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1.  Notch Signaling Specifies Megakaryocyte Development from Hematopoietic Stem Cells 
Cell stem cell  2008;3(3):314-326.
SUMMARY
In the hematopoietic system, Notch signaling specifies T cell lineage fate, in part through negative regulation of B cell and myeloid lineage development. However, we unexpectedly observed the development of megakaryocytes when using heterotypic cocultures of hematopoietic stem cells with OP9 cells expressing Delta-like1, but not with parental OP9 cells. This effect was abrogated by inhibition of Notch signaling either with γ-secretase inhibitors or by expression of the dominant-negative Master-mind-like1. The importance of Notch signaling for megakaryopoietic development in vivo was confirmed by using mutant alleles that either activate or inhibit Notch signaling. These findings indicate that Notch is a positive regulator of megakaryopoiesis and plays a more complex role in cell-fate decisions among myeloid progenitors than previously appreciated.
doi:10.1016/j.stem.2008.07.010
PMCID: PMC3970322  PMID: 18786418
2.  Complementary Genomic Screens Identify SERCA as a Therapeutic Target in NOTCH1 Mutated Cancer 
Cancer cell  2013;23(3):390-405.
SUMMARY
Notch1 is a rational therapeutic target in several human cancers, but as a transcriptional regulator, it poses a drug discovery challenge. To identify Notch1 modulators, we performed two cell-based, high-throughput screens for small-molecule inhibitors and cDNA enhancers of a NOTCH1 allele bearing a leukemia-associated mutation. SERCA calcium channels emerged at the intersection of these complementary screens. SERCA inhibition preferentially impairs the maturation and activity of mutated Notch1 receptors and induces a G0/G1 arrest in NOTCH1-mutated human leukemia cells. A small-molecule SERCA inhibitor has on-target activity in two mouse models of human leukemia and interferes with Notch signaling in Drosophila. These studies “credential” SERCA as a therapeutic target in cancers associated with NOTCH1 mutations.
doi:10.1016/j.ccr.2013.01.015
PMCID: PMC3709972  PMID: 23434461
3.  Canonical Notch signaling is dispensable for the maintenance of adult hematopoietic stem cells 
Cell stem cell  2008;2(4):356-366.
Summary
Gain-of-function experiments have demonstrated the potential of Notch signals to expand primitive hematopoietic progenitors, but whether Notch physiologically regulates hematopoietic stem cell (HSC) homeostasis in vivo is unclear. To answer this question, we evaluated the effect of global deficiencies of canonical Notch signaling in rigorous HSC assays. Hematopoietic progenitors expressing dominant negative Mastermind-like1 (DNMAML), a potent inhibitor of Notch-mediated transcriptional activation, achieved stable long-term reconstitution of irradiated hosts and showed a normal frequency of progenitor fractions enriched for long-term HSCs. Similar results were observed with cells lacking CSL/RBPJ, a DNA-binding factor that is required for canonical Notch signaling. Notch-deprived progenitors provided normal long-term reconstitution after secondary competitive transplantation. Furthermore, Notch target genes were expressed at low levels in primitive hematopoietic progenitors. Taken together, these results rule out an essential physiological role for cell-autonomous canonical Notch signals in HSC maintenance.
doi:10.1016/j.stem.2008.02.011
PMCID: PMC3717373  PMID: 18397755
4.  Modulating the Strength and Threshold of NOTCH Oncogenic Signals by mir-181a-1/b-1 
PLoS Genetics  2012;8(8):e1002855.
Oncogenes, which are essential for tumor initiation, development, and maintenance, are valuable targets for cancer therapy. However, it remains a challenge to effectively inhibit oncogene activity by targeting their downstream pathways without causing significant toxicity to normal tissues. Here we show that deletion of mir-181a-1/b-1 expression inhibits the development of Notch1 oncogene-induced T cell acute lymphoblastic leukemia (T-ALL). mir-181a-1/b-1 controls the strength and threshold of Notch activity in tumorigenesis in part by dampening multiple negative feedback regulators downstream of NOTCH and pre-T cell receptor (TCR) signaling pathways. Importantly, although Notch oncogenes utilize normal thymic progenitor cell genetic programs for tumor transformation, comparative analyses of mir-181a-1/b-1 function in normal thymocyte and tumor development demonstrate that mir-181a-1/b-1 can be specifically targeted to inhibit tumor development with little toxicity to normal development. Finally, we demonstrate that mir-181a-1/b-1, but not mir-181a-2b-2 and mir-181-c/d, controls the development of normal thymic T cells and leukemia cells. Together, these results illustrate that NOTCH oncogene activity in tumor development can be selectively inhibited by targeting the molecular networks controlled by mir-181a-1/b-1.
