Publications by authors named "Warren S Pear"

120 Publications

Notch dimerization and gene dosage are important for normal heart development, intestinal stem cell maintenance, and splenic marginal zone B-cell homeostasis during mite infestation.

PLoS Biol 2020 10 5;18(10):e3000850. Epub 2020 Oct 5.

Division of Developmental Biology, Department of Pediatrics, University of Cincinnati College of Medicine and Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States of America.

Cooperative DNA binding is a key feature of transcriptional regulation. Here we examined the role of cooperativity in Notch signaling by CRISPR-mediated engineering of mice in which neither Notch1 nor Notch2 can homo- or heterodimerize, essential for cooperative binding to sequence-paired sites (SPS) located near many Notch-regulated genes. Although most known Notch-dependent phenotypes were unaffected in Notch1/2 dimer-deficient mice, a subset of tissues proved highly sensitive to loss of cooperativity. These phenotypes include heart development, compromised viability in combination with low gene dose, and the gut, developing ulcerative colitis in response to 1% dextran sulfate sodium (DSS). The most striking phenotypes-gender imbalance and splenic marginal zone B-cell lymphoma-emerged in combination with gene dose reduction or when challenged by chronic fur mite infestation. This study highlights the role of the environment in malignancy and colitis and is consistent with Notch-dependent anti-parasite immune responses being compromised in Notch dimer-deficient animals.
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http://dx.doi.org/10.1371/journal.pbio.3000850DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7561103PMC
October 2020

Trib1 regulates T cell differentiation during chronic infection by restraining the effector program.

J Exp Med 2020 05;217(5)

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

In chronic infections, the immune response fails to control virus, leading to persistent antigen stimulation and the progressive development of T cell exhaustion. T cell effector differentiation is poorly understood in the context of exhaustion, but targeting effector programs may provide new strategies for reinvigorating T cell function. We identified Tribbles pseudokinase 1 (Trib1) as a central regulator of antiviral T cell immunity, where loss of Trib1 led to a sustained enrichment of effector-like KLRG1+ T cells, enhanced function, and improved viral control. Single-cell profiling revealed that Trib1 restrains a population of KLRG1+ effector CD8 T cells that is transcriptionally distinct from exhausted cells. Mechanistically, we identified an interaction between Trib1 and the T cell receptor (TCR) signaling activator, MALT1, which disrupted MALT1 signaling complexes. These data identify Trib1 as a negative regulator of TCR signaling and downstream function, and reveal a link between Trib1 and effector versus exhausted T cell differentiation that can be targeted to improve antiviral immunity.
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http://dx.doi.org/10.1084/jem.20190888DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7201917PMC
May 2020

TooManyCells identifies and visualizes relationships of single-cell clades.

Nat Methods 2020 04 2;17(4):405-413. Epub 2020 Mar 2.

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA.

Identifying and visualizing transcriptionally similar cells is instrumental for accurate exploration of the cellular diversity revealed by single-cell transcriptomics. However, widely used clustering and visualization algorithms produce a fixed number of cell clusters. A fixed clustering 'resolution' hampers our ability to identify and visualize echelons of cell states. We developed TooManyCells, a suite of graph-based algorithms for efficient and unbiased identification and visualization of cell clades. TooManyCells introduces a visualization model built on a concept intentionally orthogonal to dimensionality-reduction methods. TooManyCells is also equipped with an efficient matrix-free divisive hierarchical spectral clustering different from prevalent single-resolution clustering methods. TooManyCells enables multiresolution and multifaceted exploration of single-cell clades. An advantage of this paradigm is the immediate detection of rare and common populations that outperforms popular clustering and visualization algorithms, as demonstrated using existing single-cell transcriptomic data sets and new data modeling drug-resistance acquisition in leukemic T cells.
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http://dx.doi.org/10.1038/s41592-020-0748-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439807PMC
April 2020

Transcription factor and cytokine regulation of eosinophil lineage commitment.

Curr Opin Hematol 2020 01;27(1):27-33

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, Perelman School of Medicine at the University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.

Purpose Of Review: Lineage commitment is governed by instructive and stochastic signals, which drive both active induction of the lineage program and repression of alternative fates. Eosinophil lineage commitment is driven by the ordered interaction of transcription factors, supported by cytokine signals. This review summarizes key findings in the study of eosinophil lineage commitment and examines new data investigating the factors that regulate this process.

Recent Findings: Recent and past studies highlight how intrinsic and extrinsic signals modulate transcription factor network and lineage decisions. Early action of the transcription factors C/EBPα and GATA binding protein-1 along with C/EBPε supports lineage commitment and eosinophil differentiation. This process is regulated and enforced by the pseudokinase Trib1, a regulator of C/EBPα levels. The cytokines interleukin (IL)-5 and IL-33 also support early eosinophil development. However, current studies suggest that these cytokines are not specifically required for lineage commitment.

