A Test to Quantify NOTCH Pathway Activity in T Cell Acute Lymphoblastic Leukemia Patients

Background The NOTCH signaling pathway is pivotal for various physiological processes including immune responses, and has been implicated in the pathogenesis in many diseases including T-cell acute lymphoblastic leukemia (T-ALL). Over 70% of T-ALL patient samples contain mutations in NOTCH1 and/or FBXW7 that result in the activation of the NOTCH pathway. Various targeted drugs are available that inhibit NOTCH signaling, but their effectiveness varies due to variable NOTCH pathway activities among individual patients. Moreover, patients' leukemic cells that lack these mutations may still require NOTCH signaling. A quick and robust quantification of NOTCH pathway activity in primary patient samples would identify patients who could benefit from NOTCH targeted treatment. Aims In primary human T-ALL samples, we aimed to determine the NOTCH pathway activity in relation to active, intracellular NOTCH1 (ICN1) levels and in relation to NOTCH1 and/or FBXW7 mutations. Additionally, we investigated whether the NOTCH pathway activity score is more accurate than a mutation-based activity prediction. Methods Our test to assess functional NOTCH pathway activity in various cell types was applied to primary human T-ALL samples. The NOTCH test infers a quantitative NOTCH pathway activity score from mRNA levels of conserved direct NOTCH target genes based on a Bayesian network model. This model describes the causal relation between up- or downregulation of NOTCH target genes and the presence of an active or inactive NOTCH transcription complex. The Bayesian model was calibrated on publically available Affymetrix U133 Plus2.0 microarray datasets of samples with an active or inactive NOTCH pathway. Following validation on multiple cell types and malignancies, we scored NOTCH pathway activation in our well-characterized cohort of 117 T-ALL patient samples and related it to clinical and biological parameters including outcome. Results The NOTCH pathway model was calibrated using a microarray dataset containing high-grade serous ovarian cancer-which has high NOTCH activity-and normal ovarian tissue samples that lack NOTCH activity. Validation of the test using datasets from primary cells and cell lines of various origins revealed that it measures the NOTCH activity status in different cellular contexts. In primary diagnostic T-ALL samples, we observed a significant relationship between NOTCH pathway activity scores and active, intracellular cleaved NOTCH1 (ICN1) protein levels and the presence of NOTCH1-activating mutations. We next scored NOTCH pathway activity over the four T-ALL subgroups ETP-ALL, TLX, Proliferative and TALLMO. The TLX subgroup had the highest NOTCH activity levels compared to the other subgroups, consistent with the high percentage of TLX cases with NOTCH1/FBXW7 mutations. Strikingly, the significance of the correlation between ICN1 levels and NOTCH pathway activity was mainly attributed to the strong NOTCH1-activating mutations that include NOTCH1 juxtamembrane domain mutations, or hetero-dimerization mutations combined with PEST domain or FBXW7 mutations. When assessing the event-free survival and relapse-free survival curves, we observed that patients with the lowest (lower than the 25th-percentile) NOTCH pathway activity scores had the shortest event-free survival compared to the others (p<0.05, log-rank test). Summary/Conclusion High NOTCH pathway activation was mostly present in-but not limited to-T-ALL samples harboring strong NOTCH1 mutations, indicating that additional mechanisms can activate NOTCH signaling. Combined with the observation that the measured NOTCH pathway activity relates to ICN1 protein levels, this indicates that the pathway activity score more accurately reflects NOTCH pathway activity than the predicted activity based on NOTCH1 mutations alone. Disclosures Holtzer: Philips Research: Employment. Verhaegh:Philips Research: Employment. van de Stolpe:The Netherlands: Employment; Eindhoven: Employment; Philips Research: Employment.

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A Molecular Test for Quantifying Functional Notch Signaling Pathway Activity in Human Cancer

Cancers ◽

10.3390/cancers12113142 ◽

2020 ◽

Vol 12 (11) ◽

pp. 3142

Author(s):

Kirsten Canté-Barrett ◽

Laurent Holtzer ◽

Henk van Ooijen ◽

Rico Hagelaar ◽

Valentina Cordo’ ◽

...

