Notch Receptor Activation Research Articles

Engineering synthetic agonists for targeted activation of Notch signaling.

Notch signaling regulates cell fate decisions and has context-dependent tumorigenic or tumor suppressor functions. Although there are several classes of Notch inhibitors, the mechanical force requirement for Notch receptor activation has hindered attempts to generate soluble agonists. To address this problem, we engineered synthetic Notch agonist (SNAG) proteins by tethering affinity-matured Notch ligands to antibodies or cytokines that internalize their targets. This bispecific format enables SNAGs to "pull" on mechanosensitive Notch receptors, triggering their activation in the presence of a desired biomarker. We successfully developed SNAGs targeting six independent surface markers, including the tumor antigens PDL1, CD19, and HER2, and the immunostimulatory receptor CD40. SNAGs targeting CD40 increase expansion of central memory γδ T cells from peripheral blood, highlighting their potential to improve the phenotype and yield of low-abundance T cell subsets. These insights have broad implications for the pharmacological activation of mechanoreceptors and will expand our ability to modulate Notch signaling in biotechnology.

Temporal and Spatial Variations in Zebrafish Hairy/E(spl) Gene Expression in Response to Mib1-Mediated Notch Signaling During Neurodevelopment.

Notch signaling is a conserved pathway crucial for nervous system development. Disruptions in this pathway are linked to neurodevelopmental disorders, neurodegenerative diseases, and brain tumors. Hairy/E(spl) (HES) genes, major downstream targets of Notch, are commonly used as markers for Notch activation. However, these genes can be activated, inhibited, or function independently of Notch signaling, and their response to Notch disruption varies across tissues and developmental stages. MIB1/Mib1 is an E3 ubiquitin ligase that enables Notch receptor activation by processing ligands like Delta and Serrate. We investigated Notch signaling disruption using the zebrafish Mib1 mutant line, mib1ta52b, focusing on changes in the expression of Hairy/E(spl) (her) genes. Our findings reveal significant variability in her gene expression across different neural cell types, regions, and developmental stages following Notch disruption. This variability questions the reliability of Hairy/E(spl) genes as universal markers for Notch activation, as their response is highly context-dependent. This study highlights the complex and context-specific nature of Notch signaling regulation. It underscores the need for a nuanced approach when using Hairy/E(spl) genes as markers for Notch activity. Additionally, it provides new insights into Mib1's role in Notch signaling, contributing to a better understanding of its involvement in Notch signaling-related disorders.

Spatio-Temporal Regulation of Notch Activation in Asymmetrically Dividing Sensory Organ Precursor Cells in Drosophila melanogaster Epithelium.

The Notch communication pathway, discovered in Drosophila over 100 years ago, regulates a wide range of intra-lineage decisions in metazoans. The division of the Drosophila mechanosensory organ precursor is the archetype of asymmetric cell division in which differential Notch activation takes place at cytokinesis. Here, we review the molecular mechanisms by which epithelial cell polarity, cell cycle and intracellular trafficking participate in controlling the directionality, subcellular localization and temporality of mechanosensitive Notch receptor activation in cytokinesis.

Force-free activation of Notch with DNA origami

The Notch signaling pathway is a highly conserved, fundamental process to embryogenesis and neurogenesis. While force-induced conformational change is known to activate Notch receptors, Smyrlaki et al. recently used DNA origami to reveal an additional, force-independent mode of Notch activation via soluble presentation of spatially controlled ligand nanopatterns.

A magnetically powered nanomachine with a DNA clutch.

