An optogenetic system for precise spatiotemporal control of notch signaling activity

Notch signaling activity governs widespread cellular differentiation in higher animals, including humans, and is involved in several congenital diseases and different forms of cancer. Notch signals are mediated by the transcriptional regulator RBPJ in a complex with activated Notch (NICD). Analysis of Notch pathway regulation in humans is hampered by a partial redundancy of the four Notch receptor copies, yet RBPJ is solitary, allowing its study in model systems. In Drosophila melanogaster, the RBPJ orthologue is encoded by Suppressor of Hairless [Su(H)]. Using genome engineering, we replaced Su(H) by murine RBPJ in order to study its function in the fly. In fact, RBPJ largely substitutes for Su(H)’s function, yet subtle phenotypes reflect increased Notch signaling activity. Accordingly, the binding of RBPJ to Hairless (H) protein, the general Notch antagonist in Drosophila, was considerably reduced compared to that of Su(H). An H-binding defective RBPJLLL mutant matched the respective Su(H)LLL allele: homozygotes were lethal due to extensive Notch hyperactivity. Moreover, RBPJLLL protein accumulated at lower levels than wild type RBPJ, except in the presence of NICD. Apparently, RBPJ protein stability depends on protein complex formation with either H or NICD, similar to Su(H), demonstrating that the murine homologue underlies the same regulatory mechanisms as Su(H) in Drosophila. These results underscore the importance of regulating the availability of RBPJ protein to correctly mediate Notch signaling activity in the fly.

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Identification of Domains for Efficient Notch Signaling Activity in Immobilized Notch Ligand Proteins

Journal of Cellular Biochemistry ◽

10.1002/jcb.25744 ◽

2016 ◽

Vol 118 (4) ◽

pp. 785-796 ◽

Cited By ~ 8

Author(s):

Ledi Liu ◽

Hiroe Wada ◽

Natsuki Matsubara ◽

Katsuto Hozumi ◽

Motoyuki Itoh

Keyword(s):

Notch Signaling ◽

Notch Ligand ◽

Signaling Activity

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Notchless encodes a novel WD40-repeat-containing protein that modulates Notch signaling activity

The EMBO Journal ◽

10.1093/emboj/17.24.7351 ◽

1998 ◽

Vol 17 (24) ◽

pp. 7351-7360 ◽

Cited By ~ 46

Author(s):

J. Royet

Keyword(s):

Notch Signaling ◽

Wd40 Repeat ◽

Signaling Activity

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TRPM7 Induces Tumorigenesis and Stemness Through Notch Activation in Glioma

Frontiers in Pharmacology ◽

10.3389/fphar.2020.590723 ◽

2020 ◽

Vol 11 ◽

Author(s):

Jingwei Wan ◽

Alyssa Aihui Guo ◽

Pendelton King ◽

Shanchun Guo ◽

Talib Saafir ◽

...

Keyword(s):

Cell Cycle ◽

Cell Growth ◽

Notch Signaling ◽

Glioma Cell ◽

Kinase Domain ◽

Normal Brain ◽

Diffuse Astrocytoma ◽

Notch1 Signaling ◽

Aldh1 Expression ◽

Signaling Activity

We have reported that transient receptor potential melastatin-related 7 (TRPM7) regulates glioma stem cells (GSC) growth and proliferation through Notch, STAT3-ALDH1, and CD133 signaling pathways. In this study, we determined the major contributor(s) to TRPM7 mediated glioma stemness by further deciphering each individual Notch signaling. We first determined whether TRPM7 is an oncotarget in glioblastoma multiforme (GBM) using the Oncomine database. Next, we determined whether TRPM7 silencing by siRNA TRPM7 (siTRPM7) induces cell growth arrest or apoptosis to reduce glioma cell proliferation using cell cycle analysis and annexin V staining assay. We then examined the correlations between the expression of TRPM7 and Notch signaling activity as well as the expression of GSC markers CD133 and ALDH1 in GBM by downregulating TRPM7 through siTRPM7 or upregulating TRPM7 through overexpression of human TRPM7 (M7-wt). To distinguish the different function of channel and kinase domain of TRPM7, we further determined how the α-kinase-dead mutants of TRPM7 (α-kinase domain deleted/M7-DK and K1648R point mutation/M7-KR) affect Notch activities and CD133 and ALDH1 expression. Lastly, we determined the changes in TRPM7-mediated regulation of glioma cell growth/proliferation, cell cycle, and apoptosis by targeting Notch1. The Oncomine data revealed a significant increase in TRPM7 mRNA expression in anaplastic astrocytoma, diffuse astrocytoma, and GBM patients compared to that in normal brain tissues. TRPM7 silencing reduced glioma cell growth by inhibiting cell entry into S and G2/M phases and promoting cell apoptosis. TRPM7 expression in GBM cells was found to be positively correlated with Notch1 signaling activity and CD133 and ALDH1 expression; briefly, downregulation of TRPM7 by siTRPM7 decreased Notch1 signaling whereas upregulation of TRPM7 increased Notch1 signaling. Interestingly, kinase-inactive mutants (M7-DK and M7-KR) resulted in reduced activation of Notch1 signaling and decreased expression of CD133 and ALDH1 compared to that of wtTRPM7. Finally, targeting Notch1 effectively suppressed TRPM7-induced growth and proliferation of glioma cells through cell G1/S arrest and apoptotic induction. TRPM7 is responsible for sustained Notch1 signaling activation, enhanced expression of GSC markers CD133 and ALDH1, and regulation of glioma stemness, which contributes to malignant glioma cell growth and invasion.