Summary: Together, recent evidence suggests a model where early transcription factor activity drives expression of key eosinophil genes and cytokine receptors to prime lineage commitment. Understanding the factors and signals that control eosinophil lineage commitment may guide therapeutic development for eosinophil-mediated diseases and provide examples for fate choices in other lineages.
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http://dx.doi.org/10.1097/MOH.0000000000000552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7388079PMC
January 2020

Trib1 regulates eosinophil lineage commitment and identity by restraining the neutrophil program.

Blood 2019 05 27;133(22):2413-2426. Epub 2019 Mar 27.

Department of Pathology and Laboratory Medicine and.

Eosinophils and neutrophils are critical for host defense, yet gaps in understanding how granulocytes differentiate from hematopoietic stem cells (HSCs) into mature effectors remain. The pseudokinase tribbles homolog 1 (Trib1) is an important regulator of granulocytes; knockout mice lack eosinophils and have increased neutrophils. However, how Trib1 regulates cellular identity and function during eosinophilopoiesis is not understood. expression markedly increases with eosinophil-lineage commitment in eosinophil progenitors (EoPs), downstream of the granulocyte/macrophage progenitor (GMP). Using hematopoietic- and eosinophil-lineage-specific deletion, we found that Trib1 regulates both granulocyte precursor lineage commitment and mature eosinophil identity. Conditional Trib1 deletion in HSCs reduced the size of the EoP pool and increased neutrophils, whereas deletion following eosinophil lineage commitment blunted the decrease in EoPs without increasing neutrophils. In both modes of deletion, Trib1-deficient mice expanded a stable population of Ly6G eosinophils with neutrophilic characteristics and functions, and had increased CCAAT/enhancer binding protein α (C/EBPα) p42. Using an ex vivo differentiation assay, we found that interleukin 5 (IL-5) supports the generation of Ly6G eosinophils from Trib1-deficient cells, but is not sufficient to restore normal eosinophil differentiation and development. Furthermore, we demonstrated that Trib1 loss blunted eosinophil migration and altered chemokine receptor expression, both in vivo and ex vivo. Finally, we showed that Trib1 controls eosinophil identity by modulating C/EBPα. Together, our findings provide new insights into early events in myelopoiesis, whereby Trib1 functions at 2 distinct stages to guide eosinophil lineage commitment from the GMP and suppress the neutrophil program, promoting eosinophil terminal identity and maintaining lineage fidelity.
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http://dx.doi.org/10.1182/blood.2018872218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543518PMC
May 2019

Oncogenic Notch Promotes Long-Range Regulatory Interactions within Hyperconnected 3D Cliques.

Mol Cell 2019 03 7;73(6):1174-1190.e12. Epub 2019 Feb 7.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Biomedical Informatics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Department of Cancer Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Chromatin loops enable transcription-factor-bound distal enhancers to interact with their target promoters to regulate transcriptional programs. Although developmental transcription factors such as active forms of Notch can directly stimulate transcription by activating enhancers, the effect of their oncogenic subversion on the 3D organization of cancer genomes is largely undetermined. By mapping chromatin looping genome-wide in Notch-dependent triple-negative breast cancer and B cell lymphoma, we show that beyond the well-characterized role of Notch as an activator of distal enhancers, Notch regulates its direct target genes by instructing enhancer repositioning. Moreover, a large fraction of Notch-instructed regulatory loops form highly interacting enhancer and promoter spatial clusters termed "3D cliques." Loss- and gain-of-function experiments show that Notch preferentially targets hyperconnected 3D cliques that regulate the expression of crucial proto-oncogenes. Our observations suggest that oncogenic hijacking of developmental transcription factors can dysregulate transcription through widespread effects on the spatial organization of cancer genomes.
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http://dx.doi.org/10.1016/j.molcel.2019.01.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485942PMC
March 2019

Single-cell analysis reveals fibroblast heterogeneity and myeloid-derived adipocyte progenitors in murine skin wounds.

Nat Commun 2019 02 8;10(1):650. Epub 2019 Feb 8.

Department of Developmental and Cell Biology, University of California, Irvine, Irvine, CA, 92697, USA.