Machines found in nature and human-made machines share common components, such as an engine, and an output element, such as a rotor, linked by a clutch. This clutch, as seen in biological structures such as dynein, myosin or bacterial flagellar motors, allows for temporary disengagement of the moving parts from the running engine. However, such sophistication is still challenging to achieve in artificial nanomachines. Here we present a spherical rotary nanomotor with a reversible clutch system based on precise molecular recognition of built-in DNA strands. The clutch couples and decouples the engine from the machine's rotor in response to encoded inputs such as DNA or RNA. The nanomotor comprises a porous nanocage as a spherical rotor to confine the magnetic engine particle within the nanospace (∼0.004 μm3) of the cage. Thus, the entropically driven irreversible disintegration of the magnetic engine and the spherical rotor during the disengagement process is eliminated, and an exchange of microenvironmental inputs is possible through the nanopores. Our motor is only 200 nm in size and the clutch-mediated force transmission powered by an embedded ferromagnetic nanocrystal is high enough (∼15.5 pN at 50 mT) for the in vitro mechanical activation of Notch and integrin receptors, demonstrating its potential as nano-bio machinery.

Detection of native, activated Notch receptors in normal human apocrine-bearing skin and in hidradenitis suppurativa.

Notch signalling has generated considerable interest as a pathogenetic factor and a drug target in a range of human diseases. The gamma-secretase complex is crucial in the activation of Notch receptors by cleaving the intracellular domain allowing nuclear translocation. In recent years several mutations in gamma-secretase components have been discovered in patients with familial hidradenitis suppurativa (HS). This has led to hypotheses that impaired Notch signalling could be an important driver for HS in general, not only in the monogenic variants. However, no study has examined insitu Notch activation per se in HS, and some reports with conflicting results have instead been based on expression of Notch receptors or indirect measures of Notch target gene expression. In this study we established immunostaining protocols to identify native, activated Notch receptors in human skin tissue. The ability to detect changes in Notch activation was confirmed with an exvivo skin organ model in which signal was reduced or obliterated in tissue exposed to a gamma-secretase inhibitor. Using these methods on skin biopsies from healthy volunteers and a general HS cohort we demonstrated for the first time the distribution of active Notch signalling in human apocrine-bearing skin. Quantification of activated NOTCH1 & NOTCH2 revealed similar levels in non-lesional and peri-lesional HS to that of healthy controls, thus ruling out a general defect in Notch activation in HS patients. We did find a variable but significant reduction of activated Notch in epidermis of lesional HS with a distribution that appeared related to the extent of surrounding tissue inflammation.

Abstract A053: Notch completes CD4 T cell help in CD8 T cells and ensures timely departure

Abstract Efficient activation, expansion and differentiation of CD8 T cells depend on help from CD4+ T cells. Such help is especially critical for CD8 T cell responses against tumor antigens. Priming of CD8 T cells in lymph nodes (LN) involves two steps that are geographically and temporally separate. Naïve CD8 T cells are first preactivated by a migratory dendritic cell (DC) carrying antigen from tissues. Preactivated CD8 T cells must then find and interact with lymph node-resident XCR1+ DC that have been licensed for optimal CD8 T cell activation via CD4 T cell-mediated activation of CD40. The efferent signals provided by licensed CD8+XCR1+ DC to install the CD4 T cell help program in CD8 T cells are incompletely known. Moreover, it is not clear whether and, if so, how premature lymph node exit by preactivated CD8 T cells before receipt of help is avoided. Here, we show that completion of CD4 help delivery is coupled to the ability to leave lymph nodes through activation of Notch receptors on CD8 T cells. Expression of Notch receptors is induced on naïve CD8 T cells during the first step of priming, while CD40 ligation elicits expression of Notch ligands on XCR1+ lymph node resident DCs. Using a DNA vaccination model that allows induction of CD8 T cell responses in the presence or absence of CD4 T cell help, we show that the Notch1/2 receptors are necessary in CD8+ T cells for CD4+ T cell help-dependent proliferation and acquisition of effector capability. Correspondingly, RNA sequencing revealed that more than half of the genetic CD4 T cell help program in CD8 T cells depends on Notch. Strikingly, activated Notch1/2 deficient and unhelped CD8 T cells remain largely stuck in lymph nodes owing to a lack of expression of the lymph node exit receptor S1PR5. Control of expression of S1PR5 by Notch therefore ensures that preactivated CD8 T cells remain in lymph nodes until completion of their activation process by receiving the CD4 T cell help signal. Citation Format: Adrien Remi Gabriel Laurent, Julien Karrich, Nikolina Babala, Derk Amsen. Notch completes CD4 T cell help in CD8 T cells and ensures timely departure [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A053.