Keyword(s):

Notch Signaling ◽

Target Genes ◽

Human Cancer ◽

Notch Pathway ◽

Event Free Survival ◽

Free Survival ◽

Pathway Activity ◽

Notch Activity ◽

Activating Mutations ◽

Notch1 Mutations

Background: The Notch signal transduction pathway is pivotal for various physiological processes, including immune responses, and has been implicated in the pathogenesis of many diseases. The effectiveness of various targeted Notch pathway inhibitors may vary due to variabilities in Notch pathway activity among individual patients. The quantitative measurement of Notch pathway activity is therefore essential to identify patients who could benefit from targeted treatment. Methods: We here describe a new assay that infers a quantitative Notch pathway activity score from the mRNA levels of generally conserved direct NOTCH target genes. Following the calibration and biological validation of our Notch pathway activity model over a wide spectrum of human cancer types, we assessed Notch pathway activity in a cohort of T-ALL patient samples and related it to biological and clinical parameters, including outcome. Results: We developed an assay using 18 select direct target genes and high-grade serous ovarian cancer for calibration. For validation, seven independent human datasets (mostly cancer series) were used to quantify Notch activity in agreement with expectations. For T-ALL, the median Notch pathway activity was highest for samples with strong NOTCH1-activating mutations, and T-ALL patients of the TLX subtype generally had the highest levels of Notch pathway activity. We observed a significant relationship between ICN1 levels and the absence/presence of NOTCH1-activating mutations with Notch pathway activity scores. Patients with the lowest Notch activity scores had the shortest event-free survival compared to other patients. Conclusions: High Notch pathway activity was not limited to T-ALL samples harboring strong NOTCH1 mutations, including juxtamembrane domain mutations or hetero-dimerization combined with PEST-domain or FBXW7 mutations, indicating that additional mechanisms may activate Notch signaling. The measured Notch pathway activity was related to intracellular NOTCH levels, indicating that the pathway activity score more accurately reflects Notch pathway activity than when it is predicted on the basis of NOTCH1 mutations. Importantly, patients with low Notch pathway activity had a significantly shorter event-free survival compared to patients showing higher activity.

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Development of a Notch pathway assay and quantification of functional Notch pathway activity in T-cell acute lymphoblastic leukemia

10.1101/2020.07.10.183731 ◽

2020 ◽

Cited By ~ 1

Author(s):

Kirsten Canté-Barrett ◽

Laurent Holtzer ◽

Henk van Ooijen ◽

Rico Hagelaar ◽

Valentina Cordo ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Notch Pathway ◽

Signal Transduction Pathway ◽

Activity Score ◽

Mrna Levels ◽

Pathway Activity ◽

Cell Acute Lymphoblastic Leukemia

AbstractThe Notch signal transduction pathway is pivotal for various physiological processes including immune responses, and has been implicated in the pathogenesis of many diseases including T-cell acute lymphoblastic leukemia. Various targeted drugs are available that inhibit Notch pathway signaling, but their effectiveness varies due to variable Notch pathway activity among individual patients. Quantitative measurement of Notch pathway activity is therefore essential to identify patients who could benefit from targeted treatment. We here describe a new assay that infers a quantitative Notch pathway activity score from mRNA levels of conserved direct NOTCH target genes. Following biological validation, we assessed Notch pathway activity in a cohort of TALL patient samples and related it to biological and clinical parameters including outcome. High Notch pathway activity was not limited to T-ALL samples harbouring strong NOTCH1 mutations, including juxtamembrane domain mutations or hetero-dimerization combined with PEST-domain or FBXW7 mutations, indicating that additional mechanisms may activate NOTCH signaling. The measured Notch pathway activity related to intracellular NOTCH levels, indicating that the pathway activity score more accurately reflects Notch pathway activity than predicted on the basis of NOTCH1 mutations. Importantly, patients with low Notch pathway activity had a significantly shorter event-free survival compared to patients showing higher activity.

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Notch restricts lymphatic vessel sprouting induced by vascular endothelial growth factor

Blood ◽

10.1182/blood-2010-11-317800 ◽

2011 ◽

Vol 118 (4) ◽

pp. 1154-1162 ◽

Cited By ~ 73

Author(s):

Wei Zheng ◽

Tuomas Tammela ◽

Masahiro Yamamoto ◽

Andrey Anisimov ◽

Tanja Holopainen ◽

...