During wound healing in adult mouse skin, hair follicles and then adipocytes regenerate. Adipocytes regenerate from myofibroblasts, a specialized contractile wound fibroblast. Here we study wound fibroblast diversity using single-cell RNA-sequencing. On analysis, wound fibroblasts group into twelve clusters. Pseudotime and RNA velocity analyses reveal that some clusters likely represent consecutive differentiation states toward a contractile phenotype, while others appear to represent distinct fibroblast lineages. One subset of fibroblasts expresses hematopoietic markers, suggesting their myeloid origin. We validate this finding using single-cell western blot and single-cell RNA-sequencing on genetically labeled myofibroblasts. Using bone marrow transplantation and Cre recombinase-based lineage tracing experiments, we rule out cell fusion events and confirm that hematopoietic lineage cells give rise to a subset of myofibroblasts and rare regenerated adipocytes. In conclusion, our study reveals that wounding induces a high degree of heterogeneity among fibroblasts and recruits highly plastic myeloid cells that contribute to adipocyte regeneration.
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http://dx.doi.org/10.1038/s41467-018-08247-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6368572PMC
February 2019

Downregulating Notch counteracts Kras-induced ERK activation and oxidative phosphorylation in myeloproliferative neoplasm.

Leukemia 2019 03 11;33(3):671-685. Epub 2018 Sep 11.

McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI, USA.

The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function NOTCH1 mutations are prevalent in human T-cell leukemia, while loss of Notch signaling is reported in myeloid leukemias. Here, we report a novel oncogenic function of Notch signaling in oncogenic Kras-induced myeloproliferative neoplasm (MPN). We find that downregulation of Notch signaling in hematopoietic cells via DNMAML expression or Pofut1 deletion significantly blocks MPN development in Kras mice in a cell-autonomous manner. Further mechanistic studies indicate that inhibition of Notch signaling upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in Kras cells. Moreover, mitochondrial metabolism is greatly enhanced in Kras cells but significantly reprogrammed by DNMAML close to that in control cells. Consequently, cell proliferation and expanded myeloid compartment in Kras mice are significantly reduced. Consistent with these findings, combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target both ERK signaling and aberrant metabolism in oncogenic Ras-driven myeloid leukemia.
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http://dx.doi.org/10.1038/s41375-018-0248-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405304PMC
March 2019

Lineage-Determining Transcription Factor TCF-1 Initiates the Epigenetic Identity of T Cells.

Immunity 2018 02;48(2):243-257.e10

Department of Genetics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

T cell development is orchestrated by transcription factors that regulate the expression of genes initially buried within inaccessible chromatin, but the transcription factors that establish the regulatory landscape of the T cell lineage remain unknown. Profiling chromatin accessibility at eight stages of T cell development revealed the selective enrichment of TCF-1 at genomic regions that became accessible at the earliest stages of development. TCF-1 was further required for the accessibility of these regulatory elements and at the single-cell level, it dictated a coordinate opening of chromatin in T cells. TCF-1 expression in fibroblasts generated de novo chromatin accessibility even at chromatin regions with repressive marks, inducing the expression of T cell-restricted genes. These results indicate that a mechanism by which TCF-1 controls T cell fate is through its widespread ability to target silent chromatin and establish the epigenetic identity of T cells.
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http://dx.doi.org/10.1016/j.immuni.2018.01.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5824646PMC
February 2018

MAFB enhances oncogenic Notch signaling in T cell acute lymphoblastic leukemia.

Sci Signal 2017 11 14;10(505). Epub 2017 Nov 14.

Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.

Activating mutations in the gene encoding the cell-cell contact signaling protein Notch1 are common in human T cell acute lymphoblastic leukemias (T-ALLs). However, expressing mutant alleles in mice fails to efficiently induce the development of leukemia. We performed a gain-of-function screen to identify proteins that enhanced signaling by leukemia-associated Notch1 mutants. The transcription factors MAFB and ETS2 emerged as candidates that individually enhanced Notch1 signaling, and when coexpressed, they synergistically increased signaling to an extent similar to that induced by core components of the Notch transcriptional complex. In mouse models of T-ALL, MAFB enhanced leukemogenesis by the naturally occurring Notch1 mutants, decreased disease latency, and increased disease penetrance. Decreasing MAFB abundance in mouse and human T-ALL cells reduced the expression of Notch1 target genes, including and , and sustained MAFB knockdown impaired T-ALL growth in a competitive setting. MAFB bound to ETS2 and interacted with the acetyltransferases PCAF and P300, highlighting its importance in recruiting coactivators that enhance Notch1 signaling. Together, these data identify a mechanism for enhancing the oncogenic potential of weak Notch1 mutants in leukemia models, and they reveal the MAFB-ETS2 transcriptional axis as a potential therapeutic target in T-ALL.
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http://dx.doi.org/10.1126/scisignal.aam6846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5885022PMC
November 2017

A B Cell Regulome Links Notch to Downstream Oncogenic Pathways in Small B Cell Lymphomas.