200 Detection of native, activated Notch receptors in normal human apocrine-bearing skin and in hidradenitis suppurativa

200 Detection of native, activated Notch receptors in normal human apocrine-bearing skin and in hidradenitis suppurativa

Modulation of Notch Signaling by Small-Molecular Compounds and Its Potential in Anticancer Studies.

Notch signaling is responsible for conveying messages between cells through direct contact, playing a pivotal role in tissue development and homeostasis. The modulation of Notch-related processes, such as cell growth, differentiation, viability, and cell fate, offer opportunities to better understand and prevent disease progression, including cancer. Currently, research efforts are mainly focused on attempts to inhibit Notch signaling in tumors with strong oncogenic, gain-of-function (GoF) or hyperactivation of Notch signaling. The goal is to reduce the growth and proliferation of cancer cells, interfere with neo-angiogenesis, increase chemosensitivity, potentially target cancer stem cells, tumor dormancy, and invasion, and induce apoptosis. Attempts to pharmacologically enhance or restore disturbed Notch signaling for anticancer therapies are less frequent. However, in some cancer types, such as squamous cell carcinomas, preferentially, loss-of-function (LoF) mutations have been confirmed, and restoring but not blocking Notch functions may be beneficial for therapy. The modulation of Notch signaling can be performed at several key levels related to NOTCH receptor expression, translation, posttranslational (proteolytic) processing, glycosylation, transport, and activation. This further includes blocking the interaction with Notch-related nuclear DNA transcription. Examples of small-molecular chemical compounds, that modulate individual elements of Notch signaling at the mentioned levels, have been described in the recent literature.

Iron-responsive element of Divalent metal transporter 1 (Dmt1) controls Notch-mediated cell fates.

Notch receptor activation is regulated by the intramembrane protease γ-secretase, which cleaves and liberates the Notch intracellular domain (Nicd) that regulates gene transcription. While γ-secretase cleavage is necessary, we demonstrate it is insufficient for Notch activation and requires vesicular trafficking. Here, we report Divalent metal transporter 1 (Dmt1, Slc11A2) as a novel and essential regulator of Notch signalling. Dmt1-deficient cells are defective in Notch signalling and have perturbed endolysosomal trafficking and function. Dmt1 encodes for two isoforms, with and without an iron response element (ire). We show that isoform-specific silencing of Dmt1-ire and Dmt1+ire has opposite consequences on Notch-dependent cell fates in cell lines and intestinal organoids. Loss of Dmt1-ire suppresses Notch activation and promotes differentiation, whereas loss of Dmt1+ire causes Notch activation and maintains stem-progenitor cell fates. Dmt1 isoform expression correlates with Notch and Wnt signalling in Apc-deficient intestinal organoids and human colorectal cancers. Consistently, Dmt1-ire silencing induces Notch-dependent differentiation in colorectal cancer cells. These data identify Dmt1 isoforms as binary switches controlling Notch cell fate decisions in normal and tumour cells.

Regulation of Notch1 Signalling by Long Non-Coding RNAs in Cancers and Other Health Disorders.

Notch1 signalling plays a multifaceted role in tissue development and homeostasis. Currently, due to the pivotal role of Notch1 signalling, the relationship between NOTCH1 expression and the development of health disorders is being intensively studied. Nevertheless, Notch1 signalling is not only controlled at the transcriptional level but also by a variety of post-translational events. First is the ligand-dependent mechanical activation of NOTCH receptors and then the intracellular crosstalk with other signalling molecules-among those are long non-coding RNAs (lncRNAs). In this review, we provide a detailed overview of the specific role of lncRNAs in the modulation of Notch1 signalling, from expression to activity, and their connection with the development of health disorders, especially cancers.