Author Summary
Oncogenes elicit driving signals required for tumor initiation, development, and maintenance and are valuable targets for cancer therapy. However, oncogenes often have essential functions in normal cellular physiology and produce intracellular proteins that are difficult to inhibit with small molecule drugs without causing significant toxicity to normal tissues. Thus, one of the challenges in cancer therapy is to identify downstream networks that can be targeted to specifically dampen the oncogenic signals in tumor cells without harming normal tissues. In this study we demonstrate that deletion of a microRNA (miRNA) gene, mir-181a-1/b-1, specifically inhibits the activity of the Notch oncogene in tumorigenesis without causing significant defects in normal development. Although earlier studies have elegantly shown the essential role of NOTCH and pre-TCR signals in NOTCH-induced tumorigenesis, neither NOTCH nor pre-TCR signals can be targeted effectively for treatment of T-ALL with available drugs due to their weak therapeutic effects and severe toxicities. Our findings illustrate that dissecting the downstream targets of miRNAs can reveal the molecular networks that can be targeted to control tumor transformation caused by oncogenes. More importantly, our results illustrate that comparative studies on the pathways utilized by normal cells and tumor cells may reveal novel insights into how tumorigenic pathways may be selectively inhibited with limited damage to normal tissues.
doi:10.1371/journal.pgen.1002855
PMCID: PMC3415433  PMID: 22916024
5.  Increased Expression of PcG Protein YY1 Negatively Regulates B Cell Development while Allowing Accumulation of Myeloid Cells and LT-HSC Cells 
PLoS ONE  2012;7(1):e30656.
Ying Yang 1 (YY1) is a multifunctional Polycomb Group (PcG) transcription factor that binds to multiple enhancer binding sites in the immunoglobulin (Ig) loci and plays vital roles in early B cell development. PcG proteins have important functions in hematopoietic stem cell renewal and YY1 is the only mammalian PcG protein with DNA binding specificity. Conditional knock-out of YY1 in the mouse B cell lineage results in arrest at the pro-B cell stage, and dosage effects have been observed at various YY1 expression levels. To investigate the impact of elevated YY1 expression on hematopoetic development, we utilized a mouse in vivo bone marrow reconstitution system. We found that mouse bone marrow cells expressing elevated levels of YY1 exhibited a selective disadvantage as they progressed from hematopoietic stem/progenitor cells to pro-B, pre-B, immature B and re-circulating B cell stages, but no disadvantage of YY1 over-expression was observed in myeloid lineage cells. Furthermore, mouse bone marrow cells expressing elevated levels of YY1 displayed enrichment for cells with surface markers characteristic of long-term hematopoietic stem cells (HSC). YY1 expression induced apoptosis in mouse B cell lines in vitro, and resulted in down-regulated expression of anti-apoptotic genes Bcl-xl and NFκB2, while no impact was observed in a mouse myeloid line. B cell apoptosis and LT-HSC enrichment induced by YY1 suggest that novel strategies to induce YY1 expression could have beneficial effects in the treatment of B lineage malignancies while preserving normal HSCs.
doi:10.1371/journal.pone.0030656
PMCID: PMC3264595  PMID: 22292011
6.  Notch signalling in T cell lymphoblastic leukaemia/lymphoma and other haematological malignancies 
The Journal of pathology  2010;223(2):262-273.
Notch receptors participate in a highly conserved signalling pathway that regulates normal development and tissue homeostasis in a context- and dose-dependent manner. Deregulated Notch signalling has been implicated in many diseases, but the clearest example of a pathogenic role is found in T cell lymphoblastic leukaemia/lymphoma (T-LL), in which the majority of human and murine tumours have acquired mutations that lead to aberrant increases in Notch1 signalling. Remarkably, it appears that the selective pressure for Notch mutations is virtually unique among cancers to T-LL, presumably reflecting a special context-dependent role for Notch in normal T cell progenitors. Nevertheless, there are some recent reports suggesting that Notch signalling has subtle yet important roles in other forms of hematologic malignancy as well. Here, we review the role of Notch signalling in various blood cancers, focusing on T-LL with an eye toward targeted therapeutics.
doi:10.1002/path.2789
PMCID: PMC2996483  PMID: 20967796
Notch signaling; oncogene; T-cell lymphoblastic leukemia/lymphoma; chromosomal rearrangements; targeted therapy
7.  NOTCH1 and NOTCH3 coordinate esophageal squamous differentiation through a CSL-dependent transcriptional network 
Gastroenterology  2010;139(6):2113-2123.
Background & Aims
The Notch receptor family regulates cell fate through cell-cell communication. CSL (CBF-1/RBP-jκ, Su(H), Lag-1) drives canonical Notch-mediated gene transcription during cell lineage specification, differentiation and proliferation in the hematopoietic system, the intestine, the pancreas and the skin. However, the functional roles of Notch in esophageal squamous epithelial biology remain unknown.