Cell Rep 2017 Oct;21(3):784-797

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address:

Gain-of-function Notch mutations are recurrent in mature small B cell lymphomas such as mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL), but the Notch target genes that contribute to B cell oncogenesis are largely unknown. We performed integrative analysis of Notch-regulated transcripts, genomic binding of Notch transcription complexes, and genome conformation data to identify direct Notch target genes in MCL cell lines. This B cell Notch regulome is largely controlled through Notch-bound distal enhancers and includes genes involved in B cell receptor and cytokine signaling and the oncogene MYC, which sustains proliferation of Notch-dependent MCL cell lines via a Notch-regulated lineage-restricted enhancer complex. Expression of direct Notch target genes is associated with Notch activity in an MCL xenograft model and in CLL lymph node biopsies. Our findings provide key insights into the role of Notch in MCL and other B cell malignancies and have important implications for therapeutic targeting of Notch-dependent oncogenic pathways.
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http://dx.doi.org/10.1016/j.celrep.2017.09.066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687286PMC
October 2017

The common oncogenomic program of NOTCH1 and NOTCH3 signaling in T-cell acute lymphoblastic leukemia.

PLoS One 2017 12;12(10):e0185762. Epub 2017 Oct 12.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, United States of America.

Notch is a major oncogenic driver in T cell acute lymphoblastic leukemia (T-ALL), in part because it binds to an enhancer that increases expression of MYC. Here, we exploit the capacity of activated NOTCH1 and NOTCH3 to induce T-ALL, despite substantial divergence in their intracellular regions, as a means to elucidate a broad, common Notch-dependent oncogenomic program through systematic comparison of the transcriptomes and Notch-bound genomic regulatory elements of NOTCH1- and NOTCH3-dependent T-ALL cells. ChIP-seq studies show a high concordance of functional NOTCH1 and NOTCH3 genomic binding sites that are enriched in binding motifs for RBPJ, the transcription factor that recruits activated Notch to DNA. The interchangeability of NOTCH1 and NOTCH3 was confirmed by rescue of NOTCH1-dependent T-ALL cells with activated NOTCH3 and vice versa. Despite remarkable overall similarity, there are nuanced differences in chromatin landscapes near critical common Notch target genes, most notably at a Notch-dependent enhancer that regulates MYC, which correlates with responsiveness to Notch pathway inhibitors. Overall, a common oncogenomic program driven by binding of either Notch is sufficient to maintain T-ALL cell growth, whereas cell-context specific differences appear to influence the response of T-ALL cells to Notch inhibition.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185762PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638296PMC
October 2017

Genome-wide identification and characterization of Notch transcription complex-binding sequence-paired sites in leukemia cells.

Sci Signal 2017 May 2;10(477). Epub 2017 May 2.

Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.

Notch transcription complexes (NTCs) drive target gene expression by binding to two distinct types of genomic response elements, NTC monomer-binding sites and sequence-paired sites (SPSs) that bind NTC dimers. SPSs are conserved and have been linked to the Notch responsiveness of a few genes. To assess the overall contribution of SPSs to Notch-dependent gene regulation, we determined the DNA sequence requirements for NTC dimerization using a fluorescence resonance energy transfer (FRET) assay and applied insights from these in vitro studies to Notch-"addicted" T cell acute lymphoblastic leukemia (T-ALL) cells. We found that SPSs contributed to the regulation of about a third of direct Notch target genes. Although originally described in promoters, SPSs are present mainly in long-range enhancers, including an enhancer containing a newly described SPS that regulates expression. Our work provides a general method for identifying SPSs in genome-wide data sets and highlights the widespread role of NTC dimerization in Notch-transformed leukemia cells.
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http://dx.doi.org/10.1126/scisignal.aag1598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931361PMC
May 2017

Regeneration of fat cells from myofibroblasts during wound healing.

Science 2017 02 5;355(6326):748-752. Epub 2017 Jan 5.

The Saban Research Institute of Children's Hospital Los Angeles and Department of Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90027, USA.

Although regeneration through the reprogramming of one cell lineage to another occurs in fish and amphibians, it has not been observed in mammals. We discovered in the mouse that during wound healing, adipocytes regenerate from myofibroblasts, a cell type thought to be differentiated and nonadipogenic. Myofibroblast reprogramming required neogenic hair follicles, which triggered bone morphogenetic protein (BMP) signaling and then activation of adipocyte transcription factors expressed during development. Overexpression of the BMP antagonist Noggin in hair follicles or deletion of the BMP receptor in myofibroblasts prevented adipocyte formation. Adipocytes formed from human keloid fibroblasts either when treated with BMP or when placed with human hair follicles in vitro Thus, we identify the myofibroblast as a plastic cell type that may be manipulated to treat scars in humans.
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http://dx.doi.org/10.1126/science.aai8792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5464786PMC
February 2017

Protein Tyrosine Phosphatase PRL2 Mediates Notch and Kit Signals in Early T Cell Progenitors.