A molecular guide to systemic therapy in salivary gland carcinoma.

Salivary gland carcinomas (SGC) are a rare and variable group of head and neck cancers with historically poor response to cytotoxic chemotherapy and immunotherapy in the recurrent, advanced, and metastatic settings. In the last decade, a number of targetable molecular alterations have been identified in SGCs including HER2 upregulation, androgen receptor overexpression, Notch receptor activation, NTRK gene fusions, and RET alterations which have dramatically improved treatment outcomes in this disease. Here, we review the landscape of precision therapy in SGC including current options for systemic management, ongoing clinical trials, and promising future directions.

P53 regulates the effects of DAPT on Rac1 activation and migration of non-small-cell lung cancer cells

The use of γ-secretase inhibitors to inhibit the activation of Notch receptors can effectively inhibit the malignant process of tumors. Here, we demonstrate that p53 can modulate the effect of DAPT (a γ-secretase inhibitor) on the activation of small GTPase Rac1, thereby affecting cell migration of non-small-cell lung cancer H1299 and A549 cells. After treatment with 20 μM DAPT, activation of Rac1 was increased in H1299 cells but not in A549 cells. We further found that the migration ability of H1299 cells was increased, whereas that of A549 cells was reduced. The effect of DAPT on H1299 migration was repressed by Rac1-T17N, a dominant inactivated mutant of Rac1. H1299 is a p53-deficient cell line. When p53 protein was overexpressed in H1299 cells with a pEGFP-p53 plasmid, DAPT treatment no longer activated Rac1 and increased migration ability. Moreover, DAPT promoted the migration of H1299 cells by increasing the activity of Rac1 through the non-canonical Notch pathway. Taken together, these results indicate that the expression of p53 protein in lung cancer cells regulates the effect of DAPT on cell migration by modulating the activation of Rac1, suggesting that p53 may affect the therapeutic effects of Notch inhibitors in lung cancer patients.

Distinct and redundant roles for zebrafish her genes during mineralization and craniofacial patterning.

The Notch pathway is a cell-cell communication system which is critical for many developmental processes, including craniofacial development. Notch receptor activation induces expression of several well-known canonical targets including those encoded by the hes and her genes in mammals and zebrafish, respectively. The function of these genes, individually and in combination, during craniofacial development is not well understood. Here, we used zebrafish genetics to investigate her9 and her6 gene function during craniofacial development. We found that her9 is required for osteoblasts to efficiently mineralize bone, while cartilage is largely unaffected. Strikingly, gene expression studies in her9 mutants indicate that although progenitor cells differentiate into osteoblasts at the appropriate time and place, they fail to efficiently lay down mineralized matrix. This mineralization role of her9 is likely independent of Notch activation. In contrast, her9 also functions redundantly with her6 downstream of Jagged1b-induced Notch activation during dorsoventral craniofacial patterning. These studies disentangle distinct and redundant her gene functions during craniofacial development, including an unexpected, Notch independent, requirement during bone mineralization.

Induction of Human T Cell Development In Vitro with OP9-DL4-7FS Cells Expressing Human Cytokines.

