which revealed tetratricopeptide repeats (TPR). To identify proteins that interact with the TPR, we performed mass spectrometry using the TPR as bait. Our top hit was Notch1. We showed that Zmiz1 and endogenous Notch1 complex proteins associated by co-IP and reciprocal co-IP. GST pulldown assays and NMR with purified recombinant proteins showed that the interaction was direct. Using NMR of 15N/13C-labelled proteins, we identified the interacting domains to be the TPR and the RAM domain of Notch1. ChIP-seq in a T-ALL cell line showed that Zmiz1 co-bound 75% of all Notch1 binding sites, including the newly discovered distal 3’ Myc enhancer. To explain the selectivity of Zmiz1, we performed motif analysis. Zmiz1 selectively homed to a subset of Notch1-regulated enhancers enriched for HLH, Tcf, Runx, and Ets motifs. Co-IP confirmed a Notch1-independent interaction between Zmiz1 and certain transcription factors binding these motifs. Zmiz1 and Notch1 recruited each other to the 3’ Myc enhancer and cooperatively induced H3K27 acetylation. RNA-seq showed that Zmiz1 selectively regulated 27% of all Notch1-regulated genes. Zmiz1 induced Myc more strongly than any other Notch target gene. Our data support a new model in which the Notch1 complex relies on Zmiz1 to amplify an important subset of its signals by direct regulation and recruitment to an even larger transcriptional complex. The selectivity of Zmiz1 for oncogenic signals of Notch1, in particular Myc, may be clinically relevant. Deletion of Zmiz1 inhibited T-cell development and leukemogenesis, but did not cause histological intestinal toxicity, weight loss, or myeloproliferative disease. Thus, targeting Zmiz1 may avoid some of the adverse consequences of Notch inhibitors while at the same time combating resistance and leukemic growth. We here report a new direct and selective regulator of Notch1, which has clear translational potential as manipulating Notch signals may treat conditions of T-cell deficiency, dysregulation, and cancer. Disclosures No relevant conflicts of interest to declare.
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Nichol, Donna, Carrie Shawber, Michael J. Fitch, Kathryn Bambino, Anshula Sharma, Jan Kitajewski, and Heidi Stuhlmann. "Impaired angiogenesis and altered Notch signaling in mice overexpressing endothelial Egfl7." Blood 116, no. 26 (December 23, 2010): 6133–43. http://dx.doi.org/10.1182/blood-2010-03-274860.
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Abstract Epidermal growth factor-like domain 7 (Egfl7) is important for regulating tubulogenesis in zebrafish, but its role in mammals remains unresolved. We show here that endothelial overexpression of Egfl7 in transgenic mice leads to partial lethality, hemorrhaging, and altered cardiac morphogenesis. These defects are accompanied by abnormal vascular patterning and remodeling in both the embryonic and postnatal vasculature. Egfl7 overexpression in the neonatal retina results in a hyperangiogenic response, and EGFL7 knockdown in human primary endothelial cells suppresses endothelial cell proliferation, sprouting, and migration. These phenotypes are reminiscent of Notch inhibition. In addition, our results show that EGFL7 and endothelial-specific NOTCH physically interact in vivo and strongly suggest that Egfl7 antagonizes Notch in both the postnatal retina and in primary endothelial cells. Specifically, Egfl7 inhibits Notch reporter activity and down-regulates the level of Notch target genes when overexpressed. In conclusion, we have uncovered a critical role for Egfl7 in vascular development and have shown that some of these functions are mediated through modulation of Notch signaling.
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Jundt, Franziska, Rudolf A. Rupec, Bernd Rebholz, Bernd Doerken, Irmgard Foerster, Ralf Huss, and Klaus Pfeffer. "The Notch Ligand Jagged1 Causes a Myeloproliferative Disorder in Mice Lacking IκBα." Blood 106, no. 11 (November 16, 2005): 1226. http://dx.doi.org/10.1182/blood.v106.11.1226.1226.
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Abstract Hematopoiesis occurs in the liver and the bone marrow during murine development. Newborn mice with a ubiquitous deletion of IκBα develop a severe hematological disorder characterized by an increase of granulocyte/erythroid/monocyte/macrophage colony-forming units (CFU-GEMM) and hypergranulopoiesis. Here, we provide evidence that this particular myeloproliferative disturbance is mediated by continuously deregulated perinatal expression of the Notch ligand Jagged1 in IκBα-deficient hepatocytes. Signaling through Notch-family cell surface receptors and their ligands has been shown to be involved in cell fate decisions of stem cells during hematopoietic/mesenchymal differentiation. However, the role of Notch signaling in myelopoiesis is still under discussion as results gained using different experimental conditions are contradictory. Due to embryonic lethality of Notch1- and Jagged1-deficient mice, alterations of myelopoiesis are difficult to be adressed. In this study, we investigated the function of IκBα and its role within the Jagged/Notch signaling pathway during myelopoiesis. Therefore, a novel mouse line with a conditional (floxed) allele of ikba was established. Ubiquitous deletion of IκBα after cross-breeding with Deleter-Cre mice results in hypergranulopoiesis comparable to the conventional deletion of the allele. A detailed analysis revealed a myeloproliferative syndrome with increased numbers of cycling progenitor cells. The morphological analysis of liver and bone marrow of IκBα-deficient mice showed hypercellularity. The cellular components were dominated by myeloid lineages and represented mostly granulocyts with dysplastic features, characterized by pseudo-Pelger-Huet formation. Myelodysplasia could also be detected in megakaryopoiesis by the presence of micromegakaryocytes. Alterations in erythropoiesis were detectable by condensed chromatin and an asychrony of the nucleocytoplasmic ratio in the red cell precursor population. Together, our results indicate that ubiquitous loss of IκBα results in hypergranulopoiesis progressing to a myelodysplastic syndrome. Systematic analysis of transcription factors, growth factor receptors and NF-κB-regulated cell-survival genes was performed to determine molecular mechanisms underlying hypergranulopoiesis. Our data suggested that Notch1-dependent signals were responsible for the myeloproliferative disorder as Notch1 was upregulated in neutrophils and the Notch ligand Jagged1 in non-hematopoietic cells, namly hepatocytes. Myeloproliferation could be inhibited by blocking the Notch1 ligand Jagged1. Interestingly, deletion of IκBα in neutrophils and macrophages or hematopoietic stem cells did not result in dysregulation of myelopoiesis despite constitutive NF-κB activation in these cells. This establishes the relevance of non-hematopoietic expression of Jagged1 for the control and regulation of myelopoiesis. In summary, we show that cell-fate decisions leading to a premalignant hematopoietic disorder can be initiated by non-hematopoietic cells with inactive IκBα.
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Al Jaam, Bilal, Katy Heu, Florian Pennarubia, Alexandre Segelle, Laetitia Magnol, Agnès Germot, Sébastien Legardinier, Véronique Blanquet, and Abderrahman Maftah. "Reduced Notch signalling leads to postnatal skeletal muscle hypertrophy in Pofut1 cax/cax mice." Open Biology 6, no. 9 (September 2016): 160211. http://dx.doi.org/10.1098/rsob.160211.
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Postnatal skeletal muscle growth results from the activation of satellite cells and/or an increase in protein synthesis. The Notch signalling pathway maintains satellite cells in a quiescent state, and once activated, sustains their proliferation and commitment towards differentiation. In mammals, POFUT1-mediated O -fucosylation regulates the interactions between NOTCH receptors and ligands of the DELTA/JAGGED family, thus initiating the activation of canonical Notch signalling. Here, we analysed the consequences of downregulated expression of the Pofut1 gene on postnatal muscle growth in mutant Pofut1 cax/cax (cax, compact axial skeleton) mice and differentiation of their satellite cell-derived myoblasts (SCDMs). Pofut1 cax/cax mice exhibited muscle hypertrophy, no hyperplasia and a decrease in satellite cell numbers compared with wild-type C3H mice. In agreement with these observations, Pofut1 cax/cax SCDMs differentiated earlier concomitant with reduced Pax7 expression and decrease in PAX7 + /MYOD − progenitor cells. In vitro binding assays showed a reduced interaction of DELTA-LIKE 1 ligand (DLL1) with NOTCH receptors expressed at the cell surface of SCDMs, leading to a decreased Notch signalling as seen by the quantification of cleaved NICD and Notch target genes. These results demonstrated that POFUT1-mediated O- fucosylation of NOTCH receptors regulates myogenic cell differentiation and affects postnatal muscle growth in mice.