Stem Cells 2017 04 19;35(4):1053-1064. Epub 2017 Jan 19.

Department of Pediatrics, Herman B Wells Center for Pediatric Research.

The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors. Stem Cells 2017;35:1053-1064.
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http://dx.doi.org/10.1002/stem.2559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5367971PMC
April 2017

The Varied Roles of Notch in Cancer.

Annu Rev Pathol 2017 Jan 5;12:245-275. Epub 2016 Dec 5.

Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.

Notch receptors influence cellular behavior by participating in a seemingly simple signaling pathway, but outcomes produced by Notch signaling are remarkably varied depending on signal dose and cell context. Here, after briefly reviewing new insights into physiologic mechanisms of Notch signaling in healthy tissues and defects in Notch signaling that contribute to congenital disorders and viral infection, we discuss the varied roles of Notch in cancer, focusing on cell autonomous activities that may be either oncogenic or tumor suppressive.
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http://dx.doi.org/10.1146/annurev-pathol-052016-100127DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5933931PMC
January 2017

High selective pressure for Notch1 mutations that induce Myc in T-cell acute lymphoblastic leukemia.

Blood 2016 11 26;128(18):2229-2240. Epub 2016 Sep 26.

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, Institute of Medicine and Engineering, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA; and.

Activating NOTCH1 mutations are frequent in human T-cell acute lymphoblastic leukemia (T-ALL) and Notch inhibitors (γ-secretase inhibitors [GSIs]) have produced responses in patients with relapsed, refractory disease. However, sustained responses, although reported, are uncommon, suggesting that other pathways can substitute for Notch in T-ALL. To address this possibility, we first generated Kras transgenic mice with T-cell-specific expression of the pan-Notch inhibitor, dominant-negative Mastermind (DNMAML). These mice developed leukemia, but instead of accessing alternative oncogenic pathways, the tumor cells acquired Notch1 mutations and subsequently deleted DNMAML, reinforcing the notion that activated Notch1 is particularly transforming within the context of T-cell progenitors. We next took a candidate approach to identify oncogenic pathways downstream of Notch, focusing on Myc and Akt, which are Notch targets in T-cell progenitors. Kras mice transduced with Myc developed T-ALLs that were GSI-insensitive and lacked Notch1 mutations. In contrast, Kras mice transduced with myristoylated AKT developed GSI-sensitive T-ALLs that acquired Notch1 mutations. Thus, Myc can substitute for Notch1 in leukemogenesis, whereas Akt cannot. These findings in primary tumors extend recent work using human T-ALL cell lines and xenografts and suggest that the Notch/Myc signaling axis is of predominant importance in understanding both the selective pressure for Notch mutations in T-ALL and response and resistance of T-ALL to Notch pathway inhibitors.
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http://dx.doi.org/10.1182/blood-2016-01-692855DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095757PMC
November 2016

Delta/Notch-Like EGF-Related Receptor (DNER) Is Not a Notch Ligand.

PLoS One 2016 13;11(9):e0161157. Epub 2016 Sep 13.

Department of Neurology, University of Pennsylvania, Perelman School of Medicine, Philadelphia, United States of America.

Delta/Notch-like EGF-related receptor (DNER) has been reported to act as a Notch ligand, despite lacking a Delta/Serrate/Lag (DSL) binding domain common to all other known ligands. The established Notch ligand Delta-like 1 (DLL1), but not DNER, activated Notch1 in a luciferase assay, prevented the differentiation of myoblasts through Notch signaling, and bound Notch-fc in a cell-based assay. DNER is not a Notch ligand and its true function remains unknown.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0161157PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5021350PMC
August 2017

Trib2 Suppresses Tumor Initiation in Notch-Driven T-ALL.

PLoS One 2016 18;11(5):e0155408. Epub 2016 May 18.

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, Institute of Medicine and Engineering, Institute for Immunology, Center for Personalized Diagnostics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, United States of America.