Methods
Normal esophageal keratinocytes were stimulated with calcium chloride to induce terminal differentiation. The squamous epithelia were reconstituted in organotypic three-dimensional culture, a form of human tissue engineering. Notch was inhibited in culture with a γ-secretase inhibitor or dominant negative mastermind-like1 (DNMAML1). The roles of Notch receptors were evaluated by in vitro gain-of-function and loss-of-function experiments. Additionally, DNMAML1 was targeted to the mouse esophagus by cytokeratin K14 promoter-driven Cre (K14Cre) recombination of Lox-STOP-Lox-DNMAML1. Notch-regulated gene expression was determined by reporter transfection, chromatin immunoprecipitation (ChIP) assays, quantitative reverse-transcription polymerase chain reactions (RT-PCR), Western blotting, immunofluorescence and immunohistochemistry.
Results
NOTCH1 (N1) was activated at the onset of squamous differentiation in the esophagus. Intracellular domain of N1 (ICN1) directly activated NOTCH3 (N3) transcription, inducing HES5 and early differentiation markers such as involucrin (IVL) and cytokeratin CK13 in a CSL-dependent fashion. N3 enhanced ICN1 activity and was required for squamous differentiation. Loss of Notch signaling in K14Cre;DNMAML1 mice perturbed esophageal squamous differentiation and resulted in N3 loss and basal cell hyperplasia.
Conclusions
Notch signaling is important for esophageal epithelial homeostasis. In particular, the crosstalk of N3 with N1 during differentiation provides novel, mechanistic insights into Notch signaling and squamous epithelial biology.
doi:10.1053/j.gastro.2010.08.040
PMCID: PMC2997138  PMID: 20801121
NOTCH1; NOTCH3; esophageal epithelium; squamous differentiation
8.  Notch Ankyrin Repeat Domain Variation Influences Leukemogenesis and Myc Transactivation 
PLoS ONE  2011;6(10):e25645.
Background
The functional interchangeability of mammalian Notch receptors (Notch1-4) in normal and pathophysiologic contexts such as cancer is unsettled. We used complementary in vivo, cell-based and structural analyses to compare the abilities of activated Notch1-4 to support T cell development, induce T cell acute lymphoblastic leukemia/lymphoma (T-ALL), and maintain T-ALL cell growth and survival.
Principal Findings
We find that the activated intracellular domains of Notch1-4 (ICN1-4) all support T cell development in mice and thymic organ culture. However, unlike ICN1-3, ICN4 fails to induce T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) and is unable to rescue the growth of Notch1-dependent T-ALL cell lines. The ICN4 phenotype is mimicked by weak gain-of-function forms of Notch1, suggesting that it stems from a failure to transactivate one or more critical target genes above a necessary threshold. Experiments with chimeric receptors demonstrate that the Notch ankyrin repeat domains differ in their leukemogenic potential, and that this difference correlates with activation of Myc, a direct Notch target that has an important role in Notch-associated T-ALL.
Conclusions/Significance
We conclude that the leukemogenic potentials of Notch receptors vary, and that this functional difference stems in part from divergence among the highly conserved ankyrin repeats, which influence the transactivation of specific target genes involved in leukemogenesis.
doi:10.1371/journal.pone.0025645
PMCID: PMC3192765  PMID: 22022427
9.  New roles for Notch in tuberous sclerosis 
Tuberous sclerosis complex (TSC) is a dominantly inherited disease that is characterized by the growth of multiple benign tumors that are often difficult to treat. TSC is caused by mutations that inactivate the TSC1 or TSC2 genes, which normally function to inhibit activation of mammalian target of rapamycin signaling. In this issue of the JCI, two studies reported by Karbowniczek et al. and Ma et al. link TSC inactivation with activated Notch signaling (see the related articles beginning on pages 93 and 103, respectively). Using a variety of approaches, both studies show that inactivation of TSC leads to Notch1 activation. Furthermore, studies in tumor cells suggest that inhibiting Notch slows growth of the tumor cells. Although much remains to be learned about the precise mechanisms by which TSC loss leads to Notch activation, the newly identified link of TSC to Notch provides the rationale for testing Notch inhibitors in TSC-associated tumors.
doi:10.1172/JCI41897
PMCID: PMC2798710  PMID: 20038806
10.  Srcasm inhibits Fyn-induced cutaneous carcinogenesis with modulation of Notch 1 and p53 
Cancer research  2009;69(24):9439-9447.