For nearly a generation now, OP9-DL1 and OP9-DL4 cells have provided an efficient and reliable cell system to generate T cells from mouse and human hematopoietic stem cells (HSCs) and pluripotent stem cells. OP9-DL1 and OP9-DL4 were originally derived from the OP9 mouse bone marrow stromal cell line, which was transduced to ectopically express Delta-like 1 or 4 proteins, respectively. OP9-DL cells mimic the thymic microenvironment in that when cocultured with mouse or human (h) HSCs, they interact with and activate Notch receptors present on HSCs, required for T cell differentiation. The HSC/OP9-DL cocultures require additional cytokines that are necessary for survival and proliferation of hematopoietic cells. For hHSCs, these factors are interleukin-7 (IL-7), stem cell factor (SCF), and FMS-like tyrosine kinase 3 ligand (FLT3L) that are normally exogenously added to the cocultures. In this chapter, we describe methods for establishing a novel and improved version of OP9-DL4 cells, called OP9-DL4-7FS cells that circumvent the addition of these costly cytokines, by transducing OP9-DL4 cell line to express human IL-7, FLT3L, and SCF (7FS). Herein, we describe the protocol for the generation of OP9-DL4-7FS cells and the conditions for OP9-DL4-7FS/hHSC coculture to support T cell lineage initiation and expansion while comparing it to the now "classic" OP9-DL4 coculture. The use of OP9-DL4-7FS cell system will provide an improved and cost-effective method to the commonly used OP9-DL/HSC coculture for studying both mouse and human T cell development.

Hydroxylation of the NOTCH1 intracellular domain regulates Notch signaling dynamics

Notch signaling plays a pivotal role in the development and, when dysregulated, it contributes to tumorigenesis. The amplitude and duration of the Notch response depend on the posttranslational modifications (PTMs) of the activated NOTCH receptor – the NOTCH intracellular domain (NICD). In normoxic conditions, the hydroxylase FIH (factor inhibiting HIF) catalyzes the hydroxylation of two asparagine residues of the NICD. Here, we investigate how Notch-dependent gene transcription is regulated by hypoxia in progenitor T cells. We show that the majority of Notch target genes are downregulated upon hypoxia. Using a hydroxyl-specific NOTCH1 antibody we demonstrate that FIH-mediated NICD1 hydroxylation is reduced upon hypoxia or treatment with the hydroxylase inhibitor dimethyloxalylglycine (DMOG). We find that a hydroxylation-resistant NICD1 mutant is functionally impaired and more ubiquitinated. Interestingly, we also observe that the NICD1-deubiquitinating enzyme USP10 is downregulated upon hypoxia. Moreover, the interaction between the hydroxylation-defective NICD1 mutant and USP10 is significantly reduced compared to the NICD1 wild-type counterpart. Together, our data suggest that FIH hydroxylates NICD1 in normoxic conditions, leading to the recruitment of USP10 and subsequent NICD1 deubiquitination and stabilization. In hypoxia, this regulatory loop is disrupted, causing a dampened Notch response.

NOTCH gene alterations in metastatic colorectal cancer in the Nationwide Cancer Genome Screening Project in Japan (SCRUM-Japan GI-SCREEN).

Activated Notch receptor signaling has been implicated in tumor growth and progression in colorectal cancer (CRC). However, the pathogenic relevance of NOTCH gene alterations remains unclear. The aim of this study was to clarify mutational landscapes and assess their clinical significance in patients with metastatic CRC. Pre-chemotherapy tumor tissues obtained from 1154 metastatic CRC patients in the Nationwide Cancer Genome Screening Project in Japan between April 2017 and March 2019 were studied using the Oncomine Comprehensive Assay. The frequencies of NOTCH1, NOTCH2, and NOTCH3 nonsynonymous sequence variants were 11.5%, 4.4%, and 10.4%, respectively. The majority of variants were missense of unknown significance that were distributed across all domains of all three NOTCH genes. The gain-of-function mutations in NOTCH reported in multiple malignancies were not identified. The NOTCH amplification rate was less than 1%. No NOTCH fusions were detected. In patients who were registered before, or within 1year of, first-line chemotherapy, overall survival for 51 patients with only NOTCH3 variants was significantly longer than for 540 patients with no NOTCH variants (median, 40.2months vs 27.7months; P = 0.04). Multivariate analysis revealed that variant NOTCH3 was an independent prognostic factor for increased survival (hazard ratio 0.61, 95% confidence interval, 0.39-0.94; P = 0.03) besides poor prognostic factors associated with mutant TP53, KRAS, and BRAF, as well as amplified MYC. NOTCH genes are unlikely to harbor driver mutations and amplifications in patients with metastatic CRC. NOTCH3 variant should be further investigated as a favorable prognostic marker.