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Crcareva, Aleksandra, Toshiki Saito, Keiki Kumano, Mamiko Sakata-Yanagimoto, Hisamaru Hirai, and Shigeu Chiba. "Notch2 Regulates Macrophage-Related Genes in Marginal Zone B Cells." Blood 104, no. 11 (November 16, 2004): 4197. http://dx.doi.org/10.1182/blood.v104.11.4197.4197.
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Abstract [Background] While Notch1 plays critical roles in early T cell development, Notch2 is indispensable for B cell development. Conditional inactivation of Notch2 in the hematopoietic compartment leads to loss of the marginal zone B (MZB) cells and defect in the particular fraction of follicular B (FOB) cells characterized as T2 B cells. Because of their position bordering the marginal sinuses and red pulp in the spleen, MZB cells are amongst the first cells that come in contact with blood-borne substances and thus thought to have critical roles in the defense against bacterial pathogens which are presumably scavenged in this anatomical site. Here we demonstrate that Notch2 regulates the expression of genes that specifically play a role in macrophages. [Methods] Splenic T2 B cells, supposed to contain precursors of MZB cells, were sorted from Notch2 conditional knockout (N2cKO) and wild type (WT) mouse and conveyed microarray analysis. Then, different fractions of B cells in different organs from N2cKO and WT mouse were sorted and the expression of CD36, whose upregulation by Notch2 signaling was implicated by the microarray, and CD14, which has a specific function in macrophages, was examined, using real time PCR and FACS analyses. [Results] Out of 20,000 genes that were covered by the microarray analysis, only 9 had reduced expression levels with N2cKO/WT ratio < 0.5 in quadricate experiments. Surprisingly, beside Deltex1 and Hes1 that are known to be direct Notch target genes, the majority of the genes had relation with macrophages, i.e., preferential expression or particular function. Among these macrophage-related genes, we further concentrated on CD36, being known to have scavenger function in endothelial cells and macrophages. We found that among different fractions of B cells from different organs in WT mice, CD36 was specifically expressed at higher levels in MZB and T2 B cells. We further found that the level of CD36 expression was reduced in MZB and T2 B cells in the Notch2 heterozygotes (and N2cKO mice in a gene dosage-dependent manner). Since these observations suggested that Notch2 signaling regulates the expression of macrophage-related genes in MZB cells, we examined whether Notch2 signaling also regulates the expression of CD14, a representative gene preferentially expressed in macrophages coding for the LPS/LBS receptor. In the WT mouse, we found that CD14 is expressed in MZB cells at a level comparable with that in macrophages, while we did not detect its expression in FOB cells when analyzed by FACS. We again observed decreased CD14 expression in MZB cells from Notch2 heterozygotes, and in T2 B cells from the Notch2 heterozygotes and cKO mice in a gene dosage-dependent manner. [Conclusions] MZB cells have been considered to play important roles at the border of the acquired and innate immunity. The data presented here support this paradigm, and further suggest that Notch2 signaling is crucial for the transcriptional regulation of genes being related to the innate immune function in MZB cells.
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Koizumi, K. i., M. Nakajima, S. Yuasa, Y. Saga, T. Sakai, T. Kuriyama, T. Shirasawa, and H. Koseki. "The role of presenilin 1 during somite segmentation." Development 128, no. 8 (April 15, 2001): 1391–402. http://dx.doi.org/10.1242/dev.128.8.1391.
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The Notch signalling pathway plays essential roles during the specification of the rostral and caudal somite halves and subsequent segmentation of the paraxial mesoderm. We have re-investigated the role of presenilin 1 (Ps1; encoded by Psen1) during segmentation using newly generated alleles of the Psen1 mutation. In Psen1-deficient mice, proteolytic activation of Notch1 was significantly affected and the expression of several genes involved in the Notch signalling pathway was altered, including Δ-like3, Hes5, lunatic fringe (Lfng) and Mesp2. Thus, Ps1-dependent activation of the Notch pathway is essential for caudal half somite development. We observed defects in Notch signalling in both the caudal and rostral region of the presomitic mesoderm. In the caudal presomitic mesoderm, Ps1 was involved in maintaining the amplitude of cyclic activation of the Notch pathway, as represented by significant reduction of Lfng expression in Psen1-deficient mice. In the rostral presomitic mesoderm, rapid downregulation of the Mesp2 expression in the presumptive caudal half somite depends on Ps1 and is a prerequisite for caudal somite half specification. Chimaera analysis between Psen1-deficient and wild-type cells revealed that condensation of the wild-type cells in the caudal half somite was concordant with the formation of segment boundaries, while mutant and wild-type cells intermingled in the presomitic mesoderm. This implies that periodic activation of the Notch pathway in the presomitic mesoderm is still latent to segregate the presumptive rostral and caudal somite. A transient episode of Mesp2 expression might be needed for Notch activation by Ps1 to confer rostral or caudal properties. In summary, we propose that Ps1 is involved in the functional manifestation of the segmentation clock in the presomitic mesoderm.
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Fragoso, Ana Rita, Tin Mao, Song Wang, Steven Schaffert, Hyeyoung Min, Warren S. Pear, and Chang-Zheng Chen. "Essential Role for Mir-181a1/b1 In T-Cell Acute Lymphoblastic Leukemia." Blood 116, no. 21 (November 19, 2010): 470. http://dx.doi.org/10.1182/blood.v116.21.470.470.
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Abstract Abstract 470 MiRNA-mediated gene regulation represents a fundamental layer of post-transcriptional control of gene expression with diverse functional roles in normal development and tumorigenesis. Whereas some studies have shown that over-expression of miRNA genes may contribute to cancer development and progression, it is yet to be rigorously tested by the loss-of-function genetic approaches whether miRNA genes are required for cancer development and maintenance in mice. Here we show that mir-181a1/b1 coordinates Notch and pre-TCR signals during normal thymocyte differentiation and plays an essential role in development and onset of T-cell acute lymphoblastic leukemia (T-ALL) induced by some Notch mutations. Using gain-of-function and loss-of-function approaches, we demonstrated that mir-181a1/b1 controls Notch and pre-TCR receptor signals during the early stages of T cell development in the thymus by repressing multiple negative regulators of both pathways, including Nrarp, PTPN-22, SHP2, DUSP5, and DUSP6. These results illustrate that a single miRNA can coordinate multiple signaling pathways by modulating the timing and strength of signaling at different stages. Intriguingly, synergistic signaling between Notch and pre-TCR pathways is necessary for the development of T-ALL, and miR-181 family miRNAs are aberrantly expressed in T-ALL patients. These observations raise the possibility that mir-181a1/b1 might contribute to the onset or maintenance of T-ALL by targeting similar pathways in tumor cells as it does in normal thymic progenitor cells. In support of this notion, we found that loss of mir-181a1/b1 significantly delayed the onset and development of T-ALL induced by intracellular domain of Notch1 (ICN1) and caused a 32% increase in the median survival time from 41 days to 54 days in T-ALL mice. Importantly, we noted that loss of mir-181a1/b1 more efficiently repressed the leukemogeneic potential of cells with lower levels of ICN1 expression, suggesting that mir-181a1/b1 may be more effective in inhibiting T-ALL development induced by a Notch mutant with weaker signal strength. Indeed, we demonstrated that loss of mir-181a1b1 essentially blocked T-ALL development induced by the weaker Notch mutant and dramatically decreased mortality from 60% to 10% in these T-ALL mice. Since human Notch mutations identified in T-ALL patients generally have weaker signaling strength and lower oncogenic potential than that of ICN1, our findings indicate that mir-181a1/b1 may play an essential role in development of normal thymic progenitors and Notch-induced T-ALL and may be targeted to treat T-ALL patients harboring Notch mutations. Disclosures: No relevant conflicts of interest to declare.