Trib2 is highly expressed in human T cell acute lymphoblastic leukemia (T-ALL) and is a direct transcriptional target of the oncogenic drivers Notch and TAL1. In human TAL1-driven T-ALL cell lines, Trib2 is proposed to function as an important survival factor, but there is limited information about the role of Trib2 in primary T-ALL. In this study, we investigated the role of Trib2 in the initiation and maintenance of Notch-dependent T-ALL. Trib2 had no effect on the growth and survival of murine T-ALL cell lines in vitro when expression was blocked by shRNAs. To test the function of Trib2 on leukemogenesis in vivo, we generated Trib2 knockout mice. Mice were born at the expected Mendelian frequencies without gross developmental anomalies. Adult mice did not develop pathology or shortened survival, and hematopoiesis, including T cell development, was unperturbed. Using a retroviral model of Notch-induced T-ALL, deletion of Trib2 unexpectedly decreased the latency and increased the penetrance of T-ALL development in vivo. Immunoblotting of primary murine T-ALL cells showed that the absence of Trib2 increased C/EBPα expression, a known regulator of cell proliferation, and did not alter AKT or ERK phosphorylation. Although Trib2 was suggested to be highly expressed in T-ALL, transcriptomic analysis of two independent T-ALL cohorts showed that low Trib2 expression correlated with the TLX1-expressing cortical mature T-ALL subtype, whereas high Trib2 expression correlated with the LYL1-expressing early immature T-ALL subtype. These data indicate that Trib2 has a complex role in the pathogenesis of Notch-driven T-ALL, which may vary between different T-ALL subtypes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0155408PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4871414PMC
July 2017

Structural Basis for Substrate Selectivity of the E3 Ligase COP1.

Structure 2016 05 31;24(5):687-696. Epub 2016 Mar 31.

Department of Cancer Biology, Dana Farber Cancer Institute, Boston, MA 02215, USA; Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115, USA; Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA. Electronic address:

COP1 proteins are E3 ubiquitin ligases that regulate phototropism in plants and target transcription factors for degradation in mammals. The substrate-binding region of COP1 resides within a WD40-repeat domain that also binds to Trib proteins, which are adaptors for C/EBPα degradation. Here we report structures of the human COP1 WD40 domain in isolation, and complexes of the human and Arabidopsis thaliana COP1 WD40 domains with the binding motif of Trib1. The human and Arabidopsis WD40 domains are seven-bladed β propellers with an inserted loop on the bottom face of the first blade. The Trib1 peptide binds in an extended conformation to a highly conserved surface on the top face of the β propeller, indicating a general mode for recognition of peptide motifs by COP1. Together, these studies identify the structural basis and key interactions for motif recognition by COP1, and hint at how Trib1 autoinhibition is overcome to target C/EBPα for degradation.
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http://dx.doi.org/10.1016/j.str.2016.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4856590PMC
May 2016

Tribbles in normal and malignant haematopoiesis.

Biochem Soc Trans 2015 Oct;43(5):1112-5

Department of Pathology and Laboratory Medicine, Institute for Immunology, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, U.S.A.

The tribbles protein family, an evolutionarily conserved group of pseudokinases, have been shown to regulate multiple cellular events including those involved in normal and malignant haematopoiesis. The three mammalian Tribbles homologues, Trib1, Trib2 and Trib3 are characterized by conserved motifs, including a pseudokinase domain and a C-terminal E3 ligase-binding domain. In this review, we focus on the role of Trib (mammalian Tribbles homologues) proteins in mammalian haematopoiesis and leukaemia. The Trib proteins show divergent expression in haematopoietic cells, probably indicating cell-specific functions. The roles of the Trib proteins in oncogenesis are also varied and appear to be tissue-specific. Finally, we discuss the potential mechanisms by which the Trib proteins preferentially regulate these processes in multiple cell types.
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http://dx.doi.org/10.1042/BST20150117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4613494PMC
October 2015

Tribbles at the cross-roads….

Biochem Soc Trans 2015 Oct;43(5):1049-50

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, Perelman School of Medicine, University of Pennsylvania, PA 19104, U.S.A.

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http://dx.doi.org/10.1042/BST20150122DOI Listing
October 2015

IL-1 signaling modulates activation of STAT transcription factors to antagonize retinoic acid signaling and control the TH17 cell-iTreg cell balance.

Nat Immunol 2015 Mar 2;16(3):286-95. Epub 2015 Feb 2.

Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA.