Src-family tyrosine kinases (SFKs) regulate cell proliferation, and increased SFK activity is common in human carcinomas, including cutaneous squamous cell carcinomas and its precursors. The elevated SFK activity in cutaneous SCCs was modeled using keratin 14-Fyn Y528F transgenic mice, which spontaneously form punctate keratotic lesions, scaly plaques, and large tumors resembling actinic keratoses (AKs), carcinoma in situ (SCIS), and SCCs, respectively.
Lesional tissue demonstrated increased levels of activated SFKs, PDK-1, STAT-3, and Erk1/2 while Notch 1/NICD protein and transcript levels were decreased. p53 levels also were decreased in SCIS and SCCs.
Raising Srcasm levels using a K14-Fyn Y528F/K14-Srcasm double transgenic model markedly inhibited cutaneous neoplasia. In contrast, increased expression of a non-phosphorylatable Srcasm mutant maintained the neoplastic phenotype. Raising Srcasm levels decreased levels of Fyn, activated SFKs, Erk 1/2, PDK-1, and phospho-STAT3, and raised Notch 1/NICD and p53 levels.
Analysis of human specimens revealed that levels of Fyn and activated SFKs were elevated in SCCs compared with adjacent non-lesional epidermis. In addition, Notch 1 and Srcasm protein and transcript levels were decreased in human SCCs compared to non-lesional epidermis. Therefore, the SCCs produced by the Fyn Y528F mice resemble their human counterparts at the molecular level.
K14-Fyn Y528F mice represent a robust model of cutaneous carcinogenesis that manifests precancerous lesions and SCCs resembling human disease. The Fyn/Srcasm signaling nexus modulates activity of STAT-3, PDK-1, Erk 1/2, Notch 1 and p53. Further study of Fyn and Srcasm should provide insights into the mechanisms regulating keratinocyte proliferation and skin carcinogenesis.
doi:10.1158/0008-5472.CAN-09-2976
PMCID: PMC2794931  PMID: 19934324
11.  Active Notch1 Confers a Transformed Phenotype to Primary Human Melanocytes 
Cancer research  2009;69(13):5312-5320.
The importance of MAPK signaling in melanoma is underscored by the prevalence of activating mutations in N-Ras and B-Raf; yet, clinical development of inhibitors of this pathway has been largely ineffective, suggesting that alternative oncogenes may also promote melanoma. Notch is an interesting candidate that has only been correlated with melanoma development and progression; a thorough assessment of tumor-initiating effects of activated Notch on human melanocytes would clarify the mounting correlative evidence and perhaps identify a novel target for an otherwise untreatable disease. Analysis of a substantial panel of cell lines and patient lesions demonstrated that Notch activity is significantly higher in melanomas than their non-transformed counterparts. The use of a constitutively-active, truncated Notch transgene construct (NIC) was exploited to determine if Notch activation is a ‘driving’ event in melanocytic transformation or instead a ‘passenger’ event associated with melanoma progression. NIC-infected melanocytes displayed increased proliferative capacity and biological features more reminiscent of melanoma such as dysregulated cell adhesion and migration. Gene expression analyses supported these observations and aided in the identification of MCAM, an adhesion molecule associated with acquisition of the malignant phenotype, as a direct target of Notch transactivation. NIC-positive melanocytes grew at clonal density, proliferated in limiting media conditions, and also exhibited anchorage-independent growth suggesting that Notch, alone, is a transforming oncogene in human melanocytes, a phenomenon not previously described for any melanoma oncogene; this new information yields valuable insight into the basic epidemiology of melanoma and launches a realm of possibilities for drug intervention in this deadly disease.
doi:10.1158/0008-5472.CAN-08-3767
PMCID: PMC2755513  PMID: 19549918
Notch; melanoma; transformation; MCAM; therapy
12.  HIV-1 Rev–binding protein accelerates cellular uptake of iron to drive Notch-induced T cell leukemogenesis in mice 
The Journal of Clinical Investigation  2010;120(7):2537-2548.
Somatic activating mutations in Notch1 contribute to the pathogenesis of T cell acute lymphoblastic lymphoma (T-ALL), but how activated Notch1 signaling exerts this oncogenic effect is not completely understood. Here we identify HIV-1 Rev–binding protein (Hrb), a component of the clathrin-mediated endocytosis machinery, as a critical mediator of Notch-induced T-ALL development in mice. Hrb was found to be a direct transcriptional target of Notch1, and Hrb loss reduced the incidence or delayed the onset of T-ALL in mouse models in which activated Notch1 signaling either contributes to or drives leukemogenesis. Consistent with this observation, Hrb supported survival and proliferation of hematopoietic and T cell precursor cells in vitro. We demonstrated that Hrb accelerated the uptake of transferrin, which was required for upregulation of the T cell protooncogene p21. Indeed, iron-deficient mice developed Notch1-induced T-ALL substantially more slowly than control mice, further supporting a critical role for iron uptake during leukemogenesis. Taken together, these results reveal that Hrb is a critical Notch target gene that mediates lymphoblast transformation and disease progression via its ability to satisfy the enhanced demands of transformed lymphoblasts for iron. Further, our data suggest that Hrb may be targeted to improve current treatment or design novel therapies for human T-ALL patients.
doi:10.1172/JCI41277
PMCID: PMC2898592  PMID: 20516639
13.  Distinct roles for PTEN in prevention of T cell lymphoma and autoimmunity in mice 
The Journal of Clinical Investigation  2010;120(7):2497-2507.