Notch-dependent and -independent transcription are modulated by tissue movements at gastrulation.

Cells sense and integrate external information from diverse sources that include mechanical cues. Shaping of tissues during development may thus require coordination between mechanical forces from morphogenesis and cell-cell signalling to confer appropriate changes in gene expression. By live-imaging Notch-induced transcription in real time, we have discovered that morphogenetic movements during Drosophila gastrulation bring about an increase in activity-levels of a Notch-responsive enhancer. Mutations that disrupt the timing of gastrulation resulted in concomitant delays in transcription up-regulation that correlated with the start of mesoderm invagination. As a similar gastrulation-induced effect was detected when transcription was elicited by the intracellular domain NICD, it cannot be attributed to forces exerted on Notch receptor activation. A Notch-independent vnd enhancer also exhibited a modest gastrulation-induced activity increase in the same stripe of cells. Together, these observations argue that gastrulation-associated forces act on the nucleus to modulate transcription levels. This regulation was uncoupled when the complex linking the nucleoskeleton and cytoskeleton (LINC) was disrupted, indicating a likely conduit. We propose that the coupling between tissue-level mechanics, arising from gastrulation, and enhancer activity represents a general mechanism for ensuring correct tissue specification during development and that Notch-dependent enhancers are highly sensitive to this regulation.

Notch1 deficiency alters the migratory behavior of developing T cells and calcium signaling in the thymus of medaka.

The differentiation of T cells from lymphoid progenitors in the thymus follows sequential developmental stages that constantly require interaction with thymic epithelial cells. Several distinct aspects of early T cell development depend on the activation of Notch receptors on thymocytes, while the selection of thymocytes at later stages are believed to be Notch independent. Using reverse genetic approaches and whole-thymus live imaging in an in vivo teleost model, the medaka, we report that Notch1 signals is required for proliferation and specification of developing T cells as well as involved in their selection in the thymus. We reveal that Notch1 controls the migratory behavior of thymocytes through controlling the chemokine receptor Ccr9b and thereby influence the T cell receptor (TCR) activation. Hence, we propose that, in lower vertebrates, the function of Notch signaling extends to all stages of T cell development, except when thymocytes undergo TCRβ rearrangement.

The Membrane-Bound Notch Regulator Mnr Supports Notch Cleavage and Signaling Activity in Drosophila melanogaster.

The Notch signaling pathway is pivotal to cellular differentiation. Activation of this pathway involves proteolysis of the Notch receptor and the release of the biologically active Notch intracellular domain, acting as a transcriptional co-activator of Notch target genes. While the regulation of Notch signaling dynamics at the level of ligand–receptor interaction, endocytosis, and transcriptional regulation has been well studied, little is known about factors influencing Notch cleavage. We identified EP555 as a suppressor of the Notch antagonist Hairless (H). EP555 drives expression of CG32521 encoding membrane-bound proteins, which we accordingly rename membrane-bound Notch regulator (mnr). Within the signal-receiving cell, upregulation of Mnr stimulates Notch receptor activation, whereas a knockdown reduces it, without apparent influence on ligand–receptor interaction. We provide evidence that Mnr plays a role in γ-secretase-mediated intramembrane cleavage of the Notch receptor. As revealed by a fly-eye-based reporter system, γ-secretase activity is stimulated by the overexpression of Mnr, and is inhibited by its knockdown. We conclude that Mnr proteins support Notch signaling activity by fostering the cleavage of the Notch receptor. With Mnr, we identified a membrane-bound factor directly augmenting Notch intra-membrane processing, thereby acting as a positive regulator of Notch signaling activity.