Keyword(s):

Notch Signaling ◽

Cell Fate ◽

Target Genes ◽

Notch Pathway ◽

Lymphatic Vessels ◽

Soluble Form ◽

Fibrin Gel ◽

3 Dimensional ◽

Endothelial Growth Factor

Abstract Notch signaling plays a central role in cell-fate determination, and its role in lateral inhibition in angiogenic sprouting is well established. However, the role of Notch signaling in lymphangiogenesis, the growth of lymphatic vessels, is poorly understood. Here we demonstrate Notch pathway activity in lymphatic endothelial cells (LECs), as well as induction of delta-like ligand 4 (Dll4) and Notch target genes on stimulation with VEGF or VEGF-C. Suppression of Notch signaling by a soluble form of Dll4 (Dll4-Fc) synergized with VEGF in inducing LEC sprouting in 3-dimensional (3D) fibrin gel assays. Expression of Dll4-Fc in adult mouse ears promoted lymphangiogenesis, which was augmented by coexpressing VEGF. Lymphangiogenesis triggered by Notch inhibition was suppressed by a monoclonal VEGFR-2 Ab as well as soluble VEGF and VEGF-C/VEGF-D ligand traps. LECs transduced with Dll4 preferentially adopted the tip cell position over nontransduced cells in 3D sprouting assays, suggesting an analogous role for Dll4/Notch in lymphatic and blood vessel sprouting. These results indicate that the Notch pathway controls lymphatic endothelial quiescence, and explain why LECs are poorly responsive to VEGF compared with VEGF-C. Understanding the role of the Notch pathway in lymphangiogenesis provides further insight for the therapeutic manipulation of the lymphatic vessels.

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Serrate and Notch specify cell fates in the heart field by suppressing cardiomyogenesis

Development ◽

10.1242/dev.127.17.3865 ◽

2000 ◽

Vol 127 (17) ◽

pp. 3865-3876

Author(s):

M.S. Rones ◽

K.A. McLaughlin ◽

M. Raffin ◽

M. Mercola

Keyword(s):

Gene Expression ◽

Notch Signaling ◽

Cell Fate ◽

Notch Pathway ◽

Cell Types ◽

Myocardial Cell ◽

Dominant Negative ◽

Cell Fates ◽

Cell Fate Decisions ◽

Heart Field

Notch signaling mediates numerous developmental cell fate decisions in organisms ranging from flies to humans, resulting in the generation of multiple cell types from equipotential precursors. In this paper, we present evidence that activation of Notch by its ligand Serrate apportions myogenic and non-myogenic cell fates within the early Xenopus heart field. The crescent-shaped field of heart mesoderm is specified initially as cardiomyogenic. While the ventral region of the field forms the myocardial tube, the dorsolateral portions lose myogenic potency and form the dorsal mesocardium and pericardial roof (Raffin, M., Leong, L. M., Rones, M. S., Sparrow, D., Mohun, T. and Mercola, M. (2000) Dev. Biol., 218, 326–340). The local interactions that establish or maintain the distinct myocardial and non-myocardial domains have never been described. Here we show that Xenopus Notch1 (Xotch) and Serrate1 are expressed in overlapping patterns in the early heart field. Conditional activation or inhibition of the Notch pathway with inducible dominant negative or active forms of the RBP-J/Suppressor of Hairless [Su(H)] transcription factor indicated that activation of Notch feeds back on Serrate1 gene expression to localize transcripts more dorsolaterally than those of Notch1, with overlap in the region of the developing mesocardium. Moreover, Notch pathway activation decreased myocardial gene expression and increased expression of a marker of the mesocardium and pericardial roof, whereas inhibition of Notch signaling had the opposite effect. Activation or inhibition of Notch also regulated contribution of individual cells to the myocardium. Importantly, expression of Nkx2. 5 and Gata4 remained largely unaffected, indicating that Notch signaling functions downstream of heart field specification. We conclude that Notch signaling through Su(H) suppresses cardiomyogenesis and that this activity is essential for the correct specification of myocardial and non-myocardial cell fates.

