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

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

Blood ◽

10.1182/blood-2019-122856 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 4661-4661

Author(s):

Kirsten Canté-Barrett ◽

Laurent Holtzer ◽

Henk van Ooijen ◽

Rico Hagelaar ◽

Valentina Cordo' ◽

...

Keyword(s):

Notch Signaling ◽

Target Genes ◽

Lymphoblastic Leukemia ◽

Notch Pathway ◽

Cell Types ◽

Activity Score ◽

Free Survival ◽

Pathway Activity ◽

Notch Activity ◽

Protein Levels

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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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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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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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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Constitutive Notch pathway activation in murine ZMYM2-FGFR1–induced T-cell lymphomas associated with atypical myeloproliferative disease

Blood ◽

10.1182/blood-2010-07-295725 ◽

2011 ◽

Vol 117 (25) ◽

pp. 6837-6847 ◽

Cited By ~ 23

Author(s):

MingQiang Ren ◽

John K. Cowell

Keyword(s):

T Cell ◽

Notch Signaling ◽

Transcriptional Activation ◽

Target Genes ◽

Hematologic Malignancy ◽

Notch Pathway ◽

Myeloproliferative Disease ◽

Constitutive Activation ◽

T Cell Lymphomas

Abstract The ZMYM2-FGFR1 (formerly known as ZNF198-FGFR1) fusion kinase induces stem cell leukemia–lymphoma syndrome (SCLL), a hematologic malignancy characterized by rapid transformation to acute myeloid leukemia and T-lymphoblastic lymphoma. In the present study, we demonstrate frequent, constitutive activation of Notch1 and its downstream target genes in T-cell lymphomas that arose in a murine model of ZMYM2-FGFR1 SCLL. Notch up-regulation was also demonstrated in human SCLL- and FGFR1OP2-FGFR1-expressing KG-1 cells. To study the role of Notch in T-cell lymphomagenesis, we developed a highly tumorigenic cell line from ZMYM2-FGFR1–expressing cells. Pharmacologic inhibition of Notch signaling in these cells using γ-secretase inhibitors significantly delayed leukemogenesis in vivo. shRNA targeting of Notch1, as well as c-promoter–binding factor 1 (CBF1) and mastermind-like 1 (MAML1), 2 essential cofactors involved in transcriptional activation of Notch target genes, also significantly delayed or inhibited tumorigenesis in vivo. Mutation analysis demonstrated that 5′ promoter deletions and alternative promoter usage were responsible for constitutive activation of Notch1 in all T-cell lymphomas. These data demonstrate the importance of Notch signaling in the etiology of SCLL, and suggest that targeting this pathway could provide a novel strategy for molecular therapies to treat SCLL patients.

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Human myostatin negatively regulates human myoblast growth and differentiation

AJP Cell Physiology ◽

10.1152/ajpcell.00012.2011 ◽

2011 ◽

Vol 301 (1) ◽

pp. C195-C203 ◽

Cited By ~ 66

Author(s):

Craig McFarlane ◽

Gu Zi Hui ◽

Wong Zhi Wei Amanda ◽

Hiu Yeung Lau ◽

Sudarsanareddy Lokireddy ◽

...

Keyword(s):

Notch Signaling ◽

Target Genes ◽

Transforming Growth Factor ◽

Myogenic Differentiation ◽

Muscle Growth ◽

Notch Pathway ◽

Transforming Growth Factor Β ◽

Myoblast Differentiation ◽

Myogenic Regulatory Factor ◽

Human Myoblast

Myostatin, a member of the transforming growth factor-β superfamily, has been implicated in the potent negative regulation of myogenesis in murine models. However, little is known about the mechanism(s) through which human myostatin negatively regulates human skeletal muscle growth. Using human primary myoblasts and recombinant human myostatin protein, we show here that myostatin blocks human myoblast proliferation by regulating cell cycle progression through targeted upregulation of p21. We further show that myostatin regulates myogenic differentiation through the inhibition of key myogenic regulatory factors including MyoD, via canonical Smad signaling. In addition, we have for the first time demonstrated the capability of myostatin to regulate the Notch signaling pathway during inhibition of human myoblast differentiation. Treatment with myostatin results in the upregulation of Hes1, Hes5, and Hey1 expression during differentiation; moreover, when we interfere with Notch signaling, through treatment with the γ-secretase inhibitor L-685,458, we find enhanced myotube formation despite the presence of excess myostatin. Therefore, blockade of the Notch pathway relieves myostatin repression of differentiation, and myostatin upregulates Notch downstream target genes. Immunoprecipitation studies demonstrate that myostatin treatment of myoblasts results in enhanced association of Notch1-intracellular domain with Smad3, providing an additional mechanism through which myostatin targets and represses the activity of the myogenic regulatory factor MyoD. On the basis of these results, we suggest that myostatin function and mechanism of action are very well conserved between species, and that myostatin regulation of postnatal myogenesis involves interactions with numerous downstream signaling mediators, including the Notch pathway.