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Lee, Sung-UK, Manami Maeda, Nagisa Sakurai, Freddy Radtke, and Takahiro Maeda. "LRF Is Indispensable for Hematopoietic Stem Cell Function Via Blocking Notch1-Mediated T Cell-Instructive Signals in the Bone Marrow Niche." Blood 114, no. 22 (November 20, 2009): 81. http://dx.doi.org/10.1182/blood.v114.22.81.81.
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Abstract Abstract 81 Hematopoietic stem cells (HSC) have the ability to self-renew and give rise to all hematopoietic lineage cells. Understanding signals that regulate the balance between self-renewal and differentiation of HSCs is an important issue in stem cell biology as well as regenerative medicine. Notch signals are critical regulators of the lymphoid lineage fate, but their role in adult HSC function is currently under debate. We explored the role of the LRF (Leukemia/Lymphoma Related Factor), a Notch repressor (also known as Zbtb7a, pokemon, OCZF and FBI-1) in HSC function, as it plays key roles in embryonic development, oncogenesis, and hematopoiesis. Conditional inactivation of the LRF gene in mouse HSCs (LRFF/FMx1-Cre mice) led to the development of CD4/CD8 DP (double positive) T-cells at the expense of B-cell development in the bone marrow (BM) in a Notch-dependent manner. Absolute numbers of the most primitive HSCs (LT-HSCs), defined as CD150+CD48−Flt3−Vcam-1+IL7Rα−LSK (Lin−Sca1+c-Kit+), were significantly reduced, while lymphoid-biased multi-potential progenitors (LMPPs: CD150−CD48+Flt3+Vcam-1+/−IL7Rα−LSK) and common lymphoid progenitors (CLPs: Lin−CD150−CD48+Flt3+Vcam-1−IL7Rα+) were barely detectable in LRFF/FMx1-Cre mice one month after pIpC injection. Enhanced T cell development and concomitant loss of B cell development was also seen in LRF−/− fetal liver (FL). Lin−IL7Rα+c-Kit+PIR+ (Paired Immunoglobulin-like receptors) T cell precursors were significantly increased in LRF−/− FL, indicating that Notch-mediated aberrant lymphoid fate determination also takes place during fetal hematopoiesis. To address which Notch gene(s) are targeted by LRF, we studied the HSC/progenitor population of conditional LRF knockout (LRFF/FMx1-Cre) as well as LRF/Notch1 double conditional knockout mice (LRFF/FNotch1F/FMx1-Cre). In the absence of Notch1, normal B cell development was restored in LRFF/FMx1-Cre mice. Reduction of LT-HSCs in LRFF/FMx1-Cre resulted from high Notch1 activity, as loss of Notch1 rescued LT-HSC numbers, suggesting that LRF functions to maintain HSCs and normal lymphoid fate by blocking Notch1. HSCs in active cell cycle are sensitive to 5-fluoro-uracil (5-FU) treatment, which causes remaining dormant HSCs to be recruited into the cell cycle to rapidly produce new cells and to quickly re-establish the hematopoietic system. To examine the self-renewal capacity of LRF deficient LT-HSC, LRFF/FMx1-Cre mice were treated with 5-FU after pIpC injection and the recovery of LT-HSC numbers examined. While control LT-HSC numbers recovered to pretreatment levels 3 wk after 5-FU treatment, levels in LRFF/FMx1-Cre mice remained low, accompanied by DP T cell development in the BM. Furthermore, after 5-FU treatment, LT-HSC numbers of LRFF/FNotch1F/FMx1-Cre were compatible to those of control and LRFF/FMx1-Cre mice, indicating that lack of self-renewal capacity in LRF deficient LT-HSCs was due to excessive differentiation toward T cells caused by Notch1. In support of this idea, when mice were given 5-FU weekly as a challenge to assess their HSC function in vivo, the survival percentage in LRFF/FMx1-Cre mice was much lower than in controls (0% versus 50% in 1 month, P <0.0001) and that of LRFF/FNotch1F/FMx1-Cre mice was compatible to controls. Serial bone marrow transplant experiments further demonstrated functional defects of LRF deficient HSCs, as they failed to reconstitute the hematopoietic system in secondary recipients. Microarray analysis and subsequent Gene Set Enrichment Analysis demonstrated upregulation of genes that were enriched in progenitor compartments. Since LRF can act as a transcriptional repressor, mRNA levels of Notch receptors and Notch ligands were examined using the same data set. A Notch target gene Hes1, but not Notch1 itself, was upregulated, and increased levels of Hes1 was also confirmed by real-time q-PCR in FACS-sorted LT-HSCs, as well as in 10.5 d.p.c whole embryos. These data suggest that LRF does not transcriptionally regulate Notch1, as LRF loss led to Notch1 target gene activation at the LT-HSC level without affecting Notch1 mRNA. Our genetic studies clearly indicate that LRF is indispensable for the maintenance of the HSC pool by repressing T cell-instructive signals mediated by Notch1 in the BM niche. Our findings shed new light on the regulatory mechanisms underlying the balance between HSC self-renewal and differentiation. Disclosures: No relevant conflicts of interest to declare.
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Filipović, M., A. Šućur, D. Flegar, Z. Jajić, M. Ikić Matijašević, N. Lukač, N. Kovačić, et al. "POS0042 NOTCH 1 INHIBITION INCREASES OSTEOCLAST PROGENITOR ACTIVITY IN THE MOUSE MODEL OF RHEUMATOID ARTHRITIS." Annals of the Rheumatic Diseases 80, Suppl 1 (May 19, 2021): 226.3–226. http://dx.doi.org/10.1136/annrheumdis-2021-eular.2601.