Interleukin 17 (IL-17)-producing helper T cells (TH17 cells) and CD4(+) inducible regulatory T cells (iTreg cells) emerge from an overlapping developmental program. In the intestines, the vitamin A metabolite retinoic acid (RA) is produced at steady state and acts as an important cofactor to induce iTreg cell development while potently inhibiting TH17 cell development. Here we found that IL-1 was needed to fully override RA-mediated expression of the transcription factor Foxp3 and induce protective TH17 cell responses. By repressing expression of the negative regulator SOCS3 dependent on the transcription factor NF-κB, IL-1 increased the amplitude and duration of phosphorylation of the transcription factor STAT3 induced by TH17-polarizing cytokines, which led to an altered balance in the binding of STAT3 and STAT5 to shared consensus sequences in developing T cells. Thus, IL-1 signaling modulated STAT activation downstream of cytokine receptors differently to control the TH17 cell-iTreg cell developmental fate.
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http://dx.doi.org/10.1038/ni.3099DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4790724PMC
March 2015

Long-range enhancer activity determines Myc sensitivity to Notch inhibitors in T cell leukemia.

Proc Natl Acad Sci U S A 2014 Nov 4;111(46):E4946-53. Epub 2014 Nov 4.

Abramson Family Cancer Research Institute, Department of Pathology and Laboratory Medicine, and

Notch is needed for T-cell development and is a common oncogenic driver in T-cell acute lymphoblastic leukemia. The protooncogene c-Myc (Myc) is a critical target of Notch in normal and malignant pre-T cells, but how Notch regulates Myc is unknown. Here, we identify a distal enhancer located >1 Mb 3' of human and murine Myc that binds Notch transcription complexes and physically interacts with the Myc proximal promoter. The Notch1 binding element in this region activates reporter genes in a Notch-dependent, cell-context-specific fashion that requires a conserved Notch complex binding site. Acute changes in Notch activation produce rapid changes in H3K27 acetylation across the entire enhancer (a region spanning >600 kb) that correlate with Myc expression. This broad Notch-influenced region comprises an enhancer region containing multiple domains, recognizable as discrete H3K27 acetylation peaks. Leukemia cells selected for resistance to Notch inhibitors express Myc despite epigenetic silencing of enhancer domains near the Notch transcription complex binding sites. Notch-independent expression of Myc in resistant cells is highly sensitive to inhibitors of bromodomain containing 4 (Brd4), a change in drug sensitivity that is accompanied by preferential association of the Myc promoter with more 3' enhancer domains that are strongly dependent on Brd4 for function. These findings indicate that altered long-range enhancer activity can mediate resistance to targeted therapies and provide a mechanistic rationale for combined targeting of Notch and Brd4 in leukemia.
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http://dx.doi.org/10.1073/pnas.1407079111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4246292PMC
November 2014

Discovery of biomarkers predictive of GSI response in triple-negative breast cancer and adenoid cystic carcinoma.

Cancer Discov 2014 Oct 7;4(10):1154-67. Epub 2014 Aug 7.

Merck Research Laboratory, Boston, Massachusetts.

Unlabelled: Next-generation sequencing was used to identify Notch mutations in a large collection of diverse solid tumors. NOTCH1 and NOTCH2 rearrangements leading to constitutive receptor activation were confined to triple-negative breast cancers (TNBC; 6 of 66 tumors). TNBC cell lines with NOTCH1 rearrangements associated with high levels of activated NOTCH1 (N1-ICD) were sensitive to the gamma-secretase inhibitor (GSI) MRK-003, both alone and in combination with paclitaxel, in vitro and in vivo, whereas cell lines with NOTCH2 rearrangements were resistant to GSI. Immunohistochemical staining of N1-ICD in TNBC xenografts correlated with responsiveness, and expression levels of the direct Notch target gene HES4 correlated with outcome in patients with TNBC. Activating NOTCH1 point mutations were also identified in other solid tumors, including adenoid cystic carcinoma (ACC). Notably, ACC primary tumor xenografts with activating NOTCH1 mutations and high N1-ICD levels were sensitive to GSI, whereas N1-ICD-low tumors without NOTCH1 mutations were resistant.

Significance: NOTCH1 mutations, immunohistochemical staining for activated NOTCH1, and HES4 expression are biomarkers that can be used to identify solid tumors that are likely to respond to GSI-based therapies.
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http://dx.doi.org/10.1158/2159-8290.CD-13-0830DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4184927PMC
October 2014

Identifying direct Notch transcriptional targets using the GSI-washout assay.

Methods Mol Biol 2014 ;1187:247-54

Department of Pathology and Laboratory Medicine, Abramson Family Cancer Research Institute, and Institute for Immunology, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, 19104, USA.

Genetic gain- and loss-of-function studies have traditionally been used to study transcriptional networks regulated by the Notch signaling pathway; however these techniques lack the ability to resolve primary and secondary transcriptional events. In contrast, the γ-secretase inhibitor (GSI) washout assay takes advantage of the reversibility of GSI, a pharmacological inhibitor of Notch signaling, along with the ability of cycloheximide to prevent secondary transcriptional effects to identify direct Notch pathway targets. Here we review this technique and the technical considerations for adapting this assay to a cell type of choice.
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http://dx.doi.org/10.1007/978-1-4939-1139-4_19DOI Listing
March 2015

Loss of oncogenic Notch1 with resistance to a PI3K inhibitor in T-cell leukaemia.