Mutations in the tumor-suppressor gene phosphatase and tensin homolog deleted on chromosome 10 (Pten) are associated with multiple cancers in humans, including T cell malignancies. Targeted deletion of Pten in T cells induces both a disseminated “mature phenotype” lymphoma and a lymphoproliferative autoimmune syndrome in mice. Here, we have shown that these two diseases are separable and mediated by T lineage cells of distinct developmental stages. Loss of PTEN was found to be a powerful driver of lymphomagenesis within the thymus characterized by overexpression of the c-myc oncogene. In an otherwise normal thymic environment, PTEN-deficient T cell lymphomas invariably harbored RAG-dependent reciprocal t(14:15) chromosomal translocations involving the T cell receptor alpha/delta locus and c-myc, and their survival and growth was TCR dependent, but Notch independent. However, lymphomas occurred even if TCR recombination was prevented, although these lymphomas were less mature, arose later in life, and, importantly, were dependent upon Notch pathways to upregulate c-myc expression. In contrast, using the complementary methods of early thymectomy and adoptive transfers, we found that PTEN-deficient mature T cells were unable to undergo malignant transformation but were sufficient for the development of autoimmunity. These data suggest multiple and distinct regulatory roles for PTEN in the molecular pathogenesis of lymphoma and autoimmunity.
doi:10.1172/JCI42382
PMCID: PMC2898609  PMID: 20516645
14.  Tribbles homolog 2 (Trib2) inactivates C/EBPalpha and causes acute myelogenous leukemia 
Cancer cell  2006;10(5):401-411.
Summary
Tribbles homolog 2 (Trib2) was identified as a down-regulated transcript in leukemic cells undergoing growth arrest. To investigate the effects of Trib2 in hematopoietic progenitors, mice were reconstituted with hematopoietic stem cells retrovirally expressing Trib2. Trib2-transduced bone marrow cells exhibited a growth advantage ex vivo and readily established factor-dependent cell lines. In vivo, Trib2-reconstituted mice uniformly developed fatal transplantable acute myelogenous leukemia (AML). In mechanistic studies, we found that Trib2 associated with and inhibited C/EBPα. Furthermore, Trib2 expression was elevated in a subset of human AML patient samples. Together, our data identify Trib2 as an oncogene that induces AML through a mechanism involving inactivation of C/EBPα.
doi:10.1016/j.ccr.2006.09.012
PMCID: PMC2839500  PMID: 17097562
15.  Notch Directly Regulates Gata3 Expression during T Helper 2 Cell Differentiation 
Immunity  2007;27(1):100-110.
SUMMARY
Notch signaling plays multiple roles to direct diverse decisions regarding cell fate during T cell development. During helper T (Th) cell differentiation, Notch is involved in generating optimal Th2 cell responses. Here, we present data investigating how Notch mediates Th2 cell differentiation. Notch showed a CD4+ T cell intrinsic role in promoting IL-4 expression that required GATA-3. In the absence of Notch signals, Gata3 expression was markedly diminished. Introduction of an activated allele of Notch1 into CD4+ T cells led to the specific and direct upregulation of a developmentally regulated Gata3 transcript that included the exon 1a sequences. Furthermore, Notch acted in parallel with GATA-3 to synergistically activate IL-4 expression. Together, these data implicate Gata3 as a direct transcriptional Notch target that acts in concert with Notch signaling to generate optimal Th2 cell responses.
doi:10.1016/j.immuni.2007.04.018
PMCID: PMC2801546  PMID: 17658278
16.  Murine Jagged1/Notch signaling in the second heart field orchestrates Fgf8 expression and tissue-tissue interactions during outflow tract development 
The Journal of Clinical Investigation  2009;119(7):1986-1996.