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Background:Osteoclasts mediate periarticular and systemic bone loss in rheumatoid arthritis (RA). Osteoclast progenitor cells (OCPs) derived from the myeloid lineage are susceptible to regulation through Notch signaling. Murine bone marrow and splenic OCPs, identified as CD45+Ly6G-CD3-B220-NK1.1-CD11blo/+CD115+CCR2+ cells, are specifically increased in arthritis. We previously identified an increased frequency of OCPs expressing Notch receptors in arthritic mice.Objectives:Several studies suggested that Notch signaling modulation affects the course of experimental arthritis. We aimed to determine the effects of Notch receptor signaling inhibition on OCP activity and arthritis severity in murine collagen-induced arthritis (CIA).Methods:Male C57/Bl6 and DBA mice were immunized with chicken type II collagen and treated with i.p. injections of anti-Notch 1 neutralizing antibodies (1mg/kg). Notch receptor 1 through 4 expression on OCPs was analyzed by flow cytometry in periarticular bone marrow (PBM) and spleen (SPL). Gene expression of Notch receptors, ligands and transcription targets as well as osteoclast differentiation genes RANK, cFos and cFms was determined by qPCR from tissues and sorted OCPs. FACS sorted OCPs were stimulated by osteoclastogenic factors (M-CSF and RANKL), in control, IgG, Jagged (Jag)1 or Delta-like (DLL)1 coated wells, with or without anti-Notch 1 antibodies. Research was approved by the Ethics Committee.Results:We confirmed the expression of Notch receptors on OCPs by flow cytometry with Notch 1 and 2 being most abundantly expressed (around 25% and 40% positive OCPs in PBM and 35% and 20% in SPL respectively), with a significant increase of Notch 2 expression in arthritis. Seeding OCPs on DLL1 coated wells significantly increased while seeding on Jag1 coated wells significantly decreased osteoclastogenesis as reflected on the number of TRAP+ osteoclasts and expression of osteoclast differentiation genes. The addition of anti-Notch 1 antibodies to ligand-stimulated OCPs resulted in an increased number of TRAP+ osteoclasts, partially reversing Jag1 inhibition. In vivo treatment with anti-Notch 1 antibodies did not affect total OCP frequency, but increased expression of Notch 4 both in PBM and SPL as seen by flow cytometry and qPCR. Additionally, anti-Notch 1 treatment stimulated Notch transcription factors HES and HEY. Both PBM and SPL cultured OCPs from anti-Notch 1 treated mice produced a higher number of large TRAP+ osteoclasts, doubling the area covered with osteoclasts in the latter compared to untreated mice. Increased osteoclastogenesis in vitro was further confirmed by an increased expression of osteoclast differentiation genes in the treated group.Conclusion:Our results confirm that Notch signaling may represent an important therapeutic target for the regulation of osteoclast activity in arthritis. Both in vitro and in vivo anti-Notch 1 neutralizing antibodies enhanced osteoclastogenesis in CIA model, implying an inhibitory role of Notch 1 signaling in osteoclast differentiation. As Notch 2 expression is increased on OCPs of arthritic mice, we next plan to determine the effects of Notch 2 neutralization on osteoclast activity and arthritis severity.References:[1]Ikić Matijašević M, Flegar D, Kovačić N, Katavić V, Kelava T, Šućur A, et al. Increased chemotaxis and activity of circulatory myeloid progenitor cells may contribute to enhanced osteoclastogenesis and bone loss in the C57BL/6 mouse model of collagen-induced arthritis. Clin Exp Immunol. 2016;186(3):321–35.[2]Šućur A, Filipović M, Flegar D, Kelava T, Šisl D, Lukač N, et al. Notch receptors and ligands in inflammatory arthritis – a systematic review. Immunology Letters 2020 Vol. 223, p. 106–14.Acknowledgements:The work has been supported by Croatian Science Foundation projects IP-2018-01-2414, UIP-2017-05-1965 and DOK-2018-09-4276.Disclosure of Interests:None declared.
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Vollrath, Benedikt, Jeffrey Pudney, Sylvia Asa, Philip Leder, and Kevin Fitzgerald. "Isolation of a Murine Homologue of the Drosophila neuralized Gene, a Gene Required for Axonemal Integrity in Spermatozoa and Terminal Maturation of the Mammary Gland." Molecular and Cellular Biology 21, no. 21 (November 1, 2001): 7481–94. http://dx.doi.org/10.1128/mcb.21.21.7481-7494.2001.
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ABSTRACT The Drosophila neuralized gene shows genetic interactions with Notch, Enhancer of split, and other neurogenic genes and is thought to be involved in cell fate specification in the central nervous system and the mesoderm. In addition, a human homologue of the Drosophila neuralizedgene has been described as a potential tumor suppressor gene in malignant astrocytomas. We have isolated a murine homologue of theDrosophila and human Neuralized genes and, in an effort to understand its physiological function, derived mice with a targeted deletion of this gene. Surprisingly, mice homozygous for the introduced mutation do not show aberrant cell fate specifications in the central nervous system or in the developing mesoderm. This is in contrast to mice with targeted deletions in other vertebrate homologues of neurogenic genes such as Notch, Delta, andCbf-1. Male Neuralized null mice, however, are sterile due to a defect in axoneme organization in the spermatozoa that leads to highly compromised tail movement and sperm immotility. In addition, female Neuralized null animals are defective in the final stages of mammary gland maturation during pregnancy.
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Luo, Xiaodan, Pengfei Qin, Chunyan Wang, Zhenqian Huang, and Huo Tan. "Notch Is a Novel Critical Signaling Pathway Regulating Responses of T Cell and Antigen Presenting Cells in Multiple Murine aGVHD Models." Blood 126, no. 23 (December 3, 2015): 5418. http://dx.doi.org/10.1182/blood.v126.23.5418.5418.
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Abstract Introduction: Acute graft-versus-host disease (aGVHD) is a potentially life-threatening complication mediated by both host-derived antigen presenting cells (APCs) and donor T cells after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Despite prophylaxis and treatments, aGVHD stell occurs in many allo-HSCT patients. The role of Notch1 signal inhibition becomes more and more important in aGVHD study. This study is to investigate the role of Notch1 inhibition by γ-secretase inhibitor DAPT in murine aGVHD model. Methods: We established a C57BL/6 BALB/c murine aGVHD model. γ-secretase inhibitor-DAPT is used to inhibit Notch1 signal in vivo and in vitro before transplantation. The degree of clinical and histopathologic GVHD is assessed by aGVHD scores and body weight. The functions of host-derived APCs and donor T cells are analyzed by flow cytometry, ELISA and PCR. Results: All mice survived at least 14 days after transplantation and all of them developed aGVHD (n=20). The expression of Hes-1, as one of the target genes of Notch1 signal pathway, decreased significantly after DAPT inhibition. Body weight of mice in control groups decreased significantly compared to mice with Notch1 inhibition by DAPT after transplantation. Notch1 inhibited recipients produced markedly decreased amounts of the pro-inflammatory cytokines IFN-γ. The expressions of CD4 and Foxp3 increased while CD11c, CD80 and CD86 decreased after Notch1 inhibition. Conclusions: These results indicate that Notch is a novel critical signaling pathway regulating responses of T cell and antigen presenting cells in multiple murine aGVHD models. Notch signaling inhibition appears to limit the harmful effects of aGVHD. Disclosures No relevant conflicts of interest to declare.
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Schwarzer, Rolf, Julia Godau, Hermann Einsele, and Franziska Jundt. "The Notch Target Genes Hey1 and Hes7 Transcriptionally Suppress Gli1 Expression and Hedgehog Signaling in Hodgkin-Reed/Sternberg Cells of Classical Hodgkin Lymphoma: A Novel Mechanism of Drug Resistance." Blood 124, no. 21 (December 6, 2014): 275. http://dx.doi.org/10.1182/blood.v124.21.275.275.
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Abstract Tumor cell proliferation and survival of Hodgkin/Reed-Sternberg (HRS) cells are triggered through Jagged1 ligand-induced Notch1 signaling via homotypic and heterotypic cell-cell interactions in classical Hodgkin lymphoma. The developmental pathway Notch partly mediates its effects in HRS cells by stimulation of alternative NF-kB signaling. We further demonstrated that high-level expression of the essential Notch coactivator Mastermind-like 2 and downregulation of the Notch inhibitor Deltex1 contribute to aberrant activation of Notch signaling in HRS cells. Our data suggested that targeting the Notch pathway is a rational treatment strategy in classical Hodgkin lymphoma. In this study we analyzed Notch inhibition by use of the gamma secretase inhibitor GSI XII in a Hodgkin lymphoma xenotransplantation model. To this end the HRS cell line L540cy (1 x 107 cells/per mouse) was transplanted into NOD/SCID mice. After tumor growth (0.3 cm³ mean tumor volume) mice were treated daily with increasing doses of GSI XII (5-10 mg/kg). Surprisingly, L540cy cells were completely drug-resistant in vivo in contrast to high GSI XII sensitivity in vitro. To dissect potential mechanisms of drug resistance we performed human StellARrayTM quantitative polymerase chain reaction (qPCR) arrays to analyze Notch target genes in GSI XII-treated compared to untreated L540cy cells. Interestingly, inhibition of Notch activity resulted in strong mRNA upregulation of the transcription factor glioma-associated oncogene 1 (Gli1), a final effector of the developmental signaling pathway Hedgehog (HH). Chromatin immunoprecipitation (ChIP) further revealed that both negative regulatory Notch target proteins Hey1 and Hes7 directly bind three different N-boxes present in the GLI1 first intron to suppress GLI1 mRNA expression in untreated L540cy cells. In general, the HH pathway is activated through ligand binding of secreted Sonic Hedgehog (SHH). As a result Gli transcription factors translocate to the nucleus and induce target gene expression such as GLI1 or CCND1. Despite high secretion of SHH by HRS cells after two days in culture (conditioned medium), HH signaling was inactive in untreated L540cy cells. Only after release of the negative regulatory Notch targets of the hairy and enhancer of split (HES) family through Notch inhibition and concomitant increase of Gli1 expression, HH signaling was activated by SHH. HH signaling mediated drug resistance of L540cy cells in conditioned medium compared to fresh medium (SHH negative) and thereby compensated for reduced Notch activity in vitro. We hypothesized that this mechanism might contribute to GSI XII drug resistance in vivo. To proof our hypothesis we coinhibited the Notch and HH pathways in L540cy cells. As expected inhibition of the HH pathway alone by use of cyclopamine did not significantly reduce growth of L540cy cells. However, simultaneous targeting of L540cy tumors through GSI XII and cyclopamine efficiently controlled tumor cell growth. Our data indicate a first molecular link between Notch and HH in HRS cells mediating drug resistance. We suggest inhibition of both developmental pathways for effective HRS tumor growth control. Disclosures No relevant conflicts of interest to declare.