Nature 2014 Sep 20;513(7519):512-6. Epub 2014 Jul 20.

Department of Pediatrics and Benniof Children's Hospital, University of California, San Francisco, California 94143, USA.

Mutations that deregulate Notch1 and Ras/phosphoinositide 3 kinase (PI3K)/Akt signalling are prevalent in T-cell acute lymphoblastic leukaemia (T-ALL), and often coexist. Here we show that the PI3K inhibitor GDC-0941 is active against primary T-ALLs from wild-type and Kras(G12D) mice, and addition of the MEK inhibitor PD0325901 increases its efficacy. Mice invariably relapsed after treatment with drug-resistant clones, most of which unexpectedly had reduced levels of activated Notch1 protein, downregulated many Notch1 target genes, and exhibited cross-resistance to γ-secretase inhibitors. Multiple resistant primary T-ALLs that emerged in vivo did not contain somatic Notch1 mutations present in the parental leukaemia. Importantly, resistant clones upregulated PI3K signalling. Consistent with these data, inhibiting Notch1 activated the PI3K pathway, providing a likely mechanism for selection against oncogenic Notch1 signalling. These studies validate PI3K as a therapeutic target in T-ALL and raise the unexpected possibility that dual inhibition of PI3K and Notch1 signalling could promote drug resistance in T-ALL.
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http://dx.doi.org/10.1038/nature13495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4213126PMC
September 2014

Uterine Rbpj is required for embryonic-uterine orientation and decidual remodeling via Notch pathway-independent and -dependent mechanisms.

Cell Res 2014 Aug 27;24(8):925-42. Epub 2014 Jun 27.

State Key Laboratory of Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.

Coordinated uterine-embryonic axis formation and decidual remodeling are hallmarks of mammalian post-implantation embryo development. Embryonic-uterine orientation is determined at initial implantation and synchronized with decidual development. However, the molecular mechanisms controlling these events remain elusive despite its discovery a long time ago. In the present study, we found that uterine-specific deletion of Rbpj, the nuclear transducer of Notch signaling, resulted in abnormal embryonic-uterine orientation and decidual patterning at post-implantation stages, leading to substantial embryo loss. We further revealed that prior to embryo attachment, Rbpj confers on-time uterine lumen shape transformation via physically interacting with uterine estrogen receptor (ERα) in a Notch pathway-independent manner, which is essential for the initial establishment of embryo orientation in alignment with uterine axis. While at post-implantation stages, Rbpj directly regulates the expression of uterine matrix metalloproteinase in a Notch pathway-dependent manner, which is required for normal post-implantation decidual remodeling. These results demonstrate that uterine Rbpj is essential for normal embryo development via instructing the initial embryonic-uterine orientation and ensuring normal decidual patterning in a stage-specific manner. Our data also substantiate the concept that normal mammalian embryonic-uterine orientation requires proper guidance from developmentally controlled uterine signaling.
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http://dx.doi.org/10.1038/cr.2014.82DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4123295PMC
August 2014

The Notch1 transcriptional activation domain is required for development and reveals a novel role for Notch1 signaling in fetal hematopoietic stem cells.

Genes Dev 2014 Mar;28(6):576-93

Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA;

Notch1 is required to generate the earliest embryonic hematopoietic stem cells (HSCs); however since Notch-deficient embryos die early in gestation, additional functions for Notch in embryonic HSC biology have not been described. We used two complementary genetic models to address this important biological question. Unlike Notch1-deficient mice, mice lacking the conserved Notch1 transcriptional activation domain (TAD) show attenuated Notch1 function in vivo and survive until late gestation, succumbing to multiple cardiac abnormalities. Notch1 TAD-deficient HSCs emerge and successfully migrate to the fetal liver but are decreased in frequency by embryonic day 14.5. In addition, TAD-deficient fetal liver HSCs fail to compete with wild-type HSCs in bone marrow transplant experiments. This phenotype is independently recapitulated by conditional knockout of Rbpj, a core Notch pathway component. In vitro analysis of Notch1 TAD-deficient cells shows that the Notch1 TAD is important to properly assemble the Notch1/Rbpj/Maml trimolecular transcription complex. Together, these studies reveal an essential role for the Notch1 TAD in fetal development and identify important cell-autonomous functions for Notch1 signaling in fetal HSC homeostasis.
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http://dx.doi.org/10.1101/gad.227496.113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3967047PMC
March 2014