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Vertebrate retinal ganglion cells are selected from competent progenitors by the action of Notch

Development ◽

10.1242/dev.121.11.3637 ◽

1995 ◽

Vol 121 (11) ◽

pp. 3637-3650 ◽

Cited By ~ 33

Author(s):

C.P. Austin ◽

D.E. Feldman ◽

J.A. Ida ◽

C.L. Cepko

Keyword(s):

Cell Fate ◽

Ganglion Cells ◽

Notch Pathway ◽

Cell Types ◽

Projection Neurons ◽

Specific Cell ◽

Vitro Assay ◽

Notch Activity

The first cells generated during development of the vertebrate retina are the ganglion cells, the projection neurons of the retina. Although they are one of the most intensively studied cell types within the central nervous system, little is known of the mechanisms that determine ganglion cell fate. We demonstrate that ganglion cells are selected from a large group of competent progenitors that comprise the majority of the early embryonic retina and that differentiation within this group is regulated by Notch. Notch activity in vivo was diminished using antisense oligonucleotides or augmented using a retrovirally transduced constitutively active allele of Notch. The number of ganglion cells produced was inversely related to the level of Notch activity. In addition, the Notch ligand Delta inhibited retinal progenitors from differentiating as ganglion cells to the same degree as did activated Notch in an in vitro assay. These results suggest a conserved strategy for neurogenesis in the retina and describe a versatile in vitro and in vivo system with which to examine the action of the Notch pathway in a specific cell fate decision in a vertebrate.

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NBS1 interacts with Notch signaling in neuronal homeostasis

Nucleic Acids Research ◽

10.1093/nar/gkaa716 ◽

2020 ◽

Vol 48 (19) ◽

pp. 10924-10939

Author(s):

Zhong-Wei Zhou ◽

Murat Kirtay ◽

Nadine Schneble ◽

George Yakoub ◽

Mingmei Ding ◽

...

Keyword(s):

Notch Signaling ◽

Notch Pathway ◽

Direct Consequence ◽

Nijmegen Breakage Syndrome ◽

Neuron Development ◽

Genetic Ablation ◽

Notch Activity ◽

And Migration

Abstract NBS1 is a critical component of the MRN (MRE11/RAD50/NBS1) complex, which regulates ATM- and ATR-mediated DNA damage response (DDR) pathways. Mutations in NBS1 cause the human genomic instability syndrome Nijmegen Breakage Syndrome (NBS), of which neuronal deficits, including microcephaly and intellectual disability, are classical hallmarks. Given its function in the DDR to ensure proper proliferation and prevent death of replicating cells, NBS1 is essential for life. Here we show that, unexpectedly, Nbs1 deletion is dispensable for postmitotic neurons, but compromises their arborization and migration due to dysregulated Notch signaling. We find that Nbs1 interacts with NICD-RBPJ, the effector of Notch signaling, and inhibits Notch activity. Genetic ablation or pharmaceutical inhibition of Notch signaling rescues the maturation and migration defects of Nbs1-deficient neurons in vitro and in vivo. Upregulation of Notch by Nbs1 deletion is independent of the key DDR downstream effector p53 and inactivation of each MRN component produces a different pattern of Notch activity and distinct neuronal defects. These data indicate that neuronal defects and aberrant Notch activity in Nbs1-deficient cells are unlikely to be a direct consequence of loss of MRN-mediated DDR function. This study discloses a novel function of NBS1 in crosstalk with the Notch pathway in neuron development.

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Select γ-Secretase Inhibitors Induce Apoptosis in Pre-B ALL Cells and Disrupt the Balance Between Constitutive Notch Signaling and Repression.

Blood ◽

10.1182/blood.v112.11.1917.1917 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1917-1917

Author(s):

Bridget S. Wilson ◽

Xiangbing Meng ◽

Tomas Mazel ◽

Cheryl L. Willman ◽

Susan Atlas ◽

...