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Zhang, Honglai, Tao Xu, Claire Peabody, Ester Calvo Fernández, Rashmi Budhathoki, Philip Chi-En Huang, Elizabeth D. Hughes, et al. "CSIG-20. L3MBTL3 SUPPRESSES MEDULLOBLASTOMA TUMORIGENESIS THROUGH MODULATION OF THE NOTCH/RBPJ SIGNALING PATHWAY." Neuro-Oncology 22, Supplement_2 (November 2020): ii32. http://dx.doi.org/10.1093/neuonc/noaa215.132.
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Abstract The NOTCH/RBPJ pathway governs cell proliferation in many biological contexts, including SHH and Group#3medulloblastoma (MB) tumorigenesis. Using our proteomic platform, we discovered an interaction between RBPJ, a key co-factor of NOTCH for the modulation of the NOTCH/RBPJ signaling pathway, and L3MBTL3, a methyllysine reader. L3MBTL3 is recruited by RBPJ on chromatin at the enhancers of NOTCH/RBPJ target genes to repress their expression. Deletions of the L3MBTL3 locus are observed in patients with WNT and Group#3 MB and expression of L3MBTL3 in the SHH MB-derived cell DAOY inhibits cell growth, suggesting a putative tumor suppressor role for L3MBTL3 in MB. To further investigate the putative role of L3MBTL3 as a suppressor of MB tumorigenesis, we used our novel L3mbtl3 KO mouse in combination with a genetically engineered ND2:SmoA1 mouse model of SHH MB in a survival analysis. Furthermore, to identify the biological processes regulated by L3mbtl3 in MB, we analyzed by RNA-seq the transcriptome of L3mbtl3 KO mouse cerebella. Our survival analysis validated in vivo our hypothesis that L3mbtl3 is a tumor suppressor in this disease context. Indeed, our data show that [ND2:SmoA1; L3mbtl3+/-] mice have a significantly lower survival rate than ND2:SmoA1 mice (P = 0.032; Log-rank test). Moreover, our RNA-seq studies showed that L3MBTL3 regulates cell fate in the cerebellum via modulation the NOTCH/RBPJ signaling pathway. Hence, the RBPJ-L3MBTL3 interaction is at the heart of a molecular mechanism governing the repression of NOTCH/RBPJ target genes and malfunction of this molecular mechanism contributes to L3MBTL3’s tumor suppressor role in MB through aberrant “de-repression” of NOTCH/RBPJ target genes. Our discovery provides insights into the tumor suppressor role of the L3MBTL3 in MB that could be harnessed in the future for the therapeutic benefit of patients with MB.
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Sakata-Yanagimoto, Mamiko, Fumio Nakahara, Etsuko Yamaguchi-Nakagami, Keiki Kumano, Toshiki Saito, Mineo Kurokawa, Seishi Ogawa, and Shigeru Chiba. "Balance of Transcription Factors Downstream of Notch Signaling Determines the Fate of Myeloid Progenitors toward Differentiation to Mast Cells or Immortalization without Differentiation." Blood 108, no. 11 (November 16, 2006): 676. http://dx.doi.org/10.1182/blood.v108.11.676.676.
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Abstract Notch signaling represents one of the fundamental communication channels in various types of cells. While Notch activation has been shown to inhibit myeloid differentiation in a subset of hematopoietic progenitors, the role of Notch signaling in mast cell differentiation is not clear. When common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) purified from mouse bone marrow cells were stimulated with Delta1-Fc, a soluble form of Notch ligand, in the presence of stem cell factor, IL-3, IL-6, and thrombopoietin, granulocyte and macrophage differentiation, which is observed at day 7 of culture in the absence of Delta1-Fc, was markedly inhibited. Instead, Lin-c-Kit+FcεR+ mast cells dominated in the culture. Delta1-Fc did not increase mast cell generation from either CMPs or GMPs of the bone marrow of pI:pC-treated Mx-Cre x Notch2 flox/flox (N2-MxcKO) mice, in contrast to littermate Notch2 flox/flox mice treated with pI:pC, which suggests that Notch2 is responsible for the Delta1-Fc-augmented mast cell generation from CMPs and GMPs in culture. Retroviral transfer of constitutive active form of Notch2 (aN2) into CMPs and GMPs resulted in the complete loss of granulocyte-macrophage colony-forming cells and the emergence of basophilic granules-containing blast like cells, indicating the cell fate instruction. Real-time PCR analysis revealed that Delta1-Fc stimulation and aN2 introduction up-regulated the expression of Hes1, a transcriptional suppressor that is known to be a direct target of Notch activation in several cell types, within 12 h. Moreover, among GATA genes, Delta1-Fc stimulation and aN2 introduction resulted in increase of GATA3 mRNA, while expression levels of GATA1 and GATA2, which have been suggested to play a role in regulating mast cell differentiation, were unchanged. Next, we retrovirally expressed Hes1 and/or a GATA gene into CMPs and GMPs to see if the same effects were observed. Mast cells were increased only when both genes were expressed. On the other hand, when Hes1 alone was transduced, we observed rapid growth and immortalization of these cells without differentiation. C/EBPa, which is known to be suppressed in mast cell differentiation and upregulated in myeloid cell differentiation, was down-regulated within 48 h from the initiation of Hes1 retroviral transduction, suggesting that C/EBPa is a downstream target of Hes1 in this myeloid cell fate determination. Theses results indicate that, at the downstream of Notch activation, there are a C/EBPa down-regulation pathway that is Hes1-dependent and a GATA3 up-regulation pathway. Balanced regulation of these pathways should play a physiological role in myeloid and mast cell differentiation, while imbalance between these two pathways might provide a new model of myeloid transformation.
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del Toro, Raquel, Claudia Prahst, Thomas Mathivet, Geraldine Siegfried, Joshua S. Kaminker, Bruno Larrivee, Christiane Breant, et al. "Identification and functional analysis of endothelial tip cell–enriched genes." Blood 116, no. 19 (November 11, 2010): 4025–33. http://dx.doi.org/10.1182/blood-2010-02-270819.
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Abstract Sprouting of developing blood vessels is mediated by specialized motile endothelial cells localized at the tips of growing capillaries. Following behind the tip cells, endothelial stalk cells form the capillary lumen and proliferate. Expression of the Notch ligand Delta-like-4 (Dll4) in tip cells suppresses tip cell fate in neighboring stalk cells via Notch signaling. In DLL4+/− mouse mutants, most retinal endothelial cells display morphologic features of tip cells. We hypothesized that these mouse mutants could be used to isolate tip cells and so to determine their genetic repertoire. Using transcriptome analysis of retinal endothelial cells isolated from DLL4+/− and wild-type mice, we identified 3 clusters of tip cell–enriched genes, encoding extracellular matrix degrading enzymes, basement membrane components, and secreted molecules. Secreted molecules endothelial-specific molecule 1, angiopoietin 2, and apelin bind to cognate receptors on endothelial stalk cells. Knockout mice and zebrafish morpholino knockdown of apelin showed delayed angiogenesis and reduced proliferation of stalk cells expressing the apelin receptor APJ. Thus, tip cells may regulate angiogenesis via matrix remodeling, production of basement membrane, and release of secreted molecules, some of which regulate stalk cell behavior.