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K-RasG12D–induced T-cell lymphoblastic lymphoma/leukemias harbor Notch1 mutations and are sensitive to γ-secretase inhibitors

Blood ◽

10.1182/blood-2008-03-147587 ◽

2008 ◽

Vol 112 (8) ◽

pp. 3373-3382 ◽

Cited By ~ 64

Author(s):

Thomas Kindler ◽

Melanie G. Cornejo ◽

Claudia Scholl ◽

Jianing Liu ◽

Dena S. Leeman ◽

...

Keyword(s):

T Cell ◽

Target Genes ◽

Bone Marrow Cells ◽

Mapk Pathway ◽

Notch Pathway ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Double Positive ◽

The Impact ◽

Notch1 Mutations

Abstract To study the impact of oncogenic K-Ras on T-cell leukemia/lymphoma development and progression, we made use of a conditional K-RasG12D murine knockin model, in which oncogenic K-Ras is expressed from its endogenous promoter. Transplantation of whole bone marrow cells that express oncogenic K-Ras into wild-type recipient mice resulted in a highly penetrant, aggressive T-cell leukemia/lymphoma. The lymphoblasts were composed of a CD4/CD8 double-positive population that aberrantly expressed CD44. Thymi of primary donor mice showed reduced cellularity, and immunophenotypic analysis demonstrated a block in differentiation at the double-negative 1 stage. With progression of disease, approximately 50% of mice acquired Notch1 mutations within the PEST domain. Of note, primary lymphoblasts were hypersensitive to γ-secretase inhibitor treatment, which is known to impair Notch signaling. This inhibition was Notch-specific as assessed by down-regulation of Notch1 target genes and intracellular cleaved Notch. We also observed that the oncogenic K-Ras-induced T-cell disease was responsive to rapamycin and inhibitors of the RAS/MAPK pathway. These data indicate that patients with T-cell leukemia with K-Ras mutations may benefit from therapies that target the NOTCH pathway alone or in combination with inhibition of the PI3K/AKT/MTOR and RAS/MAPK pathways.

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Plasma imatinib levels and ABCB1 polymorphism influences early molecular response and failure-free survival in newly diagnosed chronic phase CML patients

Scientific Reports ◽

10.1038/s41598-020-77140-9 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Bharathi M. Rajamani ◽

Esther Sathya Bama Benjamin ◽

Aby Abraham ◽

Sukanya Ganesan ◽

Kavitha M. Lakshmi ◽

...

Keyword(s):

Myeloid Leukemia ◽

Target Genes ◽

Chronic Phase ◽

Predictive Biomarkers ◽

Imatinib Therapy ◽

Molecular Response ◽

Event Free Survival ◽

Free Survival ◽

Dosing Algorithm ◽

Sokal Score

AbstractAchieving early molecular response (EMR) has been shown to be associated with better event free survival in patients with chronic phase chronic myeloid leukemia (CP-CML) on Imatinib therapy. We prospectively evaluated the factors influencing the 2-year failure free survival (FFS) and EMR to imatinib therapy in these patients including day29 plasma Imatinib levels, genetic variants and the gene expression of target genes in imatinib transport and biotransformation. Patients with low and intermediate Sokal score had better 2-year FFS compared to those with high Sokal Score (p = 0.02). Patients carrying ABCB1-C1236T variants had high day29 plasma imatinib levels (P = 0.005), increased EMR at 3 months (P = 0.044) and a better 2 year FFS (P = 0.003) when compared to those with wild type genotype. This translates to patients with lower ABCB1 mRNA expression having a significantly higher intracellular imatinib levels (P = 0.029). Higher day29 plasma imatinib levels was found to be strongly associated with patients achieving EMR at 3 months (P = 0.022), MMR at 12 months (P = 0.041) which essentially resulted in better 2-year FFS (p = 0.05). Also, patients who achieved EMR at 3 months, 6 months and MMR at 12 months had better FFS when compared to those who did not. This study suggests the incorporation of these variables in to the imatinib dosing algorithm as predictive biomarkers of response to Imatinib therapy.

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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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