Keyword(s):

Cell Death ◽

Notch Signaling ◽

Lymphoblastic Leukemia ◽

Notch Pathway ◽

Caspase Activation ◽

Notch 1 ◽

Activating Mutations ◽

Important Group ◽

Notch Receptors ◽

Activation Status

Abstract Several γ secretase inhibitors (GSIs) were tested for the ability to induce apoptosis in precursor B acute lymphoblastic leukemia (pre-B ALL) cells. Of five GSI’s tested, treatment with two compounds resulted in effective killing of both pre-B lymphoblasts and cells from multiple pre-B ALL lines. Since Notch receptors represent an important group of γ secretase targets, we evaluated expression and activation status of Notch receptors in CD19+ lymphoblasts from pediatric pre-B ALL patients, as well as cultured pre-B ALL cells. We found that, unlike T-ALL where activating mutations are common, pre-B ALL cells appear to drive constitutive Notch signaling through autocrine signals. Blasts from 11 patients expressed 3 Notch receptors and multiple Notch counter-ligands. Expression of Notch pathway genes was also confirmed by microarray analysis of genes expressed in 207 children with high risk B precursor ALL. GSI treatment of pre-B ALL cells led to dephosphorylation of AKT and Foxo3, Bim expression and caspase activation. GSI treatment also blocked cleavage of Notch 1 and 2 to their active forms and inhibited expression of Notch targets, Hey2 and Myc. In contrast, increased expression of Hes1 and Hey1 was correlated with GSI-induced loss of the co-repressor, SMRT. GSI treatment appears to induce precursor B cell death by disrupting the balance between constitutive Notch signaling and repression.

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Notch Signaling Represses Mir-223 in T-Cell Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v118.21.4630.4630 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4630-4630

Author(s):

Samuel D Gusscott ◽

Florian Kuchenbauer ◽

Andrew P Weng

Keyword(s):

Acute Lymphoblastic Leukemia ◽

T Cell ◽

Cell Growth ◽

Notch Signaling ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Gamma Secretase ◽

Protein Levels ◽

Cell Acute Lymphoblastic Leukemia ◽

T Cell Leukemogenesis

Abstract Abstract 4630 T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer of immature T cells that often shows aberrant activation of the Notch1 signaling pathway. Several studies have utilized mRNA expression profiling to identify downstream mediators of oncogenic Notch signaling in this context. Since microRNAs (miRNAs) have in recent years been shown to play important roles in hematological maliganancy, we performed a microarray-based screen for Notch-dependent miRNA expression in T-ALL. Jurkat and P12-Ichikawa cell lines were treated with gamma-secretase inhibitor to block Notch signaling vs. DMSO control for 4 days and profiled using Exigon miRCURY LNA miRNA microarrays. Surprisingly few miRNAs were found to be regulated by this approach; however, one of the hits, miR-223, showed consistent upregulation after gamma-secretase treatment in Jurkat cells and 5 additional human T-ALL cell lines assessed by miRNA qPCR. This observation was unique to human T-ALL as murine models of T-ALL showed no evidence for Notch-dependent miR-223 expression. Given that canonical Notch signaling results in transcriptional activation, our observation that Notch signaling is associated with reduced miR-223 expression suggests an intermediary repressor may be involved. miR-223 has been reported to play an important role in normal granulopoiesis, to be expressed relatively highly in T-ALL with myeloid-like gene features, and most recently to accelerate Notch-mediated T-cell leukemogenesis. To explore potential functional consequences for Notch-dependent miR-223 repression in T-ALL, candidate miR-223 targets identified by TargetScan software were analyzed with Ingenuity Pathway Analysis software, which indicated IGF-1, insulin receptor, PTEN, and ERK5 signaling pathways as the top hits. We recently reported IGF1R signaling to be important for growth and viability of bulk T-ALL cells as well as for leukemia-initiating cell activity. Additionally, we reported that Notch signaling directly upregulates IGF1R transcription by binding to an intronic enhancer which is present between exons 21/22 in the human, but not mouse IGF1R locus. As miR-223 has previously been reported to target IGF1R mRNA and reduce its translation, we hypothesized that Notch signaling may also upregulate net IGF1R protein expression by repressing miR-223. To test this hypothesis, we transduced several human T-ALL cell lines with miR-223 retrovirus and observed a modest decrease in total IGF1R protein levels by western blot; however, no significant change was observed in surface IGF1R levels as assessed by flow cytometry. Addtionally, knockdown of miR-223 by lentiviral expression miR-223 target sequences (miR-223 “sponge”) resulted in modestly increased total IGF1R protein levels, but again showed no demonstrable effect on surface IGF1R levels. Of note, we also observed no apparent effect of either overexpression or knockdown of miR-223 on bulk cell growth or viability. We interpret these findings to suggest that Notch signaling does not have major effects on the miR transcriptome, and that up- or down-modulation of miR-223 in established T-ALL cells does not have significant effects on overall cell growth/viability. Further studies will be required to determine if miR-223 may act in concert with other Notch target genes to modulate cell physiology. Disclosures: No relevant conflicts of interest to declare.