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Xu, Song, Jinsong Hu, Dehui Xu, Isabelle Vande Broek, Xavier Leleu, Ben Van Camp, Karin Vanderkerken, and Ivan Van Riet. "Impaired Osteogenic Differentiation of Mesenchymal Stem Cells Derived From Multiple Myeloma Patients Is Associated with a Failure In the Deactivation of the NOTCH Pathway." Blood 116, no. 21 (November 19, 2010): 1894. http://dx.doi.org/10.1182/blood.v116.21.1894.1894.
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Abstract Abstract 1894 Mesenchymal stem cells (MSCs) give rise to bone marrow (BM) stromal cells and play an essential role in the formation and function of the MM microenvironment. Some recent studies revealed that MSCs from myeloma patients (MM-hMSCs) show an enhanced spontaneous and myeloma cell-induced production of cytokines and a distinctive gene expression profile, when compared to MSCs from normal donors (ND-hMSCs). However, regarding the osteogenic differentiation ability of MM-hMSCs conflicting observations were reported. In this study, we observed that MM-hMSCs, especially for those from MM patients with bone lesions, exhibited in the presence of osteogenic differentiation (OD) medium, significantly decreased alkaline phosphatase (ALP) activity, reduced expression of specific osteogenic markers (OPN, BMP2, OTX and BSP) and impaired matrix mineralization, compared to ND-hMSCs. However, MGUS-hMSCs, did not show a significantly impaired osteogenesis ability. Primary CFU-ALP assay from BM samples of diseased mice in the 5T33MM model also confirmed that the osteogenic differentiation ability of MSCs was impaired. Previous reports indicated that MM cells can suppress MSCs osteogenesis by HGF and DKK1 as observed in vitro (Giuliani et al, Cancer Res. 2007; Standal et al, Blood. 2007). Since MM-hMSCs have been cultured in vitro for several weeks and without any stimulation of MM cells, we believe that the impaired osteogenic differentiation of MM-hMSCs was due to an intrinsic abnormality. Several reports suggested that NOTCH signalling can maintain bone marrow mesenchymal progenitors in a more undifferentiated state by suppressing osteoblast differentiation (Hilton et al, Nat Med. 2008; Zanotti et al, Endocrinology. 2008). Therefore, we postulate that impaired osteogenic ability of MM-hMSCs might be (at least partly) related to abnormal NOTCH activity during osteogenesis. We found by quantitative real time PCR that NOTCH1, NOTCH2, Dll-1, Jagged-1, and NOTCH pathway downstream genes hes1, hey1, hey2, heyL were considerably decreased in ND-hMSCs after shifting them from normal culture medium to OD medium, indicating that NOTCH signalling was gradually suppressed during MSC osteogenesis. However, it was observed that the expression of NOTCH1, Jagged-1, Hes1 and Hes5 in MM-hMSCs did not decrease to the level of ND-hMSC with statistical difference. This implicates that the NOTCH signaling pathway remains in MM-hMSCs over-activated even in the presence of osteogenesis inducing signals. When the NOTCH signalling inhibitor DAPT was added to MM-hMSCs in OD medium, we found that hes1 expression was suppressed while, RUNX2 expression, a key transcription factor for osteoblastogenesis, as well as ALP activity, osteogenic genes expression and mineralization deposition were all increased. In conclusion our data indicate that MM-hMSCs exhibit in vitro lower osteogenic differentiation ability compared to ND-hMSCs, and that this impairement is associated with an inappropriate NOTCH pathway deactivation during the osteogenesis process. Targeting hMSCs in vivo by NOTCH inhibitors might have therapeutical potential to control bone disease in MM patients. Disclosures: No relevant conflicts of interest to declare.
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Lee, Sung-UK, Manami Maeda, Nagisa Sakurai, Julie Teruya-Feldstein, Freddy Radtke, and Takahiro Maeda. "LRF Regulates Self-Renewal of Hematopoietic Stem Cells by Blocking Notch1-Mediated T Cell Differentiation." Blood 112, no. 11 (November 16, 2008): 75. http://dx.doi.org/10.1182/blood.v112.11.75.75.
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Abstract The proto-oncogene LRF, encoded by the Zbtb7a gene, is a transcriptional repressor that belongs to the POK (POZ/BTB and KrŸppel) protein family. Along with its oncogenic property, recent evidence has shown that POK proteins play distinct roles in hematopoiesis and immune system development. Conditional inactivation of the LRF gene in mouse hematopoietic stem cells (HSCs) results in the development of CD4/8 double positive (DP) T cells in bone marrow (BM) at the expense of B cell development (Maeda et al. Science 2007). While LRF acts as a master regulator of B versus T lymphoid lineage fate decision by suppressing Notch-mediated signals, it is unclear as to which Notch genes LRF targets and whether LRF is required for the maintenance of HSCs per se. To address these questions, we analyzed HSC/progenitor population of conditional LRF knockout mice (LRFF/FMx1-Cre) as well as LRF/Notch1 double conditional knockout mice (LRFF/FNotch1F/FMx1-Cre). In the absence of Notch1, LRF deficient HSCs/lymphoid progenitors (LRFF/FNotch1F/FMx1-Cre) could successfully give rise to early B cells (Pro B, Pre B and immature B). There were no abnormal DP-T cells seen in the BM, suggesting that LRF primarily targets Notch1 at the HSC/progenitor stages to maintain normal lymphoid development. However the loss of the LRF gene did not rescue the phenotype of Notch1F/FMx1-Cre mice (Radtke et al. Immunity 1999). Immature B cell development in the thymus was still observed in LRFF/FNotch1F/FMx1-Cre mice, suggesting that LRF acts genetically upstream of Notch1 during the early lymphocyte development. Notably, LRFF/FNotch1F/FMx1-Cre mice still exhibit a block of terminal erythroid differentiation and macrocytic anemia as seen in LRFF/FMx1-Cre mice. Thus, LRF is required for erythropoiesis via Notch-independent mechanisms. To further identify distinct HSC/progenitor compartments, we performed multicolor-FACS analysis utilizing antibodies for SLAM family members (CD41, CD48 and CD150), c-Kit, Sca-1, Flt3, IL7R-α, Vcam-1 and lineage markers (Lin). Remarkably, no Flt3 positive HSC/progenitors were observed in LRFF/FMx1-Cre mice. While IL7R-α+ T cell precursors (IL7Rα+Lin-Sca1+c-Kit+Flt3-), which were previously reported as common lymphoid progenitors (Maeda et al. Science 2007), existed abundantly. Absolute numbers of the long-term HSCs (LT-HSCs), defined as CD150+CD48-Flt3-Vcam-1+IL7Rα-LSK (Lin-Sca1+c-Kit+), were significantly reduced in LRFF/FMx1-Cre mice one month after pIpC injection. At the same time, CD150+CD48high+Flt3-Vcam-1-IL7Rα-LSK cells, which are likely T-committed lymphoid precursors, are increased in LRFF/FMx1-Cre mice. To investigate the presence of a population of quiescent HSC/progenitors, we treated LRFF/FMx1-Cre mice with 5-fluorouracil (5-FU), a S phase-specific cytotoxic chemotherapeutic agent, and examined recovery of HSCs in BM. LT-HSCs in LRFF/FMx1-Cre mice did not repopulate as many as their counterpart one month after 5-FU treatment. Our data indicates that LRF deficient HSCs are unable to maintain its quiescent status and are on the state of cell differentiation toward T cells due to the high Notch activity. In fact, loss of the Notch1 gene partially rescued reduced LT-HSCs numbers seen in LRFF/FMx1-Cre mice.