Notch signaling is vital for proper cardiovascular development and function in both humans and animal models. Indeed, mutations in either JAGGED or NOTCH cause congenital heart disease in humans and NOTCH mutations are associated with adult valvular disease. Notch typically functions to mediate developmental interactions between adjacent tissues. Here we show that either absence of the Notch ligand Jagged1 or inhibition of Notch signaling in second heart field tissues results in murine aortic arch artery and cardiac anomalies. In mid-gestation, these mutants displayed decreased Fgf8 and Bmp4 expression. Notch inhibition within the second heart field affected the development of neighboring tissues. For example, faulty migration of cardiac neural crest cells and defective endothelial-mesenchymal transition within the outflow tract endocardial cushions were observed. Furthermore, exogenous Fgf8 was sufficient to rescue the defect in endothelial-mesenchymal transition in explant assays of endocardial cushions following Notch inhibition within second heart field derivatives. These data support a model that relates second heart field, neural crest, and endocardial cushion development and suggests that perturbed Notch-Jagged signaling within second heart field progenitors accounts for some forms of congenital and adult cardiac disease.
doi:10.1172/JCI38922
PMCID: PMC2701882  PMID: 19509466
17.  Leukemia-associated NOTCH1 alleles are weak tumor initiators but accelerate K-ras–initiated leukemia  
The Journal of Clinical Investigation  2008;118(9):3181-3194.
Gain-of-function NOTCH1 mutations are found in 50%–70% of human T cell acute lymphoblastic leukemia/lymphoma (T-ALL) cases. Gain-of-function NOTCH1 alleles that initiate strong downstream signals induce leukemia in mice, but it is unknown whether the gain-of-function NOTCH1 mutations most commonly found in individuals with T-ALL generate downstream signals of sufficient strength to induce leukemia. We addressed this question by expressing human gain-of-function NOTCH1 alleles of varying strength in mouse hematopoietic precursors. Uncommon gain-of-function NOTCH1 alleles that initiated strong downstream signals drove ectopic T cell development and induced leukemia efficiently. In contrast, although gain-of-function alleles that initiated only weak downstream signals also induced ectopic T cell development, these more common alleles failed to efficiently initiate leukemia development. However, weak gain-of-function NOTCH1 alleles accelerated the onset of leukemia initiated by constitutively active K-ras and gave rise to tumors that were sensitive to Notch signaling pathway inhibition. These data show that induction of leukemia requires doses of Notch1 greater than those needed for T cell development and that most NOTCH1 mutations found in T-ALL cells do not generate signals of sufficient strength to initiate leukemia development. Furthermore, low, nonleukemogenic levels of Notch1 can complement other leukemogenic events, such as activation of K-ras. Even when Notch1 participates secondarily, the resulting tumors show “addiction” to Notch, providing a further rationale for evaluating Notch signaling pathway inhibitors in leukemia.
doi:10.1172/JCI35090
PMCID: PMC2491459  PMID: 18677410
18.  The requirement for Notch signaling at the β-selection checkpoint in vivo is absolute and independent of the pre–T cell receptor 
The Journal of Experimental Medicine  2006;203(10):2239-2245.
Genetic inactivation of Notch signaling in CD4−CD8− double-negative (DN) thymocytes was previously shown to impair T cell receptor (TCR) gene rearrangement and to cause a partial block in CD4+CD8+ double-positive (DP) thymocyte development in mice. In contrast, in vitro cultures suggested that Notch was absolutely required for the generation of DP thymocytes independent of pre-TCR expression and activity. To resolve the respective role of Notch and the pre-TCR, we inhibited Notch-mediated transcriptional activation in vivo with a green fluorescent protein–tagged dominant-negative Mastermind-like 1 (DNMAML) that allowed us to track single cells incapable of Notch signaling. DNMAML expression in DN cells led to decreased production of DP thymocytes but only to a modest decrease in intracellular TCRβ expression. DNMAML attenuated the pre-TCR–associated increase in cell size and CD27 expression. TCRβ or TCRαβ transgenes failed to rescue DNMAML-related defects. Intrathymic injections of DNMAML− or DNMAML+ DN thymocytes revealed a complete DN/DP transition block, with production of DNMAML+ DP thymocytes only from cells undergoing late Notch inactivation. These findings indicate that the Notch requirement during the β-selection checkpoint in vivo is absolute and independent of the pre-TCR, and it depends on transcriptional activation by Notch via the CSL/RBP-J–MAML complex.
doi:10.1084/jem.20061020
PMCID: PMC2118105  PMID: 16966428
19.  Identification of a Conserved Negative Regulatory Sequence That Influences the Leukemogenic Activity of NOTCH1 
Molecular and Cellular Biology  2006;26(16):6261-6271.