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Multifaceted regulation of Notch signaling by glycosylation

Glycobiology ◽

10.1093/glycob/cwaa049 ◽

2020 ◽

Author(s):

Ashutosh Pandey ◽

Nima Niknejad ◽

Hamed Jafar-Nejad

Keyword(s):

Notch Signaling ◽

Cell Biology ◽

Normal Development ◽

Notch Pathway ◽

Cell Types ◽

Notch Signaling Pathway ◽

Protein Glycosylation ◽

Notch Receptors ◽

Current Review ◽

Notch Activation

Abstract To build a complex body composed of various cell types and tissues and to maintain tissue homeostasis in the postembryonic period, animals use a small number of highly conserved intercellular communication pathways. Among these is the Notch signaling pathway, which is mediated via the interaction of transmembrane Notch receptors and ligands usually expressed by neighboring cells. Maintaining optimal Notch pathway activity is essential for normal development, as evidenced by various human diseases caused by decreased and increased Notch signaling. It is therefore not surprising that multiple mechanisms are used to control the activation of this pathway in time and space. Over the last 20 years, protein glycosylation has been recognized as a major regulatory mechanism for Notch signaling. In this review, we will provide a summary of the various types of glycan that have been shown to modulate Notch signaling. Building on recent advances in the biochemistry, structural biology, cell biology and genetics of Notch receptors and the glycosyltransferases that modify them, we will provide a detailed discussion on how various steps during Notch activation are regulated by glycans. Our hope is that the current review article will stimulate additional research in the field of Notch glycobiology and will potentially be of benefit to investigators examining the contribution of glycosylation to other developmental processes.

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Structural and functional analysis of the repressor complex in the Notch signaling pathway ofDrosophila melanogaster

Molecular Biology of the Cell ◽

10.1091/mbc.e11-05-0420 ◽

2011 ◽

Vol 22 (17) ◽

pp. 3242-3252 ◽

Cited By ~ 31

Author(s):

Dieter Maier ◽

Patricia Kurth ◽

Adriana Schulz ◽

Andrew Russell ◽

Zhenyu Yuan ◽

...

Keyword(s):

Cell Culture ◽

Notch Signaling ◽

Target Genes ◽

Notch Pathway ◽

Structural Similarity ◽

Receptor Activation ◽

Notch Signaling Pathway ◽

Repressor Complex

In metazoans, the highly conserved Notch pathway drives cellular specification. On receptor activation, the intracellular domain of Notch assembles a transcriptional activator complex that includes the DNA-binding protein CSL, a composite of human C-promoter binding factor 1, Suppressor of Hairless of Drosophila melanogaster [Su(H)], and lin-12 and Glp-1 phenotype of Caenorhabditis elegans. In the absence of ligand, CSL represses Notch target genes. However, despite the structural similarity of CSL orthologues, repression appears largely diverse between organisms. Here we analyze the Notch repressor complex in Drosophila, consisting of the fly CSL protein, Su(H), and the corepressor Hairless, which recruits general repressor proteins. We show that the C-terminal domain of Su(H) is necessary and sufficient for forming a high-affinity complex with Hairless. Mutations in Su(H) that affect interactions with Notch and Mastermind have no effect on Hairless binding. Nonetheless, we demonstrate that Notch and Hairless compete for CSL in vitro and in cell culture. In addition, we identify a site in Hairless that is crucial for binding Su(H) and subsequently show that this Hairless mutant is strongly impaired, failing to properly assemble the repressor complex in vivo. Finally, we demonstrate Hairless-mediated inhibition of Notch signaling in a cell culture assay, which hints at a potentially similar repression mechanism in mammals that might be exploited for therapeutic purposes.

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