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Svensson, Per, Ingela Bergqvist, Stefan Norlin, and Helena Edlund. "MFng Is Dispensable for Mouse Pancreas Development and Function." Molecular and Cellular Biology 29, no. 8 (February 17, 2009): 2129–38. http://dx.doi.org/10.1128/mcb.01644-08.
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ABSTRACT Notch signaling regulates pancreatic cell differentiation, and mutations of various Notch signaling components result in perturbed pancreas development. Members of the Fringe family of β1,3-N-acetylglucosaminyltransferases, Manic Fringe (MFng), Lunatic Fringe (LFng), and Radical Fringe (RFng), modulate Notch signaling, and MFng has been suggested to regulate pancreatic endocrine cell differentiation. We have characterized the expression of the three mouse Fringe genes in the developing mouse pancreas between embryonic days 9 and 14 and show that the expression of MFng colocalized with the proendocrine transcription factor Ngn3. In contrast, the expression of LFng colocalized with the exocrine marker Ptf1a, whereas RFng was not expressed. Moreover, we show that expression of MFng is lost in Ngn3 mutant mice, providing evidence that MFng is genetically downstream of Ngn3. Gain- and loss-of-function analyses of MFng by the generation of mice that overexpress MFng in early pancreatic progenitor cells and mice with a targeted deletion of MFng provide, however, evidence that MFng is dispensable for pancreas development and function, since no pancreatic defects in these mice were observed.
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Zubeldía-Brenner, Lautaro, Catalina De Winne, Sofía Perrone, Santiago A. Rodríguez-Seguí, Christophe Willems, Ana María Ornstein, Isabel Lacau-Mengido, Hugo Vankelecom, Carolina Cristina, and Damasia Becu-Villalobos. "Inhibition of Notch signaling attenuates pituitary adenoma growth in Nude mice." Endocrine-Related Cancer 26, no. 1 (January 2019): 13–29. http://dx.doi.org/10.1530/erc-18-0337.
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Preclinical and clinical studies support that Notch signaling may play an important oncogenic role in cancer, but there is scarce information for pituitary tumors. We therefore undertook a functional study to evaluate Notch participation in pituitary adenoma growth. Tumors generated in Nude mice by subcutaneous GH3 somatolactotrope cell injection were treated in vivo with DAPT, a γ-secretase inhibitor, thus inactivating Notch signaling. This treatment led to pituitary tumor reduction, lower prolactin and GH tumor content and a decrease in angiogenesis. Furthermore, in silico transcriptomic and epigenomic analyses uncovered several tumor suppressor genes related to Notch signaling in pituitary tissue, namely Btg2, Nr4a1, Men1, Zfp36 and Cnot1. Gene evaluation suggested that Btg2, Nr4a1 and Cnot1 may be possible players in GH3 xenograft growth. Btg2 mRNA expression was lower in GH3 tumors compared to the parental line, and DAPT increased its expression levels in the tumor in parallel with the inhibition of its volume. Cnot1 mRNA levels were also increased in the pituitary xenografts by DAPT treatment. And the Nr4a1 gene was lower in tumors compared to the parental line, though not modified by DAPT. Finally, because DAPT in vivo may also be acting on tumor microenvironment, we determined the direct effect of DAPT on GH3 cells in vitro. We found that DAPT decreases the proliferative, secretory and migration potential of GH3 cells. These results position selective interruption of Notch signaling as a potential therapeutic tool in adjuvant treatments for aggressive or resistant pituitary tumors.
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Chen, Linghong, and Qais Al-Awqati. "Segmental expression of Notch and Hairy genes in nephrogenesis." American Journal of Physiology-Renal Physiology 288, no. 5 (May 2005): F939—F952. http://dx.doi.org/10.1152/ajprenal.00369.2004.
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Notch signaling pathway genes are required for nephrogenesis, raising the possibility that Notch effector Hairy-related genes should also control nephron formation. We performed in situ hybridization of Hairy transcription factors with segment-specific lectins and/or antibodies during early nephrogenesis to identify their possible roles in segment identity of the nephron. We found that among all of Notch downstream Hairy genes, only Hes1, Hes5, Hey1, and HeyL were expressed in a segment-specific manner in early nephrons and their expression pattern changed dynamically during metanephric development. Based on these patterns of expression, it was possible to propose a pairwise association of specific ligand and receptor and to suggest that the effector of this association is one of the Hairy transcription factors. We found that Hes5 is specifically expressed in the anlage of the loop of Henle, suggesting that it might be involved in the determination of its cell identity. We also examined the morphological appearance of kidneys from mice where the Hes1 or Hes5 genes were deleted and found that at least at the gross morphological level, there was little difference from wild-type kidneys. Because Hairy genes associate with other transcription factors to exert their effect, it is necessary to examine a more complete array of genetic deletions before a conclusion can be reached regarding their role in kidney development. These studies provide the basis for the future development of strategies to examine the role of individual effector molecules in the determination of the differentiation pattern of the nephron.
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Anderson, Leah J., and Richard Longnecker. "Epstein-Barr virus latent membrane protein 2A exploits Notch1 to alter B-cell identity in vivo." Blood 113, no. 1 (January 1, 2009): 108–16. http://dx.doi.org/10.1182/blood-2008-06-160937.
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Abstract Expression of latent membrane protein 2 (LMP2A) during B-cell development leads to global alterations in gene transcription similar to those seen in Hodgkin Reed-Sternberg cells of Hodgkin lymphoma (HL). Along with the consistent detection of LMP2A in Epstein-Barr virus–associated HL, this implicates a role for LMP2A in the pathogenesis of HL. We have shown that LMP2A constitutively activates the Notch1 pathway to autoregulate the LMP2A promoter. To determine whether constitutive activation of the Notch pathway is important for LMP2A-mediated alterations in B-cell development in vivo, TgE-LMP2A–transgenic mice were intercrossed with mice expressing loxP-flanked Notch1 genes and Cre recombinase. B cells from TgE Notch1lox/lox-CD19+/Cre mice have an increase in immunoglobulin M and CD43 and a decrease in CD5 expression in the bone marrow compared with TgE Notch1lox/lox mice, indicating the LMP2A signal for developmental aberrations is impaired in the absence of Notch1. Real-time reverse-transcribed polymerase chain reaction analysis reveals that LMP2A requires the Notch1 pathway to alter levels of B cell–specific transcription factors, E2A and EBF. Interestingly, Notch1 appears to be important for LMP2A-mediated survival in low interleukin-7. We propose that LMP2A and the Notch1 pathway may cooperate to induce the alterations in B-cell identity seen in Hodgkin Reed-Sternberg cells.
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García-Domínguez, Daniel J., Dominique Morello, Elsa Cisneros, Dimitris L. Kontoyiannis, and José M. Frade. "Stabilization of Dll1 mRNA by Elavl1/HuR in neuroepithelial cells undergoing mitosis." Molecular Biology of the Cell 22, no. 8 (April 15, 2011): 1227–39. http://dx.doi.org/10.1091/mbc.e10-10-0808.
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In the vertebrate neuroepithelium, the decision to differentiate is made by neural precursors soon after mitosis, when they are apically located. This process is controlled by lateral inhibitory signals triggered by the Delta/Notch pathway. During mitosis, the capacity of neural precursors to express the neurogenic genes Dll1 and Notch1 is maximal due to mRNA stabilization, but the mechanism controlling this process remains unknown. Here we show that Elav-like (Elavl1)/HuR becomes enriched in the cytoplasm of neuroepithelial cells undergoing mitosis and that this ribonucleoprotein interacts with the 3′ untranslated region (UTR) of Dll1 mRNA. This interaction is functionally relevant because RNAi against Elavl1 reduces the stability of Dll1–3′UTR-containing transcripts in mitosis-arrested neuroepithelial cells, and Elavl1 null-mutant heterozygous mice show decreased Dll1 expression in the developing brain in vivo. We also show that RNAi against Elavl1 diminishes the capacity of brain precursors to trigger lateral inhibitory signals to their neighbors, an observation consistent with the increase in the rate of neurogenesis which can be detected in vivo in the developing retina of Elavl1 heterozygous mice. We conclude that Elavl1/HuR facilitates the exposure of vertebrate neuronal precursors to apically located Delta/Notch signals.