NOTCH1 is a large type I transmembrane receptor that regulates normal T-cell development via a signaling pathway that relies on regulated proteolysis. Ligand binding induces proteolytic cleavages in NOTCH1 that release its intracellular domain (ICN1), which translocates to the nucleus and activates target genes by forming a short-lived nuclear complex with two other proteins, the DNA-binding factor CSL and a Mastermind-like (MAML) coactivator. Recent work has shown that human T-ALL is frequently associated with C-terminal NOTCH1 truncations, which uniformly remove sequences lying between residues 2524 and 2556. This region includes the highly conserved sequence WSSSSP (S4), which based on its amino acid content appeared to be a likely site for regulatory serine phosphorylation events. We show here that the mutation of the S4 sequence leads to hypophosphorylation of ICN1; increased NOTCH1 signaling; and the stabilization of complexes containing ICN1, CSL, and MAML1. Consistent with these in vitro studies, mutation of the WSSSSP sequence converts nonleukemogenic weak gain-of-function NOTCH1 alleles into alleles that cause aggressive T-ALLs in a murine bone marrow transplant model. These studies indicate that S4 is an important negative regulatory sequence and that the deletion of S4 likely contributes to the development of human T-ALL.
doi:10.1128/MCB.02478-05
PMCID: PMC1592797  PMID: 16880534
20.  An essential role for Notch in neural crest during cardiovascular development and smooth muscle differentiation 
Journal of Clinical Investigation  2007;117(2):353-363.
The cardiac outflow tract develops as a result of a complex interplay among several cell types, including cardiac neural crest cells, endothelial cells, and cardiomyocytes. In both humans and mice, mutations in components of the Notch signaling pathway result in congenital heart disease characterized by cardiac outflow tract defects. However, the specific cell types in which Notch functions during cardiovascular development remain to be defined. In addition, in vitro studies have provided conflicting data regarding the ability of Notch to promote or inhibit smooth muscle differentiation, while the physiological role for Notch in smooth muscle formation during development remains unclear. In this study, we generated mice in which Notch signaling was specifically inactivated in derivatives of the neural crest. These mice exhibited cardiovascular anomalies, including aortic arch patterning defects, pulmonary artery stenosis, and ventricular septal defects. We show that Notch plays a critical, cell-autonomous role in the differentiation of cardiac neural crest precursors into smooth muscle cells both in vitro and in vivo, and we identify specific Notch targets in neural crest that are implicated in this process. These results provide a molecular and cellular framework for understanding the role of Notch signaling in the etiology of congenital heart disease.
doi:10.1172/JCI30070
PMCID: PMC1783803  PMID: 17273555
21.  Notch signaling is an important regulator of type 2 immunity 
The Journal of Experimental Medicine  2005;202(8):1037-1042.
Notch ligands and receptors have been implicated in helper T cell (Th cell) differentiation. Whether Notch signals are involved in differentiation of T helper type 1 (Th1) cells, Th2 cells, or both, however, remains unresolved. To clarify the role of Notch in Th cell differentiation, we generated mice that conditionally inactivate Notch signaling in mature T cells. Mice that lack Notch signaling in CD4+ T cells fail to develop a protective Th2 cell response against the gastrointestinal helminth Trichuris muris. In contrast, they exhibit effective Th1 cell responses and are able to control Leishmania major infection. These data demonstrate that Notch signaling is a regulator of type 2 immunity.
doi:10.1084/jem.20050923
PMCID: PMC2213210  PMID: 16230473
22.  Notch Subunit Heterodimerization and Prevention of Ligand-Independent Proteolytic Activation Depend, Respectively, on a Novel Domain and the LNR Repeats 
Molecular and Cellular Biology  2004;24(21):9265-9273.
Notch proteins are transmembrane receptors that participate in a highly conserved signaling pathway that regulates morphogenesis in metazoans. Newly synthesized Notch receptors are proteolytically cleaved during transit to the cell surface, creating heterodimeric mature receptors comprising noncovalently associated extracellular (NEC) and transmembrane (NTM) subunits. Ligand binding activates Notch by inducing two successive proteolytic cleavages, catalyzed by metalloproteases and gamma-secretase, respectively, that permit the intracellular portion of NTM to translocate to the nucleus and activate transcription of target genes. Prior work has shown that the presence of NEC prevents ligand-independent activation of NTM, but the mechanisms involved are poorly understood. Here, we define the roles of two regions at the C-terminal end of NEC that participate in maintaining the integrity of resting Notch receptors through distinct mechanisms. The first region, a hydrophobic, previously uncharacterized portion of NEC, is sufficient to form stable complexes with the extracellular portion of NTM. The second region, consisting of the three Lin12/Notch repeats, is not needed for heterodimerization but acts to protect NTM from ligand-independent cleavage by metalloproteases. Together, these two contiguous regions of NEC impose crucial restraints that prevent premature Notch receptor activation.
doi:10.1128/MCB.24.21.9265-9273.2004
PMCID: PMC522238  PMID: 15485896
23.  NF-kB inhibitor blocks B cell development at two checkpoints 
Medical Immunology  2004;3:1.