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Regulation of Notch Signaling Activity

Current Biology ◽

10.1016/j.cub.2004.01.023 ◽

2004 ◽

Vol 14 (3) ◽

pp. R129-R138 ◽

Cited By ~ 232

Author(s):

François Schweisguth

Keyword(s):

Notch Signaling ◽

Signaling Activity

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A computational guided, functional validation of a novel therapeutic antibody proposes Notch signaling as a clinical relevant and druggable target in glioma

Scientific Reports ◽

10.1038/s41598-020-72480-y ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 1

Author(s):

Dayana Herrera-Rios ◽

Guanzhang Li ◽

Dilaware Khan ◽

Julia Tsiampali ◽

Ann-Christin Nickel ◽

...

Keyword(s):

Stem Cell ◽

Cell Signaling ◽

Notch Signaling ◽

Notch Pathway ◽

Therapy Resistance ◽

Drug Candidate ◽

Severe Toxicity ◽

Gamma Secretase ◽

Signaling Activity ◽

Notch1 Receptor

Abstract The Notch signaling network determines stemness in various tissues and targeting signaling activity in malignant brain cancers by gamma-secretase inhibitors (GSI) has shown promising preclinical success. However, the clinical translation remains challenging due to severe toxicity side effects and emergence of therapy resistance. Better anti-Notch directed therapies, specifically directed against the tumor promoting Notch receptor 1 signaling framework, and biomarkers predicting response to such therapy are of highest clinical need. We assessed multiple patient datasets to probe the clinical relevance Notch1 activation and possible differential distribution amongst molecular subtypes in brain cancers. We functionally assessed the biological effects of the first-in-human tested blocking antibody against Notch1 receptor (brontictuzumab, BRON) in a collection of glioma stem-like cell (GSC) models and compared its effects to genetic Notch1 inhibition as well as classical pharmacological Notch inhibitor treatment using gamma-secretase inhibitor MRK003. We also assess effects on Wingless (WNT) stem cell signaling activation, which includes the interrogation of genetic WNT inhibition models. Our computed transcriptional Notch pathway activation score is upregulated in neural stem cells, as compared to astrocytes; as well as in GSCs, as compared to differentiated glioblastoma cells. Moreover, the Notch signature is clinical predictive in our glioblastoma patient discovery and validation cohort. Notch signature is significantly increased in tumors with mutant IDH1 genome and tumors without 1p and 19q co-deletion. In GSCs with elevated Notch1 expression, BRON treatment blocks transcription of Notch pathway target genes Hes1/Hey1, significantly reduced the amount of cleaved Notch1 receptor protein and caused significantly impairment of cellular invasion. Benchmarking this phenotype to those observed with genetic Notch1 inhibition in corresponding cell models did result in higher reduction of cell invasion under chemotherapy. BRON treatment caused signs of upregulation of Wingless (WNT) stem cell signaling activity, and vice versa, blockage of WNT signaling caused induction of Notch target gene expression in our models. We extend the list of evidences that elevated Notch signal expression is a biomarker signature declaring stem cell prevalence and useful for predicting negative clinical course in glioblastoma. By using functional assays, we validated a first in man tested Notch1 receptor specific antibody as a promising drug candidate in the context of neuro oncology and propose biomarker panel to predict resistance and therapy success of this treatment option. We note that the observed phenotype seems only in part due to Notch1 blockage and the drug candidate leads to activation of off target signals. Further studies addressing a possible emergence of therapy resistance due to WNT activation need to be conducted. We further validated our 3D disease modeling technology to be of benefit for drug development projects.

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Glycogen synthase kinase 3β inhibition enhances Notch1 recycling

Molecular Biology of the Cell ◽

10.1091/mbc.e17-07-0474 ◽

2018 ◽

Vol 29 (4) ◽

pp. 389-395 ◽

Cited By ~ 5

Author(s):

Li Zheng ◽

Sean D. Conner

Keyword(s):

Notch Signaling ◽

Signaling Pathway ◽

Receptor Cell ◽

Glycogen Synthase ◽

Critical Role ◽

Notch Signaling Pathway ◽

Receptor Trafficking ◽

Receptor Recycling ◽

Extracellular Signals ◽

Signaling Activity

The Notch signaling pathway is essential throughout development and remains active into adulthood, where it performs a critical role in tissue homeostasis. The fact that defects in signaling can lead to malignancy illustrates the need to control Notch activity tightly. GSK3β is an established regulator of the Notch signaling pathway, although its mechanism of action remains unclear. Given the emerging role for GSK3β in receptor trafficking, we tested the idea that GSK3β controls signaling by regulating Notch transport. Consistent with published reports, we find that GSK3β inhibition enhances Notch1 signaling activity. Immunolocalization analysis reveals that Notch1 localization within a tubulovesicular compartment is altered when GSK3β activity is disrupted. We also find that receptor cell surface levels increase following acute GSK3β inhibition. This is followed by elevated Notch intracellular domain (NICD) production and a corresponding increase in signaling activity. Moreover, Notch transport assays reveal that receptor recycling rates increase when GSK3β activity is inhibited. Collectively, results presented here support a model where GSK3β regulates signaling by controlling postendocytic transport of Notch1. Given that GSK3β activity is suppressed following stimulation by multiple signal transduction pathways, our findings also suggest that cells can modulate Notch1 activity in response to extracellular signals by mobilizing Notch1 from endosomal stores.

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