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Glomski, Krzysztof, Sébastien Monette, Katia Manova, Bart De Strooper, Paul Saftig, and Carl P. Blobel. "Deletion of Adam10 in endothelial cells leads to defects in organ-specific vascular structures." Blood 118, no. 4 (July 28, 2011): 1163–74. http://dx.doi.org/10.1182/blood-2011-04-348557.
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Abstract During vertebrate angiogenesis, Notch regulates the cell-fate decision between vascular tip cells versus stalk cells. Canonical Notch signaling depends on sequential proteolytic events, whereby interaction of Notch with membrane-anchored ligands triggers proteolytic processing, first by Adam10 and then presenilins. This liberates the Notch intracellular domain, allowing it to enter the nucleus and activate Notch-dependent genes. Here we report that conditional inactivation of Adam10 in endothelial cells (A10ΔEC) recapitulates the increased branching and density of the retinal vasculature that is also caused by interfering with Notch signaling. Moreover, A10ΔEC mice have additional vascular abnormalities, including aberrant subcapsular hepatic veins, enlarged glomeruli, intestinal polyps containing endothelial cell masses, abnormal endochondral ossification, leading to stunted long bone growth and increased pathologic neovascularization following oxygen-induced retinopathy. Our findings support a model in which Adam10 is a crucial regulator of endothelial cell-fate decisions, most likely because of its essential role in canonical Notch signaling.
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Rual, J. "OS12.3 L3MBTL3 is a novel suppressor of medulloblastoma tumorigenesis." Neuro-Oncology 21, Supplement_3 (August 2019): iii22—iii23. http://dx.doi.org/10.1093/neuonc/noz126.075.
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Abstract BACKGROUND Medulloblastoma is the most common malignant brain tumor of childhood. Therapeutic approaches to medulloblastoma have led to significant improvements but are achieved at a high cost to quality of life. Alternative therapeutic approaches are needed and molecular stratification of patients with medulloblastoma has yet to be routinely implemented in the clinic. TheNotchpathway governs cell proliferation in many biological contexts, including SHHand Group#3medulloblastoma tumorigenesis. Using our proteomic platform, we discovered an interaction between RBPJ, a key co-factor of Notch for the modulation of Notch signals, and L3MBTL3, a methyllysine reader.L3MBTL3 is recruited by RBPJ on chromatin at the enhancers of Notch/RBPJ target genes to repress their expression.Deletions ofthe L3MBTL3locus are observed in patientswith WNTand Group#3medulloblastomaand expression of L3MBTL3 in the SHHmedulloblastoma-derived cell DAOY inhibits cell growth, suggesting a putative tumor suppressor role for L3MBTL3 in medulloblastoma. METHODS To further investigate the putative role of L3MBTL3 as a suppressor of medulloblastoma tumorigenesis, we used our novel L3mbtl3KO mouse in combination with a genetically engineered ND2:SmoA1mouse model of SHHmedulloblastoma in a survival analysis. RESULTS Our survival analysis validated in vivoour hypothesis that L3mbtl3is a tumor suppressor in this disease context. Indeed, our data show that [ND2:SmoA1; L3mbtl3+/-] mice have a significantly lower survival rate than ND2:SmoA1 mice (P= 0.0322; Log-rank test). Hence, the RBPJ-L3MBTL3 interaction is at the heart of a molecular mechanism governing the repression of Notch/RBPJ target genes and malfunction of this molecular mechanism contributes to L3MBTL3’s tumor suppressor role in medulloblastomathrough aberrant “de-repression” of Notch/RBPJ target genes. CONCLUSION L3MBTL3 is a novel suppressor of medulloblastoma tumorigenesis. Our discovery providesinsights into the role of the L3MBTL3 inmedulloblastomathat could be harnessed in the future for the therapeutic benefit of patientswith medulloblastoma.
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Feldman, Brian J., Tracy Hampton, and Michael L. Cleary. "A carboxy-terminal deletion mutant of Notch1accelerates lymphoid oncogenesis in E2A-PBX1transgenic mice." Blood 96, no. 5 (September 1, 2000): 1906–13. http://dx.doi.org/10.1182/blood.v96.5.1906.
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Abstract PBX1 is a proto-oncogene that plays important roles in pattern formation during development. It was discovered as a fusion with the E2A gene after chromosomal translocations in a subset of acute leukemias. The resulting E2a-Pbx1 chimeric proteins display potent oncogenic properties that appear to require dimerization with Hox DNA binding partners. To define molecular pathways that may be impacted by E2a-Pbx1, a genetic screen consisting of neonatal retroviral infection was used to identify genes that accelerate development of T-cell tumors in E2A-PBX1 transgenic mice. Retroviral insertions in the Notch1 gene were observed in 88% of tumors arising with a shortened latency. Among these, approximately half created a NotchIC allele, encoding the intracellular, signaling portion of Notch1, suggesting a synergistic interaction between the Notch and E2a-Pbx1 pathways in oncogenesis. The remaining proviral insertions involvingNotch1 occurred in a more 3′ exon, resulting in truncating mutations that deleted the carboxy-terminal region ofNotch1 containing negative regulatory sequences (Notch1ΔC). In contrast toNotchIC, forced expression ofNotch1ΔC in transgenic mice did not perturb thymocyte growth or differentiation. However, mice transgenic for both the E2A-PBX1 and Notch1ΔC genes displayed a substantially shortened latency for tumor development compared with E2A-PBX1 single transgenic mice. These studies reveal a novel mechanism for oncogenic activation ofNotch1 and demonstrate a collaborative relationship between 2 cellular oncogenes that also contribute to cell fate determination during embryonic development.
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Feldman, Brian J., Tracy Hampton, and Michael L. Cleary. "A carboxy-terminal deletion mutant of Notch1accelerates lymphoid oncogenesis in E2A-PBX1transgenic mice." Blood 96, no. 5 (September 1, 2000): 1906–13. http://dx.doi.org/10.1182/blood.v96.5.1906.h8001906_1906_1913.
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PBX1 is a proto-oncogene that plays important roles in pattern formation during development. It was discovered as a fusion with the E2A gene after chromosomal translocations in a subset of acute leukemias. The resulting E2a-Pbx1 chimeric proteins display potent oncogenic properties that appear to require dimerization with Hox DNA binding partners. To define molecular pathways that may be impacted by E2a-Pbx1, a genetic screen consisting of neonatal retroviral infection was used to identify genes that accelerate development of T-cell tumors in E2A-PBX1 transgenic mice. Retroviral insertions in the Notch1 gene were observed in 88% of tumors arising with a shortened latency. Among these, approximately half created a NotchIC allele, encoding the intracellular, signaling portion of Notch1, suggesting a synergistic interaction between the Notch and E2a-Pbx1 pathways in oncogenesis. The remaining proviral insertions involvingNotch1 occurred in a more 3′ exon, resulting in truncating mutations that deleted the carboxy-terminal region ofNotch1 containing negative regulatory sequences (Notch1ΔC). In contrast toNotchIC, forced expression ofNotch1ΔC in transgenic mice did not perturb thymocyte growth or differentiation. However, mice transgenic for both the E2A-PBX1 and Notch1ΔC genes displayed a substantially shortened latency for tumor development compared with E2A-PBX1 single transgenic mice. These studies reveal a novel mechanism for oncogenic activation ofNotch1 and demonstrate a collaborative relationship between 2 cellular oncogenes that also contribute to cell fate determination during embryonic development.