Members of the NF-kB transcription factor family are differentially expressed in the B cell lineage. Disruption of individual or two NF-kB subunits exhibits distinct defects in B lymphocyte development, activation, and survival. However, the role each NF-kB plays during B cell development has been obscured by molecular compensation. To address this issue, a trans-dominant form of IkBα was transduced into bone marrow cells to act as a pan-inhibitor of NF-kB using a retroviral system. While the development of T-lymphocytes and myeloid cell lineages was not grossly affected by the transduced IkBα gene, a significant reduction in the number and percentage of B lineage cells was apparent in IkBα transduced chimeric mice. IkBα expression decreased the percentage of pre-B and immature B cell subsets in the bone marrow and further impaired the development of follicular mature B cells and marginal zone B cells in the periphery. Introduction of the Bcl-X transgene completely restored the pre-B and immature B cell pool in the bone marrow. However, despite a significant improvement of overall viability of the B cell lineage, Bcl-X expression was insufficient to overcome the maturation block resulting from NF-kB inhibition. Together, our study suggests that NF-kB activity is required for two distinct checkpoints during B cell development: one is for pre-B/immature B cell viability, the other is to provide both survival and maturation signals to ensure the proper development of follicular mature B cells.
doi:10.1186/1476-9433-3-1
PMCID: PMC419369  PMID: 15050028
NF-kB; IkBα; Bcl-X; B cell development; retroviral gene transduction
24.  Growth Suppression of Pre-T Acute Lymphoblastic Leukemia Cells by Inhibition of Notch Signaling 
Molecular and Cellular Biology  2003;23(2):655-664.
Constitutive NOTCH signaling in lymphoid progenitors promotes the development of immature T-cell lymphoblastic neoplasms (T-ALLs). Although it is clear that Notch signaling can initiate leukemogenesis, it has not previously been established whether continued NOTCH signaling is required to maintain T-ALL growth. We demonstrate here that the blockade of Notch signaling at two independent steps suppresses the growth and survival of NOTCH1-transformed T-ALL cells. First, inhibitors of presenilin specifically induce growth suppression and apoptosis of a murine T-ALL cell line that requires presenilin-dependent proteolysis of the Notch receptor in order for its intracellular domain to translocate to the nucleus. Second, a 62-amino-acid peptide derived from a NOTCH coactivator, Mastermind-like-1 (MAML1), forms a transcriptionally inert nuclear complex with NOTCH1 and CSL and specifically inhibits the growth of both murine and human NOTCH1-transformed T-ALLs. These studies show that continued growth and survival of NOTCH1-transformed lymphoid cell lines require nuclear access and transcriptional coactivator recruitment by NOTCH1 and identify at least two steps in the Notch signaling pathway as potential targets for chemotherapeutic intervention.
doi:10.1128/MCB.23.2.655-664.2003
PMCID: PMC151540  PMID: 12509463
25.  Differential Requirement for LAT and SLP-76 in GPVI versus T Cell Receptor Signaling 
Mice deficient in the adaptor Src homology 2 domain-containing leukocyte phosphoprotein of 76 kD (SLP-76) exhibit a bleeding disorder and lack T cells. Linker for activation of T cells (LAT)-deficient mice exhibit a similar T cell phenotype, but show no signs of hemorrhage. Both SLP-76 and LAT are important for optimal platelet activation downstream of the collagen receptor, GPVI. In addition, SLP-76 is involved in signaling mediated by integrin αIIbβ3. Because SLP-76 and LAT function coordinately in T cell signal transduction, yet their roles appear to differ in hemostasis, we investigated in detail the functional consequences of SLP-76 and LAT deficiencies in platelets. Previously we have shown that LAT−/− platelets exhibit defective responses to the GPVI-specific agonist, collagen-related peptide (CRP). Consistent with this, we find that surface expression of P-selectin in response to high concentrations of GPVI ligands is reduced in both LAT- and SLP-76–deficient platelets. However, platelets from LAT−/− mice, but not SLP-76−/− mice, aggregate normally in response to high concentrations of collagen and convulxin. Additionally, unlike SLP-76, LAT is not tyrosine phosphorylated after fibrinogen binding to integrin αIIbβ3, and collagen-stimulated platelets deficient in LAT spread normally on fibrinogen-coated surfaces. Together, these findings indicate that while LAT and SLP-76 are equally required for signaling via the T cell antigen receptor (TCR) and pre-TCR, platelet activation downstream of GPVI and αIIbβ3 shows a much greater dependency on SLP-76 than LAT.
doi:10.1084/jem.20011583
PMCID: PMC2193740  PMID: 11901197
SLP-76; LAT; GPVI; integrin αIIbβ3; T cell receptor

Results 1